Giuseppe Palumbo was born in Naples Italy on march 16th 1945. He is married with two children. He got his degree in Physical Chemistry in 1969. He has worked as post doc in Naples, Italy (1969-1971) and successively at the NIH (Bethesda, MD from 1973 to 1975). In 1976 he got a permanent position as Researcher at the Centro di Endocrinologia Sperimentale del CNR (Naples). In 1985 he was at the NIH (Bethesda) for two year as Visiting Scientist. At the end of 1986 he got the chair of Biochemistry at the medical School of Catanzaro (Italy). In 1989 he was again at the NIH as Visiting Scientist and finally (1990) he returned to Naples as full Professor of General Pathology. Major interests: HDL and LDL; Thyroglobulin; analytical methods; Molecular Oncology (apoptosis, differentiation, senescence), Photodynamic therapy; Space Biology.
Abstract: Photodynamic therapy (PDT) combines a drug (a photosensitiser or photosensitising agent) with a specific type of light to kill cancer cells. It is a minimally invasive treatment, with great potential in malignant disease and premalignant conditions. Following the administration of the photosensitiser, light of the appropriate wavelength is directed onto the abnormal tissue where the drug has preferentially accumulated. Upon light activation, the photosensitiser transfers its excess energy to molecular oxygen to produce an excited state (i.e., the highly reactive singlet oxygen) that causes oxidative damage at the site of its generation. The energy transfer occurs either directly to oxygen or through an indirect mechanism that requires the formation of intermediate radical species. Many photosensitisers have been developed, but only a few have been approved for therapy in humans. Basic research in model systems (animals, cell lines) has unravelled some fundamental cellular processes involved in the cell response to PDT. The exploitation of relevant molecular observations, the discovery and introduction of new sensitisers, the progress in the light delivery systems and light dosimetry are all concurring to the increase of PDT therapeutic efficacy. However, this field has not yet reached maturity. This review briefly analyses the relevant properties of most photosensitisers and their field of application. Special attention is dedicated to the effects observed in model cancer systems; speculation and suggestions of possible future research directions are also offered.
Abstract: The endoplasmic reticulum represents the quality control site of the cell for folding and assembly of cargo proteins. A variety of conditions can alter the ability of the endoplasmic reticulum (ER) to properly fold proteins, thus resulting in ER stress. Cells respond to ER stress by activating different signal transduction pathways leading to increased transcription of chaperone genes, decreased protein synthesis, and eventually to apoptosis. In the present paper we analyzed the role that the adaptor protein tumor necrosis factor-receptor associated factor 2 (TRAF2) plays in regulating cellular responses to apoptotic stimuli from the endoplasmic reticulum. Mouse embryonic fibroblasts derived from TRAF2-/- mice were more susceptible to apoptosis induced by ER stress than the wild type counterpart. This increased susceptibility to ER stress-induced apoptosis was because of an increased accumulation of reactive oxygen species following ER stress, and was abolished by the use of antioxidant. In addition, we demonstrated that the NF-kappaB pathway protects cells from ER stress-induced apoptosis, controlling ROS accumulation. Our results underscore the involvement of TRAF2 in regulating ER stress responses and the role of NF-kappaB in protecting cells from ER stress-induced apoptosis.
Abstract: We investigated the effect of modeled microgravity (MMG) on normal vascular smooth muscle cells (VSMC) and neoplastic human breast cancer cells (MCF-7). In both cell types, MMG induced partial arrest in G2M and increased p14-3-3, HSP70, HSP60 and p21 expression. Cells synchronized by 24h starvation reentered the normal cycle within 24h if released in complete medium and exposed to normal gravity, but not if exposed to MMG. Similarly, MMG prevented VSMC and MCF-7 cells from overcoming growth arrest and re-synthesizing DNA. This study shows that cells adjust their metabolic rate in response to MMG.
Abstract: We compared the effects of monotherapy (photodynamic therapy or chemotherapy) versus combination therapy (photodynamic therapy plus a specific drug) on the non-small cell lung cancer cell line H1299. Our aim was to evaluate whether the additive/synergistic effects of combination treatment were such that the cytostatic dose could be reduced without affecting treatment efficacy. Photodynamic therapy was done by irradiating Photofrin-preloaded H1299 p53/p16-null cells with a halogen lamp equipped with a bandpass filter. The cytotoxic drugs used were cis-diammine-dichloroplatinum [II] (CDDP or cisplatin) and 2',2'-difluoro-2'-deoxycytidine (gemcitabine). Various treatment combinations yielded therapeutic effects (trypan blue dye exclusion test) ranging from additive to clearly synergistic, the most effective being a combination of photodynamic therapy and CDDP. To gain insight into the cellular response mechanisms underlying favorable outcomes, we analyzed the H1299 cell cycle profiles and the expression patterns of several key proteins after monotherapy. In our conditions, we found that photodynamic therapy with Photofrin targeted G0-G1 cells, thereby causing cells to accumulate in S phase. In contrast, low-dose CDDP killed cells in S phase, thereby causing an accumulation of G0-G1 cells (and increased p21 expression). Like photodynamic therapy, low-dose gemcitabine targeted G0-G1 cells, which caused a massive accumulation of cells in S phase (and increased cyclin A expression). Although we observed therapeutic reinforcement with both drugs and photodynamic therapy, reinforcement was more pronounced when the drug (CDDP) and photodynamic therapy exert disjointed phase-related cytotoxic activity. Thus, if photodynamic therapy is appropriately tuned, the dose of the cytostatic drug can be reduced without compromising the therapeutic response.
Abstract: PURPOSE: Treatment of tumor cells by chemotherapy activates a series of responses ranging from apoptosis to premature senescence and repair. Survival responses are characterized by inhibition of cyclin-dependent kinases. Because inhibition of cyclin-dependent kinases represents a distinctive feature of DNA damage-induced prosurvival responses, we investigated the possibility that the cyclin-dependent kinase inhibitor roscovitine modulates drug-induced responses in human adenocarcinoma cells, favoring cell survival. EXPERIMENTAL DESIGN: Sublethal concentrations of doxorubicin were used to induce premature senescence in human adenocarcinoma cells. The effect of the cyclin-dependent kinase inhibitor roscovitine on the doxorubicin-dependent cell cycle checkpoint activation and DNA repair pathways was evaluated. RESULTS: Roscovitine reinforces doxorubicin-dependent G(1) checkpoint in A549 and HEC1B cells leading to decreased frequency of double-strand breaks and to the preferential induction of senescence and enhanced clonogenic survival. However, in other tumor cell lines, such as HCT116 and H1299, combined treatment with doxorubicin and roscovitine increases the frequency of double-strand breaks and dramatically sensitizes to doxorubicin. This unexpected effect of roscovitine depends on a novel ability to inhibit DNA double-strand break repair processes and requires inactivation of the pRb pathway. CONCLUSIONS: Roscovitine, by hindering DNA repair processes, has the potential to inhibit recovery of mildly damaged tumor cells after doxorubicin treatment and to increase the susceptibility of tumor cells to chemotherapy. However, in some tumor cells, the cell cycle inhibitory function of roscovitine prevails over the DNA repair inhibitory activity, favoring premature senescence and clonogenic growth. These data indicate a novel mechanism underlying combined chemotherapy, which may have wide application in treatment of carcinomas.
Abstract: OBJECTIVE: We investigated the effects of photodynamic therapy (PDT) combined with low-dose chemotherapy on breast cancer cells. Photodynamic treatment was administered by irradiating indocyanine green-preloaded MCF-7 cells with an IR diode laser source at 805 nm; cisplatin was used for chemotherapy. METHODS: The dose-response phenomena associated with the two treatments administered individually and together were evaluated with the following tests: trypan blue dye exclusion, 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay, clonogenic survival, thymidine and methionine incorporation, and insulin-dependent and insulin-independent glucose transport. RESULTS: Viability and metabolic data demonstrated mutual reinforcement of therapeutic efficacy. However, isobolographic analysis of quantal and variable data indicated that reinforcement was additive according to trypan blue data and synergistic according to MTT data. To investigate the molecular mechanisms underlying alterations in cell proliferation and apoptosis, we evaluated (by Western blotting) the expression of proteins Bcl-2, Bax, Bcl-X(L), p21, p53, and poly(ADP-ribose) polymerase. Photodynamic treatment caused transient selective destruction of Bcl-2 and up-regulation of Bax. It also induced apoptosis in a limited fraction of cells (10-12%). Flow cytometry data showed that PDT killed mostly G(1)-phase cells, whereas cisplatin killed mostly S-phase cells. This disjointed phase-related effect may account for the favorable effects exerted by combined treatment. CONCLUSIONS: Our findings imply that low doses of cytostatic drugs may be as effective or even more effective than currently used doses if appropriately combined with PDT.
Abstract: In this paper we report the behaviour of normal vascular smooth muscle cells and transformed breast cancer cells under normal versus simulated microgravity conditions by comparing cell proliferation, Glucose transport, Methionine uptake and protein synthesis. Modeled microgravity profoundly affects cell growth (especially in normal cells) and Glucose or Methionine metabolism (although to different extent in the two cell lines). Since both cells own responsive insulin receptors, the comparison was extended to insulin-stimulated versus unstimulated conditions. We report that the detected metabolic changes were strongly enhanced when the cells were simultaneously stimulated with insulin and subjected to modeled microgravity stress. Such observations may have important returns for human health in space; they deserve further attention.
Abstract: The apoptosis regulator Bcl-2 has been shown to modulate cell-cycle progression, favouring a quiescent state over a proliferative state, in both normal and tumour cells. We show here that constitutive expression of Bcl-2 in human carcinoma cells results in a cell-cycle arrest that within a few days can become irreversible. Arrested cells acquire a senescent-like phenotype, which consists of several characteristic morphological alterations and increased activity of senescence-associated beta-galactosidase. The induction of the premature senescence programme is mediated by inhibition of Cdk2 kinase activity, and p27(KIP1) is required to maintain the senescent phenotype. We propose that the ability to activate an endogenous premature senescence programme allows Bcl-2 to suppress tumour growth. These results suggest that the down-regulation of Bcl-2 expression, which has been observed during the development and progression of human carcinoma, is related to the ability of Bcl-2 to severely hamper the growth of carcinoma cells and to induce a permanent cell-cycle arrest, with the features of senescence.
Abstract: In the human endometrial carcinoma cell line HEC1B, the overexpression of Bcl-2 leads to a G2/M cell cycle arrest. The experiments presented in this work suggest a direct interaction between the antiapoptotic protein Bcl-2 and the cyclin-dependent kinase Cdk-2 and suggest that such interaction is cell cycle dependent. While further experiments are currently under way in our laboratory to elucidate the significance of Cdk-2/Bcl-2 complexes in PCD and cell cycle regulation, we demonstrate also a specific inhibitory function of Bcl-2 on the activity of the coprecipitated kinase.
Abstract: Photosensitization of HEC1-B cells with a low concentration of hypericin and doses of light below 10 J/cm(2) caused cell death (apoptosis occurred mainly at doses between 2 and 5 J/cm(2), whereas necrosis prevailed above 6 J/cm(2)). However, pre-exposure of cells to innocuous irradiation (2 J/cm(2)) and successive challenge with a light dose that normally induced apoptosis (5 J/cm(2)) altered the expression of the proteins involved in the regulation of apoptosis, stress response and cell cycle. This change resulted in a significant increase in cell photo-tolerance.
Abstract: OBJECTIVE: In various human tumors the expression of Bcl-2 appears to vary significantly during the transformation process. Indeed, in several glandular systems, Bcl-2 levels appear to be sustained in premalignant lesions and rather low after the malignant change. Since we recently reported that transformed human endometrial cells display constitutively low levels of Bcl-2, we set out to investigate the biological meaning of this down-regulation. To this end we analyzed the effects of Bcl-2 forced overexpression in a moderately differentiated endometrial cell line of human origin. METHODS: Bcl-2 overexpression was obtained by transfecting HEC1B human endometrial adenocarcinoma cells with a suitable bcl-2 vector. The effects of Bcl-2 overepression were evaluated in several transfectants (cell clones and mixed populations) by FACS, growth rates, cloning efficiencies, and modification of the phosphorylation status of the pRb protein. Accompanying changes in the expression of the CDK inhibitor p21(WAF1/CIP1) were evaluated as well. RESULTS: Bcl-2 overexpression resulted in a reduced cell proliferation rate, decreased cloning efficiency, appreciable cell morphology changes, G2/M cell cycle arrest, remarkable accumulation of the dephosphorylated form of retinoblastoma protein, and a significant rise in p21(WAF1/CIP1). CONCLUSIONS: From these observation it may be deduced that the observed loss and down-regulation of the antiapoptotic protein in endometrial glandular human tumors is not random but possibly related to the cellular transformation process. It may be also inferred that the coincidence of a progressive fading of both Bcl-2 and cyclin inhibitor p21(WAF1/CIP1) expressions, together with accumulation of the hyperphosphorylated form of the retinoblastoma protein, may be seen as a potential indicator of ongoing malignant changes.
Abstract: We have observed that exposure of U937 cells, pre-incubated for 18 h with 0.2 microM hypericin, to 599 nm laser radiation with a fluence of 2.5 J/cm(2) renders them insensitive to higher light doses. In fact, pre-sensitized cells appear to be fully resistant to light doses that normally determine massive cellular apoptosis in experimental photo-dynamic therapy. The appearance of the photo-resistance, as measured by evaluating the changes in levels of expression of pro and anti apoptotic proteins, PARP fragmentation and cell viability is exclusively observed with exposure to light doses not exceeding 5-6 J/cm(2). Above this energy, necrosis replaces apoptosis upon photo-stimulation of U937 cells. Here, we describe the appearance of photo-resistance in hypericin-loaded U937 cells, but could not fully unravel the molecular mechanism underlying this process. However, the observed stimulation of the expression of the HSP-70 protein upon photo-induced stress may suggest its involvement in this process.
Abstract: In this paper we present some preliminary results on alteration of gene expression caused by radiation on human endometrial cells. To this purpose, we have studied the modulation of the expression of the bcl-2 gene family in two cell lines following irradiations with low energy protons and gamma-rays from a 60Co. The two epithelial cell strains, namely AN3Ca and HEC1B cells, both obtained from human neoplastic endometrial tissues, grow in culture and continue to maintain some differentiated functions typical of the original tissue. Indeed, these cells, that can be considered as representative of different stages of cellular transformation of endometrium. Because their epithelial nature and rapid growth, the expression of genes related to the maintenance of the cellular homeostasis (correction of omeostasis), as the pro and anti-apoptotic ones, is expected to be susceptible to changes in environment, including radiation. The effects have been evaluated in terms of both cell survival and changes in the expression of pro- and anti apoptotic proteins. Even though the data reported above can not be considered complete and/or definitive, nevertheless, in whole, they confirm that these cells may constitute a suitable model system to study, at molecular level, the effects of cosmic radiation on endometrium. Further observation, ensuing from these preliminary data, is that endometrial cells present different sensitivity to radiation in regard to its 'quality' and 'dosage', in accord to the original stage of differentiation.
Abstract: OBJECTIVES: Previous histochemical observations have suggested a possible involvement of the bcl-2 family genes in the acquisition of neoplastic phenotype of the endometrium. Since knowledge of the type and function of genes controlling the transformed cell may result in new diagnostic, prognostic, and therapeutic approaches, we have investigated at the molecular level the biological role of bcl-2 family genes in endometrial neoplastic cells. METHODS: To investigate the relationship between the sensitivity to apoptosis and the expression of the bcl-2 family genes, we set up a model system consisting of four human endometrial carcinoma cell lines. This system constitutes an array of two cell pairs presenting, respectively, endometrioid and adenosquamous phenotypes. G2 and G3 gradings are represented within each pair; in addition, each set contains one cell line that is apoptosis-sensitive and one that is resistant. Transfection of bcl-2 and bcl-XL into apoptosis-sensitive cells was used to monitor the biological function of protective genes. RESULTS: A differential pattern of expression of bcl-2 family genes was observed in apoptosis-sensitive versus resistant cells, independent from the histological subtype. Resistant lines exhibited high amounts of Bcl-XL and low amounts of Bcl-2. Bax expression clearly correlates with cellular susceptibility to apoptosis. Transfection of bcl-XL resulted in a dose-dependent enhancement in resistance toward apoptosis. In contrast, the main effect of bcl-2 constitutive overexpression was to drastically abate the proliferative potential of transfected cells. CONCLUSIONS: These data demonstrate, at the molecular level, that bcl-XL is selected as an apoptosis-protective gene in place of bcl-2 while bax retains its dominant proapototic role.
Abstract: We have found that 17beta-estradiol induces bcl-2 transcription in human breast cancer MCF-7 cells. To identify cis-acting elements involved in this regulation, we have analyzed hormone responsiveness of transiently transfected reporter constructs containing the bcl-2 major promoter (P(1)). Hormone inducibility was observed only when either of two sequences, located within the bcl-2 coding region and showing one and two mutations with respect to the consensus estrogen-responsive element, were inserted downstream from the P(1) promoter. Both sequences behaved as enhancers exclusively in cells expressing the estrogen receptor and were able to bind this receptor in in vitro assays. Transfections into MCF-7 cells of plasmids carrying a bcl-2 cDNA fragment which included these two elements revealed that their simultaneous presence resulted in an additive effect on reporter gene activity, whose size resembled the increase of endogenous bcl-2 mRNA level observed in untransfected cells after hormone treatment. Moreover, the identified elements were able to mediate up-regulation of bcl-2 expression by 17beta-estradiol, since exogenous bcl-2 mRNA was induced by hormone challenge of MCF-7 cells transiently transfected with a vector containing the bcl-2 coding sequence cloned under the control of a non-estrogen-responsive promoter. Finally, we show that hormone prevention of apoptosis, induced by incubating MCF-7 cells with hydrogen peroxide, was strictly related to bcl-2 up-regulation. Our results indicate that the bcl-2 major promoter does not contain cis-acting elements directly involved in transcriptional control by 17beta-estradiol and that hormone treatment inhibits programmed cell death in MCF-7 cells, inducing bcl-2 expression via two estrogen-responsive elements located within its coding region.
Abstract: The present study is concerned with the effects of an IR diode laser source emitting at 805 nm on a human leukaemic cell strain from a histiocytic lymphoma (U937) pre-loaded with the dye indocyanine green (ICG). The first aim of this work was the assessment of the earliest cellular defense events occurring upon ICG photosensitization. To this purpose, photosensitization was performed at low ICG concentration and low light energy density. The second aim was the comparative evaluation of the effects produced by continuous or fragmented irradiation. Independent of the irradiation method employed (continuous or fragmented), we could demonstrate that cells are forced to apoptosis, as indicated by the appearance in cell extracts of the 85-kDa proteolytic fragment of the poly(ADP-ribose)polymerase (PARP). This proteolysis, however, could be entirely prevented by a specific Caspase-3 inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-fmk). Indeed the only perceptible change in the expression of pro/antiapoptotic proteins produced by continuous photostimulation was a small, albeit reproducible, increase in Bax. In contrast, when the photostimulation was achieved by means of several consecutive pulses, we observed not only a remarkable increase in Bax but also a noticeable abatement in Bcl-XL expression. The potential involvement of singlet oxygen in this process has been directly demonstrated by ICG photo-mediated oxidation of dithiothreitol in water. It has also been demonstrated that this oxidation is apparently more efficient when ICG is photostimulated by light pulses.
Abstract: Apoptosis may play an important role in atherogenesis. Oxidized low-density lipoprotein (oxLDL) promotes apoptosis in the arterial wall in addition to several other proatherogenic effects. Tocopherol supplements have been suggested to protect against coronary heart disease (CHD) in epidemiological studies. The effects of oxLDL and alpha- and gamma-tocopherol on apoptotic signaling pathways are poorly understood. Thus, the goal of the study was to investigate these pathways in the presence of copper-oxidized LDL and tocopherols in human coronary smooth muscle cells (SMC). We showed that oxLDL-mediated apoptosis, assessed by DNA fragmentation, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, and caspase activation stimulated several transcription factors and proapoptotic dynamic movements of the Bcl-2 family proteins through the mitogen-activated protein kinase (MAPK) and Jun kinase pathways. alpha-Tocopherol and gamma-tocopherol significantly reduced these molecular events and cell death effectors caspase-3 and -8. Under our experimental conditions, alpha-tocopherol was significantly more effective than gamma-tocopherol, and oxLDL-mediated apoptosis increased c-Jun, cyclic AMP-responsive element-binding, Ets-like element kinase-dependent 7, and activating transcription factor-2 proteins as well as nuclear activity of the activated protein-1 complex in human coronary SMC. Moreover, our results demonstrate that tocopherols may exert their antiatherogenic effects at least in part via reduction of the MAPK and JunK cascade together with a protective profile of apoptotic genes of the Bcl-2 family. These data are consistent with the beneficial effects of tocopherols on atherogenesis seen in experimental studies and on CHD in epidemiological surveys.
Abstract: BACKGROUND AND PURPOSE: Calcium-channel blockers (CCBs) reduce systolic blood pressure and stroke-related mortality in stroke-prone spontaneously hypertensive rats (SPSHR). Brain ischemia is associated with loss of intracellular antioxidants. Increased formation of oxygen radicals and oxidation of LDL may enhance arterial vasoconstriction by various mechanisms. CCBs that also exert antioxidative properties in vitro may therefore be particularly useful. To investigate such antioxidant effects in vivo, we determined several parameters of LDL oxidation in SPSHR treated with two 1,4-dihydropyridine-type (1,4-DHP) CCBs of different lipophilic properties and compared them with antioxidant-treated and untreated controls. We also tested whether these drugs decrease the formation of oxidation-specific epitopes in arteries. METHODS: Five groups of 9 to 14 SPSHR each (aged 8 weeks) were treated with 80 mg/kg body wt per day nifedipine, 1 mg or 0.3 mg/kg body wt per day lacidipine, vitamin E (100 IU/d), or carrier for 5 weeks. A group of Wistar-Kyoto rats was used as normotensive control. Plasma samples were taken, and LDL was isolated by ultracentrifugation. Then LDL was exposed to oxygen radicals generated by xanthine/xanthine oxidase reaction (2 mmol/L xanthine+100 mU/mL xanthine oxidase), and several parameters of oxidation were determined. The presence of native apolipoprotein B and oxidation-specific epitopes in the carotid and middle cerebral arteries was determined immunocytochemically. RESULTS: 1,4-DHP CCBs completely prevented mortality. Normotensive Wistar-Kyoto rats showed less oxidation than control SPSHR. Plasma lipoperoxide levels were 0.87+/-0.27 micromol/L in control SPSHR, 0.69+/-0.19 and 0.63+/-0.20 micromol/L in the groups treated with 0.3 and 1 mg lacidipine, respectively, and 0.68+/-0.23 micromol/L in nifedipine-treated animals (P<0.05 versus control SPSHR for all values). Both CCBs significantly decreased formation of conjugated dienes and prolonged the lag time in LDL exposed to oxygen radicals. Similarly, lipoperoxides and malondialdehyde were significantly reduced (P<0.05). Reduced relative electrophoretic mobility and increased trinitrobenzenesulfonic acid reactivity of LDL from treated rats (P<0.01) also indicated that fewer lysine residues of apolipoprotein B were oxidatively modified in the presence of 1,4-DHP CCBs. Finally, these drugs reduced the intimal presence of apolipoprotein B and oxidized LDL (oxidation-specific epitopes) in carotid and middle cerebral arteries. CONCLUSIONS: In the SPSHR model, 1,4-DHP CCBs reduce plasma and LDL oxidation and formation of oxidation-specific epitopes and prolong survival independently of blood pressure modifications. Our results support the concept that the in vivo protective effect of these drugs on cerebral ischemia and stroke may in part result from inhibition of oxidative processes.
Abstract: BACKGROUND: Atherosclerotic lesions in intracranial arteries occur later and are less extensive than in extracranial arteries. To investigate potential mechanisms responsible for this difference, in particular the atherogenic response to hypercholesterolemia and LDL oxidation, we compared the extent of fatty streak formation and the composition of these very early lesions in intracranial arteries of human fetuses from normocholesterolemic and hypercholesterolemic mothers with those in extracranial arteries. METHODS AND RESULTS: Lesions were quantified by computer-assisted image analysis of 30 oil red O-stained sections, each from the middle cerebral, basilar, and common carotid arteries and the abdominal aorta of human fetuses (spontaneous abortions and premature newborns who died within 12 hours of birth; both of fetal age 6.2+/-1.3 months) from 43 hypercholesterolemic mothers and 34 normocholesterolemic mothers. Macrophages, apolipoprotein B, and 2 epitopes of oxidized LDL in lesions were determined immunocytochemically. Activities of superoxide dismutase, catalase, and glutathione peroxidase in the arterial wall were also determined. Lesion numbers and sizes were dramatically greater in the abdominal aorta (area of the largest lesion per section: 66.5+/-10.9 x10(3) microm2) and the carotid (11. 6+/-5.3 x10(3) microm2) than in the basilar and middle cerebral artery (0.4+/-0.1 and 0.8+/-0.2 x10(3) microm2, respectively; P<0. 0001). Hypercholesterolemia resulted in a significant increase of lesion size in extracranial arteries but only a marginal increase in intracranial arteries. In analogy, hypercholesterolemia induced a much greater increase in the intimal presence of macrophages, apolipoprotein B, and oxidized LDL (oxidation-specific epitopes) in extracranial than in intracranial arteries. Immunocytochemistry did not indicate that lesions of intracranial arteries contain relatively less oxidized LDL than similar-size lesions of extracranial arteries. Activities of Mn-superoxide dismutase but not of Zn-superoxide dismutase, catalase, or glutathione peroxidase were significantly higher in both intracranial arteries. CONCLUSIONS: Exposure to hypercholesterolemia during fetal development results in extensive formation of fatty streaks in extracranial but not intracranial arteries. The fact that such a difference in lesion formation occurs in the absence of many other atherogenic risk factors found later in life suggests that differences in the atherogenic response to hypercholesterolemia are an important contributor to the slower onset of the disease in intracranial vessels in adults. Fetal arteries may allow elucidation of the mechanisms responsible, for example, better protection of intracranial arteries against free radical-mediated atherogenic processes.
Abstract: BACKGROUND: The enzyme lecithin-cholesterol acyl transferase (LCAT) esterifies free cholesterol on high-density lipoprotein (HDL) and the cholesteryl ester transfer protein (CETP) transfers cholesteryl ester to very-low-density lipoprotein (VLDL) and low-density lipoproteins (LDL). Using statins, contradictory findings have been made regarding CETP activity in normolipidemic individuals and in those with familial dysbetalipoproteinemia. In contrast, LCAT activity appears to be unaffected by simvastatin. Antioxidants have also been proposed for the use of anti-atherosclerotic treatment, because the oxidation of LDL may have a key role in the pathophysiology of atherogenesis. OBJECTIVE: To investigate, in hypercholesterolemic patients, whether a combination of pravastatin with the antioxidant, vitamin E, has greater effects on the activity of CETP and of LCAT than does pravastatin alone. METHODS: This placebo-diet-controlled multicenter trial included 220 hypercholesterolemic patients who were assigned randomly to groups to receive: diet and 20-40 mg pravastatin (n = 52), diet and alpha-tocopherol (n = 60), or diet associated with placebo (n = 52). Plasma LCAT activity was determined using excess exogenous substrate, containing [3H]cholesterol. Plasma CETP activity was measured in the supernatant fraction after precipitation of endogenous apo B-containing lipoproteins with phosphotungstate-Mg2+. The exchange of cholesteryl esters between [14C]cholesteryl ester-labeled LDL and unlabeled HDL was measured during a 16-h incubation, while LCAT was inhibited. RESULTS: The addition of pravastatin to the diet induced a significant decrease in plasma CETP activity (P < 0.05); this effect was less evident in the group cotreated with vitamin E. For the first time, it was shown that CETP concentrations increased significantly after vitamin E alone (P < 0.05). No significant differences in the plasma activity of LCAT were observed among the groups. CONCLUSIONS: Pravastatin reduced CETP activity, but not that of LCAT. Addition of vitamin E prevented the decrease in CETP activity and had no effect on LCAT activity. The mechanism responsible for these effects is unknown, but could involve the prevention of radical-induced damage to CETP by vitamin E.
Abstract: The binding of Ca2+ ions to bovine and human thyroglobulin (Tg) was demonstrated qualitatively by 45Ca overlay on polyvinylidene difluoride (PVDF) membranes. A quantitative analysis of the interaction of metal ions with bovine Tg was conducted by fluorimetric titration of the protein with Tb3+ ions. These have been used with several proteins as isomorphous replacement probes for Ca2+ ions, as protein-bound Tb3+ ions fluoresce, upon irradiation in the UV region, because of energy transfer from tyrosyl and/or tryptophanyl residues. The fluorescence emission spectrum of Tg excited at 280 nm showed, upon addition of Tb3+ ions, a peak at 546 nm and a marked decrease at 335 nm, indicating an efficient Förster energy transfer between bound Tb3+ ions and closely located Tg intrinsic chromophores. Titration of Tg with Tb3+ ions, carried out by monitoring the emitted fluorescence at 546 nm, indicated the presence of 13.15 metal binding sites per Tg molecule.
Abstract: The effects of several glass-ionomer cements (Baseline and Chem Fil of De Trey; Ketac Fil and Ketac Bond of ESPE; Vitrebond and Vitremer of 3 M) on the protein synthesis of cultured gingival fibroblasts were studied. The presence of cements in the culture medium inhibited protein synthesis, although the relative effectiveness varied significantly. The cements tested have been ranked in three groups, group A (Chem Fil and Ketac Fil), group B (Ketac Bond and Baseline) and group C (Vitrebond and Vitremer), showing 50, 75 and 100% reduction in protein synthesis, respectively. Incubation of cells with medium previously conditioned by Baseline and Ketac Fil caused only transient inhibition of protein synthesis followed by almost complete recovery. This recovery was not observed when the medium was conditioned with Vitrebond. A characterization of the factors determining the inhibitory effect of one cement in each group was attempted. The cements, irrespective of the type, produced small but reproducible decreases in the pH of the medium but released fluoride ions to a different extent. Both changes in the pH of the medium and in the fluoride concentration inhibited protein synthesis by cultured gingival fibroblasts. The different action of cements can be explained, at least in part, by a differing release of fluoride ions.
Abstract: Oxidative modification of low density lipoprotein (LDL) and its byproducts may play a fundamental role in atherosclerosis. We report an in vitro analysis of LDL peroxidative compounds in an homozygous familial hypercholesterolemic (HFH) patient who subsequently died. During the autopsy, we analyzed lipids extracted directly from different atherosclerotic plaques, and we also provided an immunocytochemical analysis using the specific monoclonal antibody MDA2 (directed against malondialdeyde-lysine epitopes of oxidized LDL). The results showed that the same species of peroxidative compounds were present both in LDL in vitro and in lipids extracted directly from atherosclerotic lesions. Moreover, the immunocytochemistry analysis revealed a positive staining of atherosclerotic plaques, confirming the presence of LDL oxidation-specific epitopes. Although observation of a single case is necessarily limited, our findings are consistent with the hypothesis that oxidative modification of LDL is involved in human atherogenesis.
Abstract: BACKGROUND: Posttranslational nonenzymatic glycosylation of native low-density lipoprotein (n-LDL) occurs both in vitro and in vivo in diabetic patients. Glycosylated LDL (glc-LDL) behave similarly to oxidized LDL in some respects. In fact, unlike n-LDL, uptake the glc-LDL can occur in part by the "scavenger" receptor(s), as also demonstrated for oxidized LDL. The enzyme acetylhydrolase, carried by LDL, catabolizes platelet activating factor (PAF). This enzymatic activity is inhibited in oxidized LDL. However, it is unknown whether glc-LDL have reduced acetylhydrolase activity. OBJECTIVE: The first aim of the study was to investigate whether glc-LDL were more susceptible than n-LDL to oxidative modification, and which different oxygen radical species were involved in the phenomenon. Moreover, in order to investigate whether glycosylation may affect acetylhydrolase, we also measured this enzymatic activity in both n- and glc-LDL. METHODS: In vitro glc-LDL and n-LDL were exposed to the oxidants xanthine/xanthine oxidase (X/XO; 2 mM and 100 mU/ml, respectively), or CuSO4 (10 microM) for 18 hs at 37 degrees C. Parallel experiments were done in the presence of the superoxide radical scavenger superoxide dismutase (SOD; 330 U/ml), the hydrogen peroxide scavenger catalase (1000 U/ml), or the hydroxyl radical scavenger dimethylthiourea (10 mM) or dimethylsulfoxide (1 mM). Standards of PAF and lyso-PAF were visualized with iodine vapors after separation by thin layer chromatography. The distribution of label was determined by an imaging scanner. Labeled products were then isolated from the chromatography plate, and the amount of 3H-lyso-PAF formed was determined by liquid scintillation counting. RESULTS: Glc-LDL were more susceptible than n-LDL to lipid peroxidation (n-LDL 22.9 +/- 3.4 vs 34.8 +/- 4.2* nmoles/MDA/mg of protein in glc-LDL oxidized by X/XO and n-LDL 28.9 +/- 4.2 vs 40.4 +/- 4.1* in glc-LDL oxidized by CuSO4, *p < 0.05 vs n-LDL). SOD, but not other scavengers, prevented peroxidation, indicating an obligatory role for superoxide radicals. Oxidation of glc-LDL also induced a higher degree of apolipoprotein-B100 modifications than n-LDL, with increased electrophoresis mobility and decreased TNBS reactivity. These effects were similarly prevented by SOD. Finally, acetylhydrolase activity was significantly lower in glc-LDL than in n-LDL. CONCLUSION: Glycosylation increases LDL oxidation due to superoxide radicals, and also reduces acetylhydrolase activity. These phenomenona may contribute to enhance and/or accelerate the progression of atherosclerosis in diabetic patients.
Abstract: BACKGROUND: Oxidative modification of low-density lipoprotein (LDL) appears to play a pivotal role in atherogenesis. The specific role played by LDL peroxidation in aging is not known. Since estrogens may protect LDL from peroxidation in vitro and in vivo, we chose to investigate only men of various ages. OBJECTIVE: To determine whether LDL from healthy elderly men was differently susceptible to peroxidation than LDL of young and adult men. SUBJECTS AND METHODS: LDL was isolated from 15 normolipidemic young (aged 19-23 years), 17 adult (aged 35-55 years), and 16 elderly (aged 77-90 years) healthy men. None of the men included in the study was a smoker or a hypertensive. LDL peroxidation was achieved by exposure to 5 mumol/l copper sulfate for 18 h at 37 degrees C, and some markers of lipid peroxidation (estimating various levels of peroxidation) were evaluated. RESULTS: The levels of lipid peroxides in LDL from our elderly men were already higher under basal conditions than were those both of adult and of young men. LDL from elderly men was more susceptible to peroxidation than was that of adult and young men. Furthermore, the lag time correlated inversely to age (r = -0.68, P < 0.01), whereas lipid peroxide and malonyldialdehyde levels correlated highly to age (r = 0.79 and r = 0.77, P < 0.0002 and P < 0.0012, respectively). With aging the vitamin E content in LDL decreased whereas the arachidonic fatty acid content increased. More importantly, the relationship between the vitamin E content and the lag time made evident the parallel increase in lag time and in vitamin E level with aging. The vitamin E concentration also correlated inversely to levels of thiobarbituric acid-reactive substances in LDL from elderly patients (r = -0.61, P < 0.05). CONCLUSIONS: The present study shows that LDL peroxidation increases with age. This phenomenon may favor the progression of atherosclerosis in elderly men.
Abstract: Familial hypercholesterolemia was the first genetic disorder recognized to cause myocardial infarction. Patients with homozygous familial hypercholesterolemia have rapidly progressive coronary atherosclerosis with angina pectoris, myocardial infarction, or sudden death at a young age. Selective apheresis on dextran sulfate cellulose columns reduces mortality and may induce regression of coronary lesions. These patients have both increased levels and prolonged circulation residence time of low-density lipoprotein (LDL), which is not removed by cellular receptor. LDL oxidation may play a pivotal role in atherogenesis. LDL undergoes oxidation before being taken up by macrophages and then transformed into arterial wall foam cells. The aim of this study was to investigate LDL oxidation in eight homozygous patients with familial hypercholesterolemia during repeated LDL apheresis. LDL lipid peroxidation, estimated by conjugated-diene absorbance at 234 nm, lipid peroxides, and malondialdehyde showed an increased resistance against oxidation after repeated LDL apheresis. This phenomenon was also observed in the oxidative indexes of protein moiety of LDL (apolipoprotein-B100 fragmentation, trinitrobenzenesulfonic acid reactivity, and electrophoresis agarose mobility). Similarly, cholesteryl esterification was decreased after LDL apheresis. Thus selective LDL apheresis not only decreases the pool of LDL, but it also induces changes that render LDL less susceptible to oxidation. This phenomenon might contribute to reduce coronary atherosclerosis and thus mortality of these particular patients.
Abstract: Usual purification procedures of LDL and Lp(a) require numerous, extensive and prolonged sample handlings: this greatly increases the possibility of spontaneous oxidation. We have developed a method which, making use of two short-run ultracentrifugations in vertical rotors alternated by two rapid column-chromatography steps (SRUC), significantly shortens the preparation time to 3.5 h (LDL) and does not demand additional instrumentation or particular accuracy. Purification of Lp(a) requires a further wheat germ agglutinin chromatographic step, which can be accomplished within 30 min. More importantly, the method significantly reduces spontaneous oxidation as compared with classical isolation procedures. LDL isolated by the standard sequential method exhibits more extensive apolipoprotein B100 degradation, lipid peroxidation, and endogenous antioxidant (vitamin E) loss than the same lipoproteins obtained by means of the SRUC. This procedure may have be particularly valuable in experiments evaluating the effects of oxygen radical-induced modifications, especially in vitro.
Abstract: To determine whether oxidized LDL enhances atherogenesis by promoting monocyte recruitment into the vascular intima, we investigated whether LDL accumulation and oxidation precede intimal accumulation of monocytes in human fetal aortas (from spontaneous abortions and premature newborns who died within 12 h; fetal age 6.2+/-1.3 mo). For this purpose, a systematic assessment of fatty streak formation was carried out in fetal aortas from normocholesterolemic mothers (n = 22), hypercholesterolemic mothers (n = 33), and mothers who were hypercholesterolemic only during pregnancy (n = 27). Fetal plasma cholesterol levels showed a strong inverse correlation with fetal age (R = -0.88, P < 0.0001). In fetuses younger than 6 mo, fetal plasma cholesterol levels correlated with maternal ones (R = 0.86, P = 0.001), whereas in older fetuses no such correlation existed. Fetal aortas from hypercholesterolemic mothers and mothers with temporary hypercholesterolemia contained significantly more and larger lesions (758,651+/-87,449 and 451,255+/-37,448 micron2 per section, respectively; mean+/-SD) than aortas from normocholesterolemic mothers (61,862+/-9,555 micron2; P < 0.00005). Serial sections of the arch, thoracic, and abdominal aortas were immunostained for recognized markers of atherosclerosis: macrophages, apo B, and two different oxidation-specific epitopes (malondialdehyde- and 4-hydroxynonenal-lysine). Of the atherogenic sites that showed positive immunostaining for at least one of these markers, 58.6% were established lesions containing both macrophage/foam cells and oxidized LDL (OxLDL). 17.3% of all sites contained only native LDL, and 13.3% contained only OxLDL without monocyte/ macrophages. In contrast, only 4.3% of sites contained isolated monocytes in the absence of native or oxidized LDL. In addition, 6.3% of sites contained LDL and macrophages but few oxidation-specific epitopes. These results demonstrate that LDL oxidation and formation of fatty streaks occurs already during fetal development, and that both phenomena are greatly enhanced by maternal hypercholesterolemia. The fact that in very early lesions LDL and OxLDL are frequently found in the absence of monocyte/macrophages, whereas the opposite is rare, suggests that intimal LDL accumulation and oxidation contributes to monocyte recruitment in vivo.
Abstract: Previously, the authors reported that the serum of patients with immunoglobulin (Ig) A nephropathy stimulated peripheral leukocytes, and this effect was inhibited by nominal haptens for lectins. In vitro studies have shown that lectins can bind to rat mesangial cells and cause their activation. This study was performed to investigate whether the serum of IgA nephropathy patients contains lectins that activate mesangial cells, i.e., induce release of interleukin (IL)-6, a nephritogenic cytokine. The serum of patients was adsorbed by affinity chromatography on resins loaded with lectin-binding sugars. After adsorption, serum supernatant was collected and the resins were then eluted. Human mesangial cells were conditioned with native serum, post-adsorption supernatant, and eluate (all three at 10%) for 24 h, and the release of IL-6 was determined by ELISA. Normal serum was used as control. Incubation of mesangial cells with IgA nephropathy patients serum raised average IL-6 release from 8.5 pg/mL to 274.1 pg/mL. Adsorption in beta-D-glucose and N-acetyl-D-glucosamine caused a fall in the activity of patients' serum, to 17.0 and 63.7 pg/mL, respectively, and the activity lost was recovered in the eluate (185.2 and 142.7 pg/mL, respectively). Neither adsorption on N-acetyl-D-galactosamine nor on fucosylamine was associated with any effect on serum activity; accordingly, no activity was found in the eluates. Serum of patients with non-IgA mesangiocapillary nephritis did not stimulate mesangial cells. These results show that the serum of IgA nephropathy patients contains specific lectins that stimulate IL-6 nephropathy by mesangial cells and are, therefore, potential nephritogenic.
Abstract: Individual mRNA species from normal and neoplastic endometrium obtained from the same patient were comparatively studied by exploiting the "differential display" methodology. The mRNAs were extracted from tissues, reverse transcribed, and amplified by PCR using appropriate primers. The cDNA electrophoretic bands which were frankly different on the basis of their quantitative expression were excised from the gels and reamplified. Each of these sequences was subsequently screened for the capacity to hybridize to RNA preparations from normal endometrium and endometrial adenocarcinoma samples, first from the original patient and then from a group of selected patients. Of many, only two sequences, thereafter named N5.5 and T16.2, respectively, successfully passed the two sieving tests and were chosen for further analysis. It appears that N5.5 recognizes an mRNA which is expressed more abundantly in normal than in neoplastic (adenocarcinoma) endometrium; in contrast, T16.2 seems to preferentially recognize an mRNA which is expressed in tumoral tissues. These two sequences have been cloned and sequenced; they do not show any identity or significant similarity to any other known sequence.
Abstract: This paper describes the assessment of the homogeneity and stability of a purified and lyophilized human thyroglobulin (Tg), and characterizes its immunoreactivity. The purified and lyophilized Tg is intended to be used as a primary reference material to establish calibration of working serum based reference material. The programme involved the participation of 15 European laboratories and one laboratory from the United States. The homogeneity of the content of the ampoules was considered acceptable (< 9%). The stability was tested by accelerated temperature degradation showing predicted annual relative losses of 0.01% at -70 degrees C and 1.04% at -20 degrees C. The immunoreactivity of the Tg material as measured in different laboratories varied mostly according to the method used rather than the laboratory. The interlaboratory variability showed that the two commercial methods used in several laboratories (kit 1 and 2) had an interlaboratory variation (CV) of 15.9% (N = 5) and 7.1% (N = 3), respectively, whereas the total interlaboratory CV was 64.3% (N = 18). The immunoreactive Tg had dilution curves parallel with other Tg calibrators (those of the methods). Dilution curves of the Tg material after storage at various temperatures and time were parallel in both RIA and IRMA. In conclusion, we have prepared a Tg reference material which in extensive studies in several participating laboratories has demonstrated a sufficient homogeneity and stability as well as dilution curves parallel to the calibrators of all the immunoassays tested in the study. This reference material is considered the first step towards decreasing the interlaboratory variability between Tg immunoassays.
Abstract: This report describes the characterization of a purified human thyroglobulin (Tg) reference material, and details the procedures used in its certification. The purified Tg is intended to be used as a primary reference material to establish calibration of working serum based reference material for immunoassay procedures. The programme involved the participation of 15 European laboratories and one laboratory from the United States. The physicochemical characterization showed by polyacrylamide gel electrophoresis and immunoblotting that the purified Tg had for the major part the expected molecular size of 660 kDa with traces of lower molecular forms. The amino acid composition was close to that demonstrated for the cDNA and the content of iodine was in keeping with a moderately to highly iodinated Tg. The mass concentration in reference material RM 457 is certified to be (0.324 +/- 0.018) g/L on the basis of protein determined by the Lowry method and supported by nitrogen determination, absorbance measurement, and amino acid analysis. This reference material is considered the first step towards decreasing the interlaboratory variability between Tg methods of measurement.
Abstract: Previous studies have shown that calcium channel blockers may reduce the development of experimental atherosclerosis, and that nifedipine may slow the progression of coronary atherosclerosis in humans. The mechanisms responsible for this antiatherogenic effect are still unclear. It has been recently proposed that oxygen free radicals can induce the oxidation of human low-density lipoproteins (LDL) and that oxidized LDL may be an atherogenic stimulus. Previous studies in other systems have shown that calcium channel blockers may effectively inhibit oxygen radical-induced lipid peroxidation in vitro. Thus, the aim of the present study was to investigate whether calcium channel blockers may also reduce LDL modifications induced by oxygen radicals. Isolated human LDL were exposed to oxygen radicals generated by CuSO4 (10 microM for 18 hours) after a 30 minute pre-incubation with different concentrations (1-100 microM) of nifedipine, diltiazem, and verapamil. Lipid peroxidation was measured from malonyldihaldehyde (MDA) production. Oxygen radical-induced damage on apolipoprotein-B100 was evaluated by acrylamide and agarose gel electrophoresis. Calcium channel blockers dose-dependently prevented oxidation of both the lipid and protein components of LDL. MDA formation was reduced in LDL pre-incubated with calcium antagonists before exposure to oxygen radicals (% MDA inhibition was 89.8 +/- 6.9 with 30 microM nifedipine, 68.6 +/- 4.9 with 30 microM verapamil, and 65.6 +/- 7.1 with 30 microM diltiazem; p < 0.01 vs. controls). Similarly, apolipoprotein-B100 integrity was preserved against oxygen radical attack in the presence of calcium antagonists. Thus, calcium channel blockers reduce the oxidation of human LDL in vitro. These data suggest that reduced formation of atherogenic oxidized LDL may be an additional mechanism for the antiatherosclerotic effects of calcium channel blockers in vivo.
Abstract: The blue beam of an Argon laser (488 nm) has been focused on the cell membrane in the presence of phenol-red, an usual component of cell culture media, through a 100 x objective. At the site of the beam impact, due probably to local temperature changes, the cell membrane modifies its permeability. As a consequence of the hit, circular areas, whose radius may be apparently regulated by changing the irradiation time and/or the radiation intensity (energy), appear on the wall, last for a short time and fade spontaneously within 1-2 minutes. No evident sings of cell injury or hurt have been observed afterward. Plasmid DNA, purposely added to culture fluid, easily slips in the cytoplasm; utilizing such approach, thereafter indicated as "optoporation', we have successfully transfected two genes, namely beta-galactosidase and chloramphenicol-acetyl-transferase in murine NIH3T3 fibroblasts. Therefore optoporation represents an additional procedure for gene transfer with several advantages over already available methods: (1) it only takes advantage of the presence of phenol-red, a normal cell medium component, with no need of addition of extraneous substances; (2) it is a very mild treatment virtually suitable for any cell type and (3) it allows transfection of selected cells even in the presence of cells of different type (providing that they are morphologically distinguishable).
Abstract: Patients with homozygous familial hypercholesterolemia (FH), as a result of the increased levels and prolonged residence time of low density lipoprotein (LDL) in plasma, have a strong tendency toward accumulation of LDL-cholesterol in the arterial wall, causing premature atherosclerosis. This phenomenon may enhance per se the physiological degradation of both protein and lipid component of LDL, which be more susceptible to oxidative damage induced by oxygen radicals. It is well known that LDL may undergo oxidative modification before being taken up by macrophages which are then transformed into foam cells. It has been suggested that platelet-activating factor (PAF) may play an important role in atherogenesis and PAF catabolism is known to be mediated by serum acetylhydrolase, an enzyme that is normally associated with LDL. Thus, the present study was designed to investigate the structural properties of LDL, including acetylhydrolase activity, in homozygous FH as compared to normolipidemic subjects before and after xanthine/xanthine oxidase-mediated oxidation. We studied 8 homozygous FH patients matched with 8 normolipidemic volunteers. Lipids of LDL fraction were extracted and verified by thin layer chromatography (TLC) analysis. Fatty acids were methylated and injected into a gas chromatograph/mass spectrometer. Vitamin E in LDL was determined by high performance liquid chromatography (HPLC). As an index of susceptibility of LDL to oxidative modifications, the formation of lipid-conjugated dienes was continuously monitored at 234 nm. Lipid peroxidation was also evaluated from the amount of both lipid peroxides (LPO) and malonyldialdehyde (MDA) content. Apolipoprotein (apo) B-100 on LDL was carried on polyacrylamide and agarose gel electrophoresis. In the homozygous FH patients, the relative content of cholesteryl ester was slightly increased. Interestingly, the relative amount of arachidonic acid (20:4) was constantly increased in each lipid fraction in homozygous FH patients. The amount of vitamin E was not significantly different in the patient group from that in the control group. However, LDL from patients carried lower levels of vitamin E (nmol/mg LDL) than controls (2.7 +/- 0.4 vs. 2.9 +/- 0.3 P = NS). The results shows that lag time (min) was decreased (82 +/- 19 vs. 111 +/- 21; P < 0.05) and the maximal rate of diene production and total diene production was increased in homozygous FH patients. Mean levels of MDA were similar in both groups before oxidation, but levels after initiation of oxidation were significantly higher in the patient group. In contrast, mean levels of LPO were already higher in patients before oxidation (58 vs. 27 nmol/mg of protein; P < 0.05), and after initiation of oxidation were also significantly higher at each time points. When oxidized LDL was run on a polyacrylamide gel, an extensive apo B-100 fragmentation replaced by lower molecular mass fragments ranging from 45,000 to 205,000 m.wt., was observed only in LDL from homozygotes. Relative LDL agarose gel mobility shows that LDL from patients migrated higher than LDL of controls. Finally acetylhydrolase activity associated with LDL in patients was significantly reduced as compared to controls. Thus, in homozygous FH patients, LDL appeared more susceptible to oxidation in vitro; the indices for LDL oxidizability were all significantly different from those of controls. This phenomenon might be due to prolonged residence time of LDL in these patients, as suggested from high basal LPO levels and lower vitamin E levels carried by LDL. This hypothesis may explain together with the high content of arachidonic acid, the enhanced susceptibility of LDL from homozygous FH patients to oxidative damage.
Abstract: We have recently reported that the Arg1152-->Gln insulin receptor mutation (QK single mutant) alters a conserved motif (RK motif) immediately next to the key tyrosine phosphorylation sites (Tyr1146, Tyr1150, Tyr1151) of the receptor and constitutively activates its kinase and metabolic signaling. To investigate further the function of the RK motif, we have expressed two additional mutant insulin receptors: a single mutant, in which the second basic residue in the RK motif (Lys1153) was substituted (RA mutant); and a double mutant, in which both the Arg and the Lys residues were replaced with noncharged amino acids (QA mutant). As compared with the transfected wild-type receptors (WT), both the single and the double mutant receptors were normally synthetized and transported to the plasma membrane and bound insulin normally. Whereas the double mutant receptor exhibited preserved insulin-dependent autophosphorylation, kinase activity, and 2-deoxyglucose uptake, all of these functions were grossly impaired in the two single mutant receptors. Two-dimensional analysis of tryptic phosphopeptides from receptor beta-subunits revealed that decreased autophosphorylation of the single mutant receptors mainly involved regulatory Tyr1150,1151 and carboxyl-terminal Tyr1316,1322. At variance with the insulin-stimulated, insulin-independent tyrosine kinase activity toward poly(Glu-Tyr) 4:1 was increased 3-fold in both the double and the single mutants. All mutant receptors induced a 2-fold increase in basal 2-deoxyglucose uptake in NIH-3T3 cells. Treatment of WT transfected cells with 12-O-tetradecanoyl-phorbol-13-acetate or 8-bromo-cAMP increased insulin receptor phosphorylation by 3-fold. No phosphorylation was observed in cells expressing the two single or the double mutant receptor. Consistently, purified preparations of PKC and PKA phosphorylated the WT but not the mutant receptors in vitro. A 17-amino acid synthetic peptide encoding the receptor sequence surrounding the RK motif inhibited phosphorylation of WT insulin receptors by both protein kinases A and C. A mutant peptide in which the RK sequence was replaced by QK (to mimic the mutation in the QK receptor) exhibited no inhibitory effect. Thus, the RK insulin receptor motif is required for insulin receptor phosphorylation by protein kinases C and A and may modulate insulin-independent receptor activity. The RK motif may also have an important structural role in allowing normal insulin regulation of the kinase.
Abstract: Low-density lipoproteins (LDL) oxidized by oxygen radicals are a potent atherogenic stimulus. Chemically modified LDL are internalized by macrophages via a specific cell surface receptor that was termed the scavenger receptor, and could induce foam cell transformation. Post-translational nonenzymatic glycosylation of low density lipoprotein (LDL) occurs in vivo in diabetic patients. Glycosylated LDL (glcLDL) is degraded by macrophages in part by the classic LDL-receptor and in part by the scavenger receptor. This latter mechanism may contribute to the formation of foam cells and acceleration of atherosclerosis in diabetes mellitus. Oxygen free radicals (ORs) could induce LDL peroxidation and subsequent formation of foam cells. Glycosylation may alter protein conformation. A free radical is any chemical species that has an unpaired electron. This property renders it highly chemically reactive. When a radical reacts with a non radical another free radical is generated. This characteristic enables radicals to trigger chain reactions. Oxygen radicals are: superoxide anion (.O2-), hydroxyl radical (.OH) and hydrogen peroxide (H2O2). Thus, the aim of this study was to investigate whether glcLDL are susceptible to peroxidative modification by ORs. GlcLDL was prepared incubating LDL with 40 mM glucose in sterile phosphate-buffer-EDTA 1 mM for 10 days at 37 degrees C. Control LDL (cLDL) was similarly incubated with buffer but without glucose. After this preparation both forms of LDL were oxidized by CuSO4 (15 microM for 20 hours at 37 degrees C) or by xanthine/xanthine oxidase (X:2 mM/XO: 100 mU for 20 hours at 37 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: The basal production of IL-6 by cultured peripheral blood mononuclear cells (PBMC) from patients with IgAN, is markedly higher (A, 109 pg/ml) as compared to that of PBMC of either patients without clinical signs (I, 39 pg/ml) or appropriate controls (C, 44 pg/ml). When PBMC from healthy subjects were incubated in the presence of sera from patients A, the IL-6 production was strongly enhanced. No such an effect was observed by stimulating PBMC with sera from the other two groups of subjects (I, C). In another experiment we observed that the IL-6 production stimulated by serum from patients A could be inhibited by addition of specific monosaccharides. The inhibitory effect was rapidly abolished when the sugar-containing medium was substituted with the original one. Finally molecular components from serum of A were grossly separated by gel column chromatography. Individual fractions were incubated with PBMC of C: fractions with Mr > 30,000 highly stimulated the release of IL-6 (up to 1320 pg/ml); fractions with lower molecular weight were inactive. The data suggest the presence of an IL-6 releasing factor in the serum of IgAN patients. Although the chemical nature of such a factor is not yet established, the observations reported focus our attention to the lectins family. Since this factor seems potentially important in the understanding of the pathogenesis of IgAN, both its isolation and structural/functional characterization deserve further efforts.
Abstract: Platelet-activating factor (PAF) can exert profound inflammatory effects at very low concentrations. In plasma, PAF is hydrolyzed to lyso-PAF by acetylhydrolase, an enzyme that circulates bound to LDL. Previous studies suggest that oxygen radicals may act synergistically with PAF to potentiate tissue injury. However, mechanisms underlying this interaction have not been elucidated. In this study we investigated whether oxygen radicals may inactivate PAF acetylhydrolase. PAF acetylhydrolase activity was measured in human plasma and purified LDL before and after exposure to radicals (10-20 nmol/min per ml) generated by xanthine/xanthine oxidase. Oxygen radicals induced > 50% loss of PAF acetylhydrolase activity within 60 s and almost complete inactivation by 10 min. This phenomenon was irreversible and independent of oxidative modification of LDL. Inactivation occurred without changes in the affinity constant of the enzyme (Km was 17.9 microM under control conditions and 15.1 microM after exposure to oxygen radicals). Inactivation was prevented by the scavengers superoxide dismutase or dimethylthiourea or by the iron chelator deferoxamine. Thus, superoxide-mediated, iron-catalyzed formation of hydroxyl radicals can rapidly and irreversibly inactivate PAF acetylhydrolase. Since concomitant production of PAF and oxygen radicals can occur in various forms of tissue injury, inactivation of acetylhydrolase might represent one mechanism by which oxygen radicals may potentiate and prolong the proinflammatory effects of PAF.
Abstract: Previous studies in other systems have shown that beta-receptor blockers may effectively inhibit oxygen radical-induced lipid peroxidation. On the other hand, it has been recently proposed that oxygen free radicals can induce peroxidation of human low density lipoproteins (LDL), and that peroxidized LDL may be an atherogenic stimulus. Chemically modified LDL are internalized by macrophages via a specific cell surface receptor that was termed the scavenger receptor. This phenomenon may induce foam cells transformation in vivo. In the present study we investigated whether beta-blockers may reduce oxygen radical-mediated LDL peroxidation. Purified human LDL were oxidized by exposure to oxygen free radicals generated by xanthine (0.2 mM) and xanthine oxidase (100 mU) at 37 degrees C after a pre-incubation (30 min) in presence of different concentrations (from 1 to 30 microM) of acebutolol, metoprolol or propranolol, three agents with a different degree of lipophilicity. Peroxidation was measured from malonyldihaldehyde (MDA) production. Data have shown a significant percent inhibition of MDA formation in presence of beta-blockers (from 33 to 85%). Thus, beta-blockers reduced peroxidation of human LDL in vitro at clinically relevant concentrations. The order of potency appears to follow the degree of lipophilicity. These data suggest that, although beta-blockers are known to adversely effect lipid metabolism, these agents might on the other hand prevent atherogenesis via a mechanism of inhibition of LDL peroxidation in vivo and reduced foam cells formation.
Abstract: Signals controlling the insulin receptor endocytotic pathway have been investigated using the R1152Q insulin receptor mutant (M). This mutant receptor exhibits high levels of insulin-independent kinase activity, impaired autophosphorylation, and lack of an insulin stimulatory effect on both auto- and substrate phosphorylation. NIH-3T3 fibroblasts expressing M receptors displayed a 2.5-fold higher 125I-insulin internalization rate than wild type (WT) but lacked insulin-induced receptor internalization and down-regulation. Cell surface recycling of internalized receptors also occurred at a higher rate in M cells and was unaffected by insulin. Cell preincubation with 35 mM Tris, which inhibits the insulin receptor degradative route, elicited no effect on M receptor recycling but inhibited that of WT by 40%. In contrast, the energy depleter 2,4-dinitrophenol, which inhibits normal insulin receptor retroendocytosis, impaired M receptor recycling 4-fold more effectively than that of WT. The release of internalized intact 125I-insulin was 6-fold greater in M than in WT fibroblasts and was almost completely inhibited by dinitrophenol, whereas insulin degradation by M cells was 4-fold decreased as compared with WT. Thus, internalization and recycling of the constitutively active Gln1152 receptor kinase occur in the absence of autophosphorylation. However, tyrosine phosphorylation appears to be required for proper sorting of endocytosed insulin receptors.
Abstract: Upon incubation of bovine thyroglobulin with trypsin at high enzyme/substrate ratio, some fragments with apparent masses between 18 and 31 kDa resisted prolonged digestion. Their NH2-terminal sequences were determined. All fragments overlapped with some of the Cys-rich repeats that compose a large part of thyroglobulin and are predicted to have a rigid structure. Among the inserts that interrupt the cysteine-rich repeats, for which several data indicate a location at the surface of thyroglobulin, the insert of repeat 1.7 was unique because of its resistance to proteolysis. This insert, although exposed at the surface of the protein, may be hidden in a region of contact between thyroglobulin monomers.
Abstract: Bovine and human thyroglobulin show two closely migrating bands in reducing SDS-PAGE. Limited digestion with chymotrypsin, trypsin and thermolysin converted the slower band of the doublet into a peptide identical to the faster band, with an apparent mass of 270 kDa, in both species. The starting point of the faster band of the doublet was established at Ileu 520 with native bovine Tg and at Ser 503 with native human Tg, and at Ser 503 and Ser 504 with chymotrypsin-digested bovine and human Tg, respectively. These data explain the electrophoretic heterogeneity of thyroglobulin and unveil a region highly susceptible to proteolysis at about 500 residues from the NH2-terminus of the molecule.
Abstract: Previous studies have shown that calcium-antagonists may reduce the development of experimental atherosclerosis, and that nifedipine may slow progression of coronary atherosclerosis in man. The mechanisms responsible for this effect are still unclear. It has been recently proposed that oxygen-free radicals can induce peroxidation of human low-density lipoproteins (LDL), and that peroxidized LDL may be an atherogenic stimulus. Chemical modified LDL are internalized by macrophages via specific cell surface receptor that was termed the scavenger receptor, and could induce foam cells transformation in vivo. Previous studies on other systems have shown that calcium-antagonists may effectively inhibit oxygen radical-induced lipid peroxidation. These drugs, though differing widely in their chemical structure, are lipophilic to various degrees and presumably would concentrate in the lipid domain of the phospholipid-rich membranes. Therefore, the aim of the present study was to investigate whether calcium-channel blockers may reduce human LDL peroxidation. Purified human LDL were exposed to oxygen radicals generated by xanthine-xanthine oxidase (18 hours) after a pre-incubation (30 min) in presence of different concentrations of nifedipine, diltiazem and verapamil. Peroxidation was measured from malonyldialdehyde production. The results show that calcium-antagonists prevent LDL peroxidation. Thus, calcium-antagonists may reduce peroxidation of human LDL in vitro, at clinically relevant concentrations. These data suggest that reduced formation of atherogenic peroxidized LDL may be an additional mechanism for the anti-atherosclerotic effects of calcium-antagonists in vivo.
Abstract: Low-density lipoproteins (LDL) oxidized by oxygen radicals (OR) are a potent atherogenic stimulus. Chemically modified LDL are internalized by macrophages via a specific cell surface receptor that was termed the scavenger receptor, and may induce foam cells transformation. A free radical is any chemical species that has an unpaired electron. This property renders it highly chemically reactive. When a radical reacts with a non radical another free radical is generated. This characteristic enables radicals to trigger chain reactions. Oxygen radicals are: superoxide anion (.O2-), hydroxyl radical (.OH) and hydrogen peroxide (H2O2). It is unknown whether LDL are modified via direct lipid oxidation by OR, or whether LDL are subsequently oxidized via chain reactions after initial OR attack. To distinguish between these 2 mechanisms, LDL were exposed to OR formed by xanthine/xanthine oxidase (X/XO). Peroxidation was measured from malonyldialdehyde (MDA) levels. Parallel experiments were performed in presence of the superoxide radical scavenger superoxide dismutase (SOD; 330 U/ml), or the hydrogen peroxide scavenger catalase (CAT; 1000 U/ml), or by adding the chain-reaction inhibitor butylhydroxytoluene (BHT; 1 mM) at selected time points. SOD, but not CAT prevented LDL peroxidation, indicating an obligatory role for superoxide radicals. Superoxide generation in this model lasts only a few minutes, however, MDA levels continued to increase over several hours. Furthermore, this phenomenon was blocked when BHT was added at various times after X/XO. These data show that LDL peroxidation is triggered by initial OR generation but then involves chain reactions which do not require continuous exposure to OR.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: Ultraviolet irradiation of the [3H]colchicine-tubulin complex leads to direct photolabeling of tubulin with low but practicable efficiency. The bulk (70% to greater than 90%) of the labeling occurs on beta-tubulin and appears early after irradiation, whereas alpha-tubulin is labeled later. The labeling ratio of beta-tubulin to alpha-tubulin (beta/alpha ratio) is reduced by prolonged incubation, prolonged irradiation, urea, high ionic strength, the use of aged tubulin, dilution of tubulin, or large concentrations of colchicine or podophyllotoxin. Glycerol increases the beta/alpha ratio. Limited data with [3H]podophyllotoxin show that it covalently bound with a similar beta/alpha distribution. Vinblastine, on the other hand, exhibits preferential attachment to alpha-tubulin. The possibilities that colchicine binds at the interface between alpha-tubulin and beta-tubulin, that the drug spans this interface, and that both subunits may contribute to the binding site are suggested.
Abstract: In most highly structured native proteins, as well as in thyroglobulin, the reactivity in vitro of the various tyrosyl residues toward iodine is widely different. The present work demonstrates that of nearly 70 tyrosyl residues present in rat thyroglobulin, there is one, residue number 5 from the NH2-terminal end, which has in vivo the highest affinity toward iodine, being the first one to be iodinated. In fact, when 6-(n-propyl)-2-thiouracil (PTU)-treated, iodine-deficient animals were injected with 125I and killed shortly after, we isolated from thyroid glands poorly iodinated thyroglobulin (about 1 iodine atom/thyroglobulin molecule), nearly 90% of the radioactivity of which was found as monoiodotyrosine. Although CNBr cleavage of this protein gave several fragments after gel electrophoresis only one of these, with apparent mass 27,000 Da, contained 125I. This fragment was isolated and fully characterized. Twelve cycles of automated Edman degradation were performed; the sequence found, i.e. N-I-F-E-X-Q-V-X-A-Q-X-L, indicated that the 27,000-Da fragment is the NH2 terminus of thyroglobulin. This portion of the polypeptide chain contains several tyrosyl residues which may well all be potentially involved in the early iodination of the protein. The observation that the removal of seven amino acids from the NH2 terminus is accompanied (at the fifth step) by the total disappearance of radioactivity in the resulting shortened peptide suggested that the fifth residue was the only one iodinated under these conditions. A second, more quantitative experiment was performed on thyroglobulin obtained from 6-(n-propyl)-2-thiouracil-treated animals whose death was postponed 24 h after the injection of 125I. In this case the radioactivity was found not only in a single CNBr fragment (27,000 Da) but also in other discrete species of lower molecular mass. The mixture of these peptides was subjected to seven steps of manual Edman degradation. Fragments before and after partial degradation were run in parallel on a polyacrylamide gel and the distribution of 125I compared. Besides some change in the background, the two profiles were identical except for the absence of the 27,000-Da species. This proves that all the 125I present in the 27,000-Da species was localized at the fifth residue, the same site at which the hormone molecule is preferentially synthesized under normal conditions. This result is not unexpected and is in accord with the known properties of thyroglobulin which has a polypeptide chain designed for efficient synthesis of the hormone even at low levels of iodination.
Abstract: The method described for quantitative estimation of lipids separated on thin layer chromatography plates exploits the observation that most lipids can be stained by iodine vapor and that, in "controlled" conditions, the intensity of this staining is proportional to the actual amount of lipid in the spot. The method consists of exposing the developed plate to iodine vapor; spraying it with a suitable solvent to prevent halogen evaporation; collecting the stained lipids by scraping the spots off the plate; and determining by a rate-sensing method the absorbed iodine. The final determination is performed by measuring spectrophotometrically (at 410 nm) the rate of decolorization of a solution of Ce(IV) by As(III) in strong acidic conditions. The reaction rate, which is positively related to the concentration of iodine, is derived from the slope of the absorbance change plotted vs time. Providing that standards and samples are stained simultaneously, a quantitative estimation of lipid components of a mixture is possible in a reasonable time with excellent accuracy and reproducibility. In our hands, the method has been successfully applied to several common phospholipids, long chain fatty acids, cholesterol and deoxycholate, and triacylglycerols, in the range of 5-60 micrograms.
Abstract: The formation of dehydroalanine in thyroglobulin is the result of the side chain elimination of an iodophenyl group during the thyroid hormone formation from two iodotyrosyl residues. This amino acid is easily converted to labeled alanine (upon reduction with [3H] borohydride) or changed to labeled aspartic acid (upon addition of Na14CN and subsequent acid hydrolysis). The cleavage of the protein by CNBr produced many stainable electrophoretic bands, but the autoradiography indicated the presence of a much smaller number of radioactive species. Although three major species raised attention, because they could be all jointly labeled and were present in all preparations, only a species of 15,900 Da was fully studied. It was isolated and its sequence partially determined by Edman degradation. It was established that this species corresponded to the thyroglobulin fragment between methionines 2,432 and 2,578. This peptide contains two hormonogenic sites (positions 2,555 and 2,569) which are either tyrosyl residues or hormone residues arising from them, and five additional tyrosines all potentially involved as donor sites in the hormonogenesis. Upon treatment with N-chlorosuccinimide, the fragment was split into three smaller peptides of about 2,900, 8,500, and 4,600 Da containing 1, 2, and 2 tyrosyl residues, respectively. Only the 8,500-Da subfragment contained [3H]Ala. This finding strongly suggests that at least some of the tyrosines involved as donor sites in thyroid hormonogenesis are within this peptide and possibly map at positions 2,469 and/or 2,522. Moreover, at minimum levels of iodination, when thyroglobulin contains the lowest number of hormone molecules, dehydroalanine is mostly found in the 15,900-Da peptide.
Abstract: A secondary structure prediction has been made using the available primary sequence data of the proposed carboxy-terminal of rat thyroglobulin. The model predicts 22% alfa-helix, 28% beta-structure and 17% beta turns. Out of the 8 possible carbohydrate acceptor-sites (Asn-x-Ser/Thr), 3 (residues 136, 368, 782) are associated with peptide sequences which favour the formation of beta-turn or loop-structures and are located in high hydrophilic regions. The entire sequence is predicted to be made up of two domains: one of them is highly structured, contains the hormonogenic sites, a cluster of tyrosines and at least one carbohydrate acceptor site.
Abstract: The present investigation demonstrates that the cross-linking agent, dimethylsuberimidate, is an usefull tool to study thyroglobulin structure. In fact, while reproducible and discrete polymerization products are obtained in strictly controlled conditions, valuable information on the native assemblage of thyroglobulin subunits and the effects of its major post-translational modification (iodination) on its structure, are reported.
Abstract: Controlled freezing and thawing of 19 S calf thyroglobulin resulted in a specific and reproducible breakdown of the protein. Beside the elementary chain (300,000 Da), new, discrete bands are revealed by gel electrophoresis in sodium dodecyl sulfate under reducing conditions. These new species consist of a major peptide of 100,000 Da and several faster-migrating bands. Most of these polypeptides were purified by preparative gel electrophoresis and individually digested in formic acid with CNBr. A comparative gel electrophoresis under denaturating and reducing conditions of (i) the fragments obtained from the native protein, (ii) the electrophoretically purified elementary chain, (iii) the 100,000-Da peptide, and (iv) a smaller fragment (of about 50,000) was performed. It revealed a very close homology among the peptide maps of the intact 19 S, the elementary chain, and the 100,000-Da peptide. Furthermore, it was shown that the digestion products of the smaller fragment, present in the peptide map of the native protein, were absent in both the elementary chain and the 100,000-Da species. These results support the idea that calf thyroglobulin, even though it has an apparently complex molecular organization, contains structural motifs which are repeated in the elementary chain.
Abstract: In this paper we report on the peptide maps of the three polypeptides produced by reduction of denatured 19-S thyroglobulin from guinea pig. These maps were obtained by both chemical cleavage (CNBr) and by limited enzymatic proteolysis (Staphylococcus aureus SV 8 protease). Analysis of these peptides showed a strict correspondence among the electrophoretic bands produced by cleavage of the guinea pig thyroglobulin components. These results support the idea that thyroglobulin, even though it has an apparently complex molecular structure, contains polypeptide sequences which are repeated in the elementary chain. Since similar structures should correspond to similar functions, each of these regions may work as a functional domain for the biosynthesis of the thyroid hormones.
Abstract: Polyethylene glycol solutions (10% w/v) were used to detect the effect of mono- and divalent cations on some properties of thyroglobulin. It is shown that in presence of 10% w/v polyethylene glycol in 0.01 M Tris-HCl, pH 7.5, calcium (less than 0.05 M) modifies the solubility, the sedimentation rate, and the Stokes' radius of thyroglobulin, while monovalent cations up to 0.6 M do not effect any of these properties. These findings can be explained by an increase in molecular compactness of thyroglobulin. Furthermore, it was shown that a synthetic polymer, polyethylene glycol, could be used to detect conformational changes.
Abstract: The two half-sized subunits of 19 S thyroglobulin have been separated and analyzed. They share the same peptide composition and carbohydrate content. The only difference was the iodine level, which was about three times higher in the faster electrophoretic subunit.
Abstract: A very simple procedure which enhances sensitivity in visualizing proteins stained by all standard procedures in polyacrylamide gels is described. The process, based on the use of concentrated polyethylene glycol 6000 solutions, produces a conspicuous, reversible, and uniform size reduction of gels. The entity of the reduction depends principally on the polyethylene glycol 6000 concentration and on the acrylamide content of gels. Using an appropriate polyethylene glycol 6000 concentration, it is possible to regulate the final size and then the sensitivity as needed. Depending upon the above conditions it is possible to increase the sensitivity by a factor of 4-6 or more. Reduction of the gel volume by such a factor may eliminate the need for the cumbersome and frequently impossible preconcentration of samples. Besides the Coomassie blue stained gels, silver stained gels may also be treated in a similar manner with a consequent further enhancement in sensitivity. In addition, gels containing radiolabeled materials can be autoradiographed in times proportionally shorter, without significant sacrifice of resolution.
Abstract: 19 S thyroglobulins from several mammals were prepared under conditions designed to minimize endogenous proteolytic activities. The purified iodoproteins were then fractionated by RbCl isopycnic gradient centrifugation and fractions of varying iodine content analyzed by SDS-polyacrylamide gel electrophoresis. The dependence on the iodination level of molecular dissociability, previously reported in preparations "markedly' differing in their iodination degree, has been now ultimately assessed in all species investigated and demonstrated even for fraction differing from each other only by a few iodine atoms. Upon reduction fractions across the isopycnic gradients showed the occurrence of discrete electrophoretic bands faster than the 300,000 Da subunit: the relative amount of these peptides has been found to be linearly related to the iodine content of the molecules analyzed. This behaviour has been observed in all animal species investigated. In conclusion, our data suggest that iodination, beside the well-documented effects on thyroglobulin conformational properties, may also affect its "apparent' polypeptide chain composition. The origin of such heterogeneity is discussed.
Abstract: It is known that thyroglobulin can be dissociated into a component which appears to be a half molecule (12 S) of the undissociated molecule (19 S). In the present work, these two molecular species were isolated with a high degree of purity by preparative gel electrophoresis in sodium dodecyl sulfate and were individually reduced. The reduction pattern of the 12 S form displayed only two closely migrating bands, both having an apparent Mr near 330 000, whereas the undissociated (covalently linked) 19 S form showed a complex pattern consisting of, besides the 330 000 doublet, nonreducible material and several faster bands, resembling the pattern of the unfractionated protein. The origin of the faster-moving peptides is not known. These results have been obtained with both hog and rat thyroglobulin.
Abstract: The effects of lysolecithin and hexadecyltrimethylammonium bromide on the structure and stability of apoA-II from human high density lipoprotein have been evalued by circular dichroism and fluorescence measurements. There is a profound enhancement in the stability of apoA-II to guanidinium hydrochloride denaturation when it forms a phospholipid complex with lysolecithin micelles. This complex is not only resistant to guanidinium hydrochloride denaturation, but it can be formed in a 6 M solution of this denaturant. The behavior of apoA-II in the native human high density protein is much closer to that of the lysolecithin apoA-II complex than to that of the free apoA-II.
Abstract: The molecular properties of the single linear chain form of human apoA-II, i.e., Cm apoA-II, have been evaluated by circular dichroism, polarization of fluorescence, difference absorption, and sedimentation equilibrium. The self-association of Cm apoA-II to a dimer resembles closely that of apoA-II though the free energy change is somewhat smaller. The dimerization of Cm apoA-II is accompanied by major changes in secondary and tertiary structure. The apoA-II molecule, therefore, represents a molecular association where the intramolecular structure is strongly dependent on the quaternary structure.
Abstract: The binding of apoA-I to lysolecithin has been studied by fluorescence and circular dichroism. The influence of the conformation of apoA-I on its interaction with lysolecithin has also been evaluated. ApoA-I is bound to lysolecithin with an association greater than 10(7) whether apoA-I is native or highly unfolded in 1.8 M guanidinium hydrochloride. The association of apoA-I with lysolecithin results in an increase in secondary structure. A 25-residue fragment of apoA-I binds to lysolecithin equally strongly as the native molecule.
