Biomedical subjects
C R Brown
Publications and source records attributed to C R Brown.
Development of a maturation antigen on the plasma membrane of rat spermatozoa in the epididymis and its fate during fertilization.
The ontogeny of a surface membrane antigen on rat spermatozoa has been investigated using the monoclonal antibody, 2D6. Using indirect immunofluorescence microscopy the 2D6 antigen was first detected on spermatozoa from the proximal corpus epididymidis; no reaction was present on testicular cells. The 2D6 antibody also bound to spermatozoa flushed from the uterus of mated rats and to a sperm-derived antigen on the surface of newly fertilized eggs. When frozen sections of epididymal tissues were stained with 2D6 monoclonal antibody immunofluorescence was confined to the epithelium lining the duct in the proximal and distal corpus epididymidis. Fluorescence in the tissue was androgen-dependent. Immunoblots of proteins in luminal secretions collected by micropuncture from different sites along the epididymal duct showed that in the proximal corpus epididymidis the 2D6 monoclonal antibody recognized a 32 kD antigen, but in secretions from the distal corpus and cauda epididymidis the monoclonal antibody also recognized antigens with molecular weights of 28, 23 and 20 kD. Immunoblots of proteins from spermatozoa collected from the corpus epididymidis revealed a reaction over a 32 kD antigen, while on spermatozoa from the cauda epididymidis the 2D6 monoclonal antibody recognized only a 23 kD antigen. Two hypotheses are proposed to account for the varied reactivity of the monoclonal antibody and their relative merits are discussed.
Limited proteolysis of the porcine zona pellucida by homologous sperm acrosin.
Boar sperm acrosin isolated by affinity chromatography on p-(p'-aminophenoxypropoxy)benzamidine linked to Sepharose was tested for its proteolytic effect on the zona pellucida of freshly ovulated pig eggs. During 1 h in a physiological medium there was no observable change in the morphology of the zona pellucida but subsequent 125I labelling of the structure followed by electrophoretic analysis revealed that acrosin had asserted a limited and selective proteolysis.
Simultaneous determination of total IgE and allergen-specific IgE in serum by the MAST chemiluminescent assay system.
We have developed a chemiluminescent immunoenzymometric system. The first commercial application of this chemiluminescent assay (CLA) is the measurement of total IgE and allergen-specific IgE in human serum. The CLA system is a second-generation adaptation of the MAST RIA allergy profiling system. The MAST CLA system assay protocol consists of three steps: overnight incubation of serum, a 4-h incubation with enzyme-labeled antibody, and a 30-min chemiluminescent reaction, which produces a visible image (immunograph) on high-speed Polaroid instant film. The densities of the bands produced on the film are quantified with an inexpensive microprocessor-controlled infrared transmittance densitometer. The novel luminogenic substrates used yield a constant light output for over 2 h with an intensity at least 10-fold greater than that of commercial chemiluminescent reagents. The MAST CLA system exhibits sensitivity, specificity, and precision equal to that of the MAST RIA system (r = 0.96 for 40 serum samples analyzed with 25 allergens). As many as 35 different allergens per sample can be quantified in a single assay. The MAST CLA system requires no standard curve or volume-dependent pipetting steps, incorporates both positive and negative controls for each sample, and quantifies allergen-specific IgE at picomolar concentrations.
Diabetes mellitus in a Christchurch working population.
Using criteria recommended by the World Health Organisation, prevalence rates for diabetes mellitus have been determined in a Christchurch working population of adults aged 15-65 years. Ninety-three percent of 1047 workers employed by a large industrial group were screened for diabetes mellitus using 75 G oral glucose tolerance tests. Three point one percent of the survey group were diabetic--1.55% were known to have diabetes and a further 1.55% were new cases. In addition, 0.31% were considered to have gestational diabetes mellitus. Blood glucose elevations not diagnostic for diabetes mellitus were present in another 0.93%. By applying the criteria of the National Diabetes Data Group, slightly different prevalence rates were found--3.0% for diabetes mellitus and 1.14% for impaired glucose tolerance.
Identification of mobile and fixed antigens on the plasma membrane of rat spermatozoa using monoclonal antibodies.
We have used monoclonal antibodies to study the mobility and distribution of three different antigens on the cell surface of rat spermatozoa. We classified two of the antigens (designated 2B1 and 2D6) as 'mobile', since when detected by indirect immunofluorescence they were situated over the entire sperm flagellum and were susceptible to antibody-induced patching. Patching was critically dependent upon antibody concentrations and was much reduced at 4 degrees C. Patching of the 2B1 antigen was not induced by the 2B1 monoclonal antibody alone. Thus, 2B1 antibody labelled directly with fluorescein bound with a uniform distribution over the sperm flagellum, but this uniform fluorescence was made patchy on subsequent incubation in an unlabelled second antibody layer of anti-mouse IgG anti-serum. By 'Western blotting', the 2B1 antigen was found to be located to a 40 kD molecular weight polypeptide. The remaining 'fixed' antigen (designated 1B6) was not susceptible to antibody-induced patching, and was restricted to a discrete domain on the post-acrosomal region of the sperm surface. We discuss the relationship between mobility of sperm surface antigens and their segregation to discrete domains on the plasma membrane.
Changes in plasma membrane glycoproteins of rat spermatozoa during maturation in the epididymis.
Glycoproteins on the plasma membrane of testicular and cauda epididymidal spermatozoa have been labeled with galactose oxidase/NaB [3H]4 and sodium metaperiodate/NaB[3H]4, followed by analysis on SDS polyacrylamide gels. The major glycoprotein labeling on testicular spermatozoa has a molecular weight 110,000 whereas on cauda epididymidal spermatozoa greater than 90% of the radio-label is incorporated into proteins of molecular weight 32,000. These 32,000-mol wt X proteins are homologous with proteins of similar molecular weight purified from the epididymal secretion and which have been shown previously to be synthesized in the caput epididymidis under hormonal control. Immunofluorescence revealed that the 32,000-mol wt proteins are present on the flagellum of mature but not immature spermatozoa and that they have a patchy distribution suggesting that they are mobile within the plane of the membrane. The membrane-bound 32,000-mol wt proteins possess hydrophobic domains as revealed by charge-shift electrophoresis and they also label with a lipophilic photoaffinity probe suggesting that they are in contact with the lipid bilayer. The evidence indicates that there is a considerable reorganization of the molecular structure of the plasma membrane of spermatozoa during maturation in the epididymis and that some of the changes are brought about by a direct interaction with epididymal secretory proteins.
Changes in the protein composition of rat spermatozoa during maturation in the epididymis.
Spermatozoa from the testis and cauda epididymidis were solubilized by detergent treatment and electrophoresis on SDS polyacrylamide gels revealed that the relative amounts of 13 detergent-extractable proteins decreased during passage of spermatozoa through the epididymis, 6 increased, whilst the remainder showed little or no change. Lactoperoxidase-catalysed iodination of plasma membrane proteins showed that the components carrying most of the label in testicular spermatozoa had Mr values of 110 000, 94 000, 84 000, 55 000 and 42 000 whereas on cauda epididymal spermatozoa the Mr values were 47 000, 24 000, 17 000, 14 500 and 13 500. Substantial differences were also noted in the protein composition of rete testis fluid and cauda epididymal plasma. The results support the concept that there is a considerable reorganization of the molecular architecture of the plasma membrane of spermatozoa during maturation in the epididymis.
Purification of mouse sperm acrosin, its activation from proacrosin and effect on homologous egg investments.
When proacrosin from mouse epididymal spermatozoa was activated a single form of acrosin was produced. The enzyme was isolated by gel filtration followed by affinity chromatography using Sepharose-4B linked to an acrosin inhibitor p-(p'-aminophenoxypropoxy)benzamidine. The molecular weight of partly purified acrosin was 53 000 by gel filtration, and of the pure enzyme 39 000 by SDS-polyacrylamide gel electrophoresis. Pure mouse acrosin removed the cumulus oophorus, corona radiata and zona pellucida from the homologous egg. It is proposed that penetration of spermatozoa through egg investments, particularly through the zona pellucida, is a simpler process in the mouse than in the sheep.
Association of epididymal secretory proteins showing alpha-lactalbumin-like activity with the plasma membrane of rat spermatozoa.
Three hormonally regulated proteins with mol.wts of 18 500, 19 000 and 23 000 have been shown to associate with the plasma membrane of spermatozoa during maturation in the epididymis. All three proteins showed some alpha-lactalbumin-like activity, suggesting that they may act in concert with epididymal glycosidases and glycosyltransferases to regulate the modification of sugars on membrane-bound glycoproteins.
Comparison of hearing loss in the first year of employment in workers assigned to noise-hazard and non-noise-hazard areas at the state prison industries.
Experience in hearing loss at Utah State Prison in noise-hazard and non-noise-hazard areas suggested a matched-pair study of these two groups. The study of noise induced hearing loss and its relation to age has become more and more of scientific and economic interest in the past few years. Since the creation of OSHA and the enforcement of its standards, great strides have been undertaken at USP to protect the worker in noise areas, with both noise abatement and hearing protection. The need became apparent for a measurement of this progress and a comparison on hearing loss between the two worker groups. A retrospective chart study was designed utilizing one hundred pairs of workers matched for age, sex and initial hearing loss. The results suggest no significant difference between the two groups during one year of exposure.
Effects of ram sperm acrosin on the investments of sheep, pig, mouse and gerbil eggs.
The effects of various concentrations of highly purified preparations of ram sperm acrosin were tested in a balanced medium on the cumulus oophorus, corona radiata and zona pellucida of sheep, pig, mouse and gerbil eggs. The enzyme had a variety of effects depending upon the species of egg. These effects ranged from complete removal of all the investments from the mouse egg to a failure to remove any of the investments from the sheep egg. The in-vitro autoactivation of ram sperm proacrosin to acrosin was unmodified in the presence of various fluids from the sheep reproductive tract. It is concluded that any role for soluble ram acrosin in sperm penetration of the sheep zona pellucida is likely to be of a synergistic nature.
The location of acrosin and proacrosin in ram spermatozoa.
Explore the source record for details and available documents.
Labelling of membrane glycoproteins on rat spermatozoa collected from different regions of the epididymis.
The major glycoprotein on the plasma membrane of testicular spermatozoa labelled with the galactose oxidase/NaB3H4 technique has mol.wt. 110 000. As spermatozoa pass through the epididymis, labelling of this glycoprotein disappears and is replaced by labelling of a 32 000-mol.wt. protein. The latter protein is a major component of epididymal secretions. The evidence suggests that it is inserted into or absorbed on to the plasma membrane, and since its appearance on spermatozoa correlates with the acquisition of fertilizing capacity it should serve as a good marker for assessing maturation in vitro.
Characterization of hormonally regulated secretory proteins from the caput epididymidis of the rabbit.
1. The incorporation of [35S]methionine into protein was investigated in tissue minces from different regions of the rabbit epididymis incubated in vitro. Rates of synthesis were in the order: epididymal regions 2-5 greater than region 7 greater than region 6 greater than region 1 greater than region 8 greater than ductus deferens greater than ductuli efferentes. 2. Separation of labelled proteins on polyacrylamide gels containing sodium dodecyl sulphate followed by fluorography revealed that one protein (mol.wt. approx. 90 000) was characteristic of region 1, four proteins (one of mol.wt. 54 000 and three of mol.wt. 20 000) were synthesized principally in regions 2-5, and one protein (mol.wt. 22 500) was produced mainly in regions 6, 7 and 8. 3. Castration for 14 days decreased incorporation of [35S]methionine into total protein to less than 10% of that in controls in all regions of the epididymis. However, testosterone treatment for a further period of 14 days restored protein synthesis to normal values in regions 6, 7 and 8, but not in region 1 or regions 2-5. In regions 2-5 the synthesis of three proteins of mol.wt. 20 000 declined after castration, but was not stimulated by exogenous testosterone. Since the 20 000-mol.wt. proteins were major tissue proteins, accounting for 16-25% of the total synthesized, they were used as markers for investigating hormone action in the epididymis. 4. Castration followed immediately by testosterone treatment or ligation of the ductuli efferentes resulted in a decrease in their synthesis, suggesting that they are partially dependent on factors in testicular fluid. Purification and characterization showed them to be acidic glycoproteins with a number of biochemical and immunological properties in common. 5. It is suggested that there is a synergistic action between blood androgens and factors in testicular fluid in regulating protein synthesis in the proximal regions of the rabbit epididymis.
Estradiol-17 beta dehydrogenase from chicken liver.
NADP+-linked estradiol-17 beta dehydrogenase has been purified 300- to 400-fold from cell-free extracts of chicken liver in a 20 to 30% yield by ammonium sulfate precipitation, ion exchange chromatography, and gel filtration. The enzyme is stable for at least 3 months when stored at -20 degrees C in buffer containing glycerol (50%, v/v). Two forms, with molecular weights of 43,000 and 97,000 are present; these show one major band (Rm = 0.27) and one minor band (Rm = 0.25) on polyacrylamide disc gel electrophoresis. (Rm is defined as the ratio of the distance migrated by the protein band to that of the tracking dye.) The species of lower molecular weight is the more active, with apparent Km values for estradiol-17 beta of 25 and 17.3 microM in the presence and absence, respectively, of bovine serum albumin in the assay medium. The apparent Km for NADP+ is 7.7 microM, and the optimum pH for dehydrogenation is 9.9. The lower molecular weight form has a lambda max at 280 nm, a shoulder at 290 nm, and an A 1% 1 cm of 12.1 at 280 nm. The fluorescence spectrum corresponds to that of a tryptophan-containing protein with lambda max at 288 nm. Isoelectric focusing in gel at pH 5 to 8 shows three major bands of pI 6.9, 6.8, and 6.0. Cross-linking with dimethyl suberimidate followed by electrophoresis reveals five bands. The enzyme is affected by thio reagents and possesses no associated estradiol-sensitive transhydrogenase activity.
Two-dimensional spectroscopy of electrophoretic gels.
Optical techniques are described which permit one to analyze two-dimensional electrophoretic gels in a fashion which is analogous to the one-dimensional spectroscopy of solutions. In the methods described, an electrophoretic gel is irradiated with monochromatic light and isozyme patterns are detected by the absorption of light or the fluorescent emission of light. The system described can both generate and detect monochromatic light in a range from 200 to 1100 nm. Without the use of histochemical stains, several isozymes have been visualized by purely optical means. Five methods for the visualization of lactate dehydrogenase and five methods for the demonstration of trypsin isozymes are described. In addition, general methods have been formulated for hydrolases and oxidases. Gel spectroscopy should permit the investigation of a wide range of new isozymes.
Hormonal regulation of protein synthesis in the rat epididymis. Characterization of androgen-dependent and testicular fluid-dependent proteins.
1. Protein synthesis has been investigated in different regions of the rat epididymis by measuring incorporation of [35S]methionine in tissue minces incubated in vitro followed by analysis of labelled proteins on polyacrylamide gels containing sodium dodecyl sulphate. Rates of synthesis were highest in the proximal cauda > distal cauda > initial segment > ductuli efferentes > corpus > distal caput > proximal caput. One protein (mol.wt. 23 000) characterized the initial segment, three proteins (mol.wts. 18 500, 19 000 and 32 000) the caput and one protein (mol.wt. 47 000) the cauda. 2. After castration, [35S]methionine incorporation in all regions of the epididymis was reduced to < 10% of that in normal animals but could be restored to control levels within 5 days by testosterone treatment. Other steroids (corticosterone, oestrogen or progesterone) were ineffective. 3. The synthesis of the 18 500, 19 000, and 32 000 mol.wt. proteins in the caput and the 47 000 mol.wt. protein in the cauda were preferentially regulated by androgens, whilst the synthesis of 23 000 and approx. 80 000 mol.wt. proteins in the initial segment was dependent upon factors present in testicular fluid. 4. The androgen-dependent and testicular fluid-dependent proteins were major components of epididymal secretion. Purification and characterization of the 18 500, 19 000, 23 000 and 32 000 mol.wt. proteins showed them to be acidic glycoproteins with a carbohydrate content of 7.6-13.2%. The 47 000 mol.wt. protein, on the other hand, is highly basic. 5. A possible role for these proteins in the acquisition of motility, fertilizing capacity and storage of spermatozoa in the epididymis is discussed.