Developmental Biology Unit, Institute of Health Scieces, University of La Coruna, Campus de Oza, Building El Fortin, As Xubias Str. s/n, 15006 La Coruna, Spain
margot@udc.es
MD (1968 - Moscow State Medical Institute, Moscow, Russia); BD - embryology (1971 - Russian Academy of Medical Sciences); Ph.D. (1973 - Institute of Human Morphology, Russian Academy of Medical Sciences, Moscow, Russia); Senior Research Officer (1984 - Russian Academy of Sciences, Moscow, Russia); D.Sc. (1985 - Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia); Full Professor (1992 - Russian Academy of Sciences, Moscow, Russia); Corresponding Member of the Russian Academy of Natural Sciences (1994); Research Director of the Institute of Health Sciences at the University of La Coruna, La Coruna, Spain (1995 - ).
Abstract:
Background: Myocardin (MYOCD), a potent transcriptional coactivator of smooth muscle (SM) and cardiac genes, is
upregulated in failing myocardium in animal models and human end-stage heart failure (HF). However, the molecular and
functional consequences of myocd upregulation in HF are still unclear.
Methodology/Principal Findings: The goal of the present study was to investigate if targeted inhibition of upregulated
expression of myocd could influence failing heart gene expression and function. To this end, we used the doxorubicin (Dox)-
induced diastolic HF (DHF) model in neonatal piglets, in which, as we show, not only myocd but also myocd-dependent SMmarker
genes are highly activated in failing left ventricular (LV) myocardium. In this model, intra-myocardial delivery of
short-hairpin RNAs, designed to target myocd variants expressed in porcine heart, leads on day 2 post-delivery to: (1) a
decrease in the activated expression of myocd and myocd-dependent SM-marker genes in failing myocardium to levels seen
in healthy control animals, (2) amelioration of impaired diastolic dysfunction, and (3) higher survival rates of DHF piglets.
The posterior restoration of elevated myocd expression (on day 7 post-delivery) led to overexpression of myocd-dependent
SM-marker genes in failing LV-myocardium that was associated with a return to altered diastolic function.
Conclusions/Significance: These data provide the first evidence that a moderate inhibition (e.g., normalization) of the
activated MYOCD signaling in the diseased heart may be promising from a therapeutic point of view.
Abstract: Molecular predisposition of postnatal ventricular myocardium to chamber-dependent (concentric or eccentric) remodeling remains largely elusive. To this end, we compared gene expression in the left (LV) versus right ventricle (RV) in newborn piglets, using a differential display reverse transcription-PCR (DDRT-PCR) technique. Out of more than 5600 DDRT-PCR bands, a total of 153 bands were identified as being differentially displayed. Of these, 96 bands were enriched in the LV, whereas the remaining 57 bands were predominant in the RV. The transcripts, displaying over twofold LV-RV expression differences, were sequenced and identified by BLAST comparison to known mRNA sequences. Among the genes, whose expression was not previously recognized as being chamber-dependent, we identified a small cohort of key regulators of muscle cell growth/proliferation (MAP3K7IP2, MSTN, PHB2, APOBEC3F) and gene expression (PTPLAD1, JMJD1C, CEP290), which may be relevant to the chamber-dependent predisposition of ventricular myocardium to respond differentially to pressure (LV) and volume (RV) overloads after birth. In addition, our data demonstrate chamber-dependent alterations in expression of as yet uncharacterized novel genes, which may also be suitable candidates for association studies in animal models of LV/RV hypertrophy.
Abstract: Brain natriuretic peptide/natriuretic peptide precursor B (NPPB) is one of the most studied genes in relation to heart failure (HF) conditions. However, it is still unclear as to whether alternative splicing could create NPPB mRNA variants, which may be expressed in normal and diseased myocardium. We aimed to identify and characterize a novel alternatively spliced variant of porcine and human NPPB resulting from exon 2 skipping (designated as DeltaE2-NPPB). A variety of conventional molecular, biochemical and immunochemical methods were used to examine the expression and functional consequences of DeltaE2-NPPB in vitro and in vivo. The pig DeltaE2-NPPB mRNA is effectively translated into stable protein in cell-based assays but, in contrast to normally spliced NPPB, the DeltaE2-NPPB protein is not secreted into the media. Co-transfection assays demonstrate that DeltaE2-NPPB attenuates production and secretion of normally spliced NPPB, suggesting a negative feedback loop of NPPB signaling through generation of DeltaE2-NPPB. The inhibitory effects of DeltaE2-NPPB on the expression of NPPB are associated with sequence elements residing in exon 3 of DeltaE2-NPPB. In piglets, DeltaE2-NPPB gene expression is downregulated in both ventricles after birth, but it is markedly re-activated in the postnatal myocardium in experimental diastolic heart failure. In addition, we demonstrate that the exon-skipped NPPB variants are expressed in the postnatal and adult human myocardium and upregulated at end-stage HF due to dilated cardiomyopathy. Our work uncovers an important role of alternative exon skipping in the regulation of NPPB gene expression, thereby pinpointing a putative new mechanism for post-transcriptional regulation of NPPB production and secretion.
Abstract: The cardiac ankyrin repeat domain 1 protein (ANKRD1, also known as CARP) has been extensively characterized with regard to its proposed functions as a cardio-enriched transcriptional co-factor and stress-inducible myofibrillar protein. The present results show the occurrence of alternative splicing by intron retention events in the pig and human ankrd1 gene. In pig heart, ankrd1 is expressed as four alternatively spliced transcripts, three of which have non-excised introns: ankrd1-contained introns 6, 7 and 8 (i.e., ankrd1-i6,7,8), ankrd1-contained introns 7 and 8 (i.e., ankrd1-i7,8), and ankrd1 retained only intron 8 (i.e., ankrd1-i8). In the human heart, two orthologues of porcine intron-retaining ankrd1 variants (i.e., ankrd1-i8 and ankrd1-i7,8) are detected. We demonstrate that these newly-identified intron-retaining ankrd1 transcripts are functionally intact, efficiently translated into protein in vitro and exported to the cytoplasm in cardiomyocytes in vivo. In the piglet heart, both the intronless and intron-retaining ankrd1 mRNAs are co-expressed in a chamber-dependent manner being more abundant in the left as compared to the right myocardium. Our data further indicate co-upregulation of the ankrd1 spliced variants in myocardium in the porcine model of diastolic heart failure. Most significantly, we demonstrate that in vivo forced expression of recombinant intronless ankrd1 markedly increases the levels of intron-retaining ankrd1 variants (but not of the endogenous main transcript) in piglet myocardium, suggesting that ANKRD1 may positively regulate the expression of its own intron-containing RNAs in response to cardiac stress. Overall, our findings demonstrate that in cardiomyocytes ANKRD1 can exist in multiple isoforms which may contribute to the functional diversity of this factor in heart development and disease.
Abstract: The aim of this study was to determine the effects of forced expression of myocd-A in the left ventricular (LV) myocardium on cardiac performance in early neonatal piglets. LV transfection with the gene for homeodomain only protein (hop), an antagonist of myocd-mediated activities, was also performed. Gene delivery was performed in 6-day-old piglets using a low-traumatic, catheter-based, video-assisted procedure developed by us for direct intra-myocardial injections of plasmid DNA into 3-4 target areas of the ventral LV free wall (LVFW). Two isoforms of porcine myocd were identified, cloned and characterized: the exon 11-lacking myocd-A and its larger exon 11-containig variant, myocd-B. In neonatal piglets, myocd-A seems to be a cardio-predominant isoform enriched in the LVFW/septum, whereas the myocd-B isoform is detected not only in the heart but also in various smooth muscle cell-containing tissues. Intramyocardial myocd-A gene delivery resulted in forced transgene expression in the target areas of the LVFW as compared to controls. On day 2 post-delivery, a marked decrease of LV-end systolic pressure values (an accepted marker for impaired LV function) was observed in myocd-A-transfected piglets as compared to hop-transfected and control groups. In addition, forced myocd-A expression in the LVFW caused abnormal ECG. A significant up-regulation of the gene for fetal-predominant muscle light chain 3F myosin was detected in myocd-A-transfected LVFWs harvested on day 2 post-delivery. Extended analysis on day 7 post-delivery revealed a drop decrease in myocd-A transgene expression in target LVFW regions which was correlated with normalization of the LV systolic parameters in experimented piglets.
Abstract: It has been proposed that the ankyrin repeat domain 1 (ANKRD1) factor (also known as CARP) plays a critical role in transcriptional regulation, myofibrillar assembly and stretch sensing during heart development and cardiac insults. ANKRD1/CARP has also been reported to negatively regulate cardiac gene expression in cell-based promoter-reporter assays. Consequently, rapid up-regulation of the ankrd1 gene in myocardium in response to developmental stimuli or pathological insults has tended to be interpreted in the context of the inhibitory effects of ANKRD1 on cardiomyocyte gene expression. Surprisingly, a total ankrd1 knockout resulted in a complete lack of phenotype, suggesting that ANKRD1/CARP is not crucial for regulation of cardiac gene expression in vivo. In this essay, we summarize (1) the accumulated evidence for the apparent multifunctional properties of this enigmatic protein, (2) the distinct chamber-dependent regulation of ankrd1 expression patterns in the heart, both during development and cardiac injury, and (3) ANKRD1 involvement in networks regulating adaptation of the myocardium to stress. Whenever feasible, we present the results obtained in patients together with those obtained in the relevant animal and cellular models. A close examination of the findings still fails to define ANKRD1 as a negative regulator of cardiac gene expression in vivo, but rather indicates that its augmented expression can represent an adaptive response of the myocardium to stress both during development and various heart insults.
Abstract: We have developed a set of simple modifications of the green fluorescent protein (GFP)fragment reassembly assay in bacteria that permits: (i)fluorescent microscopy visualization of GFP reassembly only 1-2 h after induction of protein expression, thus approximating the detection of GFP reassembly to the real-time dynamics of protein complex formation in living cells; (ii) spectrofluorometric detection of reassembled GFP fluorescent signals directly in lysates from cell suspension thereby avoiding, in many cases, the need for tag-affinity isolation of protein complexes; and (iii) comparative quantification of signal intensity in numerous cell-sample lysates using a Bio-Rad IQ5 spectrofluorometric detection system (Bio-Rad Laboratories, Madrid, Spain). Collectively, the results demonstrate that the combination of microscopic and spectrofluorometric detection provides a time-saving and sensitive alternative to existing methods of fluorescence complementation analysis.
Abstract: Diastolic heart failure (DHF) was produced in 6-day-old piglets by intravenous administration of Doxorubicin, and ANKRD1 protein and mRNA levels were determined in atrial (A) and ventricular (V) chambers of failing vs control hearts. In controls, ANKRD1 showed a left-right (L-R) asymmetric distribution with protein levels 2-fold higher in the LA as compared to the RA, and 8-fold higher in the LV than the RV. In failing hearts, ANKRD1 levels were augmented about 2-fold in each ventricle but equally reduced in both atria as compared to controls. ANKRD1 downregulation in atria is discussed as a process associated with advanced DHF.
Abstract: It has been suggested that the cardiac ankyrin repeat domain 1 protein (ANKRD1), also known as CARP, can play a pathophysiological role in the contractile responsiveness of myocardium. Here, we study the potential functional roles of ANKRD1 by searching for endogenous cardiac proteins that interact preferentially with ANKRD1 in the heart-tissue extract from neonatal piglets, using non-biased pull-down approaches. These approaches identified, for the first time, a selective interaction between ANKRD1 and endogenous cardiac calsequestrin-2 (CASQ2) that is important for Ca2+ release and excitation-contraction coupling. Blot-overlay and co-immunoprecipitation assays provided further confirmation of the direct and specific interaction between the two proteins. Mapping of the peptides involved in the interaction revealed five non-overlapping binding sequences for CASQ2 on ANKRD1, as well as, three binding peptides for ANKRD1 in CASQ2. For the first time, we show by immunohistochemistry that endogenous ANKRD1 and CASQ2 are co-enriched in piglet cardiac Purkinje cells. Collectively, the results provide the first sing of a possible functional interaction between ANKRD1 and CASQ2 and suggest a potentially novel role for both proteins in cardiac Purkinje fibers.
Abstract: The mechanism of sexualization of the tubular gonad in seawater bivalves is unknown, and no information regarding the genes involved in this process is yet available, except for the identification of esterase (Est)-like "male-associated polypeptide" in the male gonad of Mytilus galloprovincialis. Our present work reveals distinct protein profiles specific for the testicular or ovarian portion of the ovotestis of Pecten maximus. Two proteins exhibiting testis- or ovary-dependent enrichment in the ovotestis have been identified and partially characterized as Est-like and fibronectin (Fn)-like polypeptides, respectively. Immunofluorescence has demonstrated a close association between the localization of these polypeptides and the gonad tubule network and interstitial stroma of the ovotestis of P. maximus. We also present evidence of Est-like and Fn-like protein enrichment, respectively, in testicular and ovarian tissue in hermaphroditic, sex-reversal, and gonochoric species of seawater bivalves. Together, the results (1) strongly suggest that sex-cell-biased expression of Est-like and Fn-like polypeptides in gonad tissue is a widespread phenomenon among bivalve mollusks, despite the high diversification of their sexual patterns, (2) confirm and expand our previous demonstration of sex-biased protein expression in M. galloprovincialis, and (3) indicate a direct link between germ cell differentiation and sexual specialization of the bivalve somatic gonad.
Abstract: BACKGROUND AND AIM: Cardiac ankyrin repeat protein (CARP), whose expression is down-regulated in response to doxorubicin (Dox) in vitro, has been proposed to be a marker of experimentally-induced cardiac hypertrophy in rodent models. In piglets, the rapid hypertrophy rate of the left ventricle (LV) as compared to that of the right ventricle (RV) represents a natural model of asymmetric ventricular enlargement. We tested whether CARP expression correlates with postnatal ventricular hypertrophy and to what extent CARP can be sensitive to Dox treatment in vivo. METHODS: CARP mRNA and protein levels were quantified (by Northern blot hybridization, semi-quantitative RT-PCR and Western blot) in the piglet heart, both during early postnatal development and upon Dox-induced cardiomyopathy (Dox-CM). RESULTS: The study revealed: (1) significantly augmented CARP mRNA and protein levels in the LV compared to the RV resulting in left vs. right asymmetry in ventricular CARP expression throughout early postnatal development; (2) dose- and chamber-dependent CARP mRNA and protein enrichment in ventricular myocardium in response to Dox; and (3) abolishment of asymmetric patterns of ventricular CARP expression at heart failure resulting from Dox-CM. CONCLUSIONS: (1) CARP is differentially regulated in the LV and RV during both postnatal development and disease; and (2) monitoring of ventricular CARP expression patterns can be used for further analysis of transition from compensated to overt heart failure.
Abstract: The implication of myocardin and homeodomain only protein (HOP) in combinatorial molecular pathways that guide heart development and cardio-specific gene expression has recently been reported. However, expression of these genes in the failing heart has not yet been investigated. This study was designed to elaborate a molecular profile of myocardin and HOP expression in the failing ventricular myocardium through the use of both explanted human heart samples and heart biopsies from neonatal piglets with doxorubicin-induced cardiomyopathy (Dox-CM). Myocardin and HOP mRNA levels were estimated by both northern blot hybridization and semiquantitative RT-PCR in human ventricular preparations in end-stage failure due to dilated cardiomyopathy (DCM), as well as in nonfailing donor hearts. Similar experiments were performed with ventricular samples from normal and Dox-treated neonatal piglets. The gene expression of brain natriuretic peptide (BNP) was used as a molecular marker of myocardial damage and failure. The study revealed the following novel findings: (1) myocardin transcripts are detected in neonatal human and pig hearts at lower levels than in mature cardiac tissues, (2) the myocardin transcript pool is significantly augmented in the failing human and porcine myocardium as compared to that in nonfailing heart samples, (3) in the failing human myocardium, increased levels of myocardin mRNA are associated with a diminished HOP transcript content, and (4) the inverse proportion in cardiac myocardin/HOP mRNA pools observed in explanted human hearts is also traceable in normal human heart and aorta. A possible dual consequence of increased myocardin and decreased HOP expression levels on serum response factor-dependent cardiac-specific expression in the normal heart and at heart failure is discussed. Therefore, increased abundance of the myocardin mRNA pool is judged to be a novel CM-related feature which, alone or in association with decreased HOP transcript levels, can be responsible for dysregulation of myocardin-mediated gene expression in failing myocardium.
Abstract: Our interest in the comparative analysis of male reproductive-tract esterases in different animal groups has led us to undertake a detailed study of the Mytilus galloprovincialis male-associated polypeptide (MAP) throughout the mussel gonad-duct tract and at spawning. The results of this work indicate that MAP is a major protein in M. galloprovincialis semen, with dual presence in both sperm cells and cell-free seminal fluid. Shortly after spawning, the released sperm mass is subdivided in diffused cloudy-like and thread-shaped 'clots', in which a soluble-phase MAP may persist as long as the clots keep their compact form. Additional experiments involving the incubation of spawned spermatozoa at increasing Triton X-100 concentrations demonstrated that MAP is also strongly associated with sperm cells. These results were further validated by immunofluorescent staining, which revealed that MAP is localized in the mid-piece region of spawned spermatozoa. This unexpected finding raises the possibility that MAP may play a role in sperm fertility in bivalves. Using whole-mount histology and micromanipulation techniques, we studied the structural patterning of the mantle gonad-duct network and assessed the sampling of luminal contents from the ducts. Of particular interest is the observation that MAP content in the luminal fluid increases from the lumen of the spermatogenic tubules to that of the collecting gonad ducts, where MAP is detected at a very high concentration. These high levels may lead to a significant presence of MAP in semen and consequently to a prolonged survival of sperm spawned at sea. In addition, data related to the potential structural similarity between mussel MAP and esterase S of the Drosophila virilis ejaculatory bulb are presented and discussed. Finally, we show that the 64kDa protein of human semen reveals positive cross-reactivity with antibodies directed against Mytilus MAP and Drosophila esterase S. Taken together, the results reveal mussel MAP as the only esterase-like protein described so far whose distribution in the gonad and semen can be specifically associated with maturation, transport, emission and survival of spermatozoa outside.
Abstract: The Saint Petersburg Society of Naturalists has reinstated the Alexander O. Kowalevsky Medal. This article announces the winners of the first medals and briefly reviews the achievements of A.O. Kowalevsky, the Russian comparative embryologist whose studies on amphioxus, tunicates and germ layer homologies pioneered evolutionary embryology and confirmed the evolutionary continuity between invertebrates and vertebrates. In re-establishing this international award, the Society is pleased to recognize both the present awardees and the memory of Kowalevsky, whose work pointed to that we now call evolutionary developmental biology.
Abstract: The impact of the organizer concept on Russian experimental embryology is shortly reviewed. Attempts to study embryonic induction in Russia may be grouped into embryological and biochemical approaches. This paper provides a framework for, and overvalue of, the contributions of Russian biologists to the problem of embryonic induction. Two model systems--lens and neural inductions--are of special significance to modern developmental biologists. Moreover, the study of eye lens induction actually gave rise to research on developmental mechanics in Russia. This was one of the reasons why we limited this article to these two model systems. After retrospective consideration of the results of the search for possible lens-inducing factor candidates, the discussion turns towards some of the examples of neural-inducing agents detected in embryonic tissues and the new questions raised by the progress that has been made in the analysis of the Spemann-Mangold organizer.
Abstract: In a few well-known cases, the biological consequences of the disruption of lim-1 homeodomain (HD) genes have demonstrated the important roles of these genes in vertebrate development, especially in the nervous tissue, kidney, and gonads. Functional assay approaches require information not only about lim-1 gene organization, but also about properties and tissue localization of Lim-1 proteins. Although lim-1 genes have been identified in certain phyla of invertebrates, no information is available on Lim-1 proteins and genes in bivalve molluscs. Our study represents the beginning stage of identification of the Lim-1-related proteins in marine bivalves. Using antibodies against the C-terminal region of the Xenopus laevis Lim-1 protein, we describe cross-reactive antigen patterns in adults and early embryos of the mussel Mytilus galloprovincialis, as well as in sea urchin and chick embryos. In adult mussels, nervous ganglia and gonads display the most prominent Lim-1 immunoreactivity. Further, the antibodies verified the prediction that mussel Lim-1 antigens, like Lim-1 HD proteins in general, can be localized in the nucleus. Moreover, antibody detection allowed us to identify the Lim-1-like antigens in unfertilized mature eggs, as well as in very early embryos of bivalve molluscs and sea urchins (Strongylocentrotus purpuratus). In mussel eggs and embryos, Lim-1 antigens are expressed in multiple forms (40, 45, and 65 kDa), as detected by SDS-PAGE followed by Western blot. Taken together, the observations emphasize the conservation of the Lim-1 protein expression pattern in the nervous tissue and gonads of different animal groups, and demonstrate that Lim-1-like polypeptides can be maternally accumulated in eggs and, therefore, are present in very early embryos before zygotic expression of the genes begins.
Abstract: This essay addresses the carboxylesterase redundancy in the male reproductive tract seemingly conserved across phyla. Evidence is provided which suggests that carboxylesterases are recruited by the male reproductive system in certain animal groups. These provide advantageous metabolic capabilities to sperm protection, sperm maturation, and sperm use. Rather than an archival record of the available data, we seek possible answers to the central question: Why is carboxylesterase over-expression adaptive with the functioning of the male reproductive tract with respect to male fertility? We discuss patterns of carboxylesterase over-expression and accumulation in different compartments of the male reproductive tract. We also provide evidence of how these patterns are associated with a long sperm path to egg through different local effects. The hyper-expression of carboxylesterases can play different physiological roles depending on its localization in the male reproductive system. However, all the "acquired" functions can serve the same purpose; creating conditions which maximize the fertilizing potential of the sperm. To confirm our concept and more clearly illuminate "moonlighting" roles of carboxylesterases in the male reproductive tract, requires a more extensive comparative analysis of a variety of carboxylesterases in a larger number of species.
Abstract: Data on expression patterns of carboxylesterases in the male reproductive tract of different animal groups (i.e. bivalve mollusks, fruitflies and rodents) are summarized to highlight some particularly interesting questions in the context of sperm differentiation, maturation and function. The male reproductive system, in spite of extreme variation in the anatomical/morphological organization in different species, is characterized by similar patterns of male-dependent carboxylesterase overexpression. The phenomenon of conserved carboxylesterase overexpression indicates similar male sex-associated functions of the enzymes. There is possible evidence of carboxylesterase recruitment by male reproductive-tract tissues indicating that it could be adaptive for spermatogenesis, sperm maturation and sperm use. Moreover, this idea can be extended to include a sperm cell lineage protection. This issue is discussed in the light of recent data on environmental reproductive xenobiotics that can provide a basis for a hypothetical explanation of carboxylesterase overexpression in the male reproductive tract. Based on a well-known role of carboxylesterases in detoxification of environmental chemicals such as organophosphate pesticides, it is proposed that various male genital tract carboxylesterases may be characterized by a similar physiological function to protect the male reproductive system against xenobiotic influences that could provoke its dysfunction, thus altering sperm differentiation and maturation.
Abstract: Mytilus mussels are characterized by annually repeated reproduction which is associated with subsequent growth, morphogenesis, breakdown and redevelopment of the gonad and reproductive tract into mantle mesenchyme. We present a description of the expression of the male-associated polypeptide (MAP; see Mikhailov et al. 1995) in different compartments of the male reproductive system as well as in mantle gonad-supporting tissue. MAP is expressed in both gonad and mantle structures in dynamic patterns that show a substantial overlap in terms of dependence on the stage of gonad development/involution. In general, the total MAP concentration directly correlates with the volume of gonad tubule/duct structures but inversely correlates with mantle connective tissue cell fraction. A maximum of MAP expression is reached in the fully ripe male gonad. MAP is localized around gonad tubules/ducts, in the gonoduct epithelium, membranes of follicle-like structures as well as in the extracellular fiber-like structures of the mantle. However, we also demonstrate unique sites of MAP accumulation in the lumen of gonad follicle-like tubules and in ductal fluid. The latter is characterized by a very high MAP concentration. MAP is also detected in sperm-containing cell suspension obtained by gonad biopsy which we interpret as a result of the adsorption of MAP on mature spermatozoa. The results obtained should be taken into consideration in the interpretation of possible MAP functions since they seem to point to MAP as a major component of ductal (seminal) fluid of the male reproductive tract. It is likely that MAP is able to complement the processes of sperm terminal differentiation and maturation. In addition, we demonstrate that the male-predominant character of MAP expression is restricted by gonad-containing tissues (i.e., mantle and visceral mass) only, although the polypeptide is also detected in other somatic organs in both males and females.
Abstract: Data on activation of crystallin synthesis during lens fiber (LF) formation in amphibians are summarized to point out the questions particularly interesting in the context of lens cell lineage-specific expression programming under different developmental conditions. LFs are known to differentiate throughout life along the same pathway that includes at least five compartments. Using the amphibian eye lens as a model, we have studied how crystallins are expressed in the course of: (1) embryonic LF formation, (2) LF differentiation in adults, and (3) LF transdifferentiation from other (non-lens) eye tissues. Our experiments showed that synthesis of crystallins during morphologically similar LF differentiation in embryonic and adult amphibian lens has different spatial-temporal patterns (i.e., is apparently activated according to different programs). Certain results obtained in our studies suggest the absence of any direct relationship between the capacity of adult newt iris cells to transdifferentiate into LFs and crystallin synthesis (<<molecular predisposition>> to such transdifferentiation) in them. Crystallins appear at the advanced stages of iris transdifferentiation into the lens and dynamics of their synthesis in the <<regenerating>> lens resembles that in the embryonic lens, although a new lens rudiment develops from the adult iris epithelium. Data on alternative patterns of the crystallin gene activation are summarized and compared with recent observations on spatial-temporal expression of Pax genes, which play an essential role in lens cell commitment and crystallin synthesis. On this basis, it is suggested that ontogenetic and tissue- or cell-specific changes in Pax gene expression may result in altered programs for activation of crystallin genes in embryonic, adult, and regenerating lens.
Abstract: We suggested that sexual differentiation of the reproductive system in gonochoric species of invertebrates can be characterized by common molecular mechanisms in spite of high morphological divergences of reproductive tract organs in different animal groups. The present study focused on this problem and report our observations on biochemical characteristics of male-associated polypeptide (MAP) identified in the gonad tissue of bivalve molluscs, Mytilus galloprovincialis, in comparison to those of male-specific carboxylesterase (esterase S) of Drosophila virilis ejaculatory bulbs. We provide evidences for the immunochemical similarity of Mytilus MAP and Drosophila esterase S. We also show that MAP is characterized by esterase activity toward both, alpha- and beta-naphthyl acetates. Using immunofluorescence, we found MAP in the gonad (mantle) connective tissue, membranes of follicles and around gonad ducts but not in sperm cells. Nevertheless, the levels of MAP expression depend on presence or absence of ripe spermatozoa in the gonad follicles. In mature gonads before spawning, MAP is expressed at high level, while in the spent gonads only traces of this polypeptide could be detected. Using Western immunoblot, MAP was not observed in spermatozoa obtained by biopsy of gonad follicles. In contrast, we found this protein in spawned sperm cells. Thus, we suggest that spawning may be required to establish the trafficking mechanisms that control whether MAP is retained or excreted by the gonad. Taken together, the results indicate that MAP of M. galloprovincialis is structurally and functionally related to esterase S of D. virilis ejaculatory bulbs.
Abstract: We have addressed the question of sexual reproductive tissue dimorphism in bivalve molluscs, Mytilus galloprovincialis Lmk, which is a stable gonochoric species although with no apparent differences in gonad morphology of both sexes. At all periods of the annual cycle the proteins specific of male/female gonads were identified. One of these proteins, "male-associated polypeptide" with apparent MW 39 kDa (MAP-39), has been biochemically and immunochemically characterized. MAP-39 concentration in male mature gonads achieved up to 10% of the total soluble protein while in female ones only traces of this protein could be detected. In male mantle, MAP-39 expression was associated with the process of gonad development and maturation as well as gamete spawning, although this polypeptide has been localized in fibroblast-like cells, membrane of follicles and connective tissue matrix of the mantle but not in germinal cells.
Abstract: A high neuralizing activity has been determined in forebrain of 7.5-day old chick embryos using Rana temporaria early gastrula ectoderm as reacting tissue (Mikhailov and Gorgolyuk, Soviet Scientific Reviews, Section of Physiology and General Biology, Vol. 1: 267-306, 1987). The corresponding protease-sensitive agent was extracted, partially purified by chromatography on DEAE-Toyopearl and Heparin-Ultragel columns, and its neuralizing activity was tested in vitro on ectoderm isolated from early gastrulae of R. temporaria, Triturus alpestris, and Xenopus laevis at different concentrations and for different periods of time (animal cap assay). Induction of neural structures was found in R. temporaria and T. alpestris explants (up to 100 and 60%, respectively), but not in cultures of X. laevis ectoderm. Under our experimental conditions, so-called "autoneuralization" of the ectoderm explants can safely be excluded. The results are discussed in relation to the neural competence of amphibian ectoderm and the mechanisms of neuralizing actions of different factors which might be involved in neural induction and patterning.
Abstract: The data are presented about isolation from the brain of 7.5-day chick embryos of a factor capable of neuralizing effect on the early gastrula ectoderm of the grass frog Rana temporaria L. Earlier this factor was defined as embryonic brain-derived neuralizing factor (EBDNF) (Mikhailov, Gorgoliuk, 1989). The isolation procedure includes (1) extraction with deionized water at pH 9.0; (2) ion exchange chromatography on a column with DEAE-adsorbent at pII 8.0; (3) affinity chromatography on Heparin-Ultragel column. EBDNF-containing fraction is eluted from the Heparin-Ultragel column with 250 mM NaCl as a separate peak. Four bands are observed on SDS-electrophoregrams of this fraction, two more prominent ones having the molecular weight of 43 and 63 kDa. The yield of EBDNF-containing fraction is about 0.01-0.02% of the wet weight of the initial brain tissue.
Abstract: This review summarizes the published and authors own data about characteristics of expression of tissue-specific lens proteins (crystallins) during differentiation of lens epithelial cells into lens fibers in adult mammals, birds, reptiles, and amphibians. Information is analyzed about the presence, synthesis, and localization of different crystallins in lens epithelium and fiber cells of lens cortex and nucleus which correspond to sequential stages of lens cell differentiation. The data available suggest that morphologically similar differentiation of lens epithelial cells into fibers in different classes of vertebrates is accompanied by different programs for activation (and/or increase) of tissue-specific protein synthesis.
Abstract: We studied effects of forskolin, an activator of adenylate cyclase activity, and dioctanoyl-cyclic adenosine monophosphate (do-cAMP) on neutralizing (N) activity of concanavalin A (Con A). Biological testing was performed using explanted animal pole ectoderm of the Rana temporaria early gastrula. Con A treatment (200 micrograms/ml, 2 h) resulted in neutralization of 70-90% explants. If the explants were previously treated with forskolin (100 microM, 1 h), Con A effect decreased to 10%. When Con A and forskolin were applied simultaneously, no N-effect was observed. The same results were obtained with simultaneous treatment of the explants with Con A and do-cAMP (10(-5) M). Moreover, treatment with forskolin of the explants previously treated with Con A inhibited their neural differentiation. We suggest that N-effect of Con A is calcium dependent; the increase in intracellular cAMP after treatment of explants with forskolin or do-cAMP interferes with intracellular Ca2+ release and this results in the inhibited N-effect of Con A.
Abstract: Using various experimental techniques, we have demonstrated that animal pole ectoderm (APE) of Rana temporaria embryos at the stage of early gastrula is a good target tissue for testing the neuralizing (N) factors. In this respect R. temporaria APE is comparable with APE of some other amphibian species. We found that concanavalin A (con A), phytohemagglutinin (PHA) and embryonic brain-derived neuralizing factor (EBDNF; a factor extracted from the chick embryonic brain and partially purified) have a pronounced N-effect on the APE of R. temporaria. In order to analyse possible mechanisms of N-action of these factors, we have cultured APE explants for 3 or 18 h in the medium containing various concentrations of con A, PHA of EBDNF. All these factors could produce neuralization in 50% explants. However, the optimal concentration and time of exposure were different. This is an evidence for different mechanisms of reception and transmission of a N-signal in each particular case. It appears that the APE consists of several cell subpopulations which differ in their threshold sensitivity to the N-effect of studied agents.
Abstract: Concanavalin A (con A), a lectin which specifically interacts with aD-mannose and aD-glucose, has a neutralizing effect on the explants of the early gastrula ectoderm of several amphibian species. Consequently, it was interesting to study con A-binding protein spectrum of the ectoderm and compare it to those of other early gastrula tissues. Animal pole ectoderm (APE), dorsal blastopore lip (DBL) and vegetal pole endoderm (VPE) were dissected from early gastrulae of Rana temporaria and Xenopus laevis. The extracts were subjected to SDS-PAGE with subsequent immunoelectroblotting on nitrocellulose membranes. The blots were sequentially treated with con A solution, horseradish peroxidase and diaminobenzidine. Spectra of the con A-binding glycoproteins were similar in APE, DBL and VPE of R. temporaria. Ten-twelve fractions with the molecular weight in the range from 30 to 150 kDa were stained in each blot. Fractions with the molecular weight of 150, 125, 104, 94 and 42 kDa showed more prominent lectin binding. Con A-binding protein spectra remained unchanged after freezing-thawing of the studied extracts, as well as after blots were treated with neuraminidase or sulphuric acid in order to remove sialic acid residues; the only exception was 42 kDa fraction. At the same time, a-methyl-D-mannoside pyranoside completely blocked con A binding by fractions of the studied extracts. In histological sections of R. temporaria early gastrula, all cells bound FITC-labelled con A. Similar data were obtained with tissues of X. laevis early gastrula. While electrophoretic pattern of X. laevis tissues drastically differed from that of R. temporaria, there were no significant differences between con A-binding protein spectra of X. laevis APE, DBL or VPE. Thus, all studied tissues of the amphibian early gastrula contain similar set of con A-binding proteins; however, only APE is capable of neutralization in response to con A action. These data favor our earlier assumption (see MikhaÄlov et al., 1989) that con A reception and transmission of the corresponding signal do not determine the characteristics of the target cells response. APE, DBL and VPE extracts were assayed also for the presence of a protein similar to cytokeratin No. 8 characteristic of simple epithelia of mammals. Experiments were performed using immunoelectroblotting with monoclonal antibodies (mAB) against cytokeratin No. 8 from rat colon (mAB E2 and E7 kindly supplied by Dr. G. A. Bannikov). In R. temporaria embryos, cytokeratin 8 was detected in APE, but not in DBL or VPE. In X. laevis gastrulae all the tissues studied contained this cytokeratin.
Abstract: The purpose of this study was to analyze immunochemically the synthesis and distribution of tissue-specific proteins, i.e., alpha-, beta- gamma- and rho-crystallins, in morphologically distinct regions of the frog (Rana temporaria L.) lens which consist of cells at various stages of differentiation, maturation and aging. Five such cell compartments can be distinguished in the lens: (1) central zone of lens epithelium (stem/clonogenic cells); (2) equatorial epithelial cells (differentiating cells); (3) lens fibers of the outer cortex (post-mitotic differentiated cells); (4) lens fibers of the deep cortex (cells without nuclei at terminal stage of differentiation); and (5) cells of the lens "nucleus" (cells formed during embryogenesis). Intact lenses and isolated lens epithelium were cultured in vitro in the presence of 35S-methionine. Then lens epithelium, outer and deep cortex, and lens nucleus were extracted with buffered saline and extracts used for immunoautoradiography. Distribution of crystallins in paraffin sections of the whole lens or isolated lens epithelium was studied using indirect immunofluorescence. Synthesis of alpha-crystallins was observed in lens epithelium and cortex, but not in lens nucleus. According to immunohistochemistry, these proteins were absent from central part of the lens epithelium: positive fluorescence was observed only in elongating cells at its periphery and in lens fibers. The data on beta-crystallins are similar except that synthesis of these proteins (traces) was detected also in lens nucleus. Synthesis of gamma-crystallins was detected in lens cortex and nucleus (traces) but not in epithelium. Immunohistochemistry showed that these proteins are absent from all regions of lens epithelium and found only in fiber cells of cortex and nucleus. Rho-crystallin was synthesized in all cell compartments of the adult lens, and all lens cells contained this protein. Our results show that cells of central lens epithelium do not contain alpha- beta- or gamma-crystallins (or the rate of their synthesis is insignificant). While cells are moving towards lens equator and elongating, synthesis of alpha- and beta-crystallins is activated. Gamma-crystallins are synthesized later, first in young lens fibers near lens equator. During embryonic development in amphibia, in contrast, gamma- and beta-crystallins are detected at earlier stages than alpha- and rho-crystallins (MikhaÄlov et al., 1988). These data suggest that different mechanisms are involved in differentiation on lens fibers from embryonic precursor cells, on one hand, and from epithelial stem cells of adult lens, on the other.
Abstract: The vertebrate lens contains so-called taxon-specific water-soluble proteins. One of them is p-crystallin with a molecular weight of 35 kDa characteristic of Ranidae family. We have identified a polypeptide with a molecular weight of 35 kDa in the eye lens of Rana temporaria which: (1) can be extracted from the lens by aqueous salt solutions, (2) has a molecular mass of 36.1 +/- 0.4 kDa (by SDS-electrophoresis) and 37 kDa (by gel filtration), (3) is heterogeneous in terms of isoelectric point (pI 6.5-8.0), (4) binds to heparin-agarose, (5) denatures in response to freezing-thawing, lyophilization and in solutions with low ionic strength. Thus, major biochemical parameters of this polypeptide differ from that of amphibian alpha, beta- and gamma-crystallins. In addition to lens, 35 kDa polypeptide was detected by immunoelectroblotting in retina, testes, liver, kidney, spleen, stomach, intestine and lungs. Its level (as percentage of water-soluble protein) is 1.1 +/- 1.4% in the lens, 1.6 +/- 0.7% in retina. 0.05% in testes and liver and 0.01% or less in other organs. Thus, despite its wide tissue distribution, 53 kDa polypeptide is expressed predominantly in lens and retina. We studied the time-course of appearance and accumulation of this polypeptide in tissues where it is expressed at high or low levels. 35 kDa polypeptide was detected for the first time during larval development: (1) in the lens (some time after the mouth opening; stages 33-34 according to Dabagian and Sleptsova, 1975), (2) in the retina (by the time of anus opening; stages 36-37), (3) in the liver (at the stage of elongated hind limb bud; stages 40-41). Definitive expression level of this protein was achieved in the lens by the beginning of metamorphosis and in the retina and liver during first months of development. Hence, during the whole period of larval development 35 kDa polypeptide content of the lens exceeds that of retina or liver. A more substantial evidence is required to confirm the identity of studied polypeptide with rho-crystallin.
Abstract: Problems of the mechanisms of embryonic induction in vertebrate development have been considered on the basis of author's experimental data. Though several polypeptide factors with certain inducing activity have been identified recently, molecular genetic mechanisms of their effect on embryonic target cells remains largely unclear. One of possible causes of very slow progress in this area of developmental biology is an inadequate system of biotesting of inducers at tissue level (ectoderm of early amphibian gastrulae) using histological criteria. A necessity for carrying out similar studies on cellular level and estimating effect of inducers using immunochemical and molecular biological methods has been postulated. Methods allowing to carry out biotesting of inducers on cell suspension or aggregate of a one type of embryonic cells have been proposed. New approaches, combining the methods of experimental embryology and molecular biology, to studies of embryonic inducers, receptors, and their mRNA, have been analyzed.
Abstract: The influence of antibodies to gangliosides of sea urchin Strongylocentrotus intermedius eggs on early embryos of this species was studied. gamma-Globulins were isolated from rabbit anti-ganglioside serum by micropreparative electrophoresis. These gamma-globulins produced anomalies in the development of embryos permeabilized in Triton X-100. The anomalies were not observed when anti-ganglioside gamma-globulins were added to the incubation medium together with gangliosides or when the permeabilized embryos were incubated with gamma-globulins of normal rabbit serum. Pretreatment of S. intermedius embryos with serotonin, tryptamine or some other indole derivatives led to the disappearance of ganglioside determinants from the cell surface and sharply increased immunofluorescence within the cell. Such pretreatment of embryos increased the amount of cell-associated gangliosides more than threefold as compared to untreated embryos. Serotonin was shown to bind specifically to sea urchin gangliosides immobilized on octyl-Sepharose. These observations suggest that cell-surface gangliosides, after binding drugs, are internalized and that serotonin and its antagonists inhibit the transport of newly synthesized gangliosides to the cell-surface membrane.
Abstract: The following mitogens: concanavalin A (con A), phytohemagglutinin (PHA), hydra growth factor (HGF) as well as neurotoxic agent kainic acid, caused neural differentiation (N) effects differed in value and also in character of dependence on concentration of the agent. The lowest effective concentration of con A was 75 micrograms/ml (15% neural differentiation, treatment during 3 h), and the effect reached maximum of 50-60% at 100-200 micrograms/ml. Con A concentration 50 micrograms/ml showed no effect but after 1% rabbit gamma-globulin was added, 17% neural differentiation was detected. N-effects observed after treatment of explants with con A (200 micrograms/ml, 3h) at 2 degrees and 21 degrees were similar (58 and 42% respectively). Minimum PHA concentration used (6 micrograms/ml, 18h) led to neural differentiation in 5% of explants. N-effect of PHA increased along with the concentration of the lectin and was most pronounced at 25 micrograms/ml. However, further increase in concentration (up to 200 micrograms/ml) resulted in decrease of its N-effect to 13%. At 12 micrograms/ml PHA exerted not only neural differentiation, but also lens-inducing (32%) action on the ectoderm. N-effect of HGF (2.5, 25 and 250 micrograms/ml) was lower as compared with the maximum effects of con A and PHA (30-35%). No correlation of HGF inducing action with its concentration was observed. Kainic acid showed weak N-effect (20-30%) at 1 and 10 micrograms/ml. Higher concentration (100 micrograms/ml) had no N-effect, but in 27% of explants "free" lentoids were found. Oubain (10(-3) and 10(-4) M) and HEPES (20 mM) did not affect the differentiation of explants.
Abstract: The published and authors' data have been summarized on (1) the spectrum and properties of crystallins in different amphibian species, (2) localization and synthesis of crystallins in different cellular compartments of the adult amphibian lens, (3) dynamics of crystallin formation during embryogenesis and (4) lens regeneration from tissues of the larval and adult amphibian eyes. The necessity of more detailed studies of crystallin synthesis during embryogenesis and lens regeneration using molecular biological and biochemical methods is stressed. The significance of this approach is illustrated by the pioneering data of Soviet scientists on crystallin polypeptides and corresponding mRNAs in development of Rana temporaria obtained with the use of DNA-RNA hybridization and immunoelectroblotting.
Abstract: We have studied in vitro differentiation of explants of the amphibian (Rana temporaria) early gastrula ectoderm after treatment with various concentrations (50-300 micrograms/ml) of 'free' and Sepharose-bound concanavalin A (Con A). The explants were incubated with Con A for 3 h at 20 degrees C; the rolling up of the explants was prevented by using special weights. We have demonstrated that: (1) free Con A has an inducing action on the explants in the concentration range 100-300 micrograms/ml medium; (2) when treated with Con A the explants produce neural tissue (50-70%), cartilage (20-40%) and, rarely, lentoids (5-10%); (3) the frequency of neural and cartilage inductions was similar at various Con A concentrations; (4) alpha-methyl-D-mannoside pyranoside inhibited the Con A effects; (5) Sepharose-bound Con A had no effect on the explants, although it was bound to the cell surface of the ectoderm inner layer. Possible mechanisms of the neutralizing and chondrogenic effects of Con A on ectodermal explants are discussed.
Abstract: Electrophoretic analysis of pattern of polypeptides making part of precipitation arcs (peaks) cut out of predried and stained immunoelectrophoregrams has been shown to be possible. The cut out precipitation arcs were extracted overnight by a starting buffer for SDS-electrophoresis, centrifuged, and supernatants were subjected to SDS-electrophoresis; electrophoregrams were stained by silver nitrate. Electrophoretic pattern of polypeptides from the stained and "native" precipitation arcs for the same antigens were shown to be identical. A good correspondence was also observed between the results of analysis of polypeptide composition of the immune precipitates and the data of immunoelectroblotting preformed with the same immune reagents. This technique allows precipitation arcs to be studied from immunoelectrophoregrams preserved at the room temperature for a decade and more.
Abstract: Eye vesicles were isolated from the early chick embryos (stage 9+ after Hamburger and Hamilton, 1951) and combined with the Rana temporaria early gastrula ectoderm (EGE) in vitro. The tissues were jointly incubated in medium 199 diluted twice with deionized water at 22 +/- 1 degree for 7-8 days or the eye vesicles were removed from the EGE ectoderm within 16-18 h. At the joint long-term incubation of these tissues, a toxic effect of the chick embryonic tissues on the EGE cells was noted. In none of the experiments, the inducing effect of the eye vesicle on the EGE was found. Similar data were obtained when the EGE was jointly cultivated with the brain (stage 9-10) and retina (stage 15) of chick embryos. The brain of the chick embryos at stage 15 exerted a weak neuralizing effect on the EGE. In the control experiments, the eye vesicles explanted with the chick embryonic ectoderm remained viable till the end of cultivation but no lentoids formed in the ectoderm. The absence of lens-inducing effect at the joint cultivation of the chick embryonic eye vesicles with the EGE is considered as a result of disturbance of the synthesis or secretion of the corresponding agents rather than a sequence of the species "incompatibility" of the inductor and reacting tissue. Hence, the use of "xenogenic" tissue recombinants is not justified when analyzing the lens-inducing activity of the eye vesicles.
Abstract: The main attention is paid to the critical analysis of experimental data on morphogenetically active substances, so called "morphogens". It is proposed to consider the morphogens as specific transmitters providing for definite phases of morphogenetic tissues interactions, rather than as vectors of "morphogenetic information". In the normal development, the most studied morphogenetic tissue interactions can be referred to as so called permissive inductions, since the cells of the vertebrate embryos (amphibians, avians) are early determined for development in the ectomeso--and endodermal directions. A slow progress in studying the morphogens can be due to the following causes. 1. Theoretical "inadequacy" of the former concepts on the essence and mechanisms of embryonic induction. The necessity to develop a new system of concepts in this area of developmental biology is stressed. 2. Incompleteness of knowledge about the properties of reacting tissues and the mechanisms of action of morphogens. The early gastrula ectoderm of amphibians, most frequently used for testing the morphogens, appears to be a heterogenous population of the cells with different properties and potencies. It is, therefore, impossible to standardize strictly the biotesting of morphogens. It is suggested that the use to this end of aggregates of cell "strains" from the gastrula ectoderm, rather than of the gastrula ectoderm itself, may be more adequate 3. Insufficiency of embryonic material for biochemical identification and isolation of natural morphogens. A study of so called heterogenous inductors might be of help; these latter can be considered as analogs of natural morphogens. But the similarity of natural and heterogenous inductors can be limited only by their final effect on target tissue. The data are provided on the chemical nature, properties and mechanisms of action for a number of natural and heterogenous inductors (vegetalizing, neuralizing, mesodermalizing, lens-inducing factors). A conclusion is drawn that specific antigens do exist normally but they should not be established as a special class of "informationally important" molecules. The information necessary for development is contained in target cells and the function of a morphogen consists in providing for a definite link in the chain of processes leading to the switching on or expression of one or another programme. Only syntheses of specific proteins can, apparently, be programmed, thus reflecting the "onset" of differentiation path for a cell.
Abstract: The possibility of direct combining the protein fractions after electrophoresis in polyacrylamide gel (PAAG) with the ectoderm of avian embryos in vitro was shown. The extract of eye vesicle of 30-33 hrs chick embryos were subjected to electrophoresis in 20% PAAG in capillaries (diameter 300 mu). The columns of PAAG were cut in equal parts and these pieces with protein fractions were combined with the trunk ectoderm of 2 days old chick embryos. The preliminary analysis has shown that some fractions of the eye vesicle exert a selective neutralizing effect on the trunk ectoderm (formation of neural tubes). The PAAG itself had no toxic or inducing effect on the trunk ectoderm cells. The lens-inducing effect of the eye vesicle fractions was not observed.
Abstract: The lenses were studied in the Rana temporaria embryos by means of immunochemistry from the formation of lens vesicle till the end of metamorphosis. The rabbit antisera against gamma- and beta-crystallins of Rana temporaria were used. The materials were fixed by acetone or Carnois fixative. beta-crystallins were shown to appear simultaneously with gamma-crystallins at stage 31--32 (Dabagyan, Sleptzova, 1975). gamma-Crystallins appeared first in the lens fibers and later (stages 36--51) in the epithelial cells. From stage 52 (tail reduction) the immunofluorescence of gamma-crystallins in the epithelium fell sharply until full disappearance. At stage 54 (end of metamorphosis) a weak fluorescence of gamma-crystallins was found in the anterior part of epithelium only. gamma-Crystallins were not found in the epithelium of adult frogs.
Abstract: The effect of fixatives on the rat bone marrow antigens was studied by a method combining immunoadsorption and immunodiffusion. This method allows to estimate rapidly and reliably the effect of fixation on immunochemical properties of the antigen under study, using polyvalence antisera. Acetone and ethanol proved to be the best fixatives for the rat bone marrow antigens since they preserved their immunochemical properties. Aldehydes (formaldehyde and glutaraldehyde) "inactivated" the bone marrow antigens.
Abstract: An attempt was undertaken to determine the chemical nature of the neuralizing and lens-inducing effect of the retina and brain extracts from 7-8 day old chick embryos. These extracts were treated with immobilized enzymes (RNAse, proteinase K) and the effect of treatment was then estimated in the organ cultures (reacting tissue - early gastrula ectoderm). The effect of the enzymes was studied in different experimental variants which allowed to exclude the effect of the own proteases and temperature on the inducing activity of the extracts under study. The neuralizing activity of the retinal extract was shown to preserve after its treatment with RNAse but to be almost fully lost (or decrease) after proteolytic hydrolysis. Proteinase suppressed as well completely the lens-inducing effect of the extract from the chick embryo brain. A conclusion is drawn that the inducing activity of the extracts under study is due to proteins, rather than to RNA.
Abstract: Silver impregnation of polyacrylamide gel sheets after isoelectrofocusing is described. This method of staining allows to increase the sensitivity of the traditional isoelectrofocusing 5 to 10 times, as compared with staining of gels by Coumassi brilliant-blue R-250 (in case of analysis of complex protein mixtures). When analyzing individual protein preparations, the sensitivity of isoelectrofocusing with silver staining is within the range of several dozens of ng per sample. A special attention is paid to methods permitting to decrease the non-specific background staining of gel by silver.
Abstract: Fractions of cavitary fluid of Ascaris (CFA) containing antigens were obtained on membranous filters. Being administered to mice they provide the development of protective immunity of animals to their subsequent infection with invasional eggs of A. suum. In mice immunized with fraction CFA XM-50 the number of A. suum larvae obtained from the lungs on the 7th day of invasion was confidentially lower than in control animals (15.277) +/- 1.825 on the 7th day of invasion was confidentially lower than in control animals (15.277 +/- 1.825 and 58.10 +/- 1.588, respectively, p less than 0.001). Immunisation of mice with fraction XM-300 caused less pronounced protective effect. Low-molecular antigens of the fraction UM-05 possess tolerant properties. In mice immunized with fraction UM-05 the number of A. suum larvae obtained from the lungs on the 7th day after invasion was greater than in control ones (68.22 +/- 6.24 and 49.39 +/- 4.13, respectively, p less than 0.001).
Abstract: Poly(A)+ RNA from the lens of the frog Rana temporaria contains three components (1200 +/- 50, 1000 +/- 50, and 900 +/- 50 bp in size) and a more heterogeneous RNA species with a length of 650-750 nucleotides. This RNA was used as a template for the AMV reverse transcriptase and Escherichia coli DNA-polymerase I and the total cDNA obtained was cloned in the PstI site of the pBR322 plasmid vector. Recombinant plasmids corresponding to abundant poly(A)+ RNA classes contain cDNA inserts from less than or equal to 200 to 1200 nucleotides in length. Part of the library (clonotheque) was divided into classes differing in the presence of absence of the restriction sites for BamHI, EcoRI and HindIII restriction endonucleases. The clones belonging to each of the five classes were characterized by the hybridization-translation test. The translation product of mRNA hybridizing with the clone pRT(1)294 has an M4 of about 22 000 and is specifically precipitated by the antiserum to lambda-crystallins of Rana temporaria. The size of the cDNA present in pRT(1)294, equal to 580 +/- 20 bp, is sufficient for coding the greater part of the lambda-crystallin amino acid sequence. On the basis of these data, we conclude that the clone pRT(1)294 codes for one of the frog lambda-crystallins.
Abstract: From eggs and embryos of the sea urchin Strongylocentrotus intermedius two gangliosides, provisionally named G-1 and G-2, were isolated in the pure state. Both gangliosides contained glucose, N-glycoloylneuraminic acid and sphingosines in a 2:2:1 ratio; G-2 contained also a sulfate group, and yielded G-1 on desulfation. By periodate oxidation/borohydride reduction, permethylation analysis, neuraminidase degradation, analysis of the aldohexitol acetates and mass-spectrometry G-1 and G-2 were shown to have hitherto unknown structures: G-1 was identified as N-glycoloylneuraminosyl-(alpha 2 leads to 6)-glucosyl-(1 leads to 8)-N-glycoloylneuraminosyl-(2 leads to 6)-glucosyl-(1 leads to 1)-ceramide, and G-2 as sulfated G-1, carrying a sulfate ester group at C-8 of the terminal sialic acid. Antisera against the two gangliosides were prepared in rabbits by immunization with ganglioside G-1 or G-2. The specificity of the antisera was revealed by immunoelectrophoresis and immunodiffusion. The antisera did not react with bovine-brain and rat-liver gangliosides, with glucosylceramide and with various hydrolytic fragments of G-1 and G-2. The surface localization of the gangliosides in embryos incubated at different cell densities was studied by immunofluorescence microscopy. The intensity of the immunofluorescence was found to increase with decreasing cell density, indicating a different surface organization in sparse and dense embryos. In the sparse embryos immunofluorescence was seen mainly in the contact regions between the blastomers.
Abstract: Two main gangliosides (G-1 and G-2) were isolated from eggs and embryos S. intermedius. They contain glucose, N-glucolylneuraminic acids, phytosphyngosine, fatty acids and alpha-hydroxy fatty acids. Molar ratios and sequence of these components are the same for both gangliosides, but G-2 contains sulphate residue which is attached to the terminal neuraminic acid. To obtain specific antisera rabbits were immunized by G-1 or G-2, which were mixed with bovine serum albumin and Freund's adjuvant. Both gangliosides possessed electrophoretic and antigenic heterogeneity. G-1 and G-2 gangliosides have common and individual antigenic determinants. Glucosylceramide of gangliosides is immunologically inactive. Individual antigenic specificity of the gangliosides depends on the presence of N-glycolylneuraminic acid (G-1) and SO3H-group (G-2). Egg gangliosides were demonstrated by immunofluorescence throughout the cell surface. After fertilization of immunofluorescent label was concentrated on one pole of the embryo only. During the development of specific fluorescence was again uniformly distributed at the blastomer surface. The most intense fluorescence was observed in the junction areas of the blastomers.
Abstract: Antigens of zona pellucida (ZP) of different mammalian species, including the man, were studied by means of various immunochemical methods. The analysis was carried out using rabbit antisera to the pig, mouse, guenon and Java macaque eggs. After immunoadsorption (by blood serum and tissue extracts) these anti-ZP-sera reacted with water-insoluble ZP components only (in immunofluorescence). The adsorbed anti-ZP-sera were specific not only to homologous ZP, but also to ZP of other mammalian species. The spectrum of antigens of the ape ZP was most closely related to that of human ZP. The pig eggs contained antigens common with the human ZP as well. The antiserum to the mouse ZP did not react with ZP of man, ape, pig, cow, rabbit and other species under study. The blood of sterile women contained the antibodies reacting (in immunofluorescence) with ZP of mouse, pig, guinea pig, sheep and man (very weakly). The data obtained suggest the complexity of antigenic mosaic of the mammalian ZP including both "cross-reacting" (common) antigens and species specific ones.
Abstract: A modification of the indirect immunofluorescent method is proposed for the detection of specific proteins in sections of the developing amphibian lens that were attached using egg albumin, stained and embedded into Canada balsam.
Abstract: Localization of specific proteins at preliminary stained with azan and embedded in balsam sections of amphibian eye lens (Rana temporaria, Xenopus laevis) was studied by indirect immunofluorescence. The analysis was performed on the lens of intact animals as well as on lens induced in amphibia gastrula ectoderm in vitro. To remove balsam the lens sections were successively washed by xylene, ethanol and saline (pH 7.1). Then they were treated according to the general principles of immunohistochemical analysis. Specific antigens have been thus shown to be detectable at lens sections which had been kept in balsam for over a year. The immunofluorescent reaction to lens proteins was negative at more prolonged storage of the sections (2--3 years).
Abstract: It has been shown that pregnancy specific beta 1-globulin (SBG) is found in the rat blood serum on the 5--7th day of pregnancy. SBG reaches the maximum by the end of pregnancy and is not detected already at the 3d--4th day after delivery. The placenta is the site of the SBG synthesis. Other organs of the pregnant rat are not able to incorporate radioactive aminoacids into the protein in vitro.
Abstract: The crystallins of ap mutants of Xenopus laevis have been studied in comparison with those of normal embryos and adults using the complex of immunochemical methods (immunoelectrophoresis, immunodiffusion, immunoadsorption, immunofluorescence, isoelectrofocusing with immunoidentification). The analysis was carried out with antisera to electrophoretic fractions of the mutant lens. 11 organ-specific antigens were found in the lens of both the normal and mutant animals. These proteins are heterogenous by electrophoretic mobility, isoelectrical point, antigenic and species specificity. Each class of crystallins contains antigens which are specific: a) for amphibians only, b) for lower vertebrates, c) for vertebrates in general. No qualitative differences were found between crystallins of the normal and mutant animals. Immunofluorescence analysis has shown that crystalins appear in the normal and mutant embryos practically at the same time. No significant differences in the appearance of specific immunofluorescence between the normal and mutant embryos were found (with various antisera). gamma-crystallins and, perhaps, a part of the primary lens fibers. Alpha-crystallins appear later. gamma-crystallins are first identified the synthesis of which manifests itself at the advanced developmental stages. The quantitative predominance of some beta--gamma-crystallins in the mutant lens detected by us (electrophoresis, isoelectrofocusing) is not related to their earlier synthesis in the embryogenesis.
Abstract: Antisera were obtained to the total extract and individual electrophoretic fractions of lens proteins: alpha-, beta-, gamma1- and gamma2-crystallins. The crystallins under study are immunochemically heterogenous: each class of lens proteins contains 2--4 antigens. Using the indirect method of fluorescent antibodies, it was established that the appearance of crystallins during development coincided with the onset of formation of the presumptive lens fibers. No crystallins were found in the lens placode and early lens vesicle. gamma-Crystallins appear later than the other lens proteins and are characteristic, mainly, for the lens fibers; at the advanced stages of organogenesis gamma-crystallins are regularly found in the epithelial cells of the developing lens as well.
Abstract: Individual lens proteins were studied during development of Rana temporaria. Antisera to alpha-, beta-crystallins of chicks and gamma-crystallins of Rana ridibunda were used as immunochemical markers. Besides the main crystallins, a new antigen was found in the R. temporaria lens tentatively called alphabeta-crystallin. It appears to be characteristic only for the amphibian lens. Using the indirect method of fluorescent antibodies, it was shown that all the antigens under study appeared in the lens of the R. temporaria tadpoles within 1--2 days (at 20 degrees). The crystallins are found initially only in the developing lens fibers and later in the lens epithelium. It was established that the lens epithelium contained gamma-crystallins which appeared somewhat earlier than alpha- and beta-crystallins, but simultaneously with alphabeta-crystallin.
Abstract: The lens induction is a two-step process and involves morphogenetic influences from the archencepalic endoderm and optic vesicle. One can suggest that the lens induction is primed by specific proteins which are synthesized and secreted by the optic vesicle cells. The proteins-inductors appear to penetrate in the cells and, while interacting (directly or via the cytoplasm) with the nuclei, "programme" the ectodermal cells towards the lens differentiation. The contact interactions and extracellular matrix are of substantial, but not crucial value for the lens induction. The synthesis of specific proteins (crystallins) is to be considered as the most objective criterion of lens differentiation. In vertebrates, there is a lag-period between the moment of lens induction and synthesis of crystallins which is the most long-term in amphibians. The chick embryos constitute an exception and the synthesis of crystallin mRNA occurs in them a few hours after the lens induction. The developing retina loses its capacity to induce lens but stimulates the processes of fiber formation and synthesis of crystallins. A factor was found in the definitive lens epithelium which may be considered as a possible regulator of lens differentiation. On the basis of experiments with heterogenous and native lens inductors, a suggestion is put forward to the effect that the activity of inducing substances is determined by a definite determinant group of the molecule, rather than by the whole molecule.
Abstract: In the lens of fishes (carp, spiny dogfish) beta-crystallins were identified which were characteristic also of reptiles, amphibians, birds and mammals (evolutionary stable beta-crystallins). The dynamics of the formation of such beta-crystallins in 5--14 days old chick embryos was studied by the indirect immunofluorescence method with antisera to fish lens. These proteins are reliably indentified first at the lens sections from 7--8days old chick embryos. At all stages under study these beta-crystallins are localized mainly in the epithelial cells and practically not found in the lens fibers. They were, however, found in the fibrous (central) part of developing lens as well by the method of immunoelectrophoresis.
Abstract: The water-soluble proteins of chick retina were studied during the formation of eye cup and at the early stages of histological differentiation of retina by the micro-method of electrophoresis in 20% polyacrilamide gel. The retina of embryos at the stages under study contains a range of proteins forming over 20 fractions in electrophoresis. The most fractions are formed by the proteins which electrophoretic mobilities exceed that of serum albumin. The early stages of retina development are characterized by the definite changes in its protein composition. These changes manifest themselves in the disappearance of the most anodic fractions beginning from the stage of contact between the optic vesicle and presumptive lens ectoderm. During the subsequent development, these proteins are detected again in the retina, the corresponding anodic fractions being most distinct at the stage of completed eye cup. Their content in the retina decreases repeatedly with the beginning of histogenesis up to their complete disappearance.
Abstract: Water-soluble antigens of chick retina were investigated using rabbit antisera to total extract and to individual electrophoretic fractions of retinal extract by methods of immunoelectrophoresis and Ouchterlony precipitation test. In the retina of the adult chick six serum and eleven tissue antigens were demonstrated. The tissue antigens of the retina comprised one organ-specific antigen and ten inter-organ antigens which were characterized by nonuniform distribution in tissues and organs of adult chick. Three antigens out of these were found only in tissues of the eye (retina, iris) and in the brain--inter-organ antigens of 'narrow' specificity. The ohter seven inter-organ antigens were found in tissues of brain and eye, as well as in various tissues and organs of hens--inter-organ antigens of 'broad' specificity. A high degree of antigen similarity between retina and iris was observed. Anti-retina sera in chick lens could detect only inter-organ antigens of 'broad' specificity. In the course of embryogenesis the first to appear in the developing retina were inter-organ antigens of 'broad' specificity (on 3rd day of incubation). Formation of antigens of this group was completed by the 9th day of incubation. On the contrary, inter-organ antigens of 'narrow' specificity appeared later, in the period of histogenesis of retina (from 5 to 18 days of incubation). The organ-specific antigen of retina was found by 7th day of incubation. One of the inter-organ retinal antigens of'narrow' specificity (retina-iris-brain) appeared in the developing chick brain at the same time as in retina--on 10th-11th day of incubation. Using the indirect immunofluorescence antibody technique this antigen was identified in the cytoplasm of retinal cells and brain neurones, but was not detected in the nerve fibres.
Abstract: The complex investigation of the bilogical properties of the triploid cell strain derived from a spontaneous abortus was carried out. Cytomorphological, autoradiographic, cytochemical, biochemical and immunochemical investigation showed that, according to most of the investigated properties, triploid cells did not differ from normal diploid cells. The cells had normal form, were well orientated, revealed expressed fibrillar apparatus and viability in the culture during 15--17 passages. The decrease of the alkaline phosphatase level, increase of acid phosphatase, lactate and malatdehydrogenase and greater nuclei area were the essential differences from the control. The cells had normal mitotic cycle parameters and the antigenic spectrum was practically identical to the normal cells.
Abstract: Antisera to diploid, trisomic and triploid embryonic fibroblast-like cells were obtained after hyperimmunization of rabbits. Immunoelectrophoretic analysis with these antisera revealed up to 9 water-soluble antigens in embryonic cells, which were present in skin fibroblasts from adult donors as well. Trisomic and triploid strains did not differ from the diploid ones by the spectrum of water-soluble antigens. The content of the number of antigens (especially of cathode fractions) in trisomic cells was significantly low as compared with those in control diploid cells, whereas in triploid ones it differed slightly. All the strains were characterized by the presence of proteins immunologically identical to alpha-globulin of human serum.
Abstract: The following antigens were found in the chick retina: one organospecific, three interorganic antigens of narrow specificity characteristic of only the tissues of the eye and brain, and seven interorganic antigens of broad specificity characteristic of, besides the tissues of the eye and brain, many other organs of the chick. The interorganic antigens of broad specificity manifest themselves the first in the retina during development. The interorganic antigens of narrow specificity and the organospecific antigen arise during the period of retinal histogenesis. One of the antigens of narrow specificity (retina--iris--brain) arises in the brain at the same time as in the retina.
