Emergence of a genotype I variant of avian infectious bronchitis virus from Northern part of India.

Infectious bronchitis virus (IBV) is one of the foremost causes of a persistent economic burden to poultry industries worldwide. IBV belongs to the genus Gammacoronavirus within the family Coronaviridae. The IBV infection leads to respiratory and nephrogenic symptoms in broiler chickens. In addition, its infection leads to reduced fertility and hatchability in layer birds. We determined the first complete genome sequence of a variant IBV from an outbreak in Haryana state of the Northern part of India using next generation sequencing. On phylogenetic analysis of the IBV isolate, it clustered with genotype I lineage 1 (GI-1). The deduced amino acid sequence of S gene of IBV isolates showed a high level of identity with strains isolated from Tamil Nadu and the reference vaccine strains. Our result suggests that the IBV virus isolated from unvaccinated chicken flocks in North India might be a revertant strain originally evolved from the live attenuated vaccine strains used in the region. Determination of the complete genome sequence of additional IBV isolates from India is necessary to understand the epidemiology of IBV in India.

Molecular cloning and in silico studies of physiologically significant trehalase from Drosophila melanogaster.

Trehalase, a physiologically important glycosidase is known for its crucial role in insect glycometabolism and stress recovery. The present study describes the molecular cloning of a gene fragment, encoding the catalytically active trehalase from Drosophila melanogaster (DmTre) and its heterologous expression in Escherichia coli. The 1275bp gene was overexpressed in two different vectors viz., pET28a and pCOLD TF and investigated for variable soluble expression, purification and activity of the recombinant enzyme with optimum pH and temperature of enzyme as 6 and 55°C, respectively. The sequence was characterized in silico by subjecting it to homology search, multiple sequence alignment and phylogenetic tree construction revealing its identity to other trehalases which belong to glycoside hydrolase family 37. The deduced amino acid sequence and modeled 3D structure of DmTre possessed all features of trehalase superfamily, including signature motifs and catalytic domain. The active site pocket of recombinant DmTre was compared with the crystal structure of E. coli trehalase identifying Glu424 and Asp226 as the putative catalytic residues. Additionally, enzyme-substrate docking suggests possible involvement of other residues in the catalysis along with Asp226. The present study holds significance in understanding the structural aspects of Drosophila trehalase in spite of unavailabilty of eukaryotic trehalase crystal structure.

Pathotypic and Sequence Characterization of Newcastle Disease Viruses from Vaccinated Chickens Reveals Circulation of Genotype II, IV and XIII and in India.

Newcastle disease virus (NDV) causes a highly contagious disease which continuously haunts the global poultry industry. The nature and molecular epidemiology of NDVs prevalent in recent outbreaks in India is poorly understood. This study aimed to characterize NDVs prevalent in vaccinated flocks in India using whole-genome sequencing and biological pathotyping. Twelve field isolates were collected from outbreaks which occurred in different parts of India and characterized as velogenic based on their intracerebral pathogenicity index (ICPI) and amino acid sequence at the F protein cleavage site. All 12 of the field isolates and five commonly used vaccine strains were selected for whole-genome sequencing using Ion Torrent PGM technology, yielding complete genome sequences for ten field isolates and all vaccine strains. The genome of all isolates was found to be 15 192 nt long with a high level of conservation across multiple genomic features with APMV-I viruses. Phylogenetic analysis and evolutionary distance calculations placed the isolates in genotypes II, IV and XIII. Revisiting other recently reported strains provided preliminary evidence of genotypes VI, VII and XVIII circulating in India. Comparison between the field and vaccine virus sequences revealed unique genomic and amino acid differences in important antigenic regions of the F and hemagglutinin-neuraminidase (HN) genes which can be targeted for site directed mutagenesis to evaluate the impact of these substitutions on virus pathogenicity. This study highlights the requirement to evaluate current vaccines through systematic protection assays to determine protection efficacy against field isolates.

Hsp90 mediates activation of the heme regulated eIF-2 alpha kinase during oxidative stress.

The heme-regulated inhibitor (HRI), a member of the eIF-2 alpha kinase family is crucial for regulating protein synthesis during stress. In addition to heme, stress proteins Hsp90 and Hsp70 are known to regulate HRI. The present study aims to determine the physical association of these Hsps in the regulation of HRI activation during oxidative stress using human K562 cells as a model. Extracts from the stress-induced cells were used for determining HRI kinase activity by measuring eIF-2 alpha phosphorylation, and Hsp-HRI interaction by immunoprecipitation and immunoblot analyses. The results indicate a significant increase in both Hsp70 and Hsp90 expression during AAPH (2,2'-azobis (2-amidinopropane) dihydrochloride)-induced oxidative stress. Further, their interaction with HRI, which correlates well with its increased HRI kinase activity leads to inhibition of protein synthesis. Thus, we demonstrate that Hsps play an important role in the regulation of initiation of protein synthesis during oxidative stress.

Plesiotherapy for non-melanoma skin cancer: innovating to overcome!

BACKGROUND: The non-surgical management of non-melanoma skin cancers is an area requiring clinical investigation. Radiotherapy has a role in treatment for a defined subset of patients. AIMS: The application of radiotherapy is subject to availability of proper equipment, non-availability of which precludes appropriate radiotherapy in most centers in third world countries. MATERIALS AND METHODS: The introduction of innovations is needed to circumvent this. Plesiotherapy is such a mode of therapy for non-melanoma skin cancer. METHODS: The introduction of innovations is needed to circumvent this. Plesiotherapy is such a mode of therapy for non-melanoma skin cancer. RESULTS: In this paper we present successful management of a cohort of non-melanoma skin cancer patients with plesiotherapy using stepping source(192) Ir HDR source. CONCLUSIONS: Plesiothrapy is an effective mode of therapy for non-melanoma skin cancer.

Effect of temperature and insecticide stresses on Aedes aegypti larvae and their influence on the susceptibility of mosquitoes to dengue-2 virus.

Two major factors, higher temperatures and the application of insecticides, can drastically alter the genetic structure of a vector mosquito population. Due to these two stresses, the majority of the population gets wiped out, but the ones that withstand the stress and survive are likely to pass on survivability, and have an altered physiology. Our study shows that exposures to higher temperatures and DDT during the larval stage affects their susceptibility as adult mosquitoes to the DEN-2 virus. The overall transcription and translation status of heat shock protein (Hsp70) in virus high- and low-susceptible was the same as that in other batches. In the case of a DDT-resistant (R-7) strain two bands were obtained during RT-PCRs after heat shock. These two alleles were obtained only with HY-1 in which R-7 males were used for the crosses, suggesting that the second allele is probably male sex linked. The higher expression of Hsp70 may provide DDT-resistant strains a better chance of survival high temperature environments, particularly in homozygotes and hybrids. It was also interesting to note that these strains have a significantly lower susceptibility to the virus. Wide-spread DDT-resistance and a rise in temperature above the average temperature during summer may result in a population with a low susceptibility to the virus. Several families of heat shock proteins are known to be expressed in mosquitoes, and may have a cumulative role in determining susceptibility to the virus, which itself is governed by several genes.

Dose-dependent differential effect of hemin on protein synthesis and cell proliferation in Leishmania donovani promastigotes cultured in vitro.

Leishmania donovani requires an exogenous source of heme for growth and transformation. In in vitro culture of the free-living promastigotes, exogenously added hemin enhances cell proliferation. In this investigation, the question of the function of heme with particular reference to protein synthesis and cell proliferation has been addressed. The results of in vitro cell culture experiments demonstrated that hemin (10 microM) alone is suitable for supporting optimum level of protein synthesis, and thereby cell proliferation of promastigotes to an extent that it can replace fetal bovine serum. However, in situ labelling experiments along with Western blots revealed that high concentration of hemin (50 microM) reduced the level of protein synthesis in general and of beta-tubulin in particular with a concomitant induction of hsp90, and induced consequent morphological changes that are observed during in situ transformation of promastigotes in mammalian macrophages. These results therefore suggest that sudden exposure to high concentration of heme in mammalian macrophages may be one of the key factors that trigger promastigote to amastigote transformation in L. donovani. Furthermore, hemin with its dual characteristic could be used as a tool to understand molecular mechanism of cell proliferation and transformation in these parasites.

Detergent-mediated destaining of coomassie brilliant blue-stained SDS polyacrylamide gels.

A simple and one-step detergent-mediated destaining procedure for SDS Polyacrylamide gels for proteins is described. Suspension (5%, w/v) of a commercially available household detergent, Vim Ultra, has been found to be very efficient in destaining polyacrylamide gels without interfering with the resolution of proteins. As compared to the routinely used solvent (methanol-acetic acid-water)-mediated destaining procedure, the present method is economical and user-friendly.

Effect of risperidone on prolactinoma growth in a psychotic woman.

OBJECTIVE: The case of a psychotic woman is described in which risperidone use was found to correspond with an increase in the size of a prolactinoma and prevented the return of serum prolactin level to baseline. METHODS: Although the patient had been treated with a high dose of bromocriptine, her prolactin level remained elevated, causing persistent galactorrhea. The patient later was treated with olanzapine and carbamazepine successfully. RESULTS: This case report highlights the role of risperidone on prolactin and discusses alternative methods of treating psychosis when the etiology is unclear, especially in younger patients. CONCLUSIONS: The authors recommend that additional studies regarding the relationship between the growth of prolactinoma and atypical antipsychotics would be worthwhile.

Temperature-induced alteration of the polytene X chromosome structure in male larvae of the strain In(1)BM2 (reinverted) of Drosophila melanogaster.

In Drosophila melanogaster, the polytene X chromosome of male third instar larva appears twice as wide as an unpaired female X chromosome or an autosome. This characteristic morphology of the male X chromosome is correlated with the increased rate of transcription of the sex-linked genes, which ensures gene dosage compensation. In male third instar larvae of the strain In(1)BM2 (reinverted), polytene nuclei manifest unusually puffy X chromosomes at 18 +/- 1 degrees C. Such 'puffy X' chromosomes are pompons, that is, despite the increased width of the chromosome, transcription remains at the wild-type level. This characteristic is a caveat to the invariable correlation between polytene chromosome puffs and transcription, and suggests that the mutant X chromosomes arise due to perturbation of a pathway that controls the structure but not the transcription of the polytene X chromosome. In this report we present evidence that the pompons of In(1)BM2 (reinverted) arise due to spiralization of the male X chromosome, which results in condensing of the chromosome. This unusual structural alteration can be induced only in male larvae of this strain, at the third instar larval stage, through temperature shifts from 24 +/- 1 degrees C to 18 +/- 1 degrees C and during recovery from cold shock. Furthermore, extract from male adult, pupae and third instar larvae can induce chromosome condensation in wild-type larvae in vitro. This new evidence not only explains the absence of correlation between chromosome width and transcription of the pompons of In(1)BM2 (reinverted), but also suggests that the chromosomal rearrangement perturbs a pathway that regulates the condensation of chromosomes.

Acetylation of alpha-crystallin with N-acetylimidazole and its influence upon the native aggregate and subunit reassembly.

PURPOSE: An attempt has been made to investigate the involvement and importance of some of the hydrogen bond forming amino acid side chains in intra and inter subunit interactions in alpha-crystallin assembly. METHODS: For this, alpha-crystallin has been acetylated, partially or completely, using N-acetylimidazole. The apparent molecular size, electrophoretic mobility, conformational properties, surface hydrophobicity and chaperone activity of the modified proteins have been determined and compared with those of unmodified native protein as well as of the aggregates reassembled from the modified subunits. RESULTS: Acetylation of the surface-exposed tyrosine side chains has been found to destabilize the integrity of the native assembly with the formation of a somewhat smaller aggregate. This acetylated aggregate appears to adopt a molten globule-like conformation as evidenced from its almost unaltered secondary structure with some detectable alterations in its tertiary structure as well as from its enhanced chaper-one activity exhibited by the reduction assay compared to the native alpha-crystallin. Reassociation studies from either partially or completely acetylated subunits indicate that acetylation perturbs the information needed for native refolding of the subunits from their unfolded state as well as that needed for the normal mode of subunit reassembly. Acetylated subunits exhibit abnormal gel electrophoretic band pattern with distinctly retarded migration compared to the unmodified subunits. However, in spite of the partial/complete acetylation of the subunits or their reassociation from the denatured state, the tryptophan fluorescence emission maxima of the modified proteins and also that of the reassociated aggregates appear to remain unaffected. CONCLUSIONS: Results tend to indicate that the unperturbed hydrogen bonding capability of the relevant side chains in alpha-crystallin is needed for the integrity of the native alpha-crystallin assembly, for the normal refolding of its denatured subunits and also for the correct mode of subunit reassembly.

The role of the 90-kDa heat-shock protein and its associated cohorts in stabilizing the heme-regulated eIF-2alpha kinase in reticulocyte lysates during heat stress.

The heme-regulated eIF-2alpha kinase (HRI) is activated not only in heme-deficient rabbit reticulocyte lysates (RRL), but also in hemin-supplemented RRL treated with heat-shock, N-ethylmaleimide (MalNEt) or heavy metal ions. We have demonstrated previously that heat-shock proteins, Hsp90, Hsp70 and FKBP52, are associated with HRI in RRL; the association of HRI with Hsp90 and FKBP52, but not Hsp70, is enhanced by hemin. To study the role of Hsp90 and its associated cohorts in the regulation of HRI, we examined the interaction of these proteins with HRI in hemin-supplemented RRLs during heat or oxidative stress. The association of HRI with Hsp90, FKBP52 and p23 was maintained in heat-, MalNEt- or Hg2(+)-treated hemin-supplemented RRL. Glycerol gradient centrifugation and gel filtration on Sephacryl S-300 indicated that neither heat shock nor MalNEt-treatment affected the apparent molecular mass of HRI in hemin supplemented RRL. In addition, active HRI was coimmunoprecipitated with 8D3 anti-Hsp90 from both heme-deficient and MalNEt-treated hemin-supplemented RRL. These results demonstrate that activation of HRI in response to heat stress and oxidative stress does not require dissociation of Hsp90 from HRI. Furthermore, HRI activity was inhibited upon addition of hemin to Hsp90-depleted heme-deficient RRL, indicating that inhibition of HRI activity by hemin is not mediated by the reassociation of Hsp90 with HRI. We also examined the dynamics of the interaction of Hsp90 with HRI. Reconstitution of the interaction of Hsp90 with HRI was stimulated by elevated temperature and required both Mg2+ and ATP. Addition of purified Hsp90 to hemin-supplemented RRL which had been treated with MalNEt to inactivate its capacity to chaperone protein renaturation, protected HRI from irreversible denaturation and aggregation upon incubation at 41 degrees C. Our results suggest that Hsp90 interacts with HRI primarily in its capacity as a molecular chaperone, stabilizing HRI from denaturation under conditions of heat stress and oxidative stress.

Lipid peroxidation of erythrocytes during anemia of the hamsters infected with Leishmania donovani.

Visceral leishmaniasis has been found to be associated with severe anemia and premature lysis of erythrocytes. Peroxidative damage of red cells has been noted in several hemolytic anemias. Present study shows enhanced formation of methemoglobin in hamsters infected with Leishmania donovani. Increased formation of malonyldialdehyde and diene conjugate has been noted in the erythrocytes of the infected animals with the progress of anemia. Results showed decreased activities of protective enzymes like superoxide dismutase, catalase and glutathione reductase against peroxidative attack. An increase in the membrane cholesterol/phospholipid ratio and a decrease in membrane fluidity of erythrocytes were observed under the diseased condition. Densitometric scan after SDS-PAGE of red cell membrane of the infected animals revealed significant degradation of band 3 and band 4.1 proteins. The results suggest that alteration in the membrane may lead to reduced life span of the red cells in experimental visceral leishmaniasis.

Lens protein phylogeny: immunocrossreactivity of vertebrate lens antigens to anti-shark crystallin antibody.

Antisera prepared against total water-soluble lens proteins of the shark, Scoliodon sorrakowah were reacted with homologous antigen and analysed reaction products by immunoelectrophoresis (IE) and two dimensional crossed antigen-antibody electrophoresis (2D-CE). On IE, shark antigens formed 5 precipitin lines including 1 alpha, 3 beta and 1 gamma crystallins and on 2D-CE 3 alpha, 6 beta and 6 gamma peaks accounting for 8%, 27% and 65% antigen in the respective group were obtained from the total crystallins. Using anti-shark antisera, the immunocrossreactivity of lens proteins from 6 Chondropterygii, 23 teleosts and 16 higher vertebrates was examined by IE. It is found that beta crystallins are the most conserved and crossreact with all vertebrate classes, whereas gamma crystallin crossreactivity is specific to the class Pisces and alpha crystallins are least conserved and their crossreactivity is confined to subclass Chondropterygii. Based on IE patterns, a phylogenetic tree is constructed which demonstrates the intrafamily closeness except in case of adaptive radiation.

Differential synthesis and cytolocalization of prosomes in chick embryos during development.

Prosomes, also called "multicatalytic proteinase" or proteasomes, were purified from chick embryos of different developmental stages by a simple, single-step procedure. These were characterized by their characteristic protein patterns determined by SDS polyacrylamide gel electrophoresis (SDS PAGE) and immunoblotting with four monoclonal antibodies, namely, anti-p27, -p28, -p29 and -p31, prepared against duck prosomes. In vitro labeling of embryos with 35S-methionine followed by SDS PAGE and fluorography of the purified prosomes revealed that their polypeptides are differentially synthesized at various stages during development. Among 12 polypeptides (p21 to p56), p21 is synthesized at the beginning of gastrulation (stage 2) followed by the synthesis of p24 at stage 3. Nine other polypeptides (p25 to p35) are synthesized at the head-fold stage (stage 6), while the synthesis of polypeptide p56 is only detected at stage 10 (10-somite stage). Indirect immunofluorescence studies, with the 4 monoclonal antibodies, demonstrated 3 distinct, developmental stage-specific patterns of cytodistribution of these four prosome polypeptides in the embryos. During early embryogenesis, these are uniformly nuclear in location, while at later stages (stage 4 onwards) they are also present in the cytoplasm. Interestingly, one of the antigens (p 28), although found uniformly in all types of tissues in the embryos up to the gastrulation stage, is undetectable in the neural tissues and nonuniformly distributed in other tissues of stage-10 embryos. These data suggest that there are subcomponents of prosomes which are synthesized as well as distributed in an independent manner during development, possibly reflecting subcomponent-specific multiple functions of these particles.

Interactions of the heme-regulated eIF-2 alpha kinase with heat shock proteins in rabbit reticulocyte lysates.

Inhibition of protein synthesis in rabbit reticulocyte lysates occurs in response to a variety of conditions including heme deficiency, addition of oxidants, and heat stress. The inhibition of translation is due to the activation of a heme-regulated protein kinase (HRI) which specifically phosphorylates the alpha-subunit of the eukaryotic initiation factor eIF-2. In this report, immunoadsorption with monoclonal antibodies (mAbs) and Western blot analysis were used to investigate the interaction of HRI, the 90-kDa heat shock protein (hsp 90), hsp 70, and the EC1 antigen in rabbit reticulocyte lysates under protein synthesizing conditions. The data indicate that hsp 90, hsp 70, and the EC1 antigen interact with HRI in rabbit reticulocyte lysate. The EC1 antigen is a protein that has been demonstrated to be associated with several steroid hormone receptor-hsp 90 complexes and reacts with the KN 382/EC1 mAb (EC1). The association of HRI with hsp 90 and the EC1 antigen in the reticulocyte lysate was found to be dependent on the presence of hemin at a concentration of 5 microM or higher; little HRI was coadsorbed by the 8D3 anti-hsp 90 mAb or the EC1 mAb in the absence of hemin. Hsp 70 remains associated with HRI in the absence of hemin, suggesting that hsp 90 and 70 may bind to HRI at different sites. The immunological properties of the hsp 70 associated with HRI indicate that it may be the constitutively express heat shock cognate protein (hsc 73). The results suggest that the association of HRI with hsp 90 and the EC1 antigen may be in a dynamic equilibrium, in which complex formation is either facilitated or stabilized by the presence of hemin, and supports the notion that these proteins in conjunction with hsp 70 may play a role in regulating HRI activity or activation in situ.

Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2 alpha (eIF-2 alpha) kinase of rabbit reticulocytes: homology to yeast GCN2 protein kinase and human double-stranded-RNA-dependent eIF-2 alpha kinase.

We have cloned the cDNA of the heme-regulated eIF-2 alpha kinase (HRI) of rabbit reticulocytes. In vitro translation of mRNA transcribed from the HRI cDNA yields a 90-kDa polypeptide that exhibits eIF-2 alpha kinase activity and is recognized by a monoclonal antibody directed against authentic HRI. The open reading frame sequence of the HRI cDNA contains all 11 catalytic domains of protein kinases with consensus sequences of protein-serine/threonine kinases in conserved catalytic domains VI and VIII. The HRI cDNA also contains an insert of approximately 140 amino acids between catalytic domains V and VI. The HRI cDNA coding sequence has extensive homology to GCN2 protein kinase of Saccharomyces cerevisiae and to human double-stranded-RNA-dependent eIF-2 alpha kinase. This observation suggests that GCN2 protein kinase may be an eIF-2 alpha kinase in yeast. In addition, HRI has an unusually high degree of homology to three protein kinases (NimA, Wee1, and CDC2) that are involved in the regulation of the cell cycle.

Tissue distribution and immunoreactivity of heme-regulated eIF-2 alpha kinase determined by monoclonal antibodies.

A highly purified preparation of heme-regulated inhibitor (HRI), an eIF-2 alpha kinase, from rabbit reticulocyte lysates has been used for generating monoclonal antibodies (mAB). Two hybridoma clones secreting HRI-specific antibodies (mAB A and mAB F) were obtained. Both antibodies immunoprecipitated biosynthetically labeled as well as phosphorylated HRI in reticulocyte lysates and also recognized denatured HRI in a Western blot. In in vitro protein kinase assays, preincubation of HRI with the antibodies significantly diminished both autokinase and eIF-2 alpha kinase activities. HRI from reticulocyte lysates could be quantitatively removed by immunoprecipitation with mAB F, and such HRI-depleted lysates were able to maintain protein synthesis under conditions of heme deficiency. With these monoclonal antibodies, HRI was detected only in the reticulocytes and bone marrow of anemic rabbits, among several rabbit tissues tested. The antibodies did not detect cross-reacting HRI in rat or human reticulocytes or in mouse erythroleukemic cells or human K562 cells even after induction of differentiation, although eIF-2 alpha kinase activity was detected in them. Polyclonal anti-rabbit HRI antibody detected HRI in rat reticulocytes. However, no cross-reacting HRI was detected by polyclonal antibody in human reticulocytes or other cell types tested. These findings suggest that HRI is not ubiquitous, and may be erythroid-specific, and that it is antigenically different in different species.

Amino acid microsequencing of internal tryptic peptides of heme-regulated eukaryotic initiation factor 2 alpha subunit kinase: homology to protein kinases.

We have purified the heme-regulated eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) kinase (HRI) from rabbit reticulocytes for amino acid microsequencing. This kinase is a single 92-kDa polypeptide and migrates in perfect alignment with 32P-labeled HRI on SDS/PAGE. Its functions of binding ATP and of autophosphorylation and eIF-2 alpha phosphorylation are inhibited by hemin. The amino acid sequences of three tryptic peptides of HRI have been obtained. A search of the data base of the National Biomedical Research Foundation reveals that these amino acid sequences are unique and that two of these three sequences show homology to protein kinases. HRI peptide P-52 contains Asp-Phe-Gly, which is the most highly conserved short stretch of amino acids in catalytic domain VII of protein kinases. HRI peptide P-74 contains the conserved amino acid residues Asp-(Met)-Tyr-Ser-(Val)-Gly-Val found in catalytic domain IX of protein kinases [Hanks, S. K., Quinn, A. M. & Hunter, T. (1988) Science 241, 42-52]. These findings are consistent with the autokinase and eIF-2 alpha kinase activities of HRI. Synthetic HRI peptide P-74 is a very potent inhibitor of eIF-2 alpha phosphorylation by HRI. Since little is known about the function of conserved domain IX, P-74 peptide may be useful in elucidating the role of this domain of protein kinases.

Differential cytolocalization of prosomes in axolotl during oogenesis and meiotic maturation.

The prosomes, a novel type of small RNA-protein complex previously characterized in avian and mammalian cells, were isolated from axolotl (Ambystoma mexicanum) oocytes and identified by sedimentation analysis and protein composition. The prosomal nature of these particles was further ascertained by immunoblot analysis with anti-duck prosome monoclonal antibodies. By in vitro [35S]methionine labelling, de novo synthesis of prosomal proteins could be detected neither during oogenesis nor meiotic maturation. The results obtained by both indirect immunofluorescence and immunoblot analyses demonstrated a dramatic change in the localization of prosomal antigens during oocyte development. They were initially detected in the oocyte cytoplasm, during oocyte growth. At the end of vitellogenesis (stages V-VI), they entered the nucleus (germinal vesicle) and were accumulated there to the highest concentration. During oocyte maturation, after nuclear envelope breakdown, prosomal antigens were found to be localized again in the cytoplasm, until fertilization. No specific localization of prosomal antigens in mature oocytes, unfertilized and fertilized eggs was observed within the oocyte cytoplasm in relation to the cytoplasmic rearrangements leading to grey crescent formation.