Biomedical subjects

D E Brooks

Publications and source records attributed to D E Brooks.

At least 91 records · Page 5Linked to original sources

PubMed1987

Androgen-regulated epididymal secretory proteins associated with post-testicular sperm development.

A considerable body of information has now been assembled with regard to the major androgen-dependent secretory proteins of the rat epididymis. The proteins have been purified, specific antibodies have been developed against them, and their primary structure has been determined from cDNA clones. The antibody and cDNA probes have, in turn, been used to study the androgenic regulation of the synthesis and secretion of the proteins, the distribution of the proteins in various tissues and animal species, and the association of the proteins with the sperm surface during epididymal maturation. Despite this intensive effort, the precise physiological functions of the proteins still remain obscure, although circumstantial evidence indicates that glycoproteins D and E may be associated with surface receptors responsible for gamete recognition. Elucidation of the physiological roles of the proteins is clearly the area that warrants the greatest attention in future work.

Androgens↗

PubMed1987

The major androgen-regulated secretory proteins of the rat epididymis bear sequence homology with members of the alpha 2u-globulin superfamily.

The primary amino acid sequence of 18.5 kDa androgen-dependent secretory proteins of the rat epididymis has been compared with protein data-base sequences. The comparison has revealed that the epididymal proteins belong to the alpha 2u-globulin superfamily which includes human, rat and mouse urinary proteins, ungulate beta-lactoglobulins, and serum retinol-binding proteins. The homology suggests that the epididymal proteins may function to transport retinoids within the male reproductive tract. The androgen-dependent characteristics of the proteins and their tissue-specific nature have been ascertained by use of the protein's cDNA as a probe. The mRNA for the proteins was found in the epididymis of the rat and mouse, but not the epididymis of guinea-pig, rabbit, bull, boar, or ram.

Alpha-Globulins↗

PubMed1986

Molecular cloning of the cDNA for androgen-dependent sperm-coating glycoproteins secreted by the rat epididymis.

cDNA clones coding for two closely related androgen-dependent sperm-coating glycoproteins secreted by the rat epididymis were selected by screening an epididymal cDNA library constructed in lambda gt 11 with affinity-purified antibody directed against the glycoproteins. The largest clone of 956 nucleotides provided coding information for a protein of 246 amino acids of which the first 19 residues comprise a putative signal peptide sequence which when cleaved would produce a mature protein of 227 residues and a molecular mass of 26 kDa. Confirmation of the identity of the clone was provided by a match between the amino acid sequence predicted from the cDNA sequence and the actual amino acid sequence determined for a tryptic peptide fragment of one of the pure glycoproteins. It is probable that the primary amino acid sequence of the two glycoproteins is identical. Northern blot and slot-blot analysis revealed that the mRNA for the glycoproteins is approximately 1250 nucleotides long and that the concentration of the mRNA in the epididymis is androgen-dependent. The glycoproteins and their mRNAs were unique to the epididymis as determined by Western and Northern blots, respectively, since signals were absent from skin, brain, liver, kidney, heart, skeletal muscle and testis. Cross-reacting proteins of slightly smaller apparent molecular mass were detected in extracts of mouse and guinea-pig epididymis, but not rabbit or bull epididymis. Comparison with existing protein data bases revealed that the epididymal glycoproteins display significant sequence homology with yeast carboxypeptidase Y.

Amino Acid Sequence↗

PubMed1986

Molecular cloning of the cDNA for two major androgen-dependent secretory proteins of 18.5 kilodaltons synthesized by the rat epididymis.

cDNA clones representing two closely related androgen-dependent secretory proteins of 18.5 kDa were selected by screening a rat epididymal cDNA library constructed in lambda gt 11 with affinity-purified antibody directed against the 18.5-kDa proteins. The entire amino acid sequence of the 18.5-kDa secretory proteins and a putative signal sequence of 18 amino acids was derived from 682 base pairs of the nucleotide sequence of overlapping cDNA clones. Confirmation of the identity of the cDNA clones was obtained by matching a partial amino acid sequence obtained for the N terminus of the pure protein with that of the sequence derived from the nucleotide code of the cDNA. Evidence is presented that the difference between the two closely related proteins may be associated with differential post-translational modification of the N terminus of the protein following cleavage of the signal sequence. Northern blot analysis revealed that the mRNA for the proteins is approximately 850 nucleotides long and that the concentration of the mRNA in the tissue is androgen-dependent. The proteins and their mRNAs were restricted to the epididymis as determined by Western and Northern blots, respectively, since signals were absent from the skin, brain, liver, kidney, heart, skeletal muscle, and testis. With the exception of a weak cross-reaction with mouse epididymis, the proteins were not detected by Western blots of extracts of guinea pig, rabbit, or bull epididymis. The two proteins account for a substantial proportion of the total protein in epididymal luminal fluid and become incorporated as components of the sperm plasma membrane where they may play a specific role in the post-testicular phase of sperm development.

Amino Acid Sequence↗

PubMed1986

Composition of gangliosides from ovine testis and spermatozoa.

Gangliosides were extracted and purified from ovine testis and ejaculated spermatozoa which contained, respectively, 57 and 9 nmol lipid-bound sialic acid per gram wet weight. Fourteen gangliosides were resolved by thin-layer chromatography of testicular gangliosides, of which eleven were purified in sufficient quantity to enable a complete compositional analysis of the carbohydrate residues to be performed. None of the gangliosides contained fucose, but several contained N-glycolylneuraminic acid as a component of the sialic acid species. Relative migration on thin-layer chromatograms relative to known standards, compositional analysis, and selective degradation by specific enzymes were used as the basis for identification. Testis contained members of the ganglio series (GM1, GD1a, GD1b, GT1b, GQ1b), hematoside series (GM3, GD3), and sialosylparagloboside in the molar ratio of 54:40:6, respectively. Testicular GM3, GM1, GD3, GD1a, GD1b and GT1b ran as double bands on thin-layer chromatography which could be accounted for by observed differences in the fatty acid moiety. In addition, the slower migrating band of each pair contained some or all of its sialic acid residues as N-glycolylneuraminic acid, whereas the faster migrating band contained exclusively N-acetylneuraminic acid, except for GM3 where N-acetylneuraminic acid was the sole species in both bands. Thin-layer chromatography of sperm gangliosides revealed seven bands comigrating with equivalent testicular gangliosides. These coincided with the slower migrating bands of testicular GM3, GM1, GD3, GD1a, both bands of GD1b, and possibly both bands of GT1b. Sperm contained only trace amounts of sialosylparagloboside but, in addition, two unidentified bands which were absent from testis were also observed. The molar ratio of the ganglio series to the hematoside series in sperm was 42:58 with GM3 accounting for 42% of total gangliosides.

Animals↗

PubMed1986

Surface charge and hydrophobic properties of fresh and cryopreserved blood phagocytes as determined by partition in two-phase aqueous polymer systems.

Cell partitioning in two-phase aqueous polymer systems was used to examine hydrophobic and surface charge-related membrane properties of fresh and cryopreserved human blood phagocytes. This technique is highly sensitive to cell surface characteristics, and the partition behavior depends exponentially on the membrane properties involved. The transition from fresh to cryopreserved and reconstituted cells was accompanied by a significant loss of net negative charge without detectable alteration in hydrophobic membrane properties as detected by the partition technique. The partition coefficient (PC), which is the proportion of cells partitioning into the upper phase, when measured for fresh cells mixed with dimethyl sulfoxide (Me2SO) and cryopreserved leukocytes. No difference was detected between the PCs of the total leukocytes and phagocytes as determined by differential leukocyte counts of the upper polymer phase. The significantly reduced PC of cells prepared by dextran (Dx) separation in both charge-sensitive and -insensitive systems is attributable to the capacity of Dx to adsorb, in part irreversibly, to cells so that those carrying Dx tend to partition with Dx in the lower phase. These results serve to illustrate the utility of partitioning as a highly sensitive method to probe leukocyte surface membrane properties.

Blood Preservation↗

PubMed1986

Synthesis and application of a poly(ethylene glycol)-antibody affinity ligand for cell separations in aqueous polymer two-phase systems.

The possibility of producing biospecific affinity ligands for separating cells in two polymer aqueous phase systems on the basis of cell surface antigens was investigated. Rabbit anti-human erythrocyte IgG was reacted with cyanuric chloride-activated monomethyl poly(ethylene glycol) (PEG) fractions (molecular weights approximately 200, 1900, and 5000) at various molar ratios of PEG to protein lysine groups. The partition coefficient of the protein in a Dextran/PEG two-phase system increased with increasing degree of modification and increasing PEG molecular weight. There was a concomitant loss in ability to agglutinate human erythrocytes. The ability of the modified IgG to bind to a DEAE-cellulose column was almost eliminated by reaction with the PEG 5000, and was decreased to a lesser extent by PEG 1900. This PEG 1900-modified IgG substantially increased the partition of fresh or fixed human erythrocytes into the PEG-rich phase of a suitable phase system, while having no effect on rabbit cell partition. The partition increase could be inhibited by unmodified anti-human red cell IgG but not by nonspecific unmodified human IgG, demonstrating that the ligand effects were specific for the cell type against which the antibody was raised. A mixture of rabbit and human erythrocytes, which ordinarily have very similar partitions in the phase systems used, could be separated on a countercurrent distribution apparatus using the modified IgG. These results demonstrate the feasibility of producing immunologically specific affinity partition ligands for cell separation.

Affinity Labels↗

PubMed1986

A fluorometric assay of the degree of modification of protein primary amines with polyethylene glycol.

An alternative primary amine assay utilizing 4-phenylspiro[furan-2(3H),1'-phthalan]-3,3'-dione (fluorescamine) is compared with the frequently applied trinitrobenzenesulfonic acid assay as a means of estimating protein primary amine modification with cyanuric chloride attachment of polyethylene glycol (PEG). The results of the two assays are compared for three different proteins and several advantages of the fluorescamine assay are emphasized. In particular, the fluorescamine assay was found to be unaffected by free PEG up to 0.08% in the assay mixture. The fluorescamine assay can be used to measure the partition coefficients of native or PEG-modified protein in two-phase aqueous polymer systems.

Amines↗

PubMed1986

Heterogeneity in the surface properties of B16 melanoma cells from sublines with differing metastatic potential detected via two-polymer aqueous-phase partition.

When mixed in aqueous solution at low concentrations, the neutral polymers dextran and poly(ethylene glycol) (PEG) rapidly form a two-phase system, consisting of a dextran-enriched lower phase and a PEG-enriched upper phase. Two B16 mouse melanoma cell lines, B16-F1 (low lung colonizing capability) and B16-F10 (high lung colonizing capability) were found to partition differentially into the upper phase in a variety of two-phase systems. Upper-phase partition depends primarily on either hydrophilic (i.e., surface charge density) or hydrophobic (i.e., affinity for the hydrocarbon chain of a PEG-fatty acid ester) cell surface properties, depending on the system used. In single-step partition studies, cells of the B16-F10 subline displayed a greater preference than B16-F1 cells for the upper phase in the hydrophilic system and less preference in systems sensitive to hydrophobic properties. Countercurrent distribution (CCD) experiments, performed with [125I]deoxyuridine DNA-labelled cells, were consistent with single-step partition results. These CCD results demonstrated that B16-F10 cells exhibited greater DNA synthesis than B16-F1 cells and that considerable heterogeneity, in both hydrophobic and hydrophilic surface properties, was present in subpopulations of cells of both sublines. The data also showed considerable enrichment of 125I-specific cell activity in certain sections of the distributions, indicating that differences in cellular DNA synthesis are reflected in the surface properties to which partition is sensitive.

Animals↗

PubMed1986

Protein composition of the luminal fluid and protein synthesis in vitro by the oviducts and uteri of ovariectomized, pro-oestrus and 5-day pregnant rats.

Serum, oviducal and uterine luminal fluid proteins from rats at three different physiological stages of reproduction were analysed by polyacrylamide gel electrophoresis. Protein synthesis and secretion were also studied by short-term incubation of tissue pieces in vitro with radioactive leucine. In ovariectomized animals and 5-day pregnant animals, uterine weight and the protein content of the luminal fluid was significantly lower than in pro-oestrus animals. These differences were much less marked in the oviduct. The protein patterns of oviducal fluid and uterine fluid were significantly different from those of blood serum. However, certain proteins, such as those of 51, 56, 68 and 80 K are clearly derived from blood serum. The protein profile of oviducal fluid and extracts of oviducal tissue showed no obvious differences at the three physiological stages. However, some differences at the three physiological stages were evident in proteins of uterine fluid and in extracts of uterine tissue.

Animals↗

PubMed1986

Differential partition of virulent Aeromonas salmonicida and attenuated derivatives possessing specific cell surface alterations in polymer aqueous-phase systems.

Two-polymer aqueous-phase systems were used to compare via partitioning the surface properties of strains of the fish pathogen Aeromonas salmonicida which differed in their ability to produce the surface protein array known as the A layer and in their ability to produce smooth lipopolysaccharide. In these two-phase systems, biological particles are known to partition between the phases in a manner related to a variety of surface properties, including hydrophobicity, charge, and lipid composition. Both the presence of the superficial protein layer and the O polysaccharide chains of lipopolysaccharide were shown to play an important role in the partitioning behavior of A. salmonicida cells. The presence of the A layer, which is crucial to the virulence of A. salmonicida, appeared to decrease the surface hydrophilicity of this pathogen and to increase, in a somewhat specific manner, its surface affinity for fatty acid esters of polyethylene glycol. The ability of two-polymer aqueous-phase systems to differentially partition A. salmonicida cells on the basis of differences in surface architecture suggests their general usefulness for the analysis of surface properties important in bacterial virulence and should permit their use in the selection of strains and mutants exhibiting specific surface characteristics.

Aeromonas↗

PubMed1986

Heat-denatured albumin-coated Dacron vascular grafts: physical characteristics and in vivo performance.

Dacron fabrics with a wide range of porosities were autoclaved for 3 minutes after being soaked in serum, 5% albumin, or 25% albumin. Porosity of compound Dacron grafts made with 25% albumin was less than 1 ml/min/cm2 regardless of the fabric base, whereas porosity of grafts made with serum or 5% albumin was proportional to the porosity of the base fabric. Porosity of the compound grafts remained stable for more than 48 hours and to pressure greater than 450 mm Hg, if the grafts were kept moist. Tubes of Marlex mesh coated with heat-denatured albumin, implanted as infrarenal aortic replacements in dogs, showed complete albumin absorption by 3 weeks. However, perigraft tissue reaction and graft incorporation were minimal and extensive false aneurysm formation resulted. Knitted filamentous Dacron 6 mm tubes coated with heat-denatured albumin were implanted as iliofemoral bypass grafts in 12 dogs, with blood-preclotted knitted filamentous Dacron grafts implanted as contralateral control grafts. Comparison of the albumin-coated grafts with the blood-preclotted control grafts showed no differences in healing or patency at 4 to 6 months. Heat-denatured 25% albumin forms a strong and hemostatic coating regardless of fabric base. Albumin-Dacron compound grafts are easily and rapidly made in the operating room, handle well, and are suitable for large and medium-sized arterial replacements without changes in healing or patency. Because of slow tissue incorporation, however, albumin-coated knitted Dacron grafts should be avoided in patients who require long-term anticoagulation therapy.

Albumins↗

PubMed1986

Unconventional aqueous humor outflow of microspheres perfused into the equine eye.

Normal drainage of aqueous humor from the anterior chamber of the equine eye occurred through conventional and unconventional routes. To determine the degree of uveoscleral outflow in the pony, 1- and 3-microns (diam) microspheres were perfused through the anterior chamber for 60 and 90 minutes. Two eyes were treated with pilocarpine before perfusion of spheres to observe any effect on outflow. Presence of spheres was detected by scanning electron microscopy and verified by light microscopy. By 60 minutes of the perfusion, 1- and 3-microns spheres thoroughly infiltrated the anterior uveal trabecular meshwork. The 3-microns spheres were notably excluded from the corneoscleral trabecular meshwork, whereas the 1-micron spheres packed into the corneoscleral trabecular meshwork and mixed with the angular aqueous plexus and intrascleral plexus vessels. After 90 minutes, 1- and 3-microns spheres penetrated the prominent supraciliary space and mixed with the suprachoroidea of the midchoroid. The 1-micron spheres infiltrated tissues more extensively than did the 3-microns spheres, packing into the anterior meshwork and supraciliary space and also moving as far posterior as the suprachoroidea of the peripapillary retina. Spheres also entered the iris, mostly at the root. Several 3-microns spheres moved into the interstitial spaces around axon bundles and vessels in the iris. The 1-micron spheres packed into these spaces in the iris root and were observed in the lumens of iris vessels. Single 1-micron spheres were observed in the lumens of choroidal vessels posteriorly. The present study supported evidence for uveoscleral and uveovortex flow in the horse and indicated that it may be more extensive than that in other species investigated (ie, the dog).

Animals↗

PubMed1986

Surgical correction of eyelid disfigurement in a stumptailed monkey (Macaca arctoides).

Self-aggression in an adult male stumptailed monkey (Macaca arctoides) resulted in severe lower eyelid distortion, conjunctivitis and epiphora. The behavior ceased with a change in environment, but the eyelid defect, conjunctivitis and epiphora persisted, requiring corrective surgery. Surgical correction was partially successful, although the animal died due to unrelated medical problems before final correction could be accomplished.

Animals↗

PubMed1985

Uterine rupture as a postpartum complication in two mares.

Uterine rupture was diagnosed in 2 postpartum mares with hemorrhagic vaginal discharge. Both mares had abdominal pain, as evidenced by pawing, kicking at the abdomen, or attempting to roll. Peritoneal fluid analysis was useful in establishing a diagnosis. One mare had many RBC in the peritoneal fluid and was anemic; this mare was managed medically with oxytocin, antibiotics, and blood transfusion. The mare was able to raise her foal to weaning age. The second mare had many RBC, degenerate neutrophils, and intracellular and extracellular bacteria in peritoneal fluid. Surgical repair of the uterus was performed, and the mare was treated for peritonitis. The mare later became pregnant.

Animals↗

PubMed1985

Characterization of a 22 kDa protein with widespread tissue distribution but which is uniquely present in secretions of the testis and epididymis and on the surface of spermatozoa.

The purification is reported of a 22 kDa protein which was first identified as one of the major components of the luminal secretion of the rat testis and epididymis. Antibodies against the 22 kDa protein cross-reacted with a protein of the same molecular weight in cytosolic extracts of other tissues from both male and female rats. However, since the protein could not be detected in blood, peritoneal fluid, saliva, milk, uterine fluid, seminal vesicle secretion, coagulating gland secretion or prostatic secretion, it would appear that the testis and epididymis may be unique in containing the protein in a soluble form within their luminal secretions. Proteins with slightly lower molecular weight were detected by the antibodies in cytosolic extracts of tissues from other animals (mice, rabbits, sheep, pigs, cattle), indicating that the protein may be conserved in a variety of species. However, in contrast to the rat, the protein was apparently not present in the testicular and epididymal secretions of these species. In addition to the occurrence of the 22 kDa protein as a soluble moiety in rat testicular and epididymal fluids, the protein was also located on sperm plasma membranes where its distribution was restricted to the surface of the flagellum. Amongst sperm surface proteins, the 22 kDa protein was the major protein containing sulphydryl groups and one of the major entities containing disulphide bonds. These properties may be of importance in the maintenance of sperm viability.

Animals↗

PubMed1985

Differential effects of gonadectomy on sensitivity to testosterone of brain centres associated with gonadotrophin negative feedback and with mating behaviour in rams.

Castrated sheep were used to study the effects of gonadectomy on sensitivity to testosterone of brain centres associated with gonadotrophin negative feedback and with mating behaviour. In the first experiment serum LH and FSH concentrations were determined in intact rams, recently castrated (2 days and 3 weeks) and long-term castrated animals (greater than 2 years, wethers) during intravenous testosterone infusion at physiological and supraphysiological levels. In intact rams, testosterone infusions effectively suppressed serum LH whilst FSH levels were suppressed only after prolonged infusion at the supraphysiological dose. Recently castrated sheep, which had higher gonadotrophin levels than intact rams, were less sensitive to testosterone feedback. Neither rate of testosterone infusion had any effect on the raised gonadotrophin levels in wethers. In a second experiment gonadotrophin concentrations and mating behaviour were determined in wethers bearing subdermal polydimethylsiloxane implants of testosterone, dihydrotestosterone and oestradiol. Testosterone implants stimulated mating behaviour in all wethers but suppressed gonadotrophins in only a proportion (three out of seven) of the animals. Both oestradiol and dihydrotestosterone suppressed LH and FSH in all wethers, whilst oestradiol, but not dihydrotestosterone, also stimulated mating behaviour. The present findings indicate that testosterone imposes continuing negative feedback on gonadotrophin secretion and that changes in the gonadotrophin regulatory system, which lead eventually to a loss in sensitivity to testosterone feedback, develop soon after gonadectomy. The results also provide the first direct evidence that longterm gonadectomy in male sheep has differential effects on sensitivity to testosterone of brain centres associated with gonadotrophin negative feedback and with mating behaviour.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

Conversion of plasma progesterone to deoxycorticosterone in nonpregnant Macaca mulatta: an animal model for the study of extraadrenal steroid 21-hydroxylase activity.

We have reported previously that during the third trimester of pregnancy a significant portion of DOC in the maternal compartment arises by extraadrenal, 21-hydroxylation of maternal plasma progesterone. Moreover, the increase in DOC observed in plasma of women during the luteal phase of the ovarian cycle, when plasma concentrations of progesterone are elevated, can be attributed to increased DOC formation in extraadrenal sites. In the present study, we sought to ascertain if progesterone is converted to DOC in adult, nonpregnant, female rhesus monkeys to establish this species as an animal model for further investigation of extraadrenal mineralocorticosteroid biosynthesis. We measured the transfer constant of conversion of progesterone to DOC in plasma [( rho]P-DOCBB) from the 3H: 14C ratio of DOC in plasma after a 4 h infusion of [3H]progesterone and [14C] DOC into anesthetized monkeys. The [rho]P-DOCBB was 0.014 +/- 0.004 (mean +/- SEM, N = 7) in the animals studied. The values obtained for [rho]P-DOCBB ranged from 0.006 to 0.04 among the animals studied, a range of values similar to that observed in pregnant and nonpregnant women, men, and adrenalectomized persons. On the basis of these data, we conclude that the rhesus monkey may be an appropriate animal model for further study of extraadrenal steroid 21-hydroxylase activity.

Adrenal Glands↗