Biomedical subjects

Jiahao Sha

Publications and source records attributed to Jiahao Sha.

26 records · Page 2Linked to original sources

PubMed2003

Rescue of defective pancreatic secretion in cystic-fibrosis cells by suppression of a novel isoform of phospholipase C.

BACKGROUND: Cystic fibrosis is caused by mutations in the gene encoding an ion-transport protein, the cystic-fibrosis transmembrane conductance regulator (CFTR). Defective secretion of anions is the primary cause of many of the clinical manifestations of cystic fibrosis, including pancreatic insufficiency. We aimed to identify a molecular mechanism from which a new method to circumvent defective pancreatic secretion could be derived. METHODS: Multiple-human-tissue RT-PCR and semiquantitative RT-PCR analyses were used to examine gene expression. An antisense technique was used in conjunction with radioimmunoassay, Fura-2 spectrofluorometry, immunohistochemistry, and the short-circuit current technique (Ussing chamber) for elucidation of gene function and its application in rescuing defective pancreatic secretion. FINDINGS: We cloned a newly identified gene, NYD-SP27, which has structural similarity to an isoform of phospholipase C. NYD-SP27 was expressed endogenously in human pancreatic-duct cells and upregulated in cystic fibrosis. Suppression of NYD-SP27, by transfection of its antisense into human cystic-fibrosis pancreatic-duct cells, resulted in augmentation of phospholipase-C-coupled calcium-ion release and protein kinase C activity, improvement in the amount of mutated CFTR reaching the plasma membrane, and restoration of cAMP-activated pancreatic anion secretion. INTERPRETATION: NYD-SP27 exerts an inhibitory effect on phospholipase-C-coupled processes that depend on calcium ions and protein kinase C, including CFTR trafficking and function. Its upregulation in pancreatic-duct cells may reveal a previously unsuspected defect in cystic fibrosis contributing to pancreatic insufficiency, and thus represents a new target for pharmacological intervention in cystic fibrosis.

Antisense Elements (Genetics)↗

PubMed2003

Identification and characterization of a novel human testis-specific Golgi protein, NYD-SP12.

A novel human testis-specific gene, NYD-SP12, was identified by hybridizing human adult or fetal testes cDNA samples with a human cDNA microarray containing 9216 clones. mRNA expression level of NYD-SP12 was 30-fold higher in human adult testes than fetal testes. Similarly, semi-quantitative RT-PCR revealed a differential expression pattern of an NYD-SP12 homologous gene in mouse adult and infant testes. PCR and hybridization analysis of NYD-SP12 mRNA from multiple human tissues indicated the expression of NYD-SP12 exclusively in the testis. In-situ hybridization revealed that the expression of this gene was confined to spermatogenic epithelium and was not found in interstitial cells. NYD-SP12 transcript was not detected in patients with spermatogenic arrest and Sertoli cell-only syndrome. NYD-SP12 cDNA (GenBank accession number: AF345909) consisted of 2070 bp. The predicted 1707 bp open-reading fragment encoded a 569 amino acid protein that was 77% identical to a mouse homologue. Furthermore, computerized SMART and Motif analysis revealed that the protein contained a Structural Classification Of Proteins (SCOP) domain in the C-terminus and a cluster of phosphorylation sites for PKC, CK and cAMP/cGMP-dependent protein kinase. Interestingly, the EGFP-NYD-SP12 fusion protein was localized to the Golgi apparatus. In conclusion, the results suggest that NYD-SP12 is involved in spermatogenesis, and that NYD-SP12-encoded protein might function in the Golgi apparatus.

Amino Acid Sequence↗

PubMed2002

NYD-SP6, a novel gene potentially involved in regulating testicular development/spermatogenesis.

Using cDNA microarray hybridization from a human testicular cDNA library, a novel gene exhibiting 30-fold difference in expression level between adult and embryo human testes was cloned and named NYD-SP6, which was 1858 bp in length with 87% nucleotide identity with the mouse homologue sequence. The deduced protein structure of NYD-SP6 was found to contain two plant homeodomain (PHD) finger domains, believed to be involved in activating transcriptional regulation. Tissue distribution analysis using Northern blot indicated that the NYD-SP6 gene was expressed in a wide range of tissues, with a high expression level in the testis. Its expression in human and mouse testes by in situ hybridization was confined to Sertoli cells and the expression was developmentally regulated as demonstrated by cDNA microarray, in situ hybridization, and semiquantitative PCR in mouse testes. GFP/NYD-SP6 protein was predominantly localized in the nucleus of the transfected CHO cells, indicating its role in transcriptional regulation. In contrast, the N-terminal truncated NYD-SP6 (tNYD-SP6) localized in the nuclear envelope, indicating this region function as a nuclear localization signal. Further Northern blot analysis of gene expression in patients with spermatogenesis arrest revealed that NYD-SP6 was absent in one patient whose spermatogenesis was blocked at the stage of spermatocytes. Taken together, these results suggest that the putative protein of NYD-SP6 may play an important role in stimulating transcription involved in testicular development and/or spermatogenesis.

Adult↗

PubMed2002

Identification and characterization of a gene coding a novel isoform of DEAD-box protein.

A gene coding a novel isoform of DEAD-box protein named testicular DEAD-box protein (tDbp), presumably involved in testicular function, was identified and characterized. Testicular DEAD-box protein was cloned from a human testis cDNA library. The cDNA microarray hybridization showed that it was expressed at a higher level in adult testis than in embryo testis. Reverse transcription-polymerase chain reaction indicated that tDbp was specifically expressed in testis, but not in some other tissues.

Amino Acid Sequence↗

PubMed2002

Identification of testis development and spermatogenesis-related genes in human and mouse testes using cDNA arrays.

We have constructed cDNA microarrays from the human testis large insert cDNA library, containing 9216 genes, together with several housekeeping genes. The cDNA microarrays were used to identify gene expression differences between human fetal and adult testes. Of >8700 hybridized clones, 731 exhibited significant differential expression characteristics. About 7500 genes were identified when the same cDNA microarrays were used for hybridization with cDNA probes from mouse testis, with 256 genes having significant differential expression between the age of 1-4 weeks. Among these genes, 101 were identified as critically related to testis development and possibly to spermatogenesis since they were found in both human and mouse testes, and expressed differentially at different stages of testis development. Of the 101 development-related genes, 59 full-length cDNAs have been sequenced previously, while the full-length cDNAs of the other 42 genes have not been published. We have obtained 11 full-length sequences of the 42 genes and deposited them in the GenBank. The conserved testis development-related genes found in both human and mouse testes may include genes that are likely to be involved in testicular functions, especially spermatogenesis, thus providing a basis for further functional characterization of the genes in mouse models.

Adult↗

PubMedDate not supplied

Expression of a novel RAD23B mRNA splice variant in the human testis.

A gene coding a novel human RAD23B protein named RAD23-like protein B, presumably involved in spermatogenesis, was identified and characterized using a complementary DNA (cDNA) microarray. In the human testis, its expression was 2.33 times higher than it was in the embryo testis, with a particularly high expression in ejaculated human spermatozoa. The full length of this gene is 1548 bp, and the putative protein is 338 amino acids long. This protein is homologous to RAD23B, which is one of two human homologs of Saccharomyces cerevisiae RAD23, and is involved in both nucleotide excision repair (NER) and ubiquitin (Ub)-dependent proteolysis. However, RAD23-like protein B lacks the Ub-like (UbL) domain that functions as a proteasome localization signal. Multiple-tissue expression profile of the messenger RNA (mRNA) that encodes the RAD23-like protein B also showed that it is highly expressed in the human testis and in ejaculated spermatozoa. Our present study indicates that this novel alternative splicing form of RAD23B is correlated with human spermatogenesis.

Amino Acid Sequence↗

PubMedDate not supplied

Novel development-related alternative splices in human testis identified by cDNA microarrays.

Alternative splicing of premessenger RNA is an important regulatory mechanism that increases the diversity of proteins transcribed from a single gene. This is particularly important in the testis because germ cell expansion and differentiation require many cellular changes and regulatory steps. To investigate novel development-related alternative splicings in the human testis, complementary DNA microarray studies were conducted with the use of probes from human fetal testes, adult testes, and human spermatozoa. Of a total of 386 Unigene clusters found to be related to the development of the testis, 67 clusters showed a total of 74 novel alternative spliceoforms. Developmental stage-dependent expression was also performed for a novel Unigene, NYD-SP20 (Hs.351068), which had 4 possible novel spliceoforms and another Unigene, CRISP2 (cysteine-rich secretory protein 2, Hs.2042), which had 3 possible novel spliceoforms. These results indicate that alternative splicing plays an important role in the complicated processes of testis development and spermatogenesis.

Adult↗

PubMedDate not supplied

Cloning and characterization of a novel transcript variant of IQGAP2 in human testis.

Using cDNA microarray hybridization from a human testicular cDNA library, one gene exhibiting 6.4-fold difference at expression level between adult and embryo human testes was cloned and named tsIQGAP2 (testis specific IQ motif containing GTPase-activating protein 2). The full cDNA length of tsIQGAP2 is 3388 bp and contains a 3215 bp open reading frame, encoding a 122.85 kDa protein of 1071 amino acids. Sequence analysis showed that tsIQGAP2 harbors all the domains (one WW domain, three IQ motifs, and one RasGAP homology domain) in HsIQGAP2 (IQ motif containing GTPase-activating protein 2) except for the N-terminal calponin homology domain. Blasting human genome database localized tsIQGAP2 to human chromosome 5q13-14 and further investigation showed that it is a novel transcript variant of HsIQGAP2. The tissue distribution of tsIQGAP2 showed that it is a testis specific gene. The above results suggest the potential role of tsIQGAP2 during spermatogenesis and the mechanism by which tsIQGAP2 regulating spermatogenesis also has been proposed.

Alternative Splicing↗