Biomedical subjects

D J Wolgemuth

Publications and source records attributed to D J Wolgemuth.

At least 37 records · Page 2Linked to original sources

PubMed1999

The cellular distribution and kinase activity of the Cdk family member Pctaire1 in the adult mouse brain and testis suggest functions in differentiation.

Pctaire1, a member of the family of cyclin-dependent kinases, has been shown to be particularly abundantly expressed in differentiated tissues such as testis and brain. However, very little is known about the cellular and subcellular distribution and function of Pctaire1 protein(s), which is the focus of this study. We show that Pctaire1 encoded two major proteins of M(r) approximately 62,000 and approximately 68,000, found predominantly in testis and brain. Within these two tissues, Pctaire1 was most abundant in the cytoplasm of terminally differentiated cells, notably, the pyramidal neurons in brain and elongated spermatids in testis. Immunoprecipitation experiments further showed that a kinase activity toward myelin basic protein was associated with Pctaire1 in the adult testis and brain and that its activity was potentially regulated through association with regulatory partner(s). These results suggest that Pctaire1 kinase might have an important role in differentiated cells such as postmitotic neurons and spermatogenic cells.

Animals↗

PubMed1999

Biochemical properties, tissue expression, and gene structure of a short chain dehydrogenase/ reductase able to catalyze cis-retinol oxidation.

We have identified a retinol dehydrogenase (cRDH) that catalyzes the oxidation of 9-cis- but not all-trans-retinol and proposed that this enzyme plays an important role in synthesis of the transcriptionally active retinoid, 9-cis-retinoic acid. There is little information regarding either the biochemical properties of cRDH or how its 9-cis-retinol substrate is formed. We now report studies of the properties and expression of human and mouse cRDH and of the characteristics and location of the murine cRDH gene. Additionally, we report mouse hepatic 9-cis-retinol concentrations and demonstrate that 9-cis-retinol is formed in a time- and protein-dependent manner upon incubation of all-trans -retinol with cell homogenate. Human and mouse cRDH display similar substrate specificities for cis-isomers of retinol and retinaldehyde. Moreover, human and mouse cRDH show marked sensitivity to inhibition by 13-cis-retinoic acid, with both being inhibited by approximately 50% by 0.15 microm 13-cis-retinoic acid (for substrate concentrations of 10 microm). Lesser inhibition is seen for 9-cis- or all-trans-retinoic acids. Immunoblot analysis using antiserum directed against human cRDH demonstrates cRDH expression in several tissues from first trimester human fetuses, indicating that cRDH is expressed early in embryogenesis. Adult mouse brain, liver, kidney, and to a lesser extent small intestine and placenta express cRDH. The murine cRDH gene consists of at least 5 exons and spans approximately 6 kb of genomic DNA. Backcross analysis mapped the mouse cRDH gene to the most distal region of chromosome 10. Taken together, these data extend our understanding of the properties of cRDH and provide additional support for our hypothesis that cRDH may play an important role in 9-cis-retinoic acid formation.

Alcohol Oxidoreductases↗

PubMed1998

Role of heat shock protein Hsp25 in the response of the orofacial nuclei motor system to physiological stress.

Although expression of the small heat shock protein family member Hsp25 has been previously observed in the central nervous system (CNS), both constitutively and upon induction, its function in the CNS remains far from clear. In the present study we have characterized the spatial pattern of expression of Hsp25 in the normal adult mouse brain as well as the changes in expression patterns induced by subjecting mice to experimental hyperthermia or hypoxia. Immunohistochemical analysis revealed a surprisingly restricted pattern of constitutive expression of Hsp25 in the brain, limited to the facial, trigeminal, ambiguus, hypoglossal and vagal motor nuclei of the brainstem. After hyperthermia or hypoxia treatment, significant increases in the levels of Hsp25 were observed in these same areas and also in fibers of the facial and trigeminal nerve tracts. Immunoblot analysis of protein lysates from brainstem also showed the same pattern of induction of Hsp25. Surprisingly, no other area in the brain showed expression of Hsp25, in either control or stressed animals. The highly restricted expression of Hsp25 implies that this protein may have a specific physiological role in the orofacial motor nuclei, which govern precise coordination between muscles of mastication and the pharynx, larynx, and face. Its rapid induction after stress further suggests that Hsp25 may serve as a specific molecular chaperone in the lower cholinergic motor neurons and along their fibers under conditions of stress or injury.

Animals↗

PubMed1998

Fetal development of the enteric nervous system of transgenic mice that overexpress the Hoxa-4 gene.

Megacolon occurs in neonatal and adult transgenic mice that overexpress the Hoxa-4 gene. Abnormalities, which are restricted to the terminal colon of these mice, include a hypoganglionosis, abnormal enteric ganglia with a structure appropriate for extra-enteric peripheral nerve and not the enteric nervous system (ENS), and gaps in the longitudinal muscle occupied by ganglia. To investigate the developmental origin of these abnormalities, we analyzed the development of the pelvis and terminal colon in Hoxa-4 transgenic mice. Morphological abnormalities were detected as early as E13. These included an enlargement of the mucosa and the bowel wall, a thickening of the enteric mesenchyme, and the ectopic location of pelvic ganglion cells, which initially clustered on the dorsolateral wall of the hindgut. As the bowel enlarged, these ectopic cells become ventrolateral and, between days E17 and E18.5, appeared to become incorporated into the gut, leaving neuron-filled gaps in the longitudinal muscle layer. The ectopic ganglia retained extra-enteric characteristics, including the presence of capillaries, basal laminae, collagen fibers, and catecholaminergic neurons, even after their incorporation into the bowel. It is proposed that the abnormal and ectopic expression of the Hoxa-4 transgene in the colon causes signalling molecule(s) of the enteric mesenchyme to be overproduced and that the overabundance of these signals leads to mucosal enlargement and misdirection of migrating pelvic neuronal progenitors.

Animals↗

PubMed1998

The identification and characterization of expression of Pftaire-1, a novel Cdk family member, suggest its function in the mouse testis and nervous system.

We have isolated a murine cDNA encoding for a novel putative Cdk-related protein kinase, which has been named Pftaire-1, by screening a testis cDNA library for new serine/threonine kinases. Pftaire-1 showed 50% and 49% amino acid identity with Cdk5 and Pctaire-3, respectively, and contains the eleven subdomains characteristic of the protein kinases. By northern blot analysis we detected two transcripts of approximately 5.5 and 4.9 kb in size. These transcripts were expressed at low level in all murine tissues tested, except in the brain, testis and embryo, where high expression was detected. Cellular localization of the mRNAs by in situ hybridization analysis shows that Pftaire-1 is expressed in late pachytene spermatocytes in the testis and in post mitotic neuronal cells both in the brain and the embryo, suggesting a role of Pftaire-1 both in the process of meiosis as well as neuron differentiation and/or function.

Amino Acid Sequence↗

PubMed1998

Mouse Odf2 cDNAs consist of evolutionary conserved as well as highly variable sequences and encode outer dense fiber proteins of the sperm tail.

The outer dense fibers (ODF) of the mammalian sperm tail comprise a unique, specialized, and very prominent structure, consisting of nine fibers surrounding the axoneme. The ODF may play an important but as yet undefined role in sperm morphology, integrity and function. Study of the ODF is hampered by insufficient knowledge of their protein composition and the genetic regulation of their synthesis. We report here on the characterization of cDNAs encoding the Odf2 proteins of outer dense fibers of mouse sperm. We isolated two cDNA clones with variable 5' regions. Variability in sequence is restricted to specific regions in the N-terminal part of the encoded proteins, whereas the C-terminal part is highly conserved in Odf2 proteins both between species and within a species. This variability is confirmed at the protein level. The outer dense fibers could be detected immunologically in total sperm tails allowing a direct comparison of their length in relation to the length of the sperm tail. Odf2 transcripts could be demonstrated in testicular RNA and are restricted to germ cells. The start of transcription is in step 5 spermatids of tubular stage V and the RNA could be detected in the cytoplasm of differentiating spermatids in all subsequent tubular stages.

Amino Acid Sequence↗

PubMed1998

Differential expression of the Oct-4 transcription factor during mouse germ cell differentiation.

The POU transcription factor Oct-4 is expressed in early mouse embryogenesis and in pluripotent ES and EC stem cell lines. After gastrulation in the embryo, Oct-4 expression is confined to the germline. The present study provides evidence that Oct-4 undergoes downregulation during oogenesis and spermatogenesis, coincident with entry into meiosis. Furthermore, analysis of maturation stages of oocytes showed that Oct-4 is upregulated de novo in the final stages of meiotic prophase I in female germ cells. These data suggest that Oct-4 downregulation in germ cells in both sexes might represent one of the molecular triggers involved in the commitment to meiosis. The upregulation of Oct-4 in oocytes at the completion of the prophase I of meiotic division further suggests a specific involvement of this transcription factor in oocyte growth or the acquisition of meiotic competence.

Animals↗

PubMed1998

Cyclin A1 is required for meiosis in the male mouse.

The mammalian A-type cyclin family consists of two members, cyclin A1 (encoded by Ccna1) and cyclin A2 (encoded by Ccna2). Cyclin A2 promotes both G1/S and G2/M transitions, and targeted deletion of Ccna2 in mouse is embryonic lethal3. Cyclin A1 is expressed in mice exclusively in the germ cell lineage and is expressed in humans at highest levels in the testis and certain myeloid leukaemia cells. To investigate the role of cyclin A1 and possible redundancy among the cyclins in vivo, we generated mice bearing a null mutation of Ccna1. Ccna1-/- males were sterile due to a block of spermatogenesis before the first meiotic division, whereas females were normal. Meiosis arrest in Ccna1-/- males was associated with increased germ cell apoptosis, desynapsis abnormalities and reduction of Cdc2 kinase activation at the end of meiotic prophase. Cyclin A1 is therefore essential for spermatocyte passage into the first meiotic division in male mice, a function that cannot be complemented by the concurrently expressed B-type cyclins.

Animals↗

PubMed1998

Expression of the murine Hoxa4 gene requires both autoregulation and a conserved retinoic acid response element.

Analysis of the regulatory regions of the Hox genes has revealed a complex array of positive and negative cis-acting elements that control the spatial and temporal pattern of expression of these genes during embryogenesis. In this study we show that normal expression of the murine Hoxa4 gene during development requires both autoregulatory and retinoic acid-dependent modes of regulation. When introduced into a Hoxa4 null background, expression of a lacZ reporter gene driven by the Hoxa4 regulatory region (Hoxa4/lacZ) is either abolished or significantly reduced in all tissues at E10. 5-E12.5. Thus, the observed autoregulation of the Drosophila Deformed gene is conserved in a mouse homolog in vivo, and is reflected in a widespread requirement for positive feedback to maintain Hoxa4 expression. We also identify three potential retinoic acid response elements in the Hoxa4 5' flanking region, one of which is identical to a well-characterized element flanking the Hoxd4 gene. Administration of retinoic acid to Hoxa4/lacZ transgenic embryos resulted in stage-dependent ectopic expression of the reporter gene in the neural tube and hindbrain. When administered to Hoxa4 null embryos, however, persistent ectopic expression was not observed, suggesting that autoregulation is required for maintenance of the retinoic acid-induced expression. Finally, mutation of the consensus retinoic acid response element eliminated the response of the reporter gene to exogenous retinoic acid, and abolished all embryonic expression in untreated embryos, with the exception of the neural tube and prevertebrae. These data add to the evidence that Hox gene expression is regulated, in part, by endogenous retinoids and autoregulatory loops.

Animals↗

PubMed1998

Expression and potential role of Fsrg1, a murine bromodomain-containing homologue of the Drosophila gene female sterile homeotic.

We have isolated a cDNA which is a murine homologue of the Drosophila gene female sterile homeotic (fsh). This homologue, which we have designated Fsrg1*, contains two bromodomains and an ET motif characteristic of the Fsh sub-class of bromodomain-containing proteins. Northern blot hybridization analysis of adult tissues revealed that Fsrg1 was expressed at low levels rather ubiquitously, but most abundantly in the testis and ovary. Polyclonal antibodies raised against an Fsrg1 fusion protein were used to characterize the Fsrg1 gene product in tissues. Constructs were also generated in which the Fsrg1 cDNA was tagged with epitopes for hemaglutinin and used in transfection experiments. Immunoblot analysis revealed that the Fsrg1 protein migrates with a relative molecular mass of approximately 110 kDa, although the cDNA sequence would predict a protein of approximately 88 kDa. The migration at approximately 110 kDa was observed for both in vivo protein and protein produced in cultured cells. The Fsrg1 protein was localized to the nucleus when expressed in cultured cells, consistent with the presence of a nuclear localization signal motif in the Fsrg1 sequence. No kinase activity was detected for this nuclear protein as assessed in either autokinase or specific substrate assays. In situ hybridization analysis revealed strikingly high expression of Fsrg1 in granulosa cells of growing follicles in the adult ovary and suggested its possible involvement in folliculogenesis. Additional clues to its potential function were provided by the demonstration of its high level of expression in epithelia of tissues which undergo hormonally-modulated remodeling.

3T3 Cells↗

PubMed1997

Identification and characterization of a stereospecific human enzyme that catalyzes 9-cis-retinol oxidation. A possible role in 9-cis-retinoic acid formation.

All-trans- and 9-cis-retinoic acid are active retinoids for regulating expression of retinoid responsive genes, serving as ligands for two classes of ligand-dependent transcription factors, the retinoic acid receptors and retinoid X receptors. Little is known, however, regarding 9-cis-retinoic acid formation. We have obtained a 1.4-kilobase cDNA clone from a normalized human breast tissue library, which when expressed in CHO cells encodes a protein that avidly catalyzes oxidation of 9-cis-retinol to 9-cis-retinaldehyde. This protein also catalyzes oxidation of 13-cis-retinol at a rate approximately 10% of that of the 9-cis isomer but does not catalyze all-trans-retinol oxidation. NAD+ was the preferred electron acceptor for oxidation of 9-cis-retinol, although NADP+ supported low rates of 9-cis-retinol oxidation. The rate of 9-cis-retinol oxidation was optimal at pHs between 7.5 and 8. Sequence analysis indicates that the cDNA encodes a protein of 319 amino acids that resembles members of the short chain alcohol dehydrogenase protein family. mRNA for the protein is most abundant in human mammary tissue followed by kidney and testis, with lower levels of expression in liver, adrenals, lung, pancreas, and skeletal muscle. We propose that this cDNA encodes a previously unknown stereospecific enzyme, 9-cis-retinol dehydrogenase, which probably plays a role in 9-cis-retinoic acid formation.

Alcohol Oxidoreductases↗

PubMed1997

Cyclin-dependent kinase 5 is associated with apoptotic cell death during development and tissue remodeling.

In a series of studies to more precisely localize the cellular sites of expression of the cyclin-dependent kinase (Cdk) family members in reproductive organs, we observed a striking expression of Cdk5 in atretic follicles in the ovary, particularly in granulosa cells that appeared to be dying. We determined that these granulosa cells were undergoing apoptotic cell death using the in situ DNA fragmentation assay. To extend the generality of the association of Cdk5 with apoptotic cells, we examined its expression as it correlated with the detection of apoptosis in a number of developmental paradigms, including regions of the embryonic nervous system, the developing eye, and the developing limb. Finally, the association of apoptosis and Cdk5 expression and associated kinase activity was examined in the limb and in an induced cell death system, that of androgen withdrawal-induced regression of the prostate gland in male mice. These observations provide new insight into the possible function of this novel Cdk during both differentiation and apoptotic cell death.

Animals↗

PubMed1997

A sequence conserved in vertebrate Hox gene introns functions as an enhancer regulated by posterior homeotic genes in Drosophila imaginal discs.

The intron of the mouse Hoxa-4 gene acts as a strong homeotic response element in Drosophila melanogaster leg imaginal discs. This activity depends on homeodomain binding sites present within a 30 bp conserved element, HB1, in the intron. A similar arrangement of homeodomain binding sites is found in many other potential homeotic target genes. HB1 activity in Drosophila imaginal discs is activated by Antennapedia and more posterior homeotic genes, but is not activated by more anterior genes. Testing a reporter gene construct with mutated binding sites in mouse embryos shows that HB1 is also active in the expression domains of posterior Hox genes in the mouse neural tube.

Animals↗

PubMed1997

The NIMA-related kinase 2, Nek2, is expressed in specific stages of the meiotic cell cycle and associates with meiotic chromosomes.

The Aspergillus nimA gene encodes a Ser/Thr protein kinase which is required for mitosis, in addition to Cdc2, and which has been suggested to have a role in chromosomal condensation. In this study, we isolated a potential murine homologue of nimA, Nek2, which was shown to be expressed most abundantly in the testis of the adult tissues examined. Its expression in the testis was restricted to the germ cells, with highest levels detected in spermatocytes at pachytene and diplotene stages. Immunohistochemical analysis revealed that Nek2 localized to nuclei, exhibiting a non-uniform distribution within the nucleus. Nek2 appeared to be associated with meiotic chromosomes, an association that was better defined by immunolocalization to hypotonically dispersed meiotic chromosomes. This localization was more apparent in regions of dense chromatin, including the sex vesicle, and was also obvious at some of the chromosome ends. The presence of Nek2 protein was not unique to male germ cells, as it was found in meiotic pachytene stage oocytes as well. Furthermore, in an in vitro experimental setting in which meiotic chromosome condensation was induced with okadaic acid, a concomitant induction of Nek2 kinase activity was observed. The expression of Nek2 in meiotic prophase is consistent with the hypothesis that in vivo, Nek2 is involved in the G2/M phase transition of the cell cycle. Our results further provide evidence that in vivo, mouse Nek2 is involved in events of meiosis, including but not limited to chromosomal condensation.

Amino Acid Sequence↗

PubMed1997

The mouse transcription factor Stat4 is expressed in haploid male germ cells and is present in the perinuclear theca of spermatozoa.

STAT (signal transducer and activator of transcription) proteins have been shown to be essential transcription factors which mediate biological effects of cytokines. Although most of the STATs have been shown to be widely expressed, Stat4 mRNA has been detected in only a few tissues, including the testis. In the present study, immunoblot analysis confirmed that the presence of Stat4 protein was similarly restricted, with the highest level observed in testis. In situ hybridization, immunoblot, and immunohistochemistry analyses revealed that in the testis, Stat4 was abundantly and exclusively expressed in male germ cells which have completed meiosis, at the round and elongating spermatid stages. Cytolocalization at various times of spermatid differentiation showed that the level of Stat4 protein increased in parallel in both cytoplasm and nuclei. No specific nuclear translocation that would have been an indicator of Stat4 activation was observed at any stage of spermatogenic differentiation. Interestingly, the Stat4 transcription factor was localized to the condensing perinuclear theca of spermatids, a localization that was confirmed by selective biochemical extraction of thecal proteins. Since the theca is known to depolymerize in the cytoplasm of the oocyte during the hours following fertilization, we hypothesized that sperm Stat4 would represent an original paternal contribution to the fertilized egg which may be involved in the onset of zygotic transcription.

Age Factors↗

PubMed1997

Vertebrate development in the environment of space: models, mechanisms, and use of the medaka.

With the advent of space travel, it is of immediate interest and importance to study the effects of exposure to various aspects of the altered environment of space, including microgravity, on Earth-based life forms. Initial studies of space travel have focused primarily on the short-term effects of radiation and microgravity on adult organisms. However, with the potential for increased lengths of time in space, it is critical to now address the effects of space on all phases of an organism's life cycle, from embryogenesis to post-natal development to reproduction. It is already possible for certain species to undergo multiple generations within the confines of the Mir Space Station. The possibility now exists for scientists to consider the consequences of even potentially subtle defects in development through multiple phases of an organism's life cycle, or even through multiple generations. In this discussion, we highlight a few of the salient observations on the effects of the space environment on vertebrate development and reproductive function. We discuss some of the many unanswered questions, in particular, in the context of the choice of appropriate models in which to address these questions, as well as an assessment of the availability of hardware already existing or under development which would be useful in addressing these questions.

Animals↗

PubMed1997

N-cadherin protein distribution in normal embryos and in embryos carrying mutations in the homeobox gene Hoxa-4.

N-cadherin is a calcium-dependent adhesion molecule with a potential role in a variety of morphogenetic events. Although a dynamic pattern of expression in the mouse embryo has been suggested by in situ hybridization analysis, to date there has been no report of N-cadherin protein expression. In this immunohistochemical study we surveyed N-cadherin protein expression in the mid-late gestation mouse embryo utilizing a recently characterized monoclonal antibody. We found N-cadherin expression in a wide array of tissues, including the brain, the eye, various cranial ganglia, the spinal cord, spinal ganglia, somites, vertebral and limb cartilage and perichondria, the developing lung and kidney, the enteric nervous system, and germ cells. These results suggest that N-cadherin protein expression, as in the chick embryo, correlates with the segregation of cells and with organogenesis. As cadherins have been proposed as targets of vertebrate Hox genes, we also examined N-cadherin expression in two lines of Hoxa-4 mutant mice. We did not observe any alterations in N-cadherin expression in either Hoxa-4 null embryos or in transgenic embryos that overexpress Hoxa-4 in the mesenchyme of the gut. However, the partial overlap in expression between Hox genes and N-cadherin, and the likelihood of redundancy in the regulation of target genes, leaves open the possibility that cadherins are direct or indirect targets of Hox genes during mouse embryogenesis.

Animals↗

PubMed1996

The developmentally restricted pattern of expression in the male germ line of a murine cyclin A, cyclin A2, suggests roles in both mitotic and meiotic cell cycles.

We have isolated cDNAs encoding a murine cyclin A, designated cyclinA2, and have examined its in vivo expression at the level of both mRNA and protein, with particular focus on the male germ line. Cyclin A2 is expressed in embryos and in a variety of adult tissues, including the testis. In the testis, however, a striking cellular specificity of expression was observed. At both the DNA and protein levels, the predominant sites of cyclin A2 expression were in the germ line stem cells, the spermatogonia, and in highest levels in preleptotene spermatocytes, cells in which premeiotic DNA synthesis occurs. The concurrent localization of cyclin A2 mRNA and protein further suggested that cyclin A2 is regulated at the level of transcription in these cells. The observed cellular specificity of cyclin A2 expression is consistent with its function during mitosis in the stem cell stage of this lineage, while the restricted meiotic stage localization suggests function in G1/S or S but not in the meiotic divisions per se.

Amino Acid Sequence↗