Abstract: We tested the tissue reactions and mechanical strength of a novel biodegradable craniomaxillofacial plating system, Inion CPS, in the course of degradation. Plates and screws composed of L-lactide, D-lactide and trimethylene carbonate were implanted to the mandible and dorsal subcutis of 12 sheep. The animals were sacrificed at 6-156 weeks. Histological evaluation was done using paraffin and methylmetacrylate techniques. Degradative and mechanical properties during the follow-up were measured both of in vivo and in vitro implants. In light microscopy, the in vivo implant material began to fragment at 52 weeks and could not be detected at 104 weeks. No significant foreign body reactions were seen in the mandibles. The dorsal subcutis disclosed mild reactions, which were, however, not of clinical significance. The implants in vitro maintained their entire mass for 26 weeks and lost 63-80% of the mass by week 104. The inherent viscosity of the implants in vitro and in vivo diminished uniformly. The screws retained their shear strength for 12-16 weeks. The plates maintained their tensile strength for at least 6 weeks. The maximum capacity of the plates in 3-point bending tests diminished gradually by 87% in 26 weeks. In conclusion, the plates and screws examined maintain adequate strength for the healing period of a bone fracture or osteotomy, producing no harmful foreign body reactions.

Abstract: The study was aimed to test the mechanical strength, structural stability, and tissue reactions of optically amorphous and crystalline polyetheretherketone (PEEK) plates during a 3-year follow-up in vivo and in vitro. The injection-moulded PEEK plates were implanted to the dorsal subcutis of 12 sheep, which were sacrificed at 6-156 weeks. Thereafter, the plates were subjected to tensile tests, and levels of crystallinity were assessed by differential scanning calorimetry (DSC). Histological evaluation was carried out using the paraffin technique. In vitro properties were examined with the tensile test and DSC at 0-156 weeks. Tissue reactions were mild and fairly similar for the amorphous and crystalline plates at corresponding points in time. The mechanical characteristics of the plates remained stable over the entire follow-up. The tensile yield load and elongation at the yield load of the crystalline plates were roughly double ( approximately 500 vs. 270 N and 2.4 vs. 1.4 mm, respectively) in comparison to the amorphous plates. The elongation at break load of the crystalline plates was smaller than that of the amorphous ones (6 vs. 10). The level of crystallinity in both the optically amorphous ( approximately 15%) and crystalline (32-34%) plates remained invariable during the follow-up. The in vitro and in vivo data coincided remarkably well. In conclusion, both optically amorphous and crystalline PEEK plates are suitable for the fixation of fractures and osteotomies.

Abstract: Dystrophin associated protein alpha-syntrophin is known to interact with voltage-gated sodium ion channel (NaCh). Dystrophin is known to be sensitive to eccentric muscle actions. For this reason, the function of the NaChs might also be affected. Molecular adaptations of dystrophin, alpha-syntrophin and NaChs were investigated after fatiguing stretch-shortening cycle (SSC) exercise, which consisted of unilateral jumps on a sledge apparatus. Muscle biopsies were taken from the vastus lateralis muscle of eight healthy subjects immediately after (IA) and 2 days after (2D) the exercise to analyze mRNA levels and immunohistochemical staining patterns. SSC exercise resulted in decreased isometric maximal voluntary contraction (IA: -31+/-9%, 2D: -14+/-16%) and a delayed increase of plasma creatine kinase activity (2D: +178+/-211%). Despite muscle soreness (P<0.001), no morphological damage was observed and no changes were found in the mRNA concentrations. However, the relative changes of the mRNA concentrations of alpha-syntrophin and NaChs were highly correlated (r=0.93, P<0.001) 2D after SSC exercise. This consistent pattern of mRNA regulation may imply a functional relationship between these two proteins. In addition, the current experiment emphasises high inter-individual variation in molecular responses to heavy exercise.

Abstract: Backround-Previous human studies have shown divergent results concerning the effects of exercise training on myocardial blood flow (MBF) at rest or during adenosine-induced hyperemia in humans. We studied whether these responses are related to alterations in adenosine A2A receptor (A2AR) density in the left-ventricular (LV) myocardium, size and work output of the athlete's heart, or to fitness level. Methods and Results-MBF at baseline and during intravenous adenosine infusion, and A2AR density at baseline were measured using positron emission tomography by novel A2AR tracer in 10 healthy male endurance athletes (ET) and 10 healthy untrained (UT) men. Structural LV parameters were measured with echocardiography. LV mass index was 71 % higher in ET than UT (193 +/- 18 g/m2 vs 114 +/- 13 g/m2, respectively). MBF per gram of tissue was significantly lower in the ET than UT at baseline, but this was only partly explained by reduced LV work load since MBF corrected for LV work was higher in ET than UT, as well as total MBF. The MBF during adenosine-induced hyperemia was reduced in ET compared to UT, and the fitter the athlete was, the lower was adenosine-induced MBF. A2AR density was not different between the groups and was not coupled to resting or adenosine-mediated MBF. Conclusions-The novel findings of the present study show that the adaptations in the heart of highly trained endurance athletes lead to relative myocardial 'overperfusion' at rest. On the other hand hyperaemic perfusion is reduced, but is not explained by A2AR density.

Abstract: BACKGROUND: Diabetes has negative, and exercise training positive, effects on the skeletal muscle vasculature, but the mechanisms are not yet fully understood. In the present experiment the effects of running exercise on the mRNA expression of pro- and antiangiogenic factors were studied in healthy and diabetic skeletal muscle. The responses in capillaries and muscle fibers, collected from the muscle with laser capture microdissection, were also studied separately. METHODS: Healthy and streptozotocin-induced diabetic mice were divided into sedentary and exercise groups. Exercise was a single bout of 1 h running on a treadmill. Gastrocnemius muscles were harvested 3 h and 6 h post exercise, and angiogenesis-related gene expressions were analyzed with real-time PCR. In addition to muscle homogenates, capillaries and muscle fibers were collected from the muscle with laser capture microdissection method and analyzed for vascular endothelial growth factor-A (VEGF-A) and thrombospondin-1 (TSP-1) mRNA expression. RESULTS: Of the proangiogenic factors, VEGF-A and VEGF receptor-2 (VEGFR-2) mRNA expression increased significantly (P < 0.05) in healthy skeletal muscle 6 h post exercise. VEGF-B also showed a similar trend (P = 0.08). No significant change was observed post exercise in diabetic muscles in the expression of VEGF-A, VEGFR-2 or VEGF-B. The expression of angiogenesis inhibitor TSP-1 and angiogenic extracellular matrix protein Cyr61 were significantly increased in diabetic muscles (P < 0.05-0.01). Capillary mRNA expression resembled that in the muscle homogenates, however, the responses were greater in capillaries compared to muscle homogenates and pure muscle fibers. CONCLUSION: The present study is the first to report the effects of a single bout of exercise on the expression of pro- and antiangiogenic factors in diabetic skeletal muscle, and it provides novel data about the separate responses in capillaries and muscle fibers to exercise and diabetes. Diabetic mice seem to have lower angiogenic responses to exercise compared to healthy mice, and they show markedly increased expression of angiogenesis inhibitor TSP-1. Furthermore, exercise-induced VEGF-A expression was shown to be greater in capillaries than in muscle fibers.

Abstract: In striated muscle, a sarcomeric noncontractile protein, titin, is proposed to form the backbone of the stress- and strain-sensing structures. We investigated the effects of diabetes, physical training, and their combination on the gene expression of proteins of putative titin stretch-sensing complexes in skeletal and cardiac muscle. Mice were divided into control (C), training (T), streptozotocin-induced diabetic (D), and diabetic training (DT) groups. Training groups performed for 1, 3, or 5 wk of endurance training on a motor-driven treadmill. Muscle samples from T and DT groups together with respective controls were collected 24 h after the last training session. Gene expression of calf muscles (soleus, gastrocnemius, and plantaris) and cardiac muscle were analyzed using microarray and quantitative PCR. Diabetes induced changes in mRNA expression of the proteins of titin stretch-sensing complexes in Z-disc (MLP, myostatin), I-band (CARP, Ankrd2), and M-line (titin kinase signaling). Training alleviated diabetes-induced changes in most affected mRNA levels in skeletal muscle but only one change in cardiac muscle. In conclusion, we showed diabetes-induced changes in mRNA levels of several fiber-type-biased proteins (MLP, myostatin, Ankrd2) in skeletal muscle. These results are consistent with previous observations of diabetes-induced atrophy leading to slower fiber type composition. The ability of exercise to alleviate diabetes-induced changes may indicate slower transition of fiber type.

Abstract: High mechanical loading was hypothesized to induce the expression of angiogenic and/or lymphangiogenic extracellular matrix (ECM) proteins in skeletal muscle. Eight men performed a strenuous exercise protocol, which consisted of 100 unilateral maximal drop jumps followed by submaximal jumping until exhaustion. Muscle biopsies were taken 30 min and 48 h postexercise from the vastus lateralis muscle and analyzed for the following parameters: mRNA and protein expression of ECM-associated CCN proteins [cysteine-rich angiogenic protein 61 (Cyr61)/CCN1, connective tissue growth factor (CTGF)/CCN2], and mRNA expression of vascular endothelial growth factors (VEGFs) and hypoxia-inducible factor-1alpha. The mRNA expression of Cyr61 and CTGF increased 30 min after the exercise (14- and 2.5-fold, respectively; P < 0.001). Cyr61 remained elevated 48 h postexercise (threefold; P < 0.05). The mRNA levels of VEGF-A, VEGF-B, VEGF-C, VEGF-D, or hypoxia-inducible factor-1alpha did not change significantly at either 30 min or 48 h postexercise; however, the variation between subjects increased markedly in VEGF-A and VEGF-B mRNA. Cyr61 protein levels were higher at both 30 min and 48 h after the exercise compared with the control (P < 0.05). Cyr61 and CTGF proteins were localized to muscle fibers and the surrounding ECM by immunohistochemistry. Fast fibers stained more intensively than slow fibers. In conclusion, mechanical loading induces rapid expression of CCN proteins in human skeletal muscle. This may be one of the early mechanisms involved in skeletal muscle remodeling after exercise, since Cyr61 and CTGF regulate the expression of genes involved in angiogenesis and ECM remodeling.

Abstract: Blood and lymphatic vessels together form the circulatory system, allowing the passage of fluids and molecules within the body. Recently we showed that lymphatic capillaries are also found in the capillary bed of skeletal muscle. Exercise is known to induce angiogenesis in skeletal muscle, but it is not known whether exercise has effects on lymphangiogenesis or lymphangiogenic growth factors. We studied lymphatic vessel density and expression of the main lymphangiogenic growth factors VEGF-C and VEGF-D and their receptor VEGFR-3 in response to acute running exercise and endurance exercise training in the skeletal muscle of healthy and diabetic mice. VEGF-C mRNA expression increased after the acute exercise bout (P < 0.05) in healthy muscles, but there was no change in diabetic muscles. VEGF-C levels were not changed either in healthy or in diabetic muscle after the exercise training. Neither acute exercise nor exercise training had an effect on the mRNA expression of VEGF-D or VEGFR-3 in healthy or diabetic muscles. Lymphatic vessel density was similar in sedentary and trained mice and was >10-fold smaller than blood capillary density. Diabetes increased the mRNA expression of VEGF-D (P < 0.01). Increased immunohistochemical staining of VEGF-D was found in degenerative muscle fibers in the diabetic mice. In conclusion, the results suggest that acute exercise or exercise training does not significantly affect lymphangiogenesis in skeletal muscle. Diabetes increased the expression of VEGF-D in skeletal muscle, and this increase may be related to muscle fiber damage.

Abstract: In coeliac disease gluten induces an immunological reaction in genetically susceptible patients, and influences on epithelial cell proliferation and differentiation in the small-bowel mucosa. Our aim was to find novel genes which operate similarly in epithelial proliferation and differentiation in an epithelial cell differentiation model and in coeliac disease patient small-bowel mucosal biopsy samples. The combination of cDNA microarray data originating from a three-dimensional T84 epithelial cell differentiation model and small-bowel mucosal biopsy samples from untreated and treated coeliac disease patients and healthy controls resulted in 30 genes whose mRNA expression was similarly affected. Nine of 30 were located directly or indirectly in the receptor tyrosine kinase pathway starting from the epithelial growth factor receptor. Removal of gluten from the diet resulted in a reversion in the expression of 29 of the 30 genes in the small-bowel mucosal biopsy samples. Further characterization by blotting and labelling revealed increased epidermal growth factor receptor and beta-catenin protein expression in the small-bowel mucosal epithelium in untreated coeliac disease patients compared to healthy controls and treated coeliac patients. We found 30 genes whose mRNA expression was affected similarly in the epithelial cell differentiation model and in the coeliac disease patient small-bowel mucosal biopsy samples. In particular, those genes involved in the epithelial growth factor-mediated signalling pathways may be involved in epithelial cell differentiation and coeliac disease pathogenesis. The epithelial cell differentiation model is a useful tool for studying gene expression changes in the crypt-villus axis.

Abstract: Diabetes alters microvascular structure and function and is a major risk factor for cardiovascular diseases. In diabetic skeletal muscle, impaired angiogenesis and reduced VEGF-A expression have been observed, whereas in healthy muscle exercise is known to have opposite effects. We studied the effects of type 1 diabetes and combined exercise training on angiogenic mRNA expression and capillarization in mouse skeletal muscle. Microarray and real-time PCR analyses showed that diabetes altered the expression of several genes involved in angiogenesis. For example, levels of proangiogenic VEGF-A, VEGF-B, neuropilin-1, VEGFR-1, and VEGFR-2 were reduced and the levels of antiangiogenic thrombospondin-1 and retinoblastoma like-2 were increased. Exercise training alleviated some of these changes, but could not completely restore them. VEGF-A protein content was also reduced in diabetic muscles. In line with the reduced levels of VEGF-A and other angiogenic factors, and increased levels of angiogenesis inhibitors, capillary-to-muscle fiber ratio was lower in diabetic mice compared to healthy controls. Exercise training could not restore capillarization in diabetic mice. In conclusion, these data illustrate that type 1 diabetes is associated with reduced skeletal muscle capillarization and the dysregulation of complex angiogenesis pathways.

Abstract: The aim of this study was to assess degradation of a novel bioactive guided tissue regeneration (GTR) membrane and to quantify the concurrent tissue responses. Pieces of membrane composed of poly-l-lactide, poly-d,l-lactide, trimethylenecarbonate and polyglycolide were dipped into an N-methyl-2-pyrroline (NMP) solution and implanted in the mandibles of 10 sheep. The animals were sacrificed at 6-104 weeks. Parallel in vitro degradation was analysed by measuring the inherent viscosity, water absorption and remaining mass. One of the 2 in vitro sets of membranes was prehandled with NMP. At 6-26 weeks in vivo, the gradually more degraded implants were surrounded by a fibrous network. At 52 and 104 weeks, the implants and fibrous networks were non-detectable. Foreign body granulomatous reactions were not observed. In vitro, the mass of the NMP-exposed membranes diminished linearly over the 2-year period down to 10%, while the non-NMP-exposed membrane maintained all their mass for the first 16 weeks. The membranes without NMP had absorbed significantly less water at weeks 4 and 8 than the other group. The inherent viscosity decreased relatively uniformly in the in vitro groups. In conclusion, the in vivo degradation was complete in 12 months with only mild histologic responses; the degradation in vitro may be slower. NMP accelerates the degradation.

Abstract: Histone acetylation plays a key role in the regulation of gene expression. The chromatin structure and accessibility of genes to transcription factors is regulated by enzymes that acetylate and deacetylate histones. The Sin3A corepressor complex recruits histone deacetylases and in many cases represses transcription. Here, we report that SAP30L, a close homolog of Sin3-associated protein 30 (SAP30), interacts with several components of the Sin3A corepressor complex. We show that it binds to the PAH3/HID (Paired Amphipathic Helix 3/Histone deacetylase Interacting Domain) region of mouse Sin3A with residues 120-140 in the C-terminal part of the protein. We provide evidence that SAP30L induces transcriptional repression, possibly via recruitment of Sin3A and histone deacetylases. Finally, we characterize a functional nucleolar localization signal in SAP30L and show that SAP30L and SAP30 are able to target Sin3A to the nucleolus.

Abstract: Diabetes induces changes in the structure and function of the extracellular matrix (ECM) in many tissues. We investigated the effects of diabetes, physical training, and their combination on the gene expression of ECM proteins in skeletal muscle. Mice were divided to control (C), training (T), streptozotocin-induced diabetic (D), and diabetic training (DT) groups. Training groups (T, DT) performed 1, 3, or 5 wk of endurance training on a treadmill. Gene expression of calf muscles was analyzed using microarray and quantitative PCR. Training group samples were collected 24 h after the last training session. Diabetes affected the gene expression of several collagens (types I, III, IV, V, VI, and XV), some noncollagenous glycoproteins, and proteoglycans (e.g., elastin, thrombospondin-1, laminin-2, decorin). Reduced gene expression of collagens in diabetic skeletal muscle was partially attenuated as a result of physical training. In diabetes, mRNA expression of the basement membrane (BM) collagens decreased and that of noncollagenous glycoproteins increased. This may change the structure of the BM in a less collagenous direction and affect its properties.

Abstract: BACKGROUND: The TGFbeta1-induced signal transduction processes involved in growth and differentiation are only partly known. The three-dimensional epithelial differentiation model, in which T84 epithelial cells are induced to differentiate either with TGFbeta1 or IMR-90 mesenchymal cell-secreted soluble factors, is previously shown to model epithelial cell differentiation seen in intestine. That model has not been used for large scale gene expression studies, such as microarray method. Therefore the gene expression changes were studied in undifferentiated and differentiated three-dimensional T84 cultures with cDNA microarray method in order to study the molecular changes and find new players in epithelial cell differentiation. RESULTS: The expression of 372 genes out of 5188 arrayed sequences was significantly altered, and 47 of them were altered by both mediators. The data were validated and the altered genes are presented in ontology classes. For the genes tested the expressions in protein level were in accordance with the mRNA results. We also found 194 genes with no known function to be potentially important in epithelial cell differentiation. The mRNA expression changes induced by TGFbeta1 were bigger than changes induced by soluble factors secreted by IMR-90 mesenchymal cells. The gene expression data was depicted in already known signaling pathway routes. CONCLUSION: Our results reveal potential new signaling pathways and several new genes affected by TGFbeta in epithelial cell differentiation. The differentiation induced by TGFbeta1 appears to be more potent than the differentiation induced by mesenchymal cells. This study indicates that our cell culture model is a suitable tool in studying regulatory mechanisms during epithelial cell differentiation in intestine. Furthermore the present results indicate that our model is a good tool for finding new players acting in the differentiation of epithelial cells.

Abstract: Glycosylation of proteins and lipids is important in cellular communication and maintenance of tissues. B3GTL (beta3-glycosyltransferase-like) is a novel glycosyltransferase that is found in multicellular animals ranging from mammals to insects and nematodes. The aim of this work was to identify and characterize the B3GTL gene in the mouse and to study its expression in various tissues. The murine gene codes for a protein which shares 84% amino acid sequence identity with its human ortholog, and contains all the primary structural features that characterize B3GTL proteins. The murine and human B3GTL genes share an identical exon/intron organization, and both genes utilize multiple polyadenylation signals. Their promoter regions show extensive conservation, implying that the two genes also share regulatory similarities. This notion was reinforced by Northern hybridization analysis of mouse tissues, which showed the tissue distribution of B3GTL mRNA to be similar to that previously found in human tissues, with the heart, kidney, and brain being major sites of expression in both species. The localization of B3GTL mRNA was studied by in situ hybridization in an extensive collection of mouse tissues, of which the granular cells of the olfactory bulb and the epithelium of the seminal vesicle displayed particularly strong signals. Together, these analyses indicate that the B3GTL mRNA is subject to strong tissue-specific and developmental regulation. The findings reported here make possible the design of a B3GTL "knock-out" mouse, provide a framework for analyzing the regulation of the gene, and provide an extensive catalog of tissues in which this novel protein acts.

Abstract: BACKGROUND: Clinical manifestation of IgA nephropathy (IgAN) strikingly occurs after respiratory tract infections. An intestinal inflammation has also been described. We hypothesized that the intestinal inflammation should manifest itself as an increase in inflammatory cells and mucosal cyclooxygenase 2 (COX-2) expression. METHODS: By using immunohistochemistry, we determined the phenotype and quantity of inflammatory cells in duodenal biopsy specimens from 17 IgAN patients. Control material comprised 18 patients undergoing gastroscopy because of dyspepsia. RESULTS: All the biopsy specimens disclosed normal villous architecture. In IgAN, CD3(+) cells and COX-2-positive cells were significantly increased and J chain-producing plasma cells were significantly decreased. CD3(+) cells coexpressed COX-2 protein and COX-2-positive cells also expressed CD45RO antigen. The number of lymphocytes correlated significantly with serum IgA and COX-2-expression with serum IgA and the degree of hematuria. COX-2-positive subepithelial fibroblasts were a conspicuous finding in IgAN. In CD68(+) and CD15(+) cells, a significant increase was seen. Many of these cells also expressed COX-2 protein. CD15(+) positivity correlated significantly with proteinuria in IgAN. CONCLUSION: Our results indicate that small bowel inflammation in IgAN shows itself as an increased number of mucosal inflammatory cells. However, polymeric IgA production is significantly decreased. An increased mucosal COX-2 expression suggests activation of the inflammatory cells and the degree of inflammation significantly correlates with serum IgA and the amount of proteinuria and hematuria. Subepithelial fibroblasts seem also to be involved in the inflammatory reaction.

Abstract: In coeliac disease in the jejunum of a genetically susceptible person wheat gliadin and related prolamins from rye and barley trigger an immunological reaction, which induces small-bowel mucosal transformations, villous atrophy and crypt hyperplasia. Though CD4+ specific T cells, intraepithelial lymphocytes, infiltrating plasma cells and autoantibodies are known to have an effect on the coeliac disease, the pathogenic mechanisms leading to the tissue injury remain to be elucidated. Our aim was to find novel gene transcripts, which might have a role in coeliac disease pathogenesis. The gene expression in duodenal biopsy samples from untreated coeliac patients (n=4), patients on gluten-free diet (n=4) and healthy controls (n=4) was studied by cDNA microarray analysis. The method allows monitoring of the expression of thousands of genes simultaneously. Compared to healthy controls, the expression of 156 and 60 genes was changed in untreated and treated coeliac disease, respectively. Between treated and untreated coeliac disease, 98 genes had altered expression. Of the 5184 genes or expressed sequence tags, altogether 263 were affected. Many of these genes are directly or indirectly connected to T-cell activation, B-cell maturation or epithelial cell differentiation. By the microarray method, numerous genes were found to evince altered mRNA expression in coeliac disease. The method holds promise in exploring the pathogenetic mechanisms in the small bowel and may reveal new target genes for the therapy of coeliac disease.

Abstract: We report the identification and primary structure of a novel human glycosyltransferase, B3GTL (beta3-glycosyltransferase-like). The 498 residue protein consists of a short cytoplasmic N-terminal "tail" (residues 1-4), a single transmembrane domain with type II topology (residues 5-28), a "stem" region (residues 29-260), and a catalytic domain (residues 261-498). The genomes of Anopheles gambiae, Drosophila melanogaster, and Caenorhabditis elegans encode potential orthologs which share 31-39% sequence identity with B3GTL, as well as the following features: a conserved catalytic domain containing a triple aspartate motif (DDD) at its core, a conserved pattern of cysteine residues, a C-terminal KDEL-like motif, and conserved residues and motifs that affiliate this novel group with a family of beta3-glycosyltransferases (GT31 in the CAZY classification). The B3GTL gene lacks canonical TATA and CAAT boxes and contains three functional polyadenylation sites. It is transcribed in a wide range of tissues and in TGF-beta-treated T84 epithelial cells.

Abstract: BACKGROUND: We have previously set up an in vitro mesenchymal-epithelial cell co-culture model which mimics the intestinal crypt villus axis biology in terms of epithelial cell differentiation. In this model the fibroblast-induced epithelial cell differentiation from secretory crypt cells to absorptive enterocytes is mediated via transforming growth factor-beta (TGF-beta), the major inhibitory regulator of epithelial cell proliferation known to induce differentiation in intestinal epithelial cells. The aim of this study was to identify novel genes whose products would play a role in this TGF-beta-induced differentiation. RESULTS: Differential display analysis resulted in the identification of a novel TGF-beta upregulated mRNA species, the Sin3-associated protein 30-like, SAP30L. The mRNA is expressed in several human tissues and codes for a nuclear protein of 183 amino acids 70% identical with Sin3 associated protein 30 (SAP30). The predicted nuclear localization signal of SAP30L is sufficient for nuclear transport of the protein although mutating it does not completely remove SAP30L from the nuclei. In the nuclei SAP30L concentrates in small bodies which were shown by immunohistochemistry to colocalize with PML bodies only partially. CONCLUSIONS: By reason of its nuclear localization and close homology to SAP30 we believe that SAP30L might have a role in recruiting the Sin3-histone deacetylase complex to specific corepressor complexes in response to TGF-beta, leading to the silencing of proliferation-driving genes in the differentiating intestinal epithelial cells.

Abstract: BACKGROUND AND AIMS: Coeliac disease is characterised by atrophy of the villi and hyperplasia of the crypts in the mucosa of the small intestine. It is caused by an environmental trigger, cereal gluten, which induces infiltration of the mucosa by inflammatory cells. We hypothesised that these inflammatory cells express cyclooxygenase 2 (COX-2), an enzyme that contributes to the synthesis of pro and anti-inflammatory prostaglandins and is known to be expressed at sites of inflammation in the stomach and colon. We have investigated expression of COX-2 in the coeliac disease affected small intestinal mucosa where it may be an indicator of either disease induction or mucosal restoration processes. PATIENTS AND METHODS: Small intestinal biopsy samples from 15 coeliac patients and 15 non-coeliac individuals were stained immunohistochemically for COX-2. Samples from 10 of the patients were also stained after these patients had been on a gluten free diet for 6-24 months. Various cell type marker antigens were used for immunohistochemical identification of the type of cell that expressed COX-2. To further verify colocalisation of the cell type marker and COX-2, double immunoperoxidase and immunofluorescence methods were employed. Immunoelectron microscopy was used to investigate the subcellular location of COX-2. RESULTS: In all samples taken from coeliac patients, clusters of cells with strong immunoreactivity for COX-2 were found in those areas of the lamina propria where the epithelium seemed to blister or was totally detached from the basement membrane. These clusters were reduced in number or totally absent in samples taken after a gluten free diet. No such clusters were seen in any control samples. The density of COX-2 positive cells lining the differentiated epithelium decreased significantly from 13.5 (5.1) cells/10(5) microm(2) (mean (SD)) in the untreated patient samples to 6.5 (2.0) cells/10(5) microm(2) after a gluten free diet (p<0.001), and was 3.3 (1.9) cells/10(5) microm(2) in control samples (p<0.001 compared with untreated or diet treated coeliac samples). Staining for COX-2 was localised to CD3+ T cells and CD68+ macrophages in the mucosal lesions but not all of these cells were positive for COX-2. Immunoelectron microscopy revealed that the ultrastructure of the COX-2 positive cells resembled that of lymphocytes, and the immunoreaction was localised to the rough endoplasmic reticulum and the nuclear envelope. CONCLUSIONS: Our results show that in coeliac disease, blistering of small intestinal epithelial cells is associated with accumulation of COX-2 positive T cells, and the number of these cells decreases after a gluten free diet. These observations suggest that COX-2 mediated prostanoid synthesis contributes to healing of the coeliac mucosa and may be involved in maintenance of intestinal integrity.

Abstract: We have previously shown that transforming growth factor-beta1 (TGF-beta1) is involved in the fibroblast-induced organization and differentiation of transformed phenotypically crypt-like T84 intestinal epithelial cells into absorptive enterocyte-like cells, when cultured within a three-dimensional collagen gel. We have used differential display polymerase chain reaction to find genes that are either up- or downregulated by TGF-beta in the T84 cells cultured in three-dimensional collagen gel and then studied how these in vitro differentially expressed genes are expressed in vivo in the small intestinal crypt-villus axis. We found that the TGF-beta1-treated T84 cells, like the villus tip enterocytes, expressed increased levels of CC3/TIP30 when compared to the undifferentiated cells. Furthermore, the expression of rab11 showed the opposite pattern, being higher in the undifferentiated cells both in vivo and in vitro. We conclude that the three-dimensional cell culture model where TGF-beta induces organization and differentiation of secretory T84 epithelial cells makes it possible to find up- and downregulated transcripts that also play a role in the human small intestinal crypt-villus axis.

Abstract: We have identified a novel gene product differentially expressed in skeletal muscle in a rat diabetes model induced by streptozotocin. Northern blot analysis showed expression in all studied tissues and a marked down-regulation in insulin-sensitive tissues (skeletal muscle, myocardium) but not in insulin-insensitive brain. cDNA sequence analysis revealed an open reading frame (ORF) of 58 aminoacids, containing one putative transmembrane domain. We designated this protein DAPIT (diabetes-associated protein in insulin-sensitive tissues). Database searches discovered several human, rat, mouse, rabbit, bovine, equine, porcine, Fugu and Xenopus ESTs with significant homology in the ORF. The aminoacid sequence of DAPIT is similar to that of a putative protein of Drosophila melanogaster and to a cAMP-generating peptide isolated from the flesh fly Neobellieria bullata. We propose that DAPIT is a novel protein that is highly conserved in vertebrates and insects.

Abstract: We sought to develop a biodegradable pancreatic stent that could be easily placed at operation into the human pancreatic duct and the degradation of which could be easily followed up. Spiral-shaped, gamma-sterilized stents were manufactured of 0.4-mm polylactide wire in which there was added 23 weight-% barium sulfate. The biodegradability of the stents was studied in vitro at two different pH values, the first resembling that of pancreatic juice and the other that of bile. The effects of enzymoactivity in the test solution and the composition of the stents (with or without barium addition) also were tested. These kinds of stents have been experimented with in two pilot patients. Degradation of the stents occurred from 24 to 52 weeks of incubation. Alkaline milieu together with the presence of pancreatic enzyme made the stents degrade twice as fast as when either alkaline milieu or enzyme was present. In the milieu resembling pancreatic juice, barium sulfate had no effect on the degradation time. Neither of the pilot patients had any postoperative complications. Biodegradable, x-ray-positive stents degrade faster in pancreatic than in biliary milieu. Their safety and efficacy in human pancreaticojejunal anastomoses need further study.

Abstract: Intestinal crypt epithelial T84 cells form luminal structures and differentiate to intestinal enterocyte-like cells in response to IMR-90 fibroblast-secreted transforming growth factor-beta when grown within three-dimensional collagen gel. In search of TGF-beta regulated genes involved in this differentiation process, we isolated a TGF-beta downregulated cDNA, human homologue of rat apoptosis antagonising transcription factor that codes for a 560-amino-acid protein. Human AATF-mRNA was expressed at high levels in human brain, heart, thymus, kidney, and placenta while in skeletal muscle and colon the expression was lower. The gene was mapped to chromosome 17q11.2-q12.

Abstract: BACKGROUND: The purpose of this study was to design a simplified polymerase chain reaction (PCR) technique for the detection of Helicobacter pylori and to compare it with conventional diagnostic methods-culture and histology of gastric biopsy specimens. In addition, the capability of this technique to detect H. pylori in the gastric mucosal biopsies of originally H. pylori-negative children with gastritis or recurrent abdominal pain was investigated. METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR) using polymerase from Thermus thermophilus was applied to detect H. pylori 16S rRNA. Twenty-five children H. pylori-positive by culture and/or histology were used as positive control subjects. Sixteen healthy H. pylori-negative children served as negative control subjects. Biopsy specimens from gastric antrum and corpus from 81 children were examined by RT-PCR. Altogether, 30 had histologic gastritis and 51 had nonspecific abdominal pain only, with no disease in histologic specimens. Histology and culture of H. pylori were negative in both patient groups. RESULTS: Reverse transcription-polymerase chain reaction detected 24 of 25 tissue-positive and 0 of 16 tissue-negative cases, indicating 96% sensitivity and 100% specificity for the test. None of the culturally and histologically H. pylori-negative samples showed H. pylori colonization when analyzed by RT-PCR. CONCLUSIONS: RT-PCR using Thermus thermophilus polymerase is a fast and simple means of detecting H. pylori in gastric biopsy specimens. It is at least as specific and sensitive as conventional methods. In pediatric patients it may be necessary to take more than two biopsy specimens to increase sensitivity in cases of local or patchy colonization.

Abstract: BACKGROUND: Previous studies suggest that intestinal mucosa may be involved in the pathogenesis of IgA nephropathy (IgAN). To further clarify this involvement, we investigated whether or not IgAN patients have small bowel mucosal findings suggestive of inflammation and stress. METHODS: Seventeen patients with IgAN underwent gastroscopic examination. Fresh small bowel biopsy specimens were frozen, and in cryosections, the proportion of alphabeta and gammadelta receptors bearing T cells and CD3+ T cells were quantitated immunohistochemically. The expression of HLA class II antigen DR (HLA-DR) and human GroEL stress-protein homologue was similarly quantitated. In an avidin-biotin peroxidase technique, the following monoclonal primary antibodies were used: anti-beta-chain (betaF1), anti-delta-chain (TCRdelta1), anti-CD3 (Leu4), anti-constant fragment of HLA-DR (L243), and anti-GroEL (ML30). Twenty-nine patients who had undergone gastroscopy because of dyspepsia served as controls. RESULTS: In all specimens, the mucosal architecture was normal. The amount of gammadelta T cells and the total amount of T cells, as indicated by CD3+ positivity, were both significantly increased in IgAN. The number of alphabeta T cells was also higher in the IgAN patients. Villous epithelium of the IgAN patients disclosed a significant increase in the expression of HLA-DR antigen and GroEL stress protein. CONCLUSIONS: Our results suggest that ongoing small bowel inflammation and stress are present in IgAN. Despite normal morphology, there is reason to believe that the small bowel mucosa is involved in the pathogenesis of IgAN.

Abstract: OBJECTIVE: Regular exercise is recommended for the non-pharmacological treatment of hypertension, but the mechanisms underlying the lowering of blood pressure remain controversial. Therefore, we studied the effects of 22-week-long training on blood pressure, arterial reactivity, and metabolic abnormalities in a model of genetic obesity and moderate hypertension. METHODS: Obese and lean Zucker rats were subjected to treadmill exercise from 8 to 30 weeks of age. Blood pressures were measured by the tail-cuff method, and urine was collected in metabolic cages. At the end of the study, the samples for biochemical determinations were taken, and reactivity of isolated mesenteric and carotid arterial rings was examined in standard organ chambers. RESULTS: The exercise prevented the elevation of blood pressure which was observed in non-exercised obese Zucker rats, and also reduced blood pressure in the lean rats. The relaxations of norepinephrine-preconstricted mesenteric and carotid arterial rings to acetylcholine and nitroprusside were clearly improved by exercise in the obese rats. In the lean rats exercise enhanced vasorelaxation to nitroprusside in the mesenteric and carotid rings, and to acetylcholine in the carotid preparations. The exercise-induced improvement of endothelium-mediated dilatation to acetylcholine was abolished by nitric oxide synthesis inhibition with NG nitro-L-arginine methyl ester, but not by cyclooxygenase inhibition with diclofenac or functional inhibition of endothelium-dependent hyperpolarization by precontractions with KCl. The urinary excretion of the systemic prostacyclin metabolite (2,3-dinor-6-ketoprostaglandin F1 alpha) was increased two-fold by exercise in the obese and lean rats, whereas that of the thromboxane A2 metabolite (11-dehydrothromboxane B2) remained unaffected. Treadmill training reduced blood glucose, cholesterol, and triglycerides, but did not affect the high levels of insulin in obese Zucker rats. CONCLUSIONS: These results suggest that the antihypertensive effect of long-term exercise in experimental obesity related hypertension is associated with improved vasodilatation. This is expressed as enhanced relaxation via endogenous and exogenous nitric oxide, and increased endothelial prostacyclin production. The improved control of arterial tone after training could be attributed to the alleviation of hyperlipidemia and insulin resistance, whereas hyperinsulinaemia per se remained unaffected.

Abstract: To investigate the role of placental glucose delivery in fetal growth, two glucose transporters (Glut3 and Glut4) were determined from term placentae. This was accomplished by immunoblotting from crude placental membrane samples from cases of fetal intrauterine growth retardation (IUGR, n = 6), macrosomia (n = 6), maternal diabetes mellitus (n = 4) and normal term (n = 8). Glut3 and Glut4 were detected in only very low numbers in all patient groups and there were no changes in their placental density, which suggests that the expression of these transporters is not involved in disorders of fetal growth. However, birth weight corresponded to placental weight, indicating that the total amount of Glut3 and Glut4 is reduced in IUGR and increased in macrosomia.

Abstract: A novel ras-related gene (rab28) was identified by a PCR-based cloning approach and subsequent screening of rat fat cell and brain cDNA libraries. The deduced amino acid sequence of the cDNA is distantly related with members of the Rab family (31-33% sequence identity, mainly restricted to the six GTP-binding motifs). Cloning of the human homologue of Rab28 by a PCR-based approach revealed the existence of two isoforms (hRab28S, hRab28L) which differ only by a 95-bp insertion within the coding region. This insertion generates an alternative sequence of the 30 C-terminal amino acids of the protein. Both C-termini of the human homologues comprise farnesylation motifs, but differ strikingly in a stretch of 13 amino acids. By PCR, mRNA of hRab28S was detected in most tissues investigated (cortex, liver, kidney, skeletal muscle, adipose tissue, testis and urothelium), whereas hRab28L was predominant in testis. Recombinant Rab28 proteins showed specific binding of radiolabeled guanosine 5'-O-[gamma-thio]triphosphate and rapidly hydrolysed [alpha-32P]GTP; there was no difference in the GTP binding characteristics of the two isoforms hRab28S and hRab28L. It is suggested that the isoforms are derived from the same gene by alternative mRNA splicing, and that their functions differ in a parameter unrelated to its basic role as a GTPase.

Abstract: The cDNA of a novel, ubiquitously expressed protein kinase (Dyrk) was cloned from a rat brain cDNA library. The deduced amino acid sequence (763 amino acids) contains a catalytic domain that is only distantly related to that of other mammalian protein kinases. Its closest relative is the protein kinase Mnb of Drosophila, which is presumably involved in postembryonic neurogenesis (85% identical amino acids within the catalytic domain). Outside the catalytic domain, the sequence comprises several striking structural features: a bipartite nuclear translocation signal, a tyrosine-rich hydrophilic motif flanking the nuclear localization signal, a PEST region, a repeat of 13 histidines, a repeat of 17 serine/threonine residues, and an alternatively spliced insertion of nine codons. A recombinant glutathione S-transferase-Dyrk fusion protein catalyzed autophosphorylation and histone phosphorylation on tyrosine and serine/threonine residues with an apparent Km of approximately 3.4 microM. Exchange of two tyrosine residues in the "activation loop" between subdomains VII and VIII for phenylalanine almost completely suppressed the activity and tyrosine autophosphorylation of Dyrk. Tyrosine autophosphorylation was also reduced by exchange of the tyrosine (Tyr-219) in a tyrosine phosphorylation consensus motif. The data suggest that Dyrk is a dual specificity protein kinase that is regulated by tyrosine phosphorylation in the activation loop and might be a component of a signaling pathway regulating nuclear functions.

Abstract: Thirty patients with chronic renal failure (CRF) and 30 age- and sex-matched controls were assessed for gastrointestinal diseases by gastroscopy, serum gastrin determination, and routine clinical and laboratory evaluation. Biopsy specimens from their gastric oxyntic mucosa were immunohistochemically stained with monoclonal antibodies against serotonin (5-hydroxytryptamine) and chromogranin A, the latter staining all gastric endocrine cells, the former disclosing serotonin-containing enterochromaffin (EC) cells only. The average EC cell density (cells/mm2) in the CRF patients was significantly lower than in the controls: 2.6 vs 12.9 (p = 0.0005). The EC cell counts also correlated negatively with serum gastrin values (p = 0.0031). The densities of the chromogranin-positive cells did not differ between CRF patients (74 cells/mm2) and controls (76 cells/mm2) (p = 0.7559). We conclude that, in addition to the previously known findings of hypoacidity, persistent hypergastrinaemia, and G and parietal cell hyperplasia, CRF also reduces the number of oxyntic EC cells. The negative correlation between EC cell density and serum gastrin levels reflects the complex interplay between different endocrinological activities in the gastrointestinal tract.

Abstract: Increased levels of mRNA transcribed from the ob gene in adipose tissue of obese/hyperinsulinaemic Zucker (fa/fa) rats were detectable as early as 3 weeks after birth and continued to rise there after in parallel with body weight and serum insulin. mRNA levels of two other fat-specific genes (ARL4, FST44) were unaltered. In C57BL/KsJ db/db mice, ob mRNA levels also increased in parallel with body weight and serum insulin, and remained elevated in older animals when insulin levels decreased. In heterozygous control animals (db/+; fa/Fa), mRNA levels were comparable with those in the homozygous controls. In normal Sprague Dawley rats, the ob mRNA increased continuously, but more slowly than in Zucker rats, in parallel with body weight and insulin levels, and reached 15 times higher levels in the heaviest rats (400 g) studied. In Sprague Dawley rats made diabetic by an injection of streptozotocin, ob mRNA levels were reduced by approximately 50% after 24 h. A 24-h fasting period reduced the ob mRNA by 50% in lean Sprague Dawley and Fa/Fa, but not in obese Zucker fa/fa rats, although insulin levels were reduced in both groups. These data indicate that ob mRNA levels increase in both normal and obese rodents in parallel with age, body weight and serum insulin, reflecting an early (Zucker rats, db-mice) or slowly developing (Sprague Dawley rats) resistance to leptin and insulin. This increase does not appear to be mediated by the recently described rapid regulation of ob mRNA by insulin, but seems to be due to a different, long-term control mechanism which signals the size of the fat depots.

Abstract: BACKGROUND & AIMS: The gut epithelium in the crypt-villus axis represents a continuous developmental system in which the role of fibroblast-epithelial interactions is obvious. The aim of this study was to establish an in vitro method whereby fibroblast-guided differentiation of crypt-like gut epithelial cells can be studied. METHODS: Intestinal epithelial cells (T84 and HT-29) were cultured within type I collagen gel together were fibroblasts without cell-to-cell contact. T84 cells were also grown in the presence of transforming growth factor beta and hepatocyte growth factor. The gels were studied using light and electron microscopy and histochemical and immunohistochemical methods. RESULTS: The epithelial cells formed unorganized cell clusters within the gels, but when given fibroblast support, 76% of the T84 cell colonies (not HT-29) organized into luminal formations, and basement membranes including laminin were well deposited. The cells in the columnar single cell-layer luminal formations (49% of all colonies) were differentiated, showing microvilli, up-regulated alkaline phosphatase brush border activity, and mucin profiles typical for small intestine. This fibroblast-induced organization and differentiation was induced by transforming growth factor beta. CONCLUSIONS: Crypt-like T84 epithelial cells are able to differentiate when grown three-dimensionally together with fibroblasts or transforming growth factor beta. This method may be used for mesenchymal-epithelial cell cross-talk studies.

Abstract: The effects of streptozotocin-induced diabetes, physical training and their combination on the activities of prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyl-transferase (GGT), both marker enzymes of collagen biosynthesis, and on the concentration of hydroxyproline (Hyp) were studied in vastus lateralis, rectus femoris and gastrocnemius muscles in rats. The experimental period was 12-16 weeks. Diabetes had an overall decreasing effect on specific PH activity in all muscles studied, whereas specific GGT activity remained at control level. Total PH and GGT activities decreased in all three muscles in the diabetic animals (P < 0.001). Training caused an increase in PH and GGT activities in gastrocnemius in non-diabetic rats, whereas training in combination with diabetes did not change specific PH or GGT activity. Diabetes increased specific Hyp concentration in vastus lateralis and gastrocnemius in trained diabetic rats (P < 0.05), whereas training decreased Hyp level significantly (P < 0.05) in vastus lateralis in non-diabetic rats, but not in diabetic animals. The results suggest that in streptozotocin-induced diabetes the decrease in collagen synthesis rate exceeds the negative total protein balance in the muscle. Although physical training may have an increasing effect on muscular collagen synthesis in non-diabetic rats, it is unable to prevent the decreasing effect of diabetes on collagen synthesis.

Abstract: The in vivo glucose uptake and the levels of two glucose transporter proteins (GLUT1 and GLUT4) were measured in heart and in various types of skeletal muscle from streptozotocin-diabetic rats. Diabetes (12-16 weeks) reduced the in vivo glucose uptake (glucose metabolic index, GMI), and the levels of GLUT1 and GLUT4 in heart by 75%, 60% and 70%, respectively. In diaphragm consisting of approximately equal amounts of type I (slow-contracting oxidative), IIa (fast-contracting oxidative) and IIb (fast-contracting glycolytic) fibers, GMI and GLUT4 levels were reduced by 60% and 40%, respectively, with no change in GLUT1 levels. In muscle consisting mainly of type I fibers (e.g., m. soleus), GMI and GLUT4 levels were reduced by 60% and 30%, respectively, whereas GLUT1 levels were unaltered. In mixed-type muscle consisting of type IIa and IIb fibers (e.g., m. plantaris and red part of m. gastrocnemius), GMI and GLUT1 levels were unchanged, whereas GLUT4 levels were decreased by 45%. In contrast, GMI was increased by 100% in type IIb fibers (e.g., the white part of m. gastrocnemius), probably reflecting the 4-fold increase in blood glucose levels, whereas GLUT4 levels were lowered by 55% with no change in GLUT1 levels. These data demonstrate a marked difference in the response of in vivo glucose uptake to long-term hypoinsulinemia between oxidative (type I) and glycolytic (type IIb) fibers. Furthermore, in contrast to the GLUT4, GLUT1 levels are regulated differentially in heart and skeletal muscle in response to streptozotocin-induced diabetes.

Abstract: A polymerase chain reaction-based cloning approach was employed in order to identify ADP-ribosylation factors (ARF) in murine 3T3-L1 cells and to study their expression before and after differentiation of cells to the adipocyte-like phenotype. Partial sequences comprising the effector domains of ARF were amplified with degenerate primers and cloned. Five of these sequences were identified as murine homologues of known human ADP-ribosylation factors (ARF 1, 2, 4, 5, and 6). In addition, partial sequences of two previously unknown isoforms were found, and complete cDNA clones were isolated from a rat fat cell library and were sequenced. Both sequences harbor a putative myristoylation site in position 2, the known consensus sequences presumably involved in GTP binding and hydrolysis, and lack cysteine residues in the C terminus. Their amino acid sequences share a 56 and 41% identity, respectively, with human ARF 1. Based on a comparison with the known ARF isoforms, the first clone appears to represent the mammalian homologue of a known sequence from Drosophila (dARL 1, 79% identity) and was therefore designated rARL 1. The second clone resembled none of the known ARF-like proteins and was designated rARL 4. mRNA of ARL 4 was undetectable in the fibroblasts but abundant in the adipocyte-like phenotype, its expression starting on day 6 of the differentiation. In contrast, ARF 1, 2, and 5 were unaltered by differentiation of the 3T3-L1 cells; mRNA levels of ARF 6, and also of ARL 1 and ARF 4, were reduced after differentiation. It is suggested that the function of ARL 4 is related to the adipocyte-like phenotype of 3T3-L1 cells.

Abstract: The effects of long-term, moderate physical exercise on in vivo glucose uptake, levels of two glucose transporter proteins (GLUT1 and GLUT4) and activities of various key enzymes of energy metabolism were measured in skeletal muscle from streptozotocin-diabetic rats. Diabetes (12-16 weeks) reduced the in vivo glucose uptake (glucose metabolic index, GMI) in muscle containing mainly type I fibres by 55% but had no effect in muscles containing mainly type IIa and IIb fibres. GMI was increased in the diabetic white skeletal muscle (mainly type IIb fibres) by more than 120%. In contrast to the complex changes in GMI, GLUT4 levels were reduced in all types of skeletal muscle from diabetic rats with no change in GLUT1 levels. Exercise training had no effects on GMI or the glucose transporter levels. Streptozotocin induced diabetes significantly reduced the oxidative capacity of skeletal muscle assayed as the activities of citrate synthase, succinate dehydrogenase and cytochrome c oxidase. Training increased the activities of oxidative enzymes, with this increase being more prominent in the diabetic animals. The present data indicate that long-term streptozotocin-induced diabetes decreases oxidative metabolic capacity and GLUT4 protein levels in skeletal muscle, but that the changes of glucose transport largely depend on the fibre type composition. Moderate training fully reverses the effect of insulinopenia and hyperglycaemia on muscle oxidative metabolism. In contrast to the previous suggestions, the expression of GLUT4 is not correlated with the capacity of oxidative metabolism in skeletal muscle of streptozotocin-diabetic rats.

Abstract: Antioxidant enzyme activities and lipid peroxidation were analysed in normal endometrium and endometrial cancer tissues from Finnish and Japanese patients. The catalase and glutathione peroxidase activities of normal endometrium were significantly lower in Finns than in Japanese. Lipid peroxidation was slightly higher in endometrial cancer as compared with normal endometrium both in the Finns and in the Japanese. When cancer tissues were compared with normal endometrium both in Finns and Japanese the activity of superoxide dismutase was significantly lower in cancer tissue than in normal endometrium. In Finns glutathione S-transferase activity was also lower in endometrial cancer tissue than in normal endometrium, and a similar tendency was also found in Japanese. This study suggests that endometrial cancer tissue is associated with an impaired enzymic antioxidant defence system.

Abstract: The effects of streptozotocin-induced diabetes (13 weeks) on the in-vivo glucose uptake and on the protein levels of glucose transporters in rat brain were studied and compared with those in cardiac muscle. Diabetes reduced the uptake of 2-[3H]deoxyglucose into lobus frontalis by 70%. However, uptake rates corrected for the 4-fold increase in serum glucose (glucose metabolic index, GMI) were essentially unaltered. The levels of glucose transporter proteins GLUT1 and GLUT3 in crude membranes from brain as assessed by immunoblotting were unaffected by diabetes, whereas GMI and levels of glucose transporters GLUT1 and GLUT4 in heart were reduced by 80 and 65%, respectively. Thus, glucose uptake and levels of glucose transporters in brain, unlike that in insulin sensitive tissues, are normal in long-term hypo-insulinaemia.

Abstract: Phenotypes of the infiltrating mononuclear cells of the lower fornix conjunctiva of nine patients with sarcoidosis and six controls were studied using monoclonal antibodies and a modified immunoperoxidase method. Four patients had sarcoidosis of recent onset (duration of 2 years or less) and five patients had a chronic disease (duration of 3 or more years). The inflammatory cells in the sarcoid conjunctival specimens were predominantly T lymphocytes, the vast majority of which were of T helper/inducer subtype expressing Leu-3a + 3b positivity. The ratio of T helper/inducer cells to T suppressor/cytotoxic cells was 3.9 on average but only 0.9 in controls. Epithelioid cell granulomas were seen in three specimens in one case of recent onset and in two chronic cases comprising a marked amount (more than 15 cells/visual field) of cells bearing phenotypes of macrophages, T cells, T helper/inducer cells and HLA-DR antigen, and in smaller quantities of T suppressor/cytotoxic cells. The mean number of all immunocompetent cell subtypes of specimens from newly diagnosed patients exceeded that of specimens from chronic patients. We believe that the sarcoid immune reaction in the conjunctiva is a dynamic process in which proliferation of immunocompetent mononuclear cells precedes the stage of granuloma formation.

Abstract: The effects of endurance training and anabolic steroid (Methandienone 1.5 mg.kg-1 p. o. daily) and their combination on regional collagen biosynthesis and concentration in the hearts of male beagle dogs were studied by measuring prolyl 4-hydroxylase (PH) activity and hydroxyproline (HYP) concentration. The PH (P less than 0.05) and HYP (P less than 0.05) were both greater in the subendocardinal layer than in the subepicardium (EPI) of the left ventricular wall in controls, whereas opposite gradients (P less than 0.05) were observed in the right ventricle. Endurance exercise caused an increase of PH activity in EPI of the left ventricular wall (P less than 0.01). The HYP concentration increased in both layers of the right ventricle in the exercise plus steroid group (P less than 0.05). The results suggest that transmural differences exist in the rate of collagen synthesis and concentration in canine cardiac ventricles and that endurance exercise may accelerate collagen synthesis in EPI of the left ventricle and the combination of exercise and anabolic steroid causes an increase in collagen concentration in the right ventricular wall.

Abstract: The effect of coronary and intraventricular pressures on the glucose uptake and its transmural distribution was studied in isolated, beating rat heart perfused using the Langendorff procedure. Left ventricular glucose uptake measured by the deoxyglucose method, and the effect of coronary (aortic) pressure was dissociated from intraventricular pressure development by draining the left ventricle. Left ventricular glucose uptake was 2.6 +/- 0.1 mumols/min per g protein (mean +/- S.E.M.) and 35 +/- 6% higher (P less than 0.001) in the subendocardium than in the subepicardium under control conditions (aortic pressure 80 cm H2O, non-drained). Elimination of the intraventricular pressure development caused no significant change in the total left ventricular glucose uptake or its transmural distribution. Increase in the aortic pressure to 150 cmH2O accelerated glucose uptake in non-drained and drained hearts by 57-75%. The increase in the glucose uptake was more pronounced in the subepicardial layer than in the subendocardial layer, so that the transmural gradient decreased by 27-32% (P less than 0.001) in non-drained and drained hearts. The results indicate that in Langendorff-perfused heart the effect of aortic pressure on the total glucose uptake and its transmural distribution across the left ventricular wall is not mediated through intraventricular pressure development, but through the coronary pressure.

Abstract: We calculated morphometrically the amount of antral gastrin-producing (G) cells and body parietal and chief cells in gastric biopsy specimens from 30 undialysed patients with chronic renal failure (CRF) and from sex- and age-matched controls. The CRF patients had raised fasting serum gastrin levels, whereas these were normal in the controls (mean, 290 +/- 283 (+/- SD) ng/l (n = 27) versus 33 +/- 36 (n = 30)). Serum gastrin values of the patients and controls correlated positively with G-cell density (r = 0.501, n = 36, p = 0.002), as did the maximal acid output of the CRF patients with parietal cell density (r = 0.617, n = 14, p = 0.019). In CRF patients the densities of G, parietal, and chief cells were higher than those in the controls (G cells, 351 +/- 151 (+/- SD) cells/mm2, n = 21 versus 211 +/- 90, n = 16, p = 0.002; parietal cells, 299 +/- 94, n = 15 versus 224 +/- 72, n = 14, p = 0.025; chief cells, 886 +/- 346, n = 15 versus 743 +/- 182, n = 14, p = 0.181). The results agree with previous findings indicating that hyposecretion of gastric acid in CRF does not derive from decreased capacity for acid secretion but rather from the inhibition of acid output. Increased parietal cell density in CRF patients gives cause to suspect that the maximum acid output might even in raised, possibly depending on the permanent hypergastrinaemic state with its trophic influence on the gastric body mucosa.

Abstract: Male rats, aged 17 weeks at the end of experiments, were divided into four groups. Two groups lived in normal cage conditions with or without extra load (20% of the body weight) and two groups were trained by running with or without extra load for 8 weeks. Oxidation rates of succinate, glutamate + malate, palmitoylcarnitine, and pyruvate, and the activities of lactate dehydrogenase, citrate synthase, isocitrate dehydrogenase and cytochrome oxidase were measured in homogenates of the right ventricle and in those of the subendocardial and subepicardial layers of the left ventricle. Oxidation rates of succinate and palmitoylcarnitine tended to be higher in the subendocardium than in the subepicardium of sedentary control animals (p less than 0.1 and p less than 0.05, respectively). Transmural differences of succinate and palmitoylcarnitine oxidation rates were even more clear after running training (p less than 0.01 and p less than 0.05, respectively), after carrying extra load (p less than 0.001 and p less than 0.001, respectively) and after training carrying extra load (p less than 0.001 and p less than 0.05, respectively). Training also enhanced pyruvate oxidation rate in the subendocardium. Oxidation rates of all substrates were lower in the right ventricle than in the left ventricle. In control animals there were no regional differences in the myocardial enzyme activities and the training- or extra-load-induced changes were modest compared with the changes in the oxidation rates. The most significant change was the training-induced enhancement in the lactate dehydrogenase activity of the subendocardium (p less than 0.001 vs subepicardium). These results show greater subendocardial than subepicardial oxidation rates of certain substrates in the normal heart. These results also suggest that the myocardium adapts to increased work by increasing the subendocardial oxidation rate of some but not all substrates, indicating further that there may be qualitative mitochondrial differences in the different regions of the heart.

Abstract: 23-month-old male rats were trained by running for 20 weeks. The oxidation rates of succinate, glutamate+malate, palmitoylcarnitine, and pyruvate and the activities of lactate dehydrogenase, citrate synthase, isocitrate dehydrogenase and cytochrome oxidase were measured in the subendocardium and subepicardium and in the right ventricle. Regional differences of substrate oxidation rates in the myocardium of old sedentary or trained rats were less than in young rats, suggesting that regional differences in the cardiac work load disappear during ageing. Training did not improve oxidation rates, in contradiction to some previous results.

Abstract: Regional glucose uptake in perfused hearts, and the activities of several glycolytic enzymes contributing to the glucose metabolism in perfused and nonperfused hearts were studied in male and female rats after 8-9 weeks of swimming training. The left ventricular glucose uptake showed a transmural gradient in the sedentary animals, the subendocardial uptake being 30% and 12% higher than that of the subepicardial layer in the males and females, respectively. Swimming exercise abolished the left ventricular glucose uptake gradient in male rats, and in female rats an opposite gradient was found, the subepicardial uptake being 23% higher than the subendocardial uptake. The activities of phosphofructokinase and 3-phosphoglyceraldehyde dehydrogenase also showed transmural gradients in the left ventricles. Training did not abolish these gradients. Training-induced changes in the activities of phosphofructokinase, 3-phosphoglyceraldehyde dehydrogenase, pyruvate kinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, citrate synthase, and malate dehydrogenase were found in certain sites of the myocardium. Perfusion of isolated hearts for 50 min with insulin-containing Krebs-Ringer buffer especially affected the activities of phosphofructokinase, lactate dehydrogenase, and citrate synthase, increasing these activities in the left ventricles and decreasing them in the atria. These results indicate that there are regional differences between male and female rats in the cardiac glucose uptake rate after swimming training.

Abstract: The effects of 60 min hypoxia and subsequent reoxygenation for 30 min on enzymatic (NADPH-dependent) and nonenzymatic (Fe2+/ascorbate-induced) lipid peroxidation capacities and on antioxidant levels were studied using Langendorff-perfused rat hearts. The assays were done on the myolayer of the right ventricle (RV) and on the subepi- and subendomyolayers of the left ventricle (epi/endo LV) after normoxic, hypoxic, and reoxygenation phases. The region injured by hypoxia/reoxygenation was located mainly in endo LV, seen as a lesser penetration of the fluorescent dye fluorescein in the myocardium. The electron microscopic findings after reoxygenation revealed swelling of the mitochondria, amorphous mitochondrial structures, and formation of paracrystallines. The myofibrillar structure of the cells was disrupted and the cells showed marked fluid accumulation. Membrane structures were marginated and formed blebs and multilamellar bodies. Ultrastructural changes were most prominent in endo LV, especially after reoxygenation. The increase in leakage of lactate in the perfusate revealed the onset of anaerobic metabolism. Abrupt release of the cytoplasmic enzymes lactate dehydrogenase and creatine kinase at the beginning of the reoxygenation phase suggested cell membrane injury. The capacity for Fe2+/ascorbate-induced lipid peroxidation slightly increased in RV and that for NADPH-dependent, enzymatic lipid peroxidation in endo LV after reoxygenation. Catalase, glutathione peroxidase, and superoxide dismutase activities remained unchanged, whereas glucose-6-phosphate dehydrogenase activity decreased after reoxygenation in RV.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: The effect of chronic exercise on the regional glucose uptake in the left ventricle of the heart was studied in resting and swimming rats using the 2-deoxyglucose method. The left ventricular glucose uptake of untrained resting controls averaged 1.7 +/- 0.1 mumol.min-1.g protein-1 and that of chronically trained resting rats 3.5 +/- 0.3 mumol.min-1.g protein-1 (P less than 0.001). During a 20-min swimming period the glucose uptake rate of untrained rats was 2.3 +/- 0.1 mumol.min-1.g protein-1 and that of trained rats 3.4 +/- 0.3 mumol.min-1.g protein-1 (P less than 0.01). The subendocardial glucose uptake was 25% higher than the subepicardial uptake in the resting control group, whereas no gradient was observed in the other groups. The product of heart rate and blood pressure during swimming increased by 60-70% in the untrained and trained groups. The increase in total left ventricular glucose uptake and its transmural distribution by training seemed to be independent of the actual oxygen consumption or supply of major alternative myocardial substrates.

Abstract: The effect of angiotensin II-induced hypertension on selected biochemical parameters was studied in Sprague-Dawley rats. Angiotensin II infusion at rates of 41.7 micrograms h-1 kg-1 and 12.5 micrograms h-1 kg-1 for 2, 5, 10 and 15 days elevated the systolic blood pressure from 143 +/- 7 mmHg to 215-230 mmHg (P less than 0.001) and 185-195 mmHg (P less than 0.001), respectively. The left ventricular weight/body weight ratio increased 10-14% (P less than 0.05) and 23-32% (P less than 0.001) after 2-15 days in rats treated at the lower and higher infusion rates, respectively. Prolyl 4-hydroxylase (PH) activity, a marker of collagen synthesis, was evenly distributed in the left ventricle. PH activity increased by about 100% in both subendocardial and subepicardial layers of the left ventricular wall after angiotensin II infusion for 10 days at 41.7 micrograms h-1 kg-1, but remained unaltered at 12.5 micrograms h-1 kg-1. No change was observed in hydroxyproline concentration. Myosin isoenzymes (V1-V3), which reflect myocardial contractility, were unevenly distributed in the left ventricular wall: the proportion of the fast-turnover isoenzyme (V1) was smaller in the subendocardial layer than in the subepicardial layer. The proportion of V1 decreased after treatment in both layers. Alkaline phosphatase activity, a marker of capillary density, was evenly distributed transmurally in the left ventricular wall. Angiotensin II caused a slight decrease in this activity in both myocardial layers. The results suggest that the elevation of blood pressure leads to transmurally evenly distributed changes in biochemical parameters reflecting collagen synthesis, capillary density and contractile properties of the myocardium.

Abstract: Rats were exercised for 6 h by swimming. Phenylalanine incorporation into myocardial proteins was increased when 2 h had elapsed after the termination of exercise. Cyclic GMP concentration did not change during the experiment, which indicates that cyclic GMP does not act directly as a trigger of myocardial protein synthesis in volume overload.

Abstract: The effect of 10 weeks of running training and termination of training on the regional distribution of cardiac glucose uptake and protein synthesis were studied in isolated perfused hearts in male rats. The left ventricular glucose uptake in hearts from sedentary rats was 1.87 +/- 0.14 mumol/min per g protein (mean +/- SE), being about 30% higher in the subendocardial than in the subepicardial layer (p less than 0.05). The gradient of left ventricular glucose uptake was similar to the controls in the rats retired from training, but was absent in the trained animals. The altered transmural glucose uptake probably reflects differences in the adaptive response of various myocardial muscle layers to a long-term intermittent increase in cardiac work load. Phenylalanine incorporation was evenly distributed through the left ventricle in all the groups, but was lowered in the left and right ventricles of the trained rats. Phenylalanine incorporation returned to the control level 5 weeks after the cessation of training.

Abstract: The effects of 8-9 weeks of running and swimming training on the transmural distribution of cardiac glucose uptake and protein synthesis in isolated perfused heart were studied in male rats. The left ventricular glucose uptake in hearts from sedentary rats was 2.5 +/- 0.3 mumoles/min per g protein (mean +/- S.D.), and about 30% higher in the subendocardial layer than in the subepicardial layer (P less than 0.01). After the running and swimming programs the total left ventricular glucose uptake was at the level of sedentary rats, but the gradient was absent. The rate of protein synthesis was evenly distributed through the left ventricular wall and similar in all experimental groups. The altered transmural distribution of glucose uptake after exercise probably reflects differences in the adaptive response of various myocardial muscle layers to a long-term intermittent increase in the cardiac work load.

Abstract: Alkaline and myofibrillar protease activities of rectus femoris, soleus, and tibialis anterior muscles and the pooled sample of gastrocnemius and plantaris muscles were analyzed in male NMRI-mice during a running-training program of 3, 10, or 20 daily 1-h sessions. The activity of citrate synthase increased during the endurance training, reflecting the increased oxidative capacity of skeletal muscles. The activities of alkaline and myofibrillar proteases continually decreased in the course of the training program in all muscles studied. Instead, the activity of beta-glucuronidase (a marker of lysosomal hydrolases) increased in all muscles. The highest activities were observed at the beginning of the training program. Present results, together with our earlier observations, show that the type of training, running as opposed to swimming, modulates the training responses in alkaline protease activities. Further, diverse adaptations in the activities of alkaline proteases and a lysosomal hydrolase suggest difference in the function of different proteolytic systems.

Abstract: We studied the effects of running-training, heavy exercise and termination of training on the heart weight, the ratio heart to body weight and the cardiac muscle activities of actomyosin ATPase, citrate synthase, succinate dehydrogenase, cytochrome c oxidase, malate dehydrogenase, adenylate kinase and beta-glucuronidase with adult male NMRI-mice. Stable hypertrophy (6-7%), estimated by the ratio heart or ventricle weight to body weight, was achieved by 28 exercises and it was dependent on the running speed (20 vs. 25 m X min-1). The withdrawal of training for 5-61 days did not permanently decrease the heart weight or the heart to body weight ratio to the level of sedentary controls. The activity of enzymes of energy metabolism or actomyosin ATPase were not affected by training, heavy exercise or terminated training. beta-glucuronidase activity slightly (20-25%) increased in the trained animals and remained at a higher level during the period of terminated training. The results suggest that the capacity for aerobic metabolism of normal mice heart is sufficient to meet the enhanced demand for ATP imposed by running-training and that the heart enlargement occurs in equal proportions with the enzymatic potential of the cardiac tissue.

Abstract: Glucose uptake is greater in the subendocardial layer of a Langendorff-perfused rat heart than in the subepicardial layer during basal conditions and also under basal conditions in the rat in vivo. The increase of aortic pressure in vitro or physical exercise in vivo causes the disappearance of the transmural gradient in glucose uptake, as does also the elimination of the mechanical work load by potassium induced cardiac arrest. Co-infusion of alternative substrates (oleate, lactate or acetate) causes an overall inhibition of cardiac glucose uptake but does not affect its proportional regional uptake. The left ventricular work load seems thus to be the major determinant of the transmural distribution of the left ventricular glucose uptake. Chronic physical exercise results in the disappearance of the transmural gradient in glucose uptake measured in isolated perfused heart. This redistribution of glucose uptake may reflect differences in the adaptative response of various myocardial layers to a long-term intermittent increase in the cardiac work load.

Abstract: Effects of a short-term vitamin E deficiency on some lipid peroxidative properties were investigated in mouse cardiac and skeletal muscles. The concentration of vitamin E decreased 35.8% in 5 weeks and 61.2% in 12 weeks in skeletal muscle. The corresponding decrease in cardiac muscle was 65.7% in 12 weeks. Simultaneously the susceptibility of muscle homogenates to in vitro lipid peroxidation increased with 48.6% (5 weeks) and 44.5% (12 weeks) in skeletal muscle and with 101.8% (12 weeks) in cardiac muscle. Highly significant negative correlations were observed between the concentration of vitamin E and in vitro lipid peroxidation in cardiac and skeletal muscles. Also the sensitivity to Fe2+-induced peroxidation was increased in skeletal muscle after the deficiency of 5 weeks. The total contents of peroxidizable lipids (Fe2+-induction) were significantly (approx. 20%) decreased after 12 weeks in cardiac and skeletal muscles. The concentration of lipofuscin was unaffected in both muscles of vitamin E-deficient mice. Vitamin E deficiency (5 weeks) decreased the activity of selenium-dependent glutathione peroxidase in skeletal muscle but did not affect the activities of catalase and beta-glucuronidase and the concentrations of protein, reduced glutathione and total sulfhydryl groups. These results show that a short-term vitamin E deficiency affects the peroxidative properties of cardiac and skeletal muscles and may thus expose the muscles to peroxidation injuries.

Abstract: Mice and rats were adjusted to daily treadmill training programs, which were heavy enough to increase the oxidative capacity of skeletal muscles. Endurance training did not affect the activities of catalase and glutathione peroxidase and the concentration of vitamin E in the lungs of mice and rats. Thus increased ventilation and oxygen utilization induced by exercise training do not modify lung antioxidants, in contrast to hyperoxia and hypoxia.

Abstract: Endurance training over 3, 10 or 20 days increased the activity of prolyl 4-hydroxylase (PH) in the left ventricle of mice. No increase was observed in the weight of the left ventricle, in galactosylhydroxylysyl glucosyltransferase activity or in hydroxyproline concentration. The increase in PH suggests that the synthesis of collagen increases during physiological adaptation of the heart to endurance exercise without changes in the ventricle weight or its total collagen content.

Abstract: This study aimed at comparing the effects of running and swimming training protocols and the termination of training on the activities of two proteases with alkaline pH-optima (alkaline protease and myofibrillar protease) in the tibialis anterior, soleus, and gastrocnemius muscles of male rats. The training on treadmill decreased the activities of alkaline and myofibrillar proteases by approx. 10-20% in the muscles studied. The activities of both proteases were unchanged in swimming-trained rats. Two weeks after the termination of running training the activity of alkaline protease was increased in gastrocnemius muscle but not in the other muscles. Swimming training increased the activity of citrate synthase in all muscles studied but training by running only in the soleus muscle. The running protocol increased the activity of beta-glucuronidase in the tibialis anterior muscle and decreased the activity in the gastrocnemius muscle. The swimming program did not affect beta-glucuronidase activities. These results show diverse effects of running and swimming training on alkaline proteolytic activities as well as on mitochondrial and lysosomal marker enzymes.

Abstract:

Abstract: Mitochondrial volume density, surface density of the outer mitochondrial membrane, the mean number and size of mitochondria, and the mean surface density of crista membranes together with the volume densities of myofibrils and sarcoplasmic space were morphometrically analyzed in cardiac muscle of two groups of sedentary control mice aged 3 and 7 months, and in two groups of mice trained either 1 month rather intensely or 4 months moderately. Of the calculated mitochondrial variables only the surface density of the outer mitochondrial membrane differed between the older controls and the older trained animals, the density being slightly smaller in the trained group. The myofibrillar volume density of the order controls was smaller than that of the younger controls, while the sarcoplasmic volume density was larger. The latter difference, possibly a function of age, was also observed in the trained groups. The results suggest that at a certain steady state level of exercise-induced cardiac muscle hypertrophy the muscle cells of trained mice do not differ markedly in ultrastructural properties from those of sedentary controls.