Biomedical subjects

K D Roberts

Publications and source records attributed to K D Roberts.

At least 55 records · Page 3Linked to original sources

PubMed1987

Radiation inactivation of hamster acrosin reveals that the biologically active unit is of low molecular size.

The relationship between structure and activity of acid-extracted and purified acrosin obtained from cauda epididymal hamster spermatozoa was studied. A four-step purification procedure of acrosin was used; it included 1.) acid extraction, 2.) gel filtration over Sephadex G-100 resin, 3.) ion exchange on CM-Sepharose CL-6B, and 4.) affinity chromatography on proflavin-Sepharose 4B. Analysis of the purified enzyme by high-performance liquid chromatography (300 SW + I-125) revealed a molecular weight of 44,000, which was identical to that obtained for acid-extracted acrosin. Slab-gel electrophoresis under nondenaturing conditions showed only one active band, as revealed with a highly sensitive assay using N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester as substrate. The radiation inactivation size of acid extracted acrosin was calculated to be 8400. This small unit could represent the active polypeptide portion of a larger monomer molecule or could represent the size of active subunits. Because acrosin is autocatalytic and highly active during fertilization, it is suggested that the active portion of the completely processed form of the enzyme is of small molecular weight.

Acids↗

PubMed1987

How should chylothorax be managed?

The management and complications of chylothorax occurring beyond the neonatal period were reviewed retrospectively. Records from 15 patients treated between 1976 and 1986 were analysed; a combination of thoracocentesis, chest drain insertion, and dietary modification were successful in abolishing chyle leakage in 10 cases. One child died from complications of cardiac surgery rather than from the chylothorax, and surgical intervention was necessary in the remaining four patients and included pleurectomy in three and thoracic duct ligation in the fourth. Lymphopenia, hypoalbuminaemia, hyponatraemia, and weight loss were the most common complications of conservative management and tended to occur in those patients with the longest duration of drainage. Postoperative recovery after pleurectomy and thoracic duct ligation was uneventful. We conclude that conservative management of chylothorax will be successful in most cases. Complications of such a policy are fairly common but rarely serious.

Child↗

PubMed1987

Formation of the hamster zona pellucida in relation to ovarian differentiation and follicular growth.

Using an immunofluorescence technique on ovarian sections, zona-immunoreactive components were detected in the cytoplasm of the oocyte from the beginning of its growth, when it is surrounded by only a thin squamous follicular cell layer, up to the end of its growth. In parallel with oocyte growth, the staining intensity decreased in the ooplasm. No staining was observed in the cytoplasm of the granulosa cells during normal follicular development in adult cyclic females. However, staining of the granulosa cells was observed at some stages of follicular development in immature females. This staining was especially evident in the ovaries of immature females (22 or 26 days old) stimulated with PMSG. In addition, the staining of the granulosa cells was consistently observed in ovaries showing an abnormal histology. Increased staining of the zona at its outer and inner regions could be distinguished in normal follicles, but when staining occurred on the granulosa cells no such pattern was observed over the zona matrix. These studies indicate that the oocyte itself but not the granulosa cells elaborates the native immunogenic material of the zona pellucida. The administration of PMSG at particular stages of ovarian differentiation interferes with follicular development leading to an abnormal extracellular assembly of the zona and its degradation (phagocytosis) by the surrounding granulosa cells.

Animals↗

PubMed1987

Immunodeficiency following neonatal thymectomy in man.

Patients undergoing cardiac bypass operations normally have a thymectomy to facilitate cannulation of the great vessels. Laboratory indices of immune function were measured in 18 children aged 9 months to 3 years who had had a thymectomy when aged 3 months or less, and in two groups of controls individually matched for age and age at operation. Total lymphocyte numbers were similar in all three groups but thymectomized children had significantly lower numbers of T cells and T cell sub-sets than controls and showed diminished responses to phytohaemagglutinin and concanavalin A. Children who have had a thymectomy early in life represent an important group in the study of the development of the immune system in man. Although the clinical consequences of early thymectomy are unclear, evidence of impairment of parameters of immunity have been found in later infancy and routine thymectomy in paediatric cardiac surgery should be avoided.

Antigens, Surface↗

PubMed1986

Preparation and properties of antibodies doubly labeled with tritium and fluorescein.

A method was developed to prepare doubly labeled antibodies whereby fluorescein-labeled antibodies were reacted with N-[ethyl-2-3H]ethylmaleimide. This tritiated conjugate exhibited the same immunoreactivity as the non-derivatized fluorescein-labeled antibody. This method provides a marker for immunocytochemical studies which takes advantage of the rapidity of fluorescent tracing combined with the stability and precision of the radioautography technique.

Animals↗

PubMed1986

Surgical correction of isolated persistent left superior vena cava draining to left atrium in a neonate.

A one day old baby who presented with cyanosis was later shown to have an isolated persistent left superior vena cava draining into the left atrium with hypoplastic right superior vena cava. This extremely rare anomaly was diagnosed by cardiac catheterisation at one week. Surgical correction at two weeks, by anastomosis of the innominate vein to the right atrium and by ligation of the left superior vena cava at its junction with the left atrium, was successful. This patient is the youngest case in which successful surgical correction of this isolated anomaly has been reported.

Echocardiography↗

PubMed1986

Surgical correction of anomalous origin of right pulmonary artery from aorta in a four day old neonate.

A two day old neonate presented with cyanosis. The echocardiogram was misinterpreted as showing transposition of the great arteries. Cardiac catheterisation was performed the next day and showed anomalous origin of the right pulmonary artery from the aorta. Surgical correction at the age of 4 days was successful. At the age of nine months the patient was symptom free with "normal" cardiac anatomy. This patient is the youngest case to have successful correction of this anomaly. The initial misinterpretation of the echocardiogram could have had serious consequences if balloon atrial septostomy had been attempted.

Aorta↗

PubMed1985

Quantification of cytosolic steroid receptors in secretory and non-secretory epithelial cells of the canine prostate.

Radiolabelled methyltrienolone, dihydrotestosterone and estradiol were used as ligands to identify and quantify androgen and estrogen receptors in freshly dispersed cells from the canine prostate. Soluble extracts (cytosols) were obtained from secretory and non-secretory epithelial cells separated on the basis of their density in Percoll gradients. For both cell types, as well as for the whole prostate, Scatchard plot analyses were linear and showed a single class of high affinity binding sites: Kd values of 3.6 +/- 2.2 X 10(-9) M and 3.0 +/- 1.2 X 10(-10) M were measured for the androgen and estrogen receptors, respectively. The number of binding sites for the cytosolic androgen receptor, expressed per mg of protein or per mg of DNA, was 2.4- to 6.7-fold higher in the non-secretory cells compared to the secretory cells. However, these two cell types contained a similar number of specific sites for the estrogens. The specificities of the androgen and estrogen receptors were shown to be identical for the two cell types: the binding of [3H]R1881 was strongly inhibited by unlabelled R1881, 5 alpha-androstane-3 alpha, 17 beta-diol and dihydrotestosterone, while 5 alpha-androstane-3 beta, 17 beta-diol, estradiol and estrone did not displace bound R1881. The addition of triamcinolone acetonide did not alter the binding of R1881 in extracts of either cell type or in the whole prostate. The binding of [3H]estradiol to the estrogen receptor was highly specific since a strong displacement was only observed with estradiol (83%).

Animals↗

PubMed1985

The purification of 3 beta-hydroxysteroid sulfotransferase of the hamster epididymis.

The purification of a hydroxysteroid sulfotransferase from the cytosolic fraction of the hamster epididymis is described using ammonium sulfate precipitation, gel filtration and PAP agarose affinity chromatography. A purification of 360-fold was achieved and resulted in the isolation of one major protein as evidenced by HPLC and SDS gel-electrophoresis. The "native" enzyme is a dimer of mol. wt 106,000 and is composed of subunits having the same molecular weight.

Animals↗

PubMed1985

The assay and partial characterization of 3 beta-hydroxysteroid sulfotransferase of the hamster epididymis.

Using a partially purified enzyme preparation obtained from hamster epididymis, a simple assay has been developed to measure the sulfurylation of dehydroisoandrosterone (DHA) and desmosterol in the presence of 3'-phosphoadenosine 5'-phospho[35S]sulfate [( 35S]PAPS). After stopping the enzymatic reaction with methanol and KCl, the 35S-labelled steroid sulfates are readily extracted into an organic phase. Optimal conditions for the sulfurylation of the two steroids were compared; optimum pH is 8.7 for DHA and 9.8 for desmosterol. Sulfoconjugation of desmosterol increases with magnesium concentrations up to 6 mM, while 40 mM concentrations of the divalent ion are required for the optimal sulfurylation of DHA. Maximum sulfurylation of these steroids requires the presence of 15 mM cysteine. Michaelis-Menten kinetics are observed with DHA which has an apparent Km of 32 microM, while desmosterol inhibits sulfotransferase activity at high concentrations. Saturation of the enzyme with PAPS results in an allosteric behaviour. Only the 3 beta-hydroxyl function of the steroid nucleus appears to be an appropriate sulfate acceptor for the epididymal hydroxysteroid sulfotransferase.

Allosteric Site↗

PubMed1984

Nonsteroidal serum factors involved in the regulation of the proliferation of canine prostatic epithelial cells in culture.

Canine prostatic epithelial cells were cultured in primary monolayers in order to define those factors that induce a proliferative response at the cellular level. Cultures were performed in a serum-free medium or in a medium supplemented either with fetal bovine serum or dog serum in the presence or absence of several sex steroids (androstenedione, testosterone, dihydrotestosterone, 3 alpha- and 3 beta-androstanediols, epitestosterone, epidihydrotestosterone, estrone, estradiol, and progesterone). Cell proliferation was observed in the absence of serum and exogenous steroids. The rate of cell division was serum dependent and steroid independent. Pretreatment of sera with charcoal had no effect on their mitogenic activities. Cells maintained in an endocrine milieu prior to tissue dispersion and throughout the whole procedure proliferate to the same extent as those deprived of hormones, whether free of serum or added supplements. The addition of insulin (2 micrograms/ml), dog prolactin (up to 25 ng/ml) and zinc (10(-8) to 10(-2) M) in a serum-free medium did not induce cell responsiveness to steroids. Dihydrotestosterone, 3 alpha-androstanediol, and estradiol alone or in combinations known to induce the growth of the canine prostate in vivo were ineffective in vitro. The proliferative responses to sera were time and concentration dependent, and dog serum was more potent than fetal bovine serum. Thus, humoral factors other than steroids, prolactin, insulin, or zinc may be of importance in the activation of epithelial cells involved in the development of prostatic hyperplasia and adenocarcinoma.

Animals↗

PubMed1984

5 Alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activities in isolated canine prostatic epithelial cells.

Kinetic parameters of two enzymes, 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase, have been calculated for freshly isolated canine prostatic epithelial cells separated into secretory and non-secretory cells on the basis of their density in Percoll gradients. For 5 alpha-reductase, a Km value of 3 X 10(-6) M was obtained in both epithelial cell types, and similar Vmax values of 1.7 X 10(-12) and 1.9 X 10(-12) moles of dihydrotestosterone formed/min/10(6) cells (P greater than 0.50) were calculated in secretory and non-secretory cells, respectively. The Km of 3 alpha-hydroxysteroid dehydrogenase (dihydrotestosterone----3 alpha-androstanediol) varied between 2.2 and 2.8 X 10(-6) M (P greater than 0.50) with respective Vmax's of 9 and 24 X 10(-12) moles of 3 alpha-androstanediol formed/min/10(6) cells (P less than 0.005) in secretory and non-secretory cells. For the reverse reaction, that is the transformation of 3 alpha-androstanediol to dihydrotestosterone, the Km's obtained were 0.4 and 0.5 X 10(-6) M (P greater than 0.50) with Vmax's of 14 and 19 X 10(-12) moles of dihydrotestosterone formed/min/10(6) cells (P less than 0.50) in secretory and non-secretory cells, respectively. Vmax values for 3 alpha-hydroxysteroid dehydrogenase are 10-fold higher than the ones for 5 alpha-reductase. Moreover, Km values for the reaction 3 alpha-androstanediol----DHT are 5-fold lower than those calculated for the reverse reaction and for 5 alpha-reductase. This could explain why the major metabolic pathway in the canine prostate gland is the conversion of 3 alpha-androstanediol to dihydrotestosterone.

3-Hydroxysteroid Dehydrogenases↗

PubMed1984

Distribution of steroid sulfotransferase in the male hamster reproductive tract.

Steroid sulfotransferase activity has been assayed in cytosol extracts obtained from the male hamster reproductive tract. Dehydroisoandrosterone and desmosterol were used as substrates in the presence of phosphoadenosine phosphosulfate-35S as sulfate donor. No significant sulfotransferase activity was found in the testis. In the epididymis, a severalfold increase in activity was found in the tissue from the caput to the caudal regions. A lower but significant activity was detected in the vas deferens. The enzyme appears to be secreted into the luminal fluid while little activity is associated with the spermatozoa. This increase in activity along the epididymis is undoubtedly responsible for the accumulation of sterol sulfates reported previously. In view of the fact that sterol sulfates are potent and specific inhibitors of acrosin, as reported for the porcine and confirmed herein for hamster acrosin, the epididymal production of steroid and sterol sulfates may represent a protective mechanism against the premature release of proteolytic activity within the male reproductive tract.

Acrosin↗

PubMed1984

Elevated secretion of androstenedione in a patient with a Leydig cell tumour.

In this study, we report the case of a 48-year old man with a well-encapsulated Leydig cell tumour, azoospermia, decreased libido and impotence. The basal peripheral blood levels of testosterone, dihydrotestosterone, 5 alpha-androstane-3 alpha,17 beta-diol and oestradiol were normal and oestrone was moderately increased. In contrast, androstenedione was extremely elevated at 521 ng/dl (normal: 88 +/- 60 ng/dl). Upon hCG stimulation, plasma testosterone increased 2.1-fold while androstenedione increased 1.4-fold. Plasma LH and FSH were also elevated and their response to LRH was exaggerated. At the time of surgery the levels of androstenedione in the spermatic vein plasma, as well as in the testicular tumour were elevated. In contrast, testosterone levels in the spermatic vein blood were decreased indicating a partial deficiency of 17 beta-hydroxysteroid dehydrogenase in the tumoural tissue. A follow-up study revealed that the contralateral testis did not respond to hCG although the sex steroid concentrations in the peripheral plasma were within normal limits. Plasma gonadotrophins remained elevated. These results demonstrate that this Leydig cell tumour secreted high amounts of androstenedione into the blood and that the contralateral testis exhibited an impaired androgenic function.

Androstenediol↗

PubMed1984

Semen selenium and human fertility.

Selenium (Se) was measured in the semen of 125 men from couples consulting for infertility. A mean concentration of 71.3 +/- 29.7 ng/ml of semen was found, with a range of 7 to 230 ng/ml. More than 85% of the Se is in the seminal plasma. There was a significant positive correlation between sperm count and semen Se. Sperm motility was maximal at semen Se levels ranging between 50 and 69 ng/ml; above and below this range, motility was decreased and the incidence of asthenospermia was high. This result suggests an optimal range for semen Se. A follow-up of 4.5 to 5 years after the initial assay of Se revealed that low semen Se levels (less than or equal to 35 ng/ml) were associated with male infertility. A Se level between approximately 40 and 70 ng/ml was optimal for reproductive performance (high pregnancy rate and low abortion rate). Semen Se levels greater than or equal to 80 ng/ml were associated with a high abortion rate and signs of ovarian dysfunction in the partner (both partners usually have the same diet and environmental exposure). These results attest to the role of Se in human reproduction, a well-established fact in several animal species. The semen Se level appears to be a useful indicator of Se status versus reproductive function. Further studies are warranted concerning the general aspects of metabolism and mechanism of action of Se in infertile couples before any therapeutic modification of intake of this element can be contemplated.

Abortion, Spontaneous↗

PubMed1983

The target sizes of the in situ and solubilized forms of human placental steroid sulfatase as measured by radiation inactivation.

Whole microsomal membrane preparations and Triton X-100-solubilized human placental steroid sulfatase were subjected to radiation inactivation analysis using gamma rays from a 60Co irradiator in order to assess the size of the physiological form of the enzyme. The data indicate that the enzyme exists as a monomer of molecular weight 78600 in Triton-containing buffers and as a polymer of molecular weight 533000 within the microsomal membrane.

Female↗

PubMed1983

Human placental steroid sulfatase: purification and properties.

Steroid sulfatase is recovered quantitatively from the 105,000 g h supernatant of human placental microsomes extracted with Triton X-100. The solubilized enzyme has been purified using conventional techniques. Throughout the purification procedure, steroid sulfatase appears to be heterogeneous as evidenced by certain, but not all, criteria. Following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the final preparation exhibits a major component and varying amounts of two minor ones. Antibodies raised in rabbits with the heterogeneous immunogen give rise to a single precipitation line when the native enzyme is analyzed by double immunodiffusion or by immunoelectrophoresis. In addition, using aged preparations of microsomes and immunoaffinity techniques, steroid sulfatase activity was found to be associated with the fastest migrating minor component. This finding would suggest that the apparent heterogeneity of purified steroid sulfatase is linked to degradation processes occurring within the microsomal preparations. Steroid sulfatase has a Stokes radius of 56 A, a sedimentation coefficient of 4.85 +/- 0.15S (in Triton-containing buffers) and binds 1.3 g of Triton X-100-per g of protein. The molecular weight of the Triton-protein complex was calculated to be 166,000 in which the glycoprotein portion contribution is about 43% (72,000). In contrast, the apparent molecular weight of the major polypeptide determined on calibrated SDS-gels is 62,000. The purified enzyme exhibits two pH optima with cholesterol sulfate as substrate, an acidic one at pH 5.0 and a second one at pH 7.5. The Km values for cholesterol sulfate, dehydroandrosterone sulfate and p-nitrophenylsulfate were 5.26, 14 and 1,320 microM, respectively.

Detergents↗

PubMed1983

Induction of acid phosphatase synthesis in canine prostatic epithelial cells in vitro.

The increase in acid phosphatase (AP) activity in cultured canine prostatic epithelial cells was investigated as a biochemical marker of in vitro cellular differentiation. The enzyme was studied in secretory and non-secretory epithelial cell populations obtained from control and cycloheximide-treated cultures over a period of 3 weeks and compared to the AP present in tissue and cellular extracts from normal canine prostates. The progressive increase in AP activity with the duration of culture was strongly inhibited by cycloheximide in both cell populations. The degree of inhibition was more pronounced late in the culture when AP activity increased at a faster rate in secretory cells. Cycloheximide inhibited protein biosynthesis by 70-80% as evidenced by a reduction in the incorporation of amino acids into acid-insoluble material. However, the specific activities of AP in the cellular extracts were similar in control and cycloheximide-treated cultures and increased sharply by 3-4-fold in the secretory cells after 12 days of culture. When extracts derived from control and cycloheximide-treated cells of various duration were submitted to electrophoresis in polyacrylamide gels (PAGE), a unique pattern of three bands of AP activity with Rf values of 0.18, 0.27 and 0.38 was obtained. In controls the AP activity in the band with an Rf of 0.18 increased preferentially during the culture period and was more important quantitatively in secretory cells. In cycloheximide-treated cultures the increase of AP activity associated with the band with an Rf of 0.18 was more strongly inhibited. The addition of tartrate to the staining mixture inhibited all three bands of AP activity. Similar results were obtained when extracts derived from freshly dispersed cells as well as from normal canine prostatic tissue were submitted to PAGE; the AP activity was resolved into 3 bands with Rf values of 0.15-0.18, 0.23-0.27 and 0.33-0.38; all three bands were inhibited by the addition of tartrate and the first band was predominant. Thus, the increase in AP activity in prostatic epithelial cells in a culture medium supplemented with serum and deprived of sex steroids is due to the de novo synthesis of a major form of the enzyme by the secretory cells.

Acid Phosphatase↗