Biomedical subjects

Hiromitsu Tanaka

Publications and source records attributed to Hiromitsu Tanaka.

At least 37 records · Page 2Linked to original sources

PubMed2004

Assembly of the silk fibroin elementary unit in endoplasmic reticulum and a role of L-chain for protection of alpha1,2-mannose residues in N-linked oligosaccharide chains of fibrohexamerin/P25.

Silk fibroin of Bombyx mori is secreted from the posterior silk gland (PSG) as a 2.3-MDa elementary unit, consisting of six sets of a disulfide-linked heavy chain (H-chain)-light chain (L-chain) heterodimer and one molecule of fibrohexamerin (fhx)/P25. Fhx/P25, a glycoprotein, associates noncovalently with the H-L heterodimers. The elementary unit was found and purified from the endoplasmic reticulum (ER) extract of PSG cells. A substantial amount of fhx/P25 unassembled into the elementary unit was also present in ER. In normal-level fibroin-producing breeds (J-139 and C108), the elementary unit contained fhx/P25 of either 30 kDa (major) or 27 kDa (minor). The 27-kDa fhx/P25 was produced from the 30-kDa form by digestion with the bacterial alpha1,2-mannosidase in vitro. The elementary unit in the ER extract contained only the 30-kDa fhx/P25, whereas both 30- and 27-kDa forms of fhx/P25 were present in the ER plus Golgi mixed extracts. In naked-pupa mutants [Nd(2), Nd-s and Nd-sD], extremely small amounts of fibroin were produced and they consisted of one molecule of 27-kDa fhx/P25 and six molecules of H-chain but no L-chain. When the Nd-sD mutant was subjected to transgenesis with the normal L-chain gene, the (H-L)6fhx1-type elementary unit containing the 30-kDa fhx/P25, was produced. These results suggest that fhx/P25 in the elementary unit is largely protected from digestion with Golgi alpha1,2-mannosidases when L-chains are present in the unit. Models suggesting a role of L-chain for the protection of alpha1,2-mannose residues of fhx/P25 are presented.

Animals↗

PubMed2004

Cloning and characterization of a mouse spergen-1 localized in sperm mitochondria.

Development of spermatozoa is a complex process involving specific morphological formation of flagella, nucleus and mitochondria. Although detailed morphological observations of these events are available, the molecular mechanisms remain to be fully elucidated. We report here the molecular cloning and characterization of mouse spergen-1 encoding a sperm specific mitochondrial protein, from a haploid germ cell-specific subtracted cDNA library of mouse testis. Isolated cDNA is c. 0.7 kb and contains a 465 bp ORF that encodes mouse spergen-1, a sperm mitochondrial protein consisting of 154 predicted amino acids. Antibodies raised against mouse Spergen-1 identified a testis-specific c. 18 Mr x 10(3) band in Western blot analysis. The protein was localized to the mitochondria of mouse sperm. Comparison of the mouse and human genomic sequences showed that 55 bps of the 5'-upstream region containing a CAAT box and binding sequence for NF-kappa B is conserved and could be important for specific expression of mouse spergen-1 in haploid germ cells.

Amino Acid Sequence↗

PubMed2004

Ketone bodies could support the motility but not the acrosome reaction of mouse sperm.

Ketone bodies, D-beta-hydroxybutyrate and acetoacetate, produced by the metabolism of fatty acids, are an important energy source for many organs, especially the heart, kidney and brain. They are utilized by the body with the help of succinyl CoA transferase (SCOT), which is ubiquitously expressed in various organs. Previously, we identified a novel SCOT-t specifically expressed in testicular germ cells and sperm, substituting somatic cell-type SCOT, however the physiological role of SCOT-t had not then been clarified. In the present study, we investigated the effects of ketone bodies, the substrate of SCOT-t, on the motility and acrosome reaction of mouse sperm. D-beta-hydroxybutyrate and acetoacetate both stimulated the motility of sperm as glucose or pyruvate. The glycolysis inhibitor stopped the motility of sperm mediated by glucose but not by D-beta-hydroxybutyrate. In contrast, ketone bodies did not stimulate the activation of the acrosome reaction of sperm, different from the effect of glucose. These results indicate that ketone bodies could be involved in sperm movement but not the acrosome reaction and the SCOT-t enzyme we have identified in sperm mitochondria may have important roles in the activity of sperm, resulting in male infertility when its function is disabled.

3-Hydroxybutyric Acid↗

PubMed2004

Mice deficient in the axonemal protein Tektin-t exhibit male infertility and immotile-cilium syndrome due to impaired inner arm dynein function.

The haploid germ cell-specific Tektin-t protein is a member of the Tektin family of proteins that form filaments in flagellar, ciliary, and axonemal microtubules. To investigate the physiological role of Tektin-t, we generated mice with a mutation in the tektin-t gene. The homozygous mutant males were infertile, while the females were fully fertile. Sperm morphology and function were abnormal, with frequent bending of the sperm flagella and marked defects in motility. In vitro fertilization assays showed that the defective spermatozoa were able to fertilize eggs. Electron microscopic examination showed that the dynein inner arm structure was disrupted in the sperm flagella of tektin-t-deficient mice. Furthermore, homozygous mutant mice had functionally defective tracheal cilia, as evidenced by altered dynein arm morphology. These results indicate that Tektin-t participates in dynein inner arm formation or attachment and that the loss of Tektin-t results in impaired motility of both flagella and cilia. Therefore, the tektin-t gene is one of the causal genes for immotile-cilium syndrome/primary ciliary dyskinesia.

Animals↗

PubMed2003

Control of mouse hils1 gene expression during spermatogenesis: identification of regulatory element by transgenic mouse.

Histone H1-like protein in spermatids 1 (Hils1) is a testis- specific histone H1-like protein exclusively expressed in haploid spermatids and should be involved in chromatin remodeling during mouse spermatogenesis. Spatial and temporal regulation of the hils1 gene expression would be critical for the formation of functional sperm, controlled at both transcriptional and translational levels. Previously, we reported that transcripts of the hils1 gene are exclusively expressed in mouse testis from 23 days of age whereas the Hils1 protein is not detected until 28 days of age, suggesting that hils1 is a member of a class of translationally regulated genes. By analyzing transgenic mice, we could demonstrate that 318-base pair (bp) 5'-proximal region corresponding to the first 70-bp proximal TATA-less promoter, and 248 bp of 5'-untranslated region is sufficient to confer testis- and spermatid-specific transcription as well as posttranscriptional control of the mouse hils1 gene in vivo.

Animals↗

PubMed2003

Haprin, a novel haploid germ cell-specific RING finger protein involved in the acrosome reaction.

The acrosome reaction (i.e. the exocytosis of the sperm vesicle) is a prerequisite for fertilization, but its molecular mechanism is largely unknown. We have identified a cDNA clone for a gene named haprin, which encodes a haploid germ cell-specific RING finger protein. This protein is a novel member of the RBCC (RING finger, B-box type zinc finger, and coiled-coil domain) motif family that has roles in several cellular processes, such as exocytosis. It is transcribed exclusively in testicular germ cells after meiotic division. Western blot and immunohistochemical analyses showed the molecular weight of Haprin protein to be Mr approximately 82,000. It was localized in the acrosomal region of elongated spermatids and mature sperm and was not present in acrosome-reacted sperm. The specific antibody against the RING finger domain of Haprin inhibited the acrosome reaction in permeabilized sperm. These results indicated that the novel RBCC protein Haprin plays a key role in the acrosome reaction and fertilization.

Acrosome↗

PubMed2003

Novel actin-like proteins T-ACTIN 1 and T-ACTIN 2 are differentially expressed in the cytoplasm and nucleus of mouse haploid germ cells.

We isolated cDNA clones for the novel actin-like proteins T-ACTIN 1 and T-ACTIN 2, which are expressed specifically in the mouse testis. These clones were from a subtracted cDNA library that was enriched for haploid germ cell-specific cDNAs. The mRNA sizes and deduced molecular masses of t-actin 1/mACTl7b and t-actin 2/mACTl7a were 2.2 kilobases (kb) and 1.8 kb, and Mr 43.1 x 10(3) and Mr 47.2 x 10(3), respectively. The two deduced amino acid sequences had 60% homology, and they had approximately 40% homology with other actins. The T-ACTINs contained some of the conserved regions seen in other actins. Although the cellular locations of these two proteins are quite different (T-ACTIN-1 was found in the cytoplasm and T-ACTIN-2 was located in the nucleus), the expression of their proteins and mRNAs is controlled during development and limited during spermiogenesis. In contrast, only T-ACTIN-2 was present in sperm heads and tails. These results suggest that T-ACTINs play important roles in sperm function and in the specific morphogenesis of spermatozoa during spermiogenesis.

Actins↗

PubMed2003

A novel protein phosphatase 2C family member (PP2Czeta) is able to associate with ubiquitin conjugating enzyme 9.

In this study we have cloned a novel member of mouse protein phosphatase 2C family, PP2Czeta, which is composed of 507 amino acids and has a unique N-terminal region. The overall similarity of the amino acid sequence between PP2Czeta and PP2Calpha was 22%. On Northern blot analysis PP2Czeta was found to be expressed specifically in the testicular germ cells. PP2Czeta expressed in COS7 cells was able to associate with ubiquitin conjugating enzyme 9 (UBC9) and the association was enhanced by co-expression of small ubiquitin-related modifier-1 (SUMO-1), suggesting that PP2Czeta exhibits its specific role through its SUMO-induced recruitment to UBC9.

Amino Acid Sequence↗

PubMed2003

Genomic structure and promoter activity of the testis haploid germ cell-specific intronless genes, Tact1 and Tact2.

The Tact1 and Tact2 genes, each of which encodes an actin-like protein, are exclusively expressed and translated in haploid germ cells in testis. To characterize the haploid germ cell-specific gene structure, a mouse genomic library was screened with a Tact1 cDNA as a probe, and four independent phage clones containing the Tact1 gene were isolated. Southern hybridization and sequencing analyses revealed that Tact1 and Tact2 were single copy genes contained on a common fragment in a head-to-head orientation, and that the distance between these genes was less than 2 kb. Comparison of the nucleotide sequences of genomic DNA and cDNA demonstrated that Tact1 and Tact2 lack introns, although all known actin or actin-related genes in mammals contain introns. Human Tact orthologues also lack introns and are located within 6.4 kb in a head-to-head orientation. These findings indicate that Tact1 and Tact2 or one of these genes arose by retroposition of a spliced mRNA transcribed from an actin progenitor gene prior to the divergence of rodents and primates. The Tact1 and Tact2 genes are unusual retroposons in that they have retained an open reading frame and are expressed in testicular germ cells, because almost all retroposons become pseudogenes. It was revealed that a 2kb sequence between the two genes bidirectionally controls haploid germ-cell specific expression by analyzing transgenic mice. Comparison of the murine Tact genes with their human orthologues showed a high level of identity between the two species in the 5'-upstream and non-coding sequences as well as in the coding region, indicating that conserved elements in these regions may be involved in the regulation of haploid germ cell-specific expression. The promoter region contains no TATA-, CCAAT- or GC-boxes, although there are potential cAMP response element (CRE)-like motifs in the 5'-upstream region and the 5'-untranslated region in Tact1 and Tact2, respectively. Transient promoter analyses indicate that CREMtau may activate Tact1 and Tact2 expression in germ cells.

Actins↗

PubMed2003

Isolation and characterization of a novel cDNA encoding a DNA-binding protein (Hils1) specifically expressed in testicular haploid germ cells.

A cDNA encoding a protein homologous with histone H1 has been cloned from a haploid germ cell specific cDNA library. Deduced amino acid sequence (170 amino acids) showed 40% identity with histone H1 globular domain. Messenger RNA of the gene was observed exclusively in the testis, and was accumulated after post-natal day 23. Western blotting analysis showed that the protein encoded by this gene is about 19 kDa in molecular weight, and it was exclusively recovered from the nuclei of testicular germ cells. Immunohistochemical analysis showed that the protein was localized to the nuclei of round and elongating spermatids, consistent with the results of immunoblot analysis. Thus, the gene product was named Hils1 (histone H1 like protein in spermatids 1). In vitro DNA-binding experiments using DNA-cellulose mini-columns showed that Hils1 was able to bind to both double and single stranded-DNAs in a non-sequence-specific manner. These findings suggest that Hils1 may play an important role in the structural changes of spermatid nuclei, such as nuclear condensation, and gene regulation of haploid germ cell differentiation.

Amino Acid Sequence↗

PubMed2003

Fabry disease: detection of undiagnosed hemodialysis patients and identification of a "renal variant" phenotype.

BACKGROUND: Fabry disease is an X-linked recessive lysosomal storage disease resulting from deficient alpha-galactosidase A (alpha-Gal A) activity. Renal failure is a major debilitating complication in classically affected males. To determine if this disorder is underdiagnosed in patients with end-stage renal disease (ESRD), the frequency of unrecognized males with Fabry disease on chronic hemodialysis was determined. METHODS: Plasma alpha-Gal A activity was measured in 514 consecutive males with ESRD on hemodialysis. Patients with low alpha-Gal A activity were evaluated clinically and their alpha-Gal A mutations were determined. RESULTS: Six (1.2%) of 514 hemodialysis patients had low plasma alpha-Gal A activities and a previously identified (E66Q, A97V, M296I) or novel (G373D) missense mutation. At ages 30 to 68 years, five patients lacked the classic manifestations of angiokeratoma, acroparesthesias, hypohidrosis, and ocular opacities, while the sixth lacked angiokeratoma and ocular changes. Five had left ventricular hypertrophy (LVH). CONCLUSION: The clinical spectrum of Fabry disease includes a "renal variant" phenotype in patients without classic symptoms who develop ESRD. Affected males undergoing hemodialysis or renal transplantation can be readily diagnosed by plasma alpha-Gal A assays. These patients and their family members may benefit from enzyme replacement therapy for the later, life-threatening cardiovascular and cerebrovascular complications of Fabry disease.

Adult↗

PubMed2003

Single nucleotide polymorphisms in the protamine-1 and -2 genes of fertile and infertile human male populations.

Although various genetic factors have been implicated in human male infertility, the causative genes for the different types of idiopathic male infertility have not been elucidated. Protamines, which are the major DNA-binding proteins in the sperm nucleus, package the DNA into the sperm head. Analysis of the human protamine-1 (PRM1) and -2 (PRM2) gene sequences in 226 sterile male patients and in 270 proven-fertile male volunteers revealed four single nucleotide polymorphisms (SNPs) in the PRM1 coding region, which did not cause any amino acid substitutions, and one SNP in the PRM2 gene, which produced translation termination. We also observed one SNP in the 3' non-coding region of the PRM1 gene, and two SNPs within the intron of the PRM2 gene. The prevalence of these SNPs was similar in both infertile patients and in proven-fertile volunteers, except that the c248t alteration in the PRM2 gene induced a nonsense codon under conditions of heterozygosity in one infertile patient. Although the PRM1 and PRM2 genes are highly conserved, the single SNP in the PRM2 gene that induces translation termination may result in male infertility due to haploinsufficiency of PRM2.

Amino Acid Sequence↗

PubMed2003

Molecular cloning and characterization of the human orthologue of the oppo 1 gene encoding a sperm tail protein.

We report here the molecular cloning and characterization of a human orthologue of oppo 1, a mouse gene encoding a male germ-cell-specific sperm tail protein, and the organization of its genomic structure. The mRNA of the human oppo 1 gene (h-oppo 1) was expressed exclusively in the testis, and the 30 kDa protein encoded by the mRNA was detected in human testis and sperm. Immunohistochemical analyses showed that human OPPO 1 protein was localized in the flagellae of ejaculated sperm. A human genomic DNA database search indicated that the h-oppo 1 gene mapped to chromosome 17. The genomic structure of h-oppo 1 showed differences in exon/intron usage, the sequence of the 5'-flanking region, and the first intron was rich in Alu repeats as compared with the mouse oppo 1 gene. Comparison of the two genomic sequences indicated that human oppo 1 has evolved independently, resulting in substantial differences in the genomic structure after the human-mouse split, whereas the sequence of the basic functional unit of the oppo 1 gene seems to have been relatively well conserved.

Amino Acid Sequence↗

PubMed2003

A lipase isolated from the silkworm Bombyx mori shows antiviral activity against nucleopolyhedrovirus.

A protein showing strong antiviral activity against Bombyx mori nucleopolyhedrovirus (BmNPV) was purified from the digestive juice of B. mori larvae. A homology search of the deduced amino acid sequence of the protein cDNA revealed 56% homology with Drosophila melanogaster lipase and 21% homology with human lipase. As lipase activity of the protein was confirmed in vitro, this protein was designated Bmlipase-1. Northern blot analysis showed that the Bmlipase-1 gene is expressed in the midgut but not in other tissues, nor is it activated by BmNPV infection. In addition, the Bmlipase-1 gene was shown not to be expressed in the molting and wandering stages, indicating that the gene is hormonally regulated. Our results suggest that an insect digestive enzyme has potential as a physiological barrier against BmNPV at the initial site of viral infection.

Amino Acid Sequence↗

PubMed2002

Structure and promoter activity of the gene encoding ornithine decarboxylase antizyme expressed exclusively in haploid germ cells in testis (OAZt/Oaz3).

Ornithine decarboxylase antizyme 1 and 2 (OAZ1 and OAZ2) are expressed ubiquitously, and control the intracellular concentration of polyamines. Their testicular isoform, OAZt/Oaz3, is specifically expressed in differentiated haploid germ cells. We have identified and characterized the gene encoding OAZt in mice. The mouse OAZt gene contains, as does the human ortholog and paralogs, five exons and four introns. Comparison of the mouse OAZt with the human ortholog gene revealed that exon sizes are identical and nucleotide sequences in exons are highly homologous (83% identity). The major transcriptional start site was determined by primer extension assay. Promoter activity was confirmed by transgenic mouse assays, using the upstream region of the mouse OAZt gene fused to a EGFP reporter gene. The OAZt essential promoter located between -133 and +242, has two CREs and an Inr, and lacks a TATA box. These elements are conserved in the human ortholog but not in the paralogs, indicating that such a short upstream region including two CREs and Inr is sufficient to drive endogenous OAZt mRNA expression in the haploid testicular germ cells.

3T3 Cells↗

PubMed2002

Nectin couples cell-cell adhesion and the actin scaffold at heterotypic testicular junctions.

Actin-based cell-cell adherens junctions (AJs) are crucial not only for mechanical adhesion but also for cell morphogenesis and differentiation. While organization of homotypic AJs is attributed mostly to classic cadherins, the adhesive mechanism of heterotypic AJs in more complex tissues remains to be clarified. Nectin, a member of a family of immunoglobulin-like adhesion molecules at various AJs, is a possible organizer of heterotypic AJs because of its unique heterophilic trans-interaction property. Recently, nectin-2 (-/-) mice have been shown to exhibit the defective sperm morphogenesis and the male-specific infertility, but the role of nectin in testicular AJs has not been investigated. We show here the heterotypic trans-interaction between nectin-2 in Sertoli cells and nectin-3 in spermatids at Sertoli-spermatid junctions (SspJs), heterotypic AJs in testes. Moreover, each nectin-based adhesive membrane domain exhibits one-to-one colocalization with each actin bundle underlying SspJs. Inactivation of the mouse nectin-2 gene causes not only impaired adhesion but also loss of the junctional actin scaffold at SspJs, resulting in aberrant morphogenesis and positioning of spermatids. Localization of afadin, an adaptor protein of nectin with the actin cytoskeleton, is also nectin-2 dependent at SspJs. These results indicate that the nectin-afadin system plays essential roles in coupling cell-cell adhesion and the cortical actin scaffold at SspJs and in subsequent sperm morphogenesis.

Actins↗

PubMed2002

Spermatogonia-dependent expression of testicular genes in mice.

Spermatogenesis is initiated by the interaction of germ cells and somatic cells in seminiferous tubules. We used cDNA microarrays and representational difference analysis to identify genes that are expressed in the testis of the jsd/jsd mutant mouse, which contains only type A spermatogonial germ cells and Sertoli cells, but not in the testis of the W/W(v) mutant mouse, where Sertoli cells but few germ cells are present. We isolated 20 known genes and 4 novel genes, including 2 genes encoding lipocalin family members (prostaglandin D synthetase and 24p3) and 2 tumor suppressors (protein tyrosine phosphatase TD14 and Sui1). All 24 of these jsd/jsd-derived genes were highly expressed in the cryptorchid testis as well as in the jsd/jsd testis. This indicates that their selective expression is not directly caused by the as-yet-uncharacterized jsd gene product, but is rather correlated to the cessation of spermatogonial differentiation. In situ hybridization analysis and flow cytometric sorting followed by reverse transcriptase-PCR revealed that these genes are expressed in both the spermatogonial germ cells and the somatic cells in the developing gonads and adult testes. As the mRNAs of these jsd/jsd-derived genes were barely detectable in the W/W(v) testis, we propose that early spermatogonial germ cells regulate the expression of a group of testicular genes.

Animals↗

PubMed2002

Use of stepwise subtraction to comprehensively isolate mouse genes whose transcription is up-regulated during spermiogenesis.

We report the isolation of 153 mouse genes whose expression is dramatically up-regulated during spermiogenesis. We used a novel variation of the subtractive hybridization technique called stepwise subtraction, wherein the subtraction process is systematically repeated in a stepwise manner. We named the genes thus identified as TISP genes (transcript induced in spermiogenesis). The transcription of 80 of these TISP genes is almost completely specific to the testis. This transcription is abruptly turned on after 17 days of age, when the mice enter puberty and spermiogenesis is initiated. Considering that the most advanced cells present at these stages of spermatogenesis are the spermatids, it is likely that we could isolate most of the spermatid-specific genes. DNA sequencing revealed that about half the TISP genes are novel and uncharacterized genes, confirming the utility of the stepwise subtraction approach for gene discovery.

Animals↗