Biomedical subjects
K D Roberts
Publications and source records attributed to K D Roberts.
Protein synthesis and secretion in the human epididymis and immunoreactivity with sperm antibodies.
The synthesis and secretion of proteins in the different regions of the human epididymis were studied in vitro. Epididymal tissues obtained from patients undergoing castration for prostatic carcinoma or from cadavers were incubated in the presence of [35S]methionine, and the resulting radiolabeled proteins were analysed on SDS-PAGE. The corpus region was found to be the most active segment in total protein synthesis. Significant qualitative and quantitative changes were observed in the pattern of proteins secreted from the different epididymal regions. To establish those epididymal proteins that interact with maturing sperm, the secreted products were immunoreacted with antibodies raised against a Triton X-100 extract of ejaculated human sperm heads. The antibodies react mainly with the head region of ejaculated spermatozoa as judged by indirect immunofluorescence. Protein A-gold labeling of freeze-fracture images showed gold particle distribution on the sperm plasma membrane. Western blot analysis of the secreted proteins revealed four bands (66, 37, 32, and 29 kDa) in the proximal regions and six additional bands (80, 76, 48, 27, 22, and 17 kDa) in the distal part of the epididymis. Immunoprecipitation of the secreted proteins with these antibodies revealed six radioactive bands of 170, 80, 76, 60, 48, and 37 kDa, which indicates that certain proteins of epididymal origin bind to the sperm plasma membrane.
Identification of heparin-binding proteins in bovine seminal plasma.
A group of four similar proteins, BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa, represent the major acidic proteins found in bovine seminal plasma (BSP). These proteins are secretory products of the seminal vesicles; they bind to spermatozoa upon ejaculation and could represent decapacitation factors. It has been shown that the glycosaminoglycans present in the female reproductive tract are involved in the capacitation of spermatozoa. Therefore, it was of interest to investigate whether BSP-A1, -A2, -A3, and -30-kDa proteins of bovine seminal fluid interact with heparin. Chromatography of alcohol precipitates of bovine seminal fluid on a heparin-Sepharose column resolved these proteins into three peaks. Peaks 1 and 2 (retarded proteins) were eluted upon extensive washing of the column with 0.05 M phosphate buffer, pH 7.4 (equilibrating buffer), and accounted for approximately 25% of the applied proteins. Proteins in peak 3 represented adsorbed proteins and were eluted with phosphate buffer containing 1 M NaCl. Proteins in each peak were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Peak 1 contained proteins with molecular weights ranging from 8 to 350 kDa, peak 2 contained a single protein with a molecular weight of 14 kDa, and peak 3 contained proteins with molecular weights of 15.5, 16, 25, and 30 kDa. The proteins in peak 3 were further resolved into unadsorbed (peak 4) and adsorbed (peak 5) proteins on a gelatin-Agarose column. Separation of the proteins of peak 3 and peak 5 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reducing agents followed by transfer to nitrocellulose and probing with antibodies against the previously well-characterized BSP proteins indicated the presence of BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Safety of protamine sulfate administration in vasectomized men.
The majority of vasectomized men develop antibodies against different sperm antigens, including protamine. Due to the fact that salmon protamine is used clinically for heparin reversal and that a cross-reactivity has been observed between human and salmon protamine, vasectomized men may be at risk for adverse reactions to protamine sulfate. In order to explore this possibility, serum samples were analyzed for the presence of anti-sperm and anti-protamine antibodies in 20 vasectomized and 20 non-vasectomized men (controls) about to undergo major heart surgery requiring heparin reversal. The patients were closely monitored for any possible reaction following the injection of protamine. Anti-sperm antibodies were detected in the serum of 15 of the vasectomized men whereas only two of the non-vasectomized subjects had this type of antibody. Twenty-five percent of the vasectomized men presented with anti-protamine antibodies; in the control group, none of the patients had developed this type of antibody. Following the administration of protamine sulfate, none of the patients in either group showed any adverse reaction to the drug. It is concluded that vasectomized men are not at increased risk towards adverse reactions following the injection of protamine sulfate.
Hepatic tumour resection with profound hypothermia and circulatory arrest.
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Colon interposition in children.
Since 1960 we have performed 80 colonic interpositions on 79 children. We present a retrospective review of their progress. Sixty-nine interpositions were for long-gap oesophageal atresia, while the remainder followed oesophagectomies, eight for strictures and two others. Overall, the transthoracic route was used in 69 per cent of cases, while 29 per cent were retrosternally placed and one was retropleural. The mean follow-up was 11.08 years. The mortality rate was 12 per cent, with one-third of deaths unrelated to the operation. Graft failure, which occurred on eight occasions (10 per cent), was due to ischaemia in four, intractable stricture in two and stomal ulceration in two. Leakage from the proximal anastomosis occurred in 31 per cent of cases and stricture formation in 27 per cent. Thirty per cent of patients complained of acid reflux, of whom one-third developed stomal ulceration. We present data concerning other complications, including long-term recurrent respiratory infections, malabsorption, gastrointestinal haemorrhage, diarrhoea, intestinal obstruction and redundancy of the graft. Staging the procedure did not affect the outcome, however the higher incidence of serious complications encountered following retrosternal interposition finally led us to abandon this procedure in favour of the transthoracic route. There has been little improvement in the growth rate, but in terms of swallowing ability the outcome was satisfactory in 94 per cent of cases.
Detection of prophospholipase A2 in human spermatozoa.
Prophospholipase A2 (proPA2) has been isolated from human spermatozoa after acid extraction and chromatography on hydrophobic WP-Butyl (C4) and ion-exchange (SP 5PW) columns. The addition of benzamidine, a noncompetitive synthetic trypsin inhibitor, to semen samples has kept a portion of the sperm phospholipase A2 (PA2) in its zymogen form and allowed its isolation after acid extraction. When radioactive phosphatidylcholine (PC) or phosphatidylethanolamine (PE) were used as substrates, an identical elution profile of this enzyme was obtained on a C4 column. The proenzyme was separated from active PA2 on the C4 column. Human sperm proPA2 exhibited a less cationic charge than active PA2 on the SP 5PW column. Porcine pancreatic proPA2 had the same chromatographic behavior on high performance liquid chromatography (HPLC) (SP 5PW) as human sperm proPA2. The purification procedure resulted in the isolation of proPA2 which, upon activation by proteolysis, presented the same chromatographic elution profile on HPLC as active PA2 of human spermatozoa and porcine pancreas. Thus, a zymogen form of PA2 exists in human spermatozoa.
Activation of phospholipase A2 of human spermatozoa by proteases.
The effect of various proteases (kallikrein, plasmin, and trypsin) on sperm phospholipase A2 activity (PA2: EC 3.1.1.4) has been studied. The addition of trypsin to spermatozoa, isolated and washed in the presence of the protease inhibitor benzamidine, increased PA2 activity optimally with trypsin concentrations of 1.0-1.5 units/assay. In kinetic studies, all of the above proteases stimulated the deacylation of phosphatidylcholine (PC); in fresh spermatozoa, trypsin showed a higher activation potential than kallikrein or plasmin. In the presence of benzamidine, the activity remained at basal levels. Endogenous protease activity due to acrosin (control) resulted in an increase in PC deacylation compared to the basal level. The maximum activation time of PA2 activity by proteases was 30 min. Natural protease inhibitors (soybean trypsin inhibitor and aprotinin) kept the PA2 activity at basal levels and a by-product of kallikrein, bradykinin, did not significantly affect the control level. Protein extracts of fresh spermatozoa exhibited the same pattern of PA2 activation upon the addition of proteases, thus indicating that the increase in PA2 activity was not merely due to the release of the enzyme from the acrosome. All of these findings suggest the presence of a precursor form of phospholipase A2 that can be activated by endogenous proteases (acrosin) as well by exogenous proteases present in seminal plasma and in follicular fluid (plasmin, kallikrein). Thus, this interrelationship of proteases and prophospholipase A2 could activate a dormant fusogenic system: the resulting effect would lead to membrane fusion by lysolipids, key components in the acrosome reaction.
Evidence for two forms of phospholipase A2 in human semen.
The molecular weight of the active unit of phospholipase A2 (PA2) in human seminal plasma and spermatozoa was determined using the radiation inactivation technique. Fresh spermatozoa possess more than one form of PA2 activity as judged by the biphasic nature of the curve obtained during enzyme inactivation. However, when stored frozen for several months followed by a period of heating for 60 min at 60 degrees C prior to irradiation, the sperm exhibited PA2 activity, which corresponded to a single low molecular mass form of 12,000 d when radioactive phosphatidylcholine (PC) was used as substrate and 8,000 d when radioactive phosphatidylethanolamine (PE) was used as substrate. In fresh seminal fluid, only one active form of PA2 was detected as judged by the linear nature of the curve obtained during enzyme inactivation by irradiation. Using PC as substrate, the active unit was again estimated to be 12,000 d, whereas it corresponded to 18,000 d when PE was used. The PA2 activity associated with normal spermatozoa exhibited a 60% decrease in activity after storage at -20 degrees C for 48 hr followed by a heating period of 10 min at 60 degrees C. Long-term storage of spermatozoa at -20 degrees C also resulted in a similar decrease in the deacylation of PC. No further loss of activity was observed during subsequent heat treatment at 60 degrees C. Seminal plasma, however, showed no loss of activity following short (48 hr at 4 degrees C or -20 degrees C) or long-term storage and subsequent heat treatment. Thus, the behavior of PA2 when the effect of temperature was studied and in radiation inactivation experiments indicates that the low molecular weight component in the seminal plasma as well as in spermatozoa is temperature resistant. However, in fresh spermatozoa, a second form of PA2 was found and was sensitive to changes in temperature.
Identification of sterol acceptors that stimulate cholesterol efflux from human spermatozoa during in vitro capacitation.
The nature of cholesterol-binding proteins acting upon human spermatozoa during in vitro capacitation was determined by measuring the efflux of [3H]cholesterol and of [3H]cholesteryl sulfate from labeled spermatozoa. Efflux of [3H]sterols was stimulated when the labeled gametes were incubated in Ham's F-10 medium supplemented with female serum or follicular fluid. Upon centrifugation of capacitated spermatozoa and application of the supernatant to density-gradient ultracentrifugation for lipoprotein analysis, both [3H]cholesterol and [3H]cholesteryl sulfate were found to be carried by very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), high-density lipoproteins (HDL), as well as the albumin fraction (d greater than 1.21) in serum. When the capacitation medium was supplemented with follicular fluid, the [3H]sterols were bound to HDL's and to the albumin fraction; when the latter fraction was analysed by molecular sieve chromatography, 60-70% of the radioactivity eluted in fractions with a mean molecular weight corresponding to that of human serum albumin. Sperm cholesterol efflux was also stimulated when serum or follicular fluid was added to a simplified medium (50 mM Tris-HCl, 0.56% NaCl, pH 7.8); efflux of [3H]cholesterol from labeled gametes progressed in a time-dependent manner, but was low in the absence of serum components. The [3H]cholesterol/cholesterol ratios were higher in the albumin and HDL fractions, indicating some degree of specificity of these sterol acceptors. It was observed that follicular fluid albumin has a [3H]sterol binding capacity that is 2-3-fold higher than that of serum albumin. Commercial human serum albumin also promoted sperm cholesterol efflux. These results provide new information concerning those components of follicular fluid which may play a role in human sperm capacitation and provide further support for the concept that loss of cholesterol from the sperm plasma membrane is an important component of the capacitation process.
Enhancement of the detection of alkali-resistant phosphoproteins in polyacrylamide gels.
Following their separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, labeled proteins obtained from cultured canine prostatic epithelial cells incubated with [35S]-methionine and [32P]phosphate were subjected to alkali treatment, a method that is currently used to detect phosphotyrosine-containing proteins. Significant amounts of 35S-labeled material were lost during the alkali treatment. The crosslinking of proteins within the gels by glutaraldehyde treatment eliminated protein losses and did not alter the efficiency of phosphoester bond hydrolysis by alkali treatment. Consequently, the time required to detect alkali-resistant phosphoproteins by autoradiography was greatly reduced. Prostatic phosphoproteins were also shown to contain phosphotyrosine, indicating the presence of tyrosine protein kinase activity in these proliferating epithelial cells.
Androgen binding as evidenced by a whole cell assay system using cultured canine prostatic epithelial cells.
The androgen receptor content in the prostate has been usually evaluated using subcellular fractions without taking into account cellular and functional heterogeneity of the gland. Using enriched populations of immature canine prostatic epithelial cells cultured in primary monolayers, a whole cell assay system was developed to measure androgen receptors. Tritiated dihydrotestosterone (DHT) and/or methyltrienolone (R1881) in serum-free medium were used as ligands and Triamcinolone acetonide (0.5 microM) was added to prevent the binding of R1881 to other types of receptors. The amount of radiolabelled ligand specifically bound to the cells was determined at equilibrium. Specific binding was proportional to the number of cells seeded. Scatchard analysis revealed the presence of at least two types of binding sites. The Kd for the high affinity binding site was 2 x 10(-9) M. Competition studies indicated that this component was specific for androgens; Methyltrienolone, Mibolerone and the antiandrogen RU 23908 were the most efficient competitors. They were followed by DHT, 5 alpha-androstane-3 alpha, 17 beta-diol, testosterone, estradiol and estrone. Progesterone, 5 alpha-androstane-3 beta, 17 beta-diol and epitestosterone were not inhibitors. The level of specific binding was 11.0 +/- 7.6 fmol of bound R1881 per 10(6) cells (n = 34) or 2075 +/- 1434 fmol per mg of DNA; these values correspond to an average of 6624 +/- 4577 sites per cell. Thus, using this whole cell assay system, specific and androgen receptors were detected in immature prostatic epithelial cells in culture. This assay will therefore be useful to study the interrelationship between androgen binding activity and specific cell functions.
Left heart bypass in the surgery of aortic coarctation in children.
Of 47 children over the age of 1 year who underwent repair of aortic coarctation during 1978-85, 22 had the operation performed with left heart bypass because the distal aortic pressure was below 50 mm Hg after the initial application of the arterial clamps. One further child had an elective left heart bypass. Satisfactory distal perfusion was achieved in all 23 patients. There were no deaths related to the use of left heart bypass but three complications occurred. One child had a mild transient hemiparesis on the ninth postoperative day and two had a pericardial effusion.
Changes in plasma ionised calcium concentration during paediatric cardiopulmonary bypass surgery.
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Male infertility associated with round-headed acrosomeless spermatozoa.
The properties of spermatozoa with round head syndrome in four unrelated patients are reported. The findings were as follows: (1) Electron microscopy demonstrated that all spermatozoa lacked an acrosome and postacrosomal sheath. (2) Acrosin activity was only 1% to 6% of that found in sperm obtained from fertile donors. (3) Phospholipase A2 activity was not significantly different from that of spermatozoa from donors of unknown fertility. (4) Electrophoresis of whole sperm extracts revealed deficiencies in major protein bands. (5) The round-headed spermatozoa failed to bind or penetrate the vitellus in the egg penetration test. (6) The rates of chemically induced nuclear chromatin decondensation of round-headed spermatozoa suggest that the acrosome content is not involved in this process.
Sterol sulfates in the epididymis; synthesis and possible function in the reproductive process.
Sterol sulfates are present in relatively high concentrations in the male reproductive tract. Cholesteryl sulfate is the major sterol sulfate in the human epididymis while desmosteryl sulfate is the major sterol sulfate in the hamster epididymis. While the testis is the major source of sterol sulfate in the human, the epididymis of the hamster is the source of demosteryl sulfate. This conjugate accumulates along the length of the epididymis and is taken up by the plasma membrane in the acrosomal region of the spermatozoa. Sulfotransferase activity increases along the epididymis and this is due to the actual synthesis of the enzyme. Sterol sulfates are potent and specific inhibitors of the proteolytic enzyme, acrosin. This property could provide protection against the premature release of proteolytic activity within the male reproductive tract. It is proposed that the removal of this inhibition occurs within the female tract via sulfatase activity in order to enable the acrosome reaction and ovum penetration to occur.
Day-case ligation of patent ductus arteriosus in premature infants.
Over a 3-year period patent ductus arterious (PDA) ligation was performed on a day-case transfer basis on 45 premature infants. The overall survival rate was 93%. We would recommend this practice as an alternative to surgery in the neonatal intensive care unit.
Uptake of an oviductal antigen by the hamster zona pellucida.
Using an antiserum raised against hamster oviductal zona pellucida, we observed specific immunogenic components of the reproductive tract on the zonae of oviductal eggs and in oviductal fluid. Results of immunohistochemical studies suggested that these oviductal components may originate from epithelial cells of the isthmus and, to a lesser extent, of the ampulla and fimbria. The oviductal immunogenic components have also been observed within the bursal cavity, which contains the ovary. These observations suggest that these oviductal components may play an important role in the first steps of the hamster reproductive process.