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Biomedical subjects

H J Cooke

Publications and source records attributed to H J Cooke.

At least 19 recordsLinked to original sources
PubMed1999

T-STAR/ETOILE: a novel relative of SAM68 that interacts with an RNA-binding protein implicated in spermatogenesis.

RBM is an RNA-binding protein encoded on the Y chromosome in mammals and is expressed only in the nuclei of male germ cells. Genetic evidence from infertile men implicates it in spermatogenesis, but its function is unknown. Of a number of potential partners for RBM identified by a yeast two-hybrid screen with testis cDNA, the most frequent isolates encoded a novel RNA-binding protein, termed T-STAR, that is closely related to SAM68, an Src-associated protein of unknown function. The mouse homologue was also cloned and designated étoile. It mapped to chromosome 15, while T-STAR mapped to the syntenic region on human chromosome 8. T-STAR/étoile is expressed primarily in the testis; in rat germ cells, the expression of both T-STAR/étoile and SAM68 is regulated during meiosis. Transfection of T-STAR/étoile fused with green fluorescent protein into HeLa cells caused an accumulation of protein in a novel compartment of the nucleus, adjacent to the nucleolus but distinct from the peri-nucleolar compartment. RBM and other hnRNP G family members are candidate downstream targets for regulation by T-STAR/ETOILE and SAM68.

3T3 Cells↗
PubMed1999

Activation of neuronal adenosine A1 receptors suppresses secretory reflexes in the guinea pig colon.

The role of adenosine A1 receptors (A1R) in reflex-evoked short-circuit current (Isc) indicative of chloride secretion was studied in the guinea pig colon. The A1R antagonist 8-cyclopentyltheophylline (CPT) enhanced reflex-evoked Isc. Adenosine deaminase and the nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine enhanced and reduced reflex-induced Isc, respectively. The A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA) inhibited reflex-evoked Isc at nanomolar concentrations, and its action was antagonized by CPT. In the presence of either N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide to block the 5-hydroxytryptamine (5-HT)-mediated pathway or piroxicam to block the prostaglandin-mediated pathway, CCPA reduced the residual reflex-evoked Isc. CCPA reduced the response to a 5-HT pulse without affecting the tetrodotoxin-insensitive Isc responses to carbachol or forskolin. Immunoreactivity for A1R was detected in the membrane (10% of neurons) and cytoplasm (90% of neurons) of neural protein gene product 9.5-immunoreactive (or S-100-negative) submucosal neurons, in glia, and in the muscularis mucosa. A1R immunoreactivity in a majority of neurons remained elevated in the cytoplasm despite preincubation with adenosine deaminase or CPT. A1R immunoreactivity colocalized in synaptophysin-immunoreactive presynaptic varicose nerve terminals. The results indicate that endogenous adenosine binding to high-affinity A1R on submucosal neurons acts as a physiological brake to suppress reflex-evoked Isc indicative of chloride secretion.

Adenosine↗
PubMed1999

Y chromosome and male infertility.

Recent genome analysis of the Y chromosome has increased the number of genes found on this chromosome markedly. Many of these genes in the part of the Y chromosome that does not undergo recombination with the X chromosome are members of gene families. Evolutionary considerations imply that genes on the Y chromosome will degenerate unless they have male advantageous or female deleterious functions. Spermatogenesis is an example of a male advantageous function and genes in three regions of the human Y chromosome have been promoted as candidate male fertility factors.

Animals↗
PubMed1998

Endogenous nitric oxide inhibits endothelin-1-induced chloride secretion in guinea pig colon.

Segments of guinea pig distal colon, stripped of the external muscle layers, were set up in flux chambers for measurement of short-circuit current (Isc) indicative of active, electrogenic ion transport. During neural blockade with tetrodotoxin, the nitric oxide scavenger, hemoglobin, and the nitric oxide synthase inhibitor, N omega-nitro-L-arginine (L-NNA), reduced Isc. The reduction in Isc in response to hemoglobin was reversed by L-arginine and blockers of chloride secretion, including bumetanide and diphenylamine-2-carboxylic acid, but not by the potassium channel blockers, barium and tetraethylammonium, nor by amiloride, an epithelial sodium channel blocker. The hemoglobin-induced reduction in Isc was not affected by blockade of prostaglandin synthesis with piroxicam. During neural blockade, the nitric oxide donors, sodium nitroprusside and NONOate, increased Isc which was abolished by piroxicam. Endothelin-1 (ET-1) also evoked an increase in Isc that was unaffected by amiloride and was inhibitable by bumetanide, chloride-free solutions, tetrodotoxin, piroxicam, and the ETA receptor antagonist, BQ123. The ETB receptor agonist, [Ala1,3,11,15]-endothelin-1, had no appreciable effect on Isc. Hemoglobin and L-NNA enhanced the ET-1-induced Isc response by about twofold without affecting prostaglandin E2 release or its secretory response. The results suggest that endogenous nitric oxide stimulates a low level of chloride secretion that is independent of prostaglandins, unlike nitric oxide donors which increase chloride secretion by releasing prostaglandins. In addition, endogenous nitric oxide suppresses ET-1-evoked chloride secretion by mechanisms that are unrelated to the release of prostaglandin E2 or its ability to stimulate epithelial cells. Endogenous nitric oxide may play an important role in modulating chloride secretion during ischemic challenge when endothelin levels are high.

Animals↗
PubMed1998

Understanding the genes involved in spermatogenesis: a progress report.

OBJECTIVE: To review the current literature on genes known to affect fertility in the human and mouse. DESIGN: A literature review was performed and key articles were chosen for focus in the areas of genes with effects only on spermatogenesis and oogenesis, with an emphasis on Y-chromosome-encoded gene families and spermatogenesis. In addition, studies describing genes deleted in transgenic mice were incorporated. RESULT(S): Several gene families on the Y chromosome are implicated in spermatogenic failure, but the link between the genetic lesion and the resulting defect is unclear. Many mouse genes involved in repair and DNA damage monitoring have specific effects on gametogenesis in and around meiosis. CONCLUSION(S): Many genes are involved only in gametogenesis, and some of these are beginning to be understood in terms of their functions. An even larger number of genes is required for gametogenesis, and other functions and mouse models give insights important for human disease.

Animals↗
PubMed1998

Characterisation of the coding sequence and fine mapping of the human DFFRY gene and comparative expression analysis and mapping to the Sxrb interval of the mouse Y chromosome of the Dffry gene.

DFFRY (the Y-linked homologue of the DFFRX Drosophila fat-facets related X gene) maps to proximal Yq11.2 within the interval defining the AZFa spermatogenic phenotype. The complete coding region of DFFRY has been sequenced and shows 89% identity to the X-linked gene at the nucleotide level. In common with DFFRX , the potential amino acid sequence contains the conserved Cys and His domains characteristic of ubiquitin C-terminal hydrolases. The human DFFRY mRNA is expressed in a wide range of adult and embryonic tissues, including testis, whereas the homologous mouse Dffry gene is expressed specifically in the testis. Analysis of three azoospermic male patients has shown that DFFRY is deleted from the Y chromosome in these individuals. Two patients have a testicular phenotype which resembles Sertoli cell-only syndrome, and the third diminished spermatogenesis. In all three patients, the deletions extend from close to the 3' end into the gene, removing the entire coding sequence of DFFRY. The mouse Dffry gene maps to the Sxrb deletion interval on the short arm of the mouse Y chromosome and its expression in mouse testis can first be detected between 7.5 and 10.5 days after birth when type A and B spermatogonia and pre-leptotene and leptotene spermatocytes are present.

Adult↗
PubMed1998

Dynamic changes in the subnuclear organisation of pre-mRNA splicing proteins and RBM during human germ cell development.

RBM is a germ-cell-specific RNA-binding protein encoded by the Y chromosome in all mammals, implying an important and evolutionarily conserved (but as yet unidentified) function during male germ cell development. In order to address this function, we have developed new antibody reagents to immunolocalise RBM in the different cell types in the human testis. We find that RBM has a different expression profile from its closest homologue hnRNPG. Despite its ubiquitous expression in all transcriptionally active germ cell types, RBM has a complex and dynamic cell biology in human germ cells. The ratio of RBM distributed between punctate nuclear structures and the remainder of the nucleoplasm is dynamically modulated over the course of germ cell development. Moreover, pre-mRNA splicing components are targeted to the same punctate nuclear regions as RBM during the early stages of germ cell development but late in meiosis this spatial association breaks down. After meiosis, pre-mRNA splicing components are differentially targeted to a specific region of the nucleus. While pre-mRNA splicing components undergo profound spatial reorganisations during spermatogenesis, neither heterogeneous ribonucleoproteins nor the transcription factor Sp1 show either developmental spatial reorganisations or any specific co-localisation with RBM. These results suggest dynamic and possibly multiple functions for RBM in germ cell development.

Cyclic AMP Response Element Modulator↗
PubMed1998

Presence of NK1 receptors on a mucosal-like mast cell line, RBL-2H3 cells.

Reverse transcription--polymerase chain reaction of mRNA from rat RBL-2H3 cells yielded a 316 base pair band consistent with that predicted for the neurokinin-1 (NK1) receptor. Saturation and competition binding with 125I-labeled Bolton-Hunter substance P, substance P fragments, and a series of selective tachykinin receptor agonists and antagonists demonstrated that RBL-2H3 cells express high affinity binding sites for substance P on their surfaces with the kinetic and pharmacological properties of NK1 receptors. The pharmacology of these 125I-labeled substance P binding sites was (from most to least potent) [Sar9,Met(O2)11]substance P > substance P 4-11 >> GR82334 << MEN 10,376. However, substance P 1-4, substance P 8-11, substance P 9-11, and [Trp7, beta-Ala8]neurokinin A 4-10 failed to compete for binding. The metabolically stable NK1 receptor agonist, [Sar9,Met(O2)11] substance P, caused a 49% increase in 5-hydroxytryptamine release above basal levels. The results demonstrate the presence of functional NK1 receptors on RBL-2H3 cells, a mucosal-like mast cell line.

Animals↗
PubMed1997

Activation of 5-HT1P receptors on submucosal afferents subsequently triggers VIP neurons and chloride secretion in the guinea-pig colon.

The role of vasoactive intestinal peptide (VIP) was investigated when mucosal stroking and 5-hydroxytryptamine (5-HT) were used to activate neural reflexes that stimulate chloride secretion in the guinea pig colon. Muscle-stripped segments of colon containing intact submucosal ganglia without myenteric ganglia were set up in modified flux chambers in order to record short-circuit current (Isc). Mucosal stroking with a brush for 1 s or a pulse of 5-HT (injection of 15 microliters of 100 microM 5-HT into 1.5 ml of mucosal solution) caused an increase in Isc that was reduced by the VIP antagonist, neurotensin6-11-VIP7-28, in a concentration-dependent manner. The Isc responses to mucosal stroking and a 5-HT pulse were reduced by 53% and 58%, respectively, by 2 microM neurotensin6-11-VIP7-28. The residual Isc response in the presence of neurotensin6-11-VIP7-28 was abolished by atropine. Blockade of 5-HT1P receptors on submucosal afferent neurons decreased Isc responses to stroking or a 5-HT pulse. The residual Isc response after 5-HT1P receptors were blocked was reduced by only 11-14% by neurotensin6-11-VIP7-28. In the presence of blockade of both 5-HT1P and VIP receptors, atropine abolished the Isc response to both stimuli. The observations suggest that the neural circuitry activated by stroking includes at least two independent pathways. One pathway contains VIP neurons which receive inputs directly or indirectly from 5-HT1P receptor-containing afferents. A second pathway involves muscarinic cholinergic transmission that is independent of 5-HT1P and VIP receptor activation.

Animals↗
PubMed1997

The mouse Dazla gene encodes a cytoplasmic protein essential for gametogenesis.

RBM and DAZ/SPGY are two families of genes located on the Y chromosome that encode proteins containing RNA-binding motifs, and both have been described as candidate human spermatogenesis genes. Transmission of deletions from father to son has been observed in the case of DAZ, but neither gene family has been shown to be essential for spermatogenesis in human males. The DAZ/SPGY genes are particularly amenable to a knockout approach, as they are found on the Y chromosome in Old World primates and apes, but in other mammals, they are represented only by an autosomal gene, DAZLA, which is also present in Old World primates and apes. It has also been shown that a Dazla homologue is essential for spermatogenesis in Drosophila. Here we show that Dazla protein is cytoplasmic in male and female germ cells, unlike the nuclear RBM protein. Disruption of the Dazla gene leads to loss of germ cells and complete absence of gamete production, demonstrating that Dazla is essential for the differentiation of germ cells.

Animals↗
PubMed1997

Efficient combination of large DNA in vitro: in gel site specific recombination (IGSSR) of PAC fragments containing alpha satellite DNA and the human HPRT gene locus.

In an attempt to combine a cloned genomic copy of a selectable gene with different cloned centromeric sequences to develop mammalian artificial chromosomes (MAC) we used site specific recombination mediated by purified Cre recombinase acting on the loxP sequence in PAC vector DNA. A new method was required to purify highly concentrated, virtually 100% intact PAC DNA which could be stored for a long period. Here we show the efficient linking of linearized PACs containing alpha satellite DNA from chromosomes X and 17 with sizes of 125 and 140 kb, respectively, to a 95 kb restriction fragment derived from a 175 kb PAC containing the intact human HPRT gene locus.

Bacteriophage P1↗
PubMed1997

The molecular genetics of male infertility.

Spermatogenesis is an elaborate process involving both cell division and differentiation, and cell-cell interactions. Defects in any of these processes can result in infertility, and in some cases these can be genetic in cause. Mapping experiments have defined at least three regions of the human Y chromosome that are required for normal spermatogenesis. Two of these contain the genes encoding the RNA binding proteins RBM and DAZ, suggesting that the control of RNA metabolism is likely to be an important control point for human spermatogenesis. A similar analysis in mice has shown that at least two regions of the mouse Y chromosome are essential for spermatogenesis. Both genetic and reverse genetic approaches have been used to identify mouse autosomal genes required for spermatogenesis. These studies have shown that genes in a number of different pathways are essential for normal spermatogenesis, and also provide putative models of human infertility.

Animals↗
PubMed1997

Hamster chromosomes containing amplified human alpha-satellite DNA show delayed sister chromatid separation in the absence of de novo kinetochore formation.

The centromeres of human chromosomes contain large amounts of the tandemly repeated alpha-satellite DNA family. Previous studies have shown that integration of alpha-satellite DNA into ectopic locations in mammalian chromosomes can result in the de novo formation of several features of centromeric function. Here we further examine the possible centromeric properties of alpha-satellite DNA by introducing it into hamster chromosomes. A large amplified region of ectopic alpha-satellite DNA was shown to direct binding of anticentromere antibodies (ACAs) and centromere protein B (CENP-B). The chromosome containing these ectopic arrays showed a high frequency of formation of anaphase bridges. Owing to the favourable morphology of these chromosomes, we were able to determine that this bridging was due to delayed sister chromatid disjunction at the location of the ectopic alpha-satellite, and not due to de novo formation of a fully functional kinetochore. A separate hamster cell line containing large tandemly repeated amplicons including the DHFR gene also displayed similar behaviour during anaphase. These results may support a role for alpha-satellite DNA in sister chromatid cohesion at centromeres. However, other repetitive DNA in favourable configurations appears to be capable of mimicking this behaviour during anaphase.

Anaphase↗
PubMed1997

5-HT activates neural reflexes regulating secretion in the guinea-pig colon.

The role of 5-hydroxytryptamine (5-HT) in neural reflexes regulating secretion was examined in muscle-stripped segments of guinea-pig colon set up in modified flux chambers. A 15-microL pulse of 5-HT (100 microM) to the mucosal bath (1.5 mL), which was continuously perfused, evoked an increase in short-circuit current (Isc). The 5-HT-induced increase in Isc was inhibited by tetrodotoxin, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), GR82334 and atropine, but not by tropisetron. 5-HTP-DP reduced the response to a 5-HT pulse over the concentration range of 1 nM to 1 microM. The Isc response to a 5-HT pulse was unaffected by the cyclooxygenase inhibitor, piroxicam. This contrasted with a reduction in the Isc response to mucosal stroking with a brush by piroxicam. The results suggest that a 5-HT pulse, like mucosal stroking, activates a secretory reflex that includes tachykinin and cholinergic neurons but, unlike mucosal stroking, does not release prostaglandins.

Animals↗
PubMed1997

Substance P as a mediator of colonic secretory reflexes.

The role of substance P in neural reflex pathways activated by stroking was investigated in muscle-stripped segments of distal colon from guinea pigs. Stroking the mucosal surface with a brush at 1 stroke/s evoked an increase in short-circuit current (Isc) indicative of chloride secretion. The response to mucosal stroking was maximally reduced by 69-75% by the antagonist GR-82334. The agonist [Sar9,Met(O2)11] substance P caused a bumetanide-sensitive increase in Isc when added to the mucosal or serosal bath. Ablation of extrinsic afferents with acute or chronic administration of capsaicin did not alter the mucosal stroking response. Reverse transcription-polymerase chain reaction and in situ hybridization revealed the presence of neurokinin1 (NK1) receptor messenger RNA in isolated colonocytes or crypt glands. Ligand binding of 125I-Bolton-Hunter-labeled substance P was inhibited by GR-82334. The 50% inhibitory concentration was 0.84 nM. The results demonstrate a role for substance P released from capsaicin-insensitive submucosal neurons and in mucosal stroking reflexes. The presence of NK1 receptors on isolated colonocytes suggests that appropriate elements are present for axon reflex activation of intestinal epithelial cells.

Animals↗