Assignment of synaptonemal complex protein 1 (SCP1) to human chromosome 1p13 by fluorescence in situ hybridization and its expression in the testis.
Explore the source record for details and available documents.
Chromosome Mapping↗
Biomedical subjects
Y Nishina
Publications and source records attributed to Y Nishina.
A receptor tyrosine kinase, Sky, and its ligand Gas 6 are expressed in gonads and support primordial germ cell growth or survival in culture.
Although a number of growth factors and their receptors are involved in the proliferation and differentiation of primordial germ cells (PGCs), the only factor that has been shown to be active in vivo is Steel factor, a ligand for c-Kit. To identify new growth factor receptors that may be required for PGCs function in vivo, we used an reverse transcription-polymerase chain reaction-based strategy to screen for protein kinase genes expressed in PGC-derived embryonic germ cells. We report here that one such gene encoding the receptor tyrosine kinase, Sky, is expressed in both PGCs and their supporting cells in male genital ridges after 11.5 dpc. Interestingly, Sky expression was not detected in female genital ridges, although transcripts were detected in supporting cells in the developing ovary at later stages. Gas 6, a ligand for Sky, was also expressed in interstitial cells which surround Sky positive cells in genital ridges, and, in addition, it supported PGC growth or survival in culture. After birth, Sky expression in testis was restricted to Sertoli cells, and Gas 6 was detected around peritubular cells and Leydig cells. These results suggest that Gas 6-Sky signaling plays a role in PGC growth, sexual differentiation, and Sertoli cell functions in vivo. Sky expression in Sertoli cells diminished by 3 weeks of age, when haploid germ cells first appear. On the other hand, the expression in Sertoli cells was markedly upregulated in the testis of germ cell-deficient W/Wv and jsd/jsd mice. The results suggest that signals from differentiated germ cells suppress Sky gene expression in Sertoli cells. High-resolution chromosomal mapping of Sky is also reported.
Animals↗
Isochromat photon map induced by scanning tunneling microscopy from gold particles.
Explore the source record for details and available documents.
Journal Article↗
Differentiation-dependent enhanced expression of protein phosphatase 2Cbeta in germ cells of mouse seminiferous tubules.
The presence of five distinct isoforms of protein phosphatase 2Cbeta (PP2Cbeta-1 approximately -5) is known. In this study, we demonstrate that the mRNA levels of PP2Cbeta-3, -4 and -5 and PP2Cbeta protein level increased during the course of the first wave of spermatogenesis in neonatal mouse testis. Northern blot and in situ hybridization analyses revealed that PP2Cbeta-3, -4 and -5 were expressed predominantly in pachytene spermatocytes and in more highly differentiated germ cells. The substrate specificity of PP2Cbeta-4 determined with artificial substrates differed from those of PP2Cbeta-3 and -5, suggesting that the difference in the structure of PP2Cbeta-3, -4 and -5 reflect their unique physiological functions in testicular germ cells.
Animals↗
C-NMR study on the interaction of medium-chain acyl-CoA dehydrogenase with acetoacetyl-CoA.
The change-transfer interaction in the complex of pig kidney medium-chain acyl-CoA dehydrogenase (MCAD) with acetoacetyl-CoA was investigated by 13C-NMR spectroscopy and molecular orbital treatment. The acyl carbons of acetoacetyl-CoA were separately 13C-labeled and 13C-NMR spectra of the complexes of MCAD with the 13C-labeled acetoacetyl-CoA were measured. Each 13C-carbon atom was observed as a distinct peak and easily distinguished from the protein background. The chemical shift values for free acetoacetyl-CoA were 198.5, 59.9, 208.8, and 32.8 ppm for C(1), C(2), C(3), and C(4), respectively, which shifted to 181.3, 103.4, 192.3, and 29.9 ppm, respectively, when acetoacetyl-CoA was complexed with MCAD. While C(4) underwent a small upfield shift, the other carbons complexed with MCAD. While C(4) underwent a small upfield shift, the other carbons experienced significant shifts; both the C(1) and C(3) carbonyl carbons shifted upfield by about 17 ppm, and the C(2) carbon was observed as a very broad peak at a position shifted downfield by more than 40 ppm. These results were compared with 13C-NMR spectra of the keto-, enol-, and enolate forms of ethyl acetoacetate labeled with 13C at the acyl carbons, and interpreted with reference to the charge-transfer model based on the optimum overlap between the lowest unoccupied molecular orbital (LUMO) of flavin and the highest occupied molecular orbital (HOMO) of the enolate state of the acetoacetyl moiety of acetoacetyl-CoA. The C(2) carbon of acetoacetyl-CoA takes on the sp2 configuration in the bound form, indicating that one of the protons at C(2) of acetoacetyl-CoA is abstracted when bound to MCAD. C(1) = O is substantially polarized in the bound form of acetoacetyl-CoA, implying the presence of a machinery that polarizes this carbonyl group at the binding site, which thereby lowers the pKa value of the alpha-proton at C(2). This machinery is of fundamental importance in the initial step of MCAD catalysis.
Acetoacetates↗
Crystallization of expressed porcine kidney D-amino acid oxidase and preliminary X-ray crystallographic characterization.
The cDNA for porcine kidney D-amino acid oxidase (DAO) was cloned by means of the reverse transcription-polymerase chain reaction system from porcine kidney RNA and over-expressed in Escherichia coli which had been transformed with a vector containing the DAO cDNA. The expressed DAO was purified to homogeneity by a three-step procedure, i.e., heat-treatment, DEAE Sepharose column chromatography, and hydroxyapatite column chromatography. The purified DAO preparation, rDAO (recombinant DAO), showed an identical UV-visible absorption spectrum and catalytic activity with those of the wild-type enzyme purified from porcine kidney. Crystallization of rDAO was performed by the hanging-drop method and crystals of suitable quality for X-ray crystallography were obtained. The crystals so obtained diffracted to 2.5 A with a conventional X-ray source, and to 2.0 A with synchrotron radiation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 110.3, b = 92.9, c = 71.6 A. A Vm value of 2.35 A3/Da indicates that there are two subunits related by a twofold non-crystallographic axis in the asymmetric unit. Two heavy atom derivatives have been identified.
Animals↗
Three-dimensional structure of porcine kidney D-amino acid oxidase at 3.0 A resolution.
The X-ray crystallographic structure of porcine kidney D-amino acid oxidase, which had been expressed in Escherichia coli transformed with a vector containing DAO cDNA, was determined by the isomorphous replacement method for the complex form with benzoate. The known amino acid sequence, FAD and benzoate were fitted to an electron density map of 3.0 A resolution with an R-factor of 21.0%. The overall dimeric structure exhibits an elongated ellipsoidal framework. The prosthetic group, FAD, was found to be in an extended conformation, the isoalloxazine ring being buried in the protein core. The ADP moiety of FAD was located in the typical beta alpha beta dinucleotide binding motif, with the alpha-helix dipole stabilizing the pyrophosphate negative charge. The substrate analog, benzoate, is located on the re-face of the isoalloxazine ring, while the si-face is blocked by hydrophobic residues. The carboxylate group of benzoate is ion-paired with the Arg283 side chain and is within interacting distance with the hydroxy moiety of Tyr228. The phenol ring of Tyr224 is located just above the benzene ring of benzoate, implying the importance of this residue for catalysis. There is no positive charge or alpha-helix dipole near N(1) of flavin. Hydrogen bonds were observed at C(2) = O, N(3)-H, C(4) = O, and N(5) of the flavin ring.
Animals↗
In vitro refolding and unfolding of subunits of electron-transferring flavoprotein: characterization of the folding intermediates and the effects of FAD and AMP on the folding reaction.
Electron-transferring flavoprotein (ETF) from pig kidney is composed of two subunits (alpha and beta, molecular weights of 33,000 and 29,000) and two small molecules, FAD and AMP. In this study, in vitro refolding and unfolding of the subunits of ETF were carried out with urea as the denaturing reagent. The refolding reaction of alpha and beta was revealed to proceed kinetically in two steps: D in equilibrium with I-->N, where D,I, and N denote the denatured, intermediate, and native forms, respectively. The features of the I forms of alpha and beta, described below, are consistent with the concept of the so-called "molten globule state," which is frequently observed in protein refolding. (i) The conversion between D and I was very rapid. (ii) The I form showed as much secondary structure as the N form as judged from the far-UV circular dichroism. (iii) The solvent accessibility of the I form, estimated by the analysis of equilibrium unfolding experiments, was intermediate between those of the D and N forms. (iv) The standard free energy of the I form is almost the same as that of the D form. The refolding reaction progressed more slowly and the environment of the tryptophan chromophore was changed more drastically in beta refolding that in alpha refolding. We previously reported that the reconstitution of holoETF from denatured subunits is speeded up by increasing the AMP concentration. In this study, the effects of AMP, FAD, and the other subunit on the single subunit folding were examined, but no effect was detected. This result suggests that AMP plays a role in a later process, namely, assembly of the four components (refolded alpha and beta, FAD, and AMP).
Adenosine Monophosphate↗
Apoptosis in adult mouse testis induced by experimental cryptorchidism.
Induction of cryptorchidism in the mouse causes infertility due to disruption of spermatogenesis including reduction of germ cells; however, the cellular mechanism responsible for the degenerative changes in cryptorchid testis is still unclear. In surgically induced bilateral cryptorchidism of 3-month-old C57BL/Tw mice, cellular changes in the cryptorchid testis were studied 1, 2, 3, 7, 14 and 21 days after the operation by electron microscopy, DNA fragmentation, in situ 3'-end labeling, serum and testicular testosterone measurements and gene expression. Although the testis showed DNA fragmentation even in intact mice, the cryptorchidism increased the degree of the fragmentation at 1 postcryptorchidism (p.c.) day. Apoptosis was encountered mainly in spermatids and spermatocytes. The number of apoptotic cells in the cryptorchid testis showed a 7-fold increase at 1 p.c day as compared to the intact testis, then it gradually decreased. Serum testosterone levels showed a significant decrease at 2 p.c. days and remained low thereafter. Expression of transforming growth factor-beta 2 (TGF-beta 2), TGF-beta 3, tumor necrosis factor-alpha receptor and Fas mRNAs increased in the cryptorchid testis within 24 h after the operation. In lpr(cg) and lpr mice lacking functional Fas, gld mice lacking functional Fas ligand and lpr(cg)-gld mice lacking both functional Fas and Fas ligand, the experimental cryptorchidism also induced apoptosis in germ cells at 1 p.c. day. The present results indicate that cryptorchidism induces apoptotic dell death in germ cells, and that testosterone reduction and the Fas system may not be significantly involved in the apoptosis of male germ cells.
Animals↗
Functional expression of two forms of rat acyl-CoA oxidase and their substrate specificities.
Using the reverse transcription of RNA followed by the polymerase chain reaction, we cloned the cDNAs for the rat acyl-CoA oxidases I and II, which are produced by alternative splicing from a single gene, and developed a system for their expression in Escherichia coli. The homogeneous preparations of these enzymes, without proteolytic procession, showed oxidase activity with acyl-CoAs having various acyl-chain lengths. The two types of the enzyme exhibited different substrate specificities with respect to the acyl-chain length, acyl-CoA oxidase I showing the optimum activity at shorter chain-length relative to acyl-CoA oxidase II.
Acyl-CoA Oxidase↗
Isolation of a novel cDNA that encodes a protein localized to the pre-acrosome region of spermatids.
We have identified a novel cDNA clone, named AZ1, obtained from a cDNA library of mRNA prepared from C3H10T1/2 cells that had been transiently exposed to 5-azacytidine, a potent demethylating reagent. The amount of transcript increased with 5-azacytidine treatment of C3H10T1/2 cells and the transcript was highly expressed in mouse testis. As the mutant mouse jsd/jsd, which has a defect in germ cell maturation, barely expressed the transcript, the message was expected to be expressed specifically in spermatocytes. The mRNA was detected at significant levels in the testes from mice aged 16 days after birth, suggesting that its expression started at the pachytene spermatocyte stage. The elucidated nucleotide sequence contained a 2841-nucleotide open reading frame, and the expected amino acid sequence had a molecular mass of 107,254 Da. Specific antibodies raised against the fusion protein including glutathione S-transferase revealed an approximately 130-kDa band of a translation product in testis and in cultured cells transfected with AZ1 cDNA in the expression vector on Western-blot analysis. The protein was localized to the pre-acrosome region of round and elongated spermatids. However, it was not detected at a more advanced stage of spermatids, i.e. just before their release from Sertoli cells. This protein may play an important role in spermatogenesis.
Acrosome↗
Characterization of the testis-specific gene 'calmegin' promoter sequence and its activity defined by transgenic mouse experiments.
We have cloned the genomic DNA of calmegin [(1992) J. Biol. Chem. 269, 7744-7749] and analyzed its promoter region. It contained GC-rich sequences and potential binding sites for AP 2 and Sp 1, but lacked the TATA sequence. The 330 bp 5' flanking sequence of calmegin genomic DNA fused with the CAT gene was used for the study of promoter activity in transgenic mice. The CAT gene activity was detected exclusively in testes, indicating that the 330 bp calmegin 5' sequence was sufficient for the testis-specific expression. The existence of testicular nuclear factors specifically bound to the putative promoter sequence was also demonstrated.
Animals↗
Electronic states and stability of selenium clusters.
Explore the source record for details and available documents.
Journal Article↗
Induction of teratocarcinoma F9 cell differentiation with cis-diammine dichloroplatinum(II) (CDDP).
cis-Diammine dichloroplatinum(II) (CDDP) is the salt of a platinum compound which has been noted to have a wide spectrum of activity against malignant disorders. We have studied the effects of CDDP on embryonal carcinoma F9 cell differentiation. In the presence of this agent in vitro, the cells showed rapid morphological changes, a marked increase in the mRNA expression of various differentiation markers accompanied by a loss of tumorigenicity. These results indicate that the differentiation of F9 cells is induced with CDDP.
Animals↗
Effects of protein phosphatase inhibition by okadaic acid on the differentiation of F9 embryonal carcinoma cells.
Okadaic acid, a protein phosphatase inhibitor, is a strong tumor promoter which apparently activates protein phosphorylation. To examine the role of protein phosphatases in stem cell growth and differentiation, embryonal carcinoma F9 cells were treated with okadaic acid. In the presence of this agent, the cells showed rapid morphological changes and arrest of proliferation at the M phase of the cell cycle, accompanied by a marked increase in the mRNA expression of various differentiation markers. Okadaic acid induced rapid increase in the mRNA levels of both c-jun and junB and results indicate that the inhibition of phosphatase by okadaic acid induces apparent activation of protein phosphorylation and may cause the expression of differentiation marker genes in F9 cells via the activation of AP-1.
Animals↗
The kinetics of induction of Hox1.6 and C-jun mRNA during three different ways of inducing differentiation in teratocarcinoma F9 cells.
Changes in Hox1.6 and c-jun gene expression were examined upon F9 cell differentiation that was induced by three independent methods: a drug treatment with retinoic acid (RA), that with sodium butyrate (NaB), and a genetic approach using the ts mutant. To obtain further information on the mechanism of teratocarcinoma cell differentiation we have examined the kinetics of the induction of Hox1.6 and c-jun mRNA whose gene products have been demonstrated to have specific roles in gene regulation. Expression of Hox1.6 mRNA was induced more rapidly than c-jun mRNA by all the above three inducing methods. Furthermore, protein synthesis was not required for the induction of Hox1.6 mRNA as well as of c-jun mRNA synthesis in all three methods. The data suggested that the transcriptional increase in the Hox1.6 mRNA was a primary response and could play an important role in F9 cell differentiation.
Butyrates↗
Substrate activating mechanism of short-chain acyl-CoA, medium-chain acyl-CoA, long-chain acyl-CoA, and isovaleryl-CoA dehydrogenases from bovine liver: a resonance Raman study on the 3-ketoacyl-CoA complexes.
We have shown previously that acetoacetyl-CoA bound to medium-chain acyl-CoA dehydrogenase from pig kidney is transformed into an enolate form, O = C(3)-C(2)H = C(1)-O-, and that the interaction between the C(4a) = N(5) moiety of flavin and the O = C(3)-C(2)H = C(1)-O- moiety of acetoacetyl-CoA is important for the charge-transfer interaction [Nishina, Y. et al. (1992) J. Biochem. 111, 699-706]. In this study, we examined four kinds of acyl-CoA dehydrogenases [short-chain acyl-CoA (SCAD), medium-chain acyl-CoA (MCAD), long-chain acyl-CoA (LCAD), and isovaleryl-CoA (IVD) dehydrogenases] from bovine liver. The Raman spectra of non-labeled and isotopically labeled acetoacetyl-CoA in keto-form revealed that the 1,716-cm-1 and 1,650-cm-1 bands were derived from the C(3) = O and the C(1) = O stretching mode, respectively. In the charge-transfer complexes of acetoacetyl-CoA with the four kinds of dehydrogenases, the resonance Raman (RR) bands corresponding to the C(3) = O and the C(1) = O of acetoacetyl-CoA were observed at around 1,643-1,622 and 1,506-1,476 cm-1, respectively, indicating that acetoacetyl-CoA was transformed into the enolate form as the result of the complexation with the enzymes. Further, in RR spectra with excitation at 632.8 nm, within the charge-transfer band of the complexes of acetoacetyl-CoA with the four acyl-CoA dehydrogenases, both bands associated with the C(4a) = N(5) moiety of oxidized flavin and the O = C(3)-C(2)H = C(1)-O- moiety of acetoacetyl-CoA were enhanced, but the benzene portion of oxidized flavin was not. These results indicate that the substrate activating mechanism is common to all four kinds of dehydrogenases, i.e., the interaction between the C(1) = O of acetoacetyl-CoA and the positively polarized atoms of the enzymes located in close proximity to the oxygen atom of C(1) = O is important, and the C(4a) = N(5) moiety of flavin participates in the interaction. Some kinds of 3-ketoacyl-CoAs were tested instead of acetoacetyl-CoA and essentially similar results were obtained. The positions of the bands derived from the C(1)-O- moiety of 3-ketoacyl-CoAs were different by ca. 30 cm-1 in two groups, i.e., ca. 1,475 cm-1 for SCAD and MCAD and ca. 1,505 cm-1 for LCAD and IVD, that is, RR spectra can classify the four dehydrogenases into two groups.
Acyl-CoA Dehydrogenase↗
Structural modulation of 2-enoyl-CoA bound to reduced acyl-CoA dehydrogenases: a resonance Raman study of a catalytic intermediate.
A catalytic intermediate, the so-called "purple complex," of acyl-CoA dehydrogenase is produced on its reaction with the substrate, acyl-CoA. The purple complex is a charge-transfer complex between the reduced enzyme and the product, enoyl-CoA. Resonance Raman spectra of the purple complexes of three acyl-CoA dehydrogenases [short-chain acyl-CoA (SCAD), medium-chain acyl-CoA (MCAD), and isovaleryl-CoA (IVD) dehydrogenases] were measured with excitation at 632.8 nm within charge-transfer absorption bands. The 1,577 cm-1 band of the SCAD purple complex formed in the reaction with butyryl-CoA is mainly associated with the C(1) = O stretching of crotonyl-CoA, judging from the isotopic frequency shifts upon 13C or 18O substitution of butyryl-CoA. The 1,627 cm-1 band of the C(1) = O moiety of crotonyl-CoA in solution shifted downward by 50 cm-1 on complexation with reduced SCAD. This large frequency shift indicates a substantial interaction between C(1) = O and the enzyme, and is further evidence for an appreciable contribution of a polarized form of the C(1) = O moiety in the enzyme-bound enoyl-CoA. This frequency shift can be explained by the hydrogen bond of C(1) = O. The 1,577 cm-1 band of the MCAD purple complex remained constant, regardless of the acyl carbon-chain length (from C4 to C16 of the substrate, acyl-CoA); the alky chain scarcely affected the interaction of the C(1) = O moiety in the active site.(ABSTRACT TRUNCATED AT 250 WORDS)
Acyl Coenzyme A↗