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T G Cooper

Publications and source records attributed to T G Cooper.

At least 199 records · Page 11Linked to original sources
PubMed1990

Secretory proteins from the epididymis and their clinical relevance.

The literature pertaining to epididymal proteins and their functions in fertilization is reviewed. Animal studies have indicated that specific epididymal proteins may be involved in aspects of sperm motility, sperm-zona binding and the acrosome reaction. If analogous proteins in the human exist, use could be made of them in the andrology clinic. Currently, only one specific epididymal protein (alpha-glucosidase) is routinely measured for semen analysis. Glucosidase secretion, in addition to reflecting inflammation of the organ, is used in conjunction with other markers of human fertility to identify patients with ductal occlusion for whom bypass operations may be useful therapy. Glucosidase inhibitors have been used to improve the assay, by establishing true semen blank values, and to quantify histochemical activity in frozen tissue sections. From its localization in the human corpus and cauda epididymidis, neutral glucosidase can not be used to identify occlusion in the proximal regions of the duct. Other proteins may be valuable markers of these regions. In the future, other specific proteins of epididymal origin found in seminal fluid could well illuminate dysfunction of the organ in cases of infertility or be end-points of the disruptive action of drugs aimed at the epididymis.

Animals↗
PubMed1990

The GLN3 gene product is required for transcriptional activation of allantoin system gene expression in Saccharomyces cerevisiae.

We show that mutation at the GLN3 locus results in decreased steady-state levels of DAL7, DUR1,2, CAR1, and URA3 mRNAs derived from cultures grown in the presence of inducer. Basal levels of these RNA species, however, were not significantly affected by a gln3 mutation. The GLN3 product appears to affect gene expression in two ways. The pleiotropic requirement of GLN3 for induced gene expression probably derives from the need of the GLN3 product for inducer uptake into the cell and its loss in gln3 mutants. We also demonstrate that transcriptional activation, mediated by the DAL5 and DAL7 upstream activation sequences, requires a functional GLN3 gene product. This observation identified transcriptional activation as the most likely point of GLN3 participation in the expression of allantoin system genes.

Allantoin↗
PubMed1990

Multiple positive and negative cis-acting elements mediate induced arginase (CAR1) gene expression in Saccharomyces cerevisiae.

Expression of the arginase (CAR1) gene in Saccharomyces cerevisiae is induced by arginine or its analog homoarginine. Induction has been previously shown to require a negatively acting upstream repression sequence, which maintains expression of the gene at a low level in the absence of inducer. The objective of this work was to identify the cis-acting elements responsible for CAR1 transcriptional activation and response to inducer. We identified three upstream activation sequences (UASs) that support transcriptional activation in a heterologous expression vector. Two of these UAS elements function in the absence of inducer, whereas the third functions only when inducer is present. One of the inducer-independent UAS elements exhibits significant homology to the Sp1 factor-binding sites identified in simian virus 40 and various mammalian genes.

Arginase↗
PubMed1990

A cis-acting element present in multiple genes serves as a repressor protein binding site for the yeast CAR1 gene.

Induction of the arginase (CAR1) gene expression in Saccharomyces cerevisiae has previously been shown to require participation of a cis-dominantly regulated upstream repression sequence (URS). Deletion of this element results in high-level expression of the CAR1 gene without inducer. To determine the structure of the CAR1 URS element, we performed a saturation mutagenesis. Results of the mutagenic analysis indicated that the CAR1 URS was a 9-base-pair palindromic sequence, 5'-AGCCGCCGA-3'. A DNA fragment containing this sequence was shown to bind one or more proteins by a gel shift assay. DNA fragments containing point mutations that completely eliminated URS function were not effective competitors in this assay, whereas those which supported URS function were effective competitors. Sequences in the 5'-flanking regions of 14 other genes were found to be homologous to the CAR1 URS. These sequences were shown to support varying degrees of URS function in the expression vector assay, to bind protein as demonstrated by the gel shift assay, and to compete with a DNA fragment containing the CAR1 URS for protein binding. These results indicate that the CAR1 URS element possesses the characteristics of a repressor binding site. Further, they are consistent with the suggestion that sites homologous to the CAR1 URS may be situated in the 5'-flanking regions of multiple unrelated yeast genes. The widespread occurrence of this element raises the possibility that it is the target site for one or more negatively acting general transcription factors.

Arginase↗
PubMed1990

Maintenance of human epididymal epithelial cell function in monolayer culture.

Cells liberated by enzyme treatment of tubules dissected out of human epididymides obtained at castration for prostatic carcinoma were cultured for up to 42 days on permeable supports. Outgrowth and monolayer formation was unrelated to the age of the patient or his treatment with anti-androgens. The cells comprising the monolayer were cuboidal and shorter than those in situ but their ultrastructure was characteristic of epithelial cells. There was no evidence of smooth muscle cells or fibroblast overgrowth. The cells manifested fluid-phase and adsorptive endocytosis from both apical and baso-lateral aspects and released into the medium alkaline and acid phosphatases and N-acetylglucosaminidase.

Acetylglucosaminidase↗
PubMed1990

Re-examination of the presence of alpha-lactalbumin in the epididymis of the rat.

Using an assay for alpha-lactalbumin in which galactosyltransferase activity was stabilized and a tissue phosphatase inhibitor was present, no evidence was found for alpha-lactalbumin-like activity in rat epididymal tissue, epididymal fluids or medium from cultured epididymal epithelial cells with either glucose or N-acetylglucosamine as acceptor. However, when assay conditions were suboptimal, apparent transfer of radioactivity to both acceptors could be demonstrated in the epididymis and other tissues. In these assays the amount of alpha-lactalbumin registered was linearly correlated to the extent of stimulation of alpha-lactalbumin added exogenously to tissue extracts as internal standards. When rete testis fluid from rats was used as source of galactosyltransferase under suboptimal conditions, no transfer to glucose was demonstrable in epididymal fluid and an apparent decreased transfer to N-acetylglucosamine could be explained by increases in (pyro)phosphatase activity. Putative alpha-lactalbumin activity in the epididymis may be an artefact of unoptimized assays.

Acetylglucosamine↗
PubMed1989

Effects of bovine mammary alpha-lactalbumin on hyperactivation and sperm-zona pellucida binding of mouse spermatozoa.

The effects of bovine mammary alpha-lactalbumin on the motility and zona-binding characteristics of mouse sperm were investigated. Two properties of sperm associated with capacitation, hyperactivated motility, and the ability of sperm to bind to the zona pellucida of oocytes were shown to be suppressed by alpha-lactalbumin. These inhibitory effects were not accompanied by changes in the percentage of motile cells or by differences in the velocity parameters of the hyperactivated and non-hyperactivated spermatozoa. Bovine serum albumin prevented and reversed the alpha-lactalbumin-induced suppression of hyperactivation. Sperm-zona pellucida binding was partially restored by lowering the alpha-lactalbumin concentration in the medium in which sperm were allowed to bind to the zona pellucida. The results suggest that mouse sperm are decapacitated by bovine mammary alpha-lactalbumin. The counteracting effect of bovine serum albumin to the suppressive action of alpha-lactalbumin on the flagellum suggests the involvement of a mechanism different from the action of alpha-lactalbumin on the sperm head inhibiting binding to the zona pellucida.

Animals↗
PubMed1989

Immature rat epididymal epithelial cells grown in static primary monolayer culture on permeable supports. I. Vectorial secretion.

A cell culture system is characterized for monolayers of immature rat epididymal epithelial cells grown on permeable supports. Cover of the filters was achieved by days 4-5 and was maintained for 9-12 days. The secretion of acid phosphatase (ACP), alkaline phosphatase (AKP) and N-acetylglucosaminidase (NAG) into apical and basal compartments of culture chambers was monitored with time in culture for cells from the proximal and distal epididymis of 37-day-old animals. There was independent secretion of the three enzymes: secretion of NAG and AKP was mainly apical, that of ACP basal; daily secretion of ACP and AKP was constant throughout culture, that of NAG declined; there was greater secretion of NAG and AKP by cells from the proximal than the distal region. The initial high apical secretion of NAG is thought to reflect loss of enzyme from unattached cells, whereas the later AKP secretion is truly directional. Secretion was not influenced by the enzymes used in cell preparation. The cytotoxic agent Thimerosal inhibited secretion of all enzymes when placed beneath the cultures, indicating that secretion depended on viable cells, but initially stimulated release of AKP when applied above the cells possibly reflecting release from the cell membrane.

Acetylglucosaminidase↗
PubMed1989

Immature rat epididymal epithelial cells grown in static primary monolayer culture on permeable supports. II. Histochemistry and ultrastructure.

N-acetylglucosaminidase (NAG), acid phosphatase (ACP) and alkaline phosphatase (AKP) were localised histochemically in fixed cells from the 37-day-old rat epididymis grown in static monolayer culture for 2-8 days. ACP and NAG were cytosolic enzymes found in perinuclear positions, whereas staining of AKP ws consistent with a membranous position. These enzymes were also examined in frozen tissue sections of the epididymis, from rats of the equivalent age, where NAG had intense activity in both supra- and infra-nuclear cytoplasm and ACP was more active apically. For the first time AKP was localised along basolateral membranes of the epithelium and in the lumen of the mid-caput region. The monolayer in culture was of principal cells only and they maintained their polarity and ultrastructural characteristics, but the height of the cells was reduced compared to that obtained in situ.

Acetylglucosaminidase↗
PubMed1989

Fluid-phase transcytosis in the primate epididymis in vitro and in vivo.

Ligated tubules from the corpus epididymidis of men and monkeys were incubated in medium containing horseradish peroxidase (HRP) as a marker for fluid-phase endocytosis. HRP was localized by light and electron microscopy after 0, 15, 30 and 60 min of incubation. Movement between the cells was prevented by tight junctions, but bypass of this barrier was apparently achieved by an intracellular vesicular mechanism leading to a time-dependent appearance of HRP in the lumen. Uptake of HRP into basal cells and capture by the lysosomal apparatus of principal cells were also observed. HRP-filled vesicles also appeared in the basal, mid and apical cytoplasm of epithelial cells in the caput 1 h after injection of the tracer into the epididymal circulation of the monkey, suggesting that this pathway also operates in vivo.

Aged↗
PubMed1989

Requirement of upstream activation sequences for nitrogen catabolite repression of the allantoin system genes in Saccharomyces cerevisiae.

Synthesis of the transport systems and enzymes mediating uptake and catabolism of nitrogenous compounds is sensitive to nitrogen catabolite repression. In spite of the widespread occurrence of the control process, little is known about its mechanism. We have previously demonstrated that growth of cells on repressive nitrogen sources results in a dramatic decrease in the steady-state levels of mRNA encoded by the allantoin and arginine catabolic pathway genes and of the transport systems associated with allantoin metabolism. The present study identified the upstream activation sequences in the 5'-flanking regions of the allantoin system genes as the cis-acting sites through which nitrogen catabolite repression is exerted.

Allantoin↗
PubMed1989

Identification of sequences responsible for transcriptional activation of the allantoate permease gene in Saccharomyces cerevisiae.

DAL5 is a constitutively expressed allantoin system gene whose product is required for allantoate transport. Its simple pattern of expression prompted us to use this gene for identifying the element(s) that mediates transcriptional activation of allantoin system genes. Deletion analysis of the DAL5 5'-flanking sequences resulted in identification of two small regions required for DAL5 expression. Analysis of these two regions with synthetic oligonucleotides localized the sequences supporting transcriptional activation to two DNA fragments of 10 to 12 base pairs, each containing one copy of the pentanucleotide 5'-GATAA-3'. The 5'-flanking region of DAL5 contained eight copies of this sequence. Synthetic constructions containing single copies of these fragments were unable to support transcriptional activation, while those containing two or more copies supported high-level activation. The 5'-GATAA-3' sequence was also found beneath the footprint of a DNA-binding protein. These observations are consistent with the suggestion that DNA fragments containing the sequence 5'-GATAA-3' play an important role in DAL5 gene expression, probably representing a portion of the binding site for a transcriptional activation factor.

Chromosome Deletion↗
PubMed1989

The DAL7 promoter consists of multiple elements that cooperatively mediate regulation of the gene's expression.

Expression of the allantoin system genes in Saccharomyces cerevisiae is induced by allophanate or its analog, oxalurate. This work provides evidence for the involvement of distinct types of cis-acting elements in the induction process. The first element was found to have the properties of an upstream activation sequence (UAS). This element was localized to a 16-base-pair (bp) DNA fragment containing a short 5-bp sequence that occurred repeatedly in the upstream region of DAL7. When present in two or more copies, the 16-bp fragment supported high-level beta-galactosidase production in a CYC1-lacZ expression vector; there was, however, no response to the allantoin pathway inducer. The second element had the properties of a negatively acting element or upstream repression sequence (URS). This element was localized to a 16-bp DNA fragment containing an 8-bp sequence that was repeated four times in the upstream region of DAL7. A fragment containing the 8-bp repeated sequence placed adjacent to the UAS-containing fragment mediated inhibition of the ability of the UAS to support lacZ expression regardless of whether inducer was present. A third element, designated an upstream induction sequence (UIS), was required for response to inducer. The UIS was localized to a small DNA fragment containing an approximately 10-bp sequence that was repeated twice in the upstream region of DAL7. When a fragment containing the 10-bp repeated sequence was placed adjacent to these UAS and URS elements, the construction (UIS-UAS-URS) supported normal oxalurate-mediated induction of beta-galactosidase synthesis. These data are consistent with the suggestion that multiple, cis-acting elements participate in the induction process.

Allantoin↗
PubMed1989

A gene product needed for induction of allantoin system genes in Saccharomyces cerevisiae but not for their transcriptional activation.

The allantoin-degradative pathway of Saccharomyces cerevisiae consists of several genes whose expression is highly induced by the presence of allophanic acid. Induced expression requires a functional DAL81 gene product. Analysis of these genes has demonstrated the presence of three cis-acting elements in the upstream regions: (i) an upstream activation sequence (UAS) required for transcriptional activation in an inducer-independent fashion, (ii) an upstream repression sequence (URS) that mediates inhibition of this transcriptional activation, and (iii) an upstream induction sequence (UIS) needed for a response to inducer. The UIS element mediates inhibition of URS-mediated function when inducer is present. We cloned and characterized the DAL81 gene and identified the element with which it was associated. The gene was found to encode a rare 3.2-kilobase-pair mRNA. The amount of DAL81-specific RNA responded neither to induction nor to nitrogen catabolite repression. Deletion of the DAL81 gene resulted in loss of induction but did not significantly affect basal level expression of the DAL7 and DUR1,2 genes or the UAS and URS functions present in plasmid constructions. These data suggest that (i) transcriptional activation of the DAL genes and their responses to inducer are mediated by different factors and cis-acting sequences and (ii) the UIS functions only when a wild-type DAL81 gene product is available.

Allantoin↗
PubMed1988

Transcytosis in the epididymis studied by local arterial perfusion.

The transport of protein across the cells of the epididymal epithelium was studied using horseradish peroxidase. Transient vascular perfusion of the epididymis of the rat and golden hamster was achieved by pulsatile retrograde infusion into the testicular artery. Peroxidase was found in the interstitium and in the epithelium, located in vesicles, vacuoles and multivesicular bodies of principal, clear and apical cells. Similar findings were obtained in mice after systemic injection of the tracer. In the rat, discharge to the lumen was confirmed by the appearance of enzyme activity in luminal fluid from the caput epididymidis after local injection. The extent of transport amounted to no more than what has been considered leakage in physiological experiments, and the time-course of appearance complemented that found by electron microscopy. The level of transcytosis after pulsatile administration of peroxidase in vivo, as judged from the occurrence of tracer in the epithelium, was much less than that obtained during constant immersion in vitro. The protein was present in multivesicular bodies of principal cells and in vesicles of clear cells at short times after presentation in vitro, when it could not have arrived by endocytosis from the lumen. This suggests that routing of basal endocytic vesicles to the lysosomal apparatus occurs.

Animals↗
PubMed1988

Structure and transcription of the allantoate permease gene (DAL5) from Saccharomyces cerevisiae.

We determined the nucleotide sequence of the DAL5 gene, which encodes a component of the allantoate transport system. Translation of the sequence revealed that the DAL5 gene product is highly hydrophobic. It possesses an alternating motif of hydrophilic sequences that can potentially be folded into alpha-helices and hydrophobic sequences that can potentially be folded into beta-pleated sheets. These are expected characteristics of an integral membrane protein, which correlate well with DAL5 gene function. S1 protection fragments generated by DAL5 transcripts exhibited high heterogeneity over a 30-base-pair range. This pattern of fragments was not affected by growth conditions of the cells or the conditions of the assay.

Allantoin↗
PubMed1987

Protein transport to the epididymal lumen.

Experiments were performed to clarify the debate over the entry of circulating proteins into the epididymal lumen by use of the marker horseradish peroxidase (HRP). Epididymal tubules from the caput epididymidis of the rat were immersed in medium TC 199 containing HRP (3.5 mg/ml) for 5 min to 3 h at 33 degrees C. Sections were examined for the presence of tracer within the epithelial cells by electron microscopy. From 5 min to 3 h, vesicles containing peroxidase reaction products were found throughout the cytoplasm of the principal cells. Vesicles occurred close to both the basal and apical membranes, and many were found opening into the interstitial space and lumen, depending on the length of incubation. By 5 min labelled vesicles were infrequently found in the apical part of the cells. Reaction product was observed in the epididymal lumen adhering to the microvilli from 30 min of incubation onwards. At all periods of incubation peroxidase was present at the base of the epithelium and between the cells, but it was never found within the tight junctional complexes, and no reaction deposits were found within epithelial cells of tubules incubated in the absence of peroxidase. It is concluded that large molecules leaving the capillaries may enter the epididymal lumen in the caput by means of fluid-phase endocytosis.

Animals↗