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Biomedical subjects

R Webb

Publications and source records attributed to R Webb.

At least 127 records · Page 7Linked to original sources

Photoaffinity labelling of the ATP-binding sites of two Ca2+,Mg-ATPase isoforms in pancreatic endoplasmic reticulum.

Pancreatic rough ER ATP-binding proteins, including two isoforms of SERCA-2b Ca2+,Mg-ATPase, were identified using specific photoaffinity labelling with 8-azido-ATP. 8-Azido-ATP irreversibly inhibited Ca2+,Mg-ATPase activity only after UV irradiation and the inhibition was prevented by inclusion of 5 mM ATP in the labelling reaction. Rough ER proteins of apparent molecular masses 141, 111, 100, 84, 69, 55 and 47 kDa were detected following photoaffinity-labelling with 8-azido-[alpha-32P]ATP. The two bands at 111 kDa and 100 kDa corresponded in molecular mass to the two SERCA-2b Ca2+,Mg-ATPase isoforms previously demonstrated immunologically [1]. Immunoprecipitation of rough ER proteins by a SERCA-2b-specific antibody showed that the two ATPase bands were photoaffinity-labelled. Photoaffinity labelling of the 111 and 100 kDa proteins was: (a) abolished when Ca2+,Mg-ATPase activity was inactivated by EDTA-treatment of rough ER membranes; (b) inhibited by the Ca2+,Mg-ATPase inhibitor vanadate; (c) not affected by thapsigargin. The data demonstrate that pancreatic rough ER contains two isoforms of the SERCA-2b Ca2+,Mg-ATPase whose ATP-binding properties are susceptible to inhibition by vanadate but not thapsigargin.

Adenosine Triphosphate↗

Effects of indirect selection for pituitary responsiveness to gonadotropin-releasing hormone on the storage and release of luteinizing hormone and follicle-stimulating hormone in prepubertal male lambs.

Divergent selection based on the LH response to a 5-micrograms dose of GnRH in 10-wk-old Finn Dorset ram lambs has created two lines of sheep that differ significantly in their response to both physiological and pharmacological dosages of GnRH. The aims of this study were to investigate whether there are between-line differences in endogenous LH and FSH secretion in ram lambs, as in the adult ewes, and to determine whether these differences are related to differences in storage of gonadotropins within the pituitary glands of the two lines. To address the first aim, endogenous LH and FSH secretion was monitored in 20 ram lambs from each line for a 6-h period at 2, 6, and 10 wk of age. To investigate potential between-line differences in gonadotropin storage, the LH responses to two acute challenges with either 0.5, 5.0, or 50.0 micrograms of GnRH were examined in 10-wk-old ram lambs from each line. The results demonstrated that the between-line difference in the LH response to a 5-micrograms GnRH challenge in ram lambs is accompanied by significant between-line differences in the regulation of endogenous gonadotropin secretion. Specifically, lambs from the High line secreted LH pulses of significantly greater amplitude than did lambs from the low line at 2 and 10 wk of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

The effect of the Booroola (FecB) gene on peripheral FSH concentrations and ovulation rates during oestrus, seasonal anoestrus and on FSH concentrations following ovariectomy in Scottish Blackface ewes.

The aim of this study was to investigate the role of FSH in the control of ovulation rate by the Booroola gene. Three Booroola genotypes (FecBFecB, FecBFec+ and Fec+Fec+) of the F2 population, from a cross between Booroola Merino and Scottish Blackface, and two Booroola genotypes (FecBFec+ and Fec+Fec+; 25% Booroola Merino and 75% Scottish Blackface), from the backcross of FecBFec+ sires to Scottish Blackface ewes, were compared. During seasonal anoestrus significant differences (P < 0.05) in hCG-stimulated ovulation rates were obtained between FecBFecB and Fec+Fec+ ewes from the F2 population, and FecBFec+ ewes were intermediate. No significant difference in hCG-stimulated ovulation rate was observed in the backcross population between FecBFec+ ewes and Fec+Fec+ ewes. There were no significant differences between genotypes in mean serum FSH concentrations during seasonal anoestrus in either backcross of F2 population. During the breeding season, two separate experiments confirmed the expected ovulation rate differences between genotypes (FecBFecB > FecBFec+ > Fec+Fec+). In both experiments, mean peripheral FSH concentrations in the F2 population were similar in FecBFec+ and Fec+Fec+ ewes, but were significantly higher (P < 0.05) in FecBFecB ewes. In the backcross population, mean peripheral FSH concentrations during the oestrous cycle were not significantly different between FecBFec+ and Fec+Fec+ ewes, despite significant differences in ovulation rate. Ovariectomy during the breeding season resulted in significantly higher (P < 0.001) mean peripheral FSH concentrations in all three genotypes. After ovariectomy, mean FSH concentrations between FecBFec+ and Fec+Fec+ ewes, form both backcross and F2 populations, were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)

Anestrus↗

Effects of chronic treatment with a gonadotrophin-releasing hormone agonist on peripheral concentrations of FSH and LH, and ovarian function in heifers.

The effect of chronic treatment with a gonadotrophin-releasing hormone agonist (GnRHa) on ovarian function in cattle was investigated by injecting heifers i.m. twice a day with saline, 5 micrograms GnRHa (Buserelin) or 10 micrograms GnRHa (n = 7) for 21 days. Blood samples were taken twice a day during the treatment period, and then three times a day for 7 days and once daily for a further 4 days. Frequent samples were also collected on day 1, day 10 and day 21 of treatment. The ovaries of all heifers were examined daily using real-time ultrasonography throughout the experimental period. No significant differences in the response were observed between two doses of GnRHa. The first GnRHa injection produced a large LH and FSH surge and this acute response was still present by day 21 of treatment, but both the magnitude and duration of response were significantly attenuated (P < 0.01). After an initial increase, LH returned to the basal concentration, which was maintained until the termination of treatment, when concentrations increased significantly, with a preovulatory surge occurring approximately 6 days later. Peripheral FSH concentrations during the oestrous cycle in control animals displayed a pattern of three waves, each of which closely preceded a wave of follicular development. Concentrations of FSH in GnRHa-treated heifers showed a normal pattern for the first wave after the start of treatment. During the next wave, concentrations increased and remained at the peak values until about 4 days after the end of treatment. An additional ovulation was induced in 11 of 14 GnRHa-treated heifers within 2-3 days of the start of treatment, and a significant (P < 0.05) increase in serum progesterone concentrations was detected 2 days later. All GnRHa-treated heifers then showed a normal follicular wave, with the development and regression of a dominant follicle. The dominant follicles from the next wave grew to only 7-9 mm in diameter and remained at this size until the end of treatment, when they resumed growth, ovulated approximately 7 days later and formed corpora lutea. We conclude that chronic treatment of heifers with GnRHa for 3 weeks suppresses pulsatile secretion of LH and blocks the development of dominant follicles beyond 9 mm in diameter, preventing the preovulatory LH surge and ovulation. However, GnRHa did not suppress the secretion of FSH within the 3 week treatment period. The maintenance of the dominant follicles for an extended period should provide an ideal model to study the control of follicular atresia in cattle in vivo.

Animals↗

Control of antral follicle development and selection in sheep and cattle.

The development of antral follicles in sheep and cattle is dependent on FSH, but large antral follicles can shift their gonadotrophic dependence from FSH to LH. The mechanisms that result in the selection of at least one ovulatory follicle from identical follicular cohorts, exposed to the same endocrine environment, still remain to be elucidated. The aim of this research was to extend in vitro results from the rodent to sheep and cattle and, using both in vivo and in vitro models, to identify factors that can enhance or attenuate the action of gonadotrophins in stimulating follicle development. Using sheep with ovarian autotransplants, we have obtained evidence to show that a number of factors inhibit ovarian function in vivo, whereas only insulin-like growth factor I (IGF-I) has a stimulatory effect. Further study of the mechanism of action of these factors at a cellular level has been made possible by the development of a serum-free granulosa cell culture system for both sheep and cattle that allows induction and maintenance of oestradiol production. Using this model system, we have confirmed many of the results from out studies in vivo and have shown that IGF-I and insulin interact at physiological concentrations to influence both cellular proliferation and oestradiol production. Overall, these data support the hypothesis that the physiological basis of follicle selection is the differential expression of factors that modulate the action of gonadotrophins on follicular cells at key points during the process of follicle development.

Animals↗

Restoration of fertility to oophorectomized sheep by ovarian autografts stored at -196 degrees C.

Cortical slices were prepared from the right ovaries of six lambs and either grafted directly to the ovarian pedicles of origin or cooled slowly to liquid nitrogen temperatures in medium containing dimethylsulphoxide. Three weeks later, the contra-lateral ovary was removed and replaced with frozen-thawed slices from the same animal. Two of the animals mated during their second oestrous cycle 3-4 months later and the remainder had at least one ovulatory cycle. The pregnancies reached full-term development, one lamb being derived from an ovulation in a fresh graft and the other from a frozen-thawed graft. None of the sheep had peripheral plasma concentrations of follicle stimulating hormone or luteinizing hormone consistently in the castrate range, and only one graft was devoid of follicles when the animals were slaughtered 9 months after the operations. Grafts with primordial follicles always contained developing follicles, which occasionally attained pre-ovulatory sizes of 7 mm in diameter. A corpus albicans was present in five grafts. Since all developing follicles had degenerated 1 week after grafting in an additional ewe, the large follicles in long-term grafts had presumably commenced growing after the operation. There were no obvious differences between fresh and frozen-thawed grafts in either appearance or weight, and all had apparently grown since implantation. Despite substantial depletion of primordial follicle numbers, the results indicated that frozen storage and replacement of a patient's own ovarian tissue might be practicable when fertility potential is threatened by chemotherapy/radiotherapy.

Animals↗

Alterations in endogenous gonadotropin secretion and pituitary responsiveness to gonadotropin-releasing hormone in adult ewes, following indirect selection in prepubertal male lambs.

Indirect selection to improve reproductive performance in the female, through use of a physiologically related trait in prepubertal males, the LH response to a GnRH challenge, has created two lines of sheep with altered pituitary gland sensitivity to both physiological and pharmacological doses of GnRH. These lines also exhibit correlated between-line differences in female reproductive performance during the first, and at the beginning of the second, breeding season. This study was designed to determine whether the observed between-line differences in female reproductive performance were related to between-line differences in endogenous gonadotropin secretion during the luteal and follicular phases of the estrous cycle and whether the between-line difference in the LH response to a GnRH challenge was maintained in adult females. The results indicated that, despite similar mean gonadotropin and steroid concentrations during the luteal and follicular phases of the estrous cycle, significant between-line differences in LH pulse amplitude were present during the follicular phase (high line > low line). In addition, the results indicated that the between-line difference in the LH response to a GnRH challenge was maintained in adult ewes, being most apparent during the follicular phase, when the magnitude of the difference was similar to that in prepubertal female lambs. Therefore, in addition to showing maintenance of the between-line difference in the selected characteristic in postpubertal females, the results demonstrate that adult ewes from the two lines differ in additional, unselected but physiologically related traits and provide a model system with which to study steroidal regulation of gonadotropin secretion in the ewe.

Aging↗

MapA, an iron-regulated, cytoplasmic membrane protein in the cyanobacterium Synechococcus sp. strain PCC7942.

Growth of Synechococcus sp. strain PCC 7942 in iron-deficient media leads to the accumulation of an approximately 34-kDa protein. The gene encoding this protein, mapA (membrane-associated protein A), has been cloned and sequenced (GenBank accession number, L01621). The mapA transcript is not detectable in normally grown cultures but is stably accumulated by cells grown in iron-deficient media. However, the promoter sequence for this gene does not resemble other bacterial iron-regulated promoters described to date. The carboxyl-terminal region of the derived amino acid sequence of MapA resembles bacterial proteins involved in iron acquisition, whereas the amino-terminal end of MapA has a high degree of amino acid identity with the abundant, chloroplast envelope protein E37. An approach employing improved cellular fractionation techniques as well as electron microscopy and immunocytochemistry was essential in localizing MapA protein to the cytoplasmic membrane of Synechococcus sp. strain PCC 7942. When these cells were grown under iron-deficient conditions, a significant fraction of MapA could also be localized to the thylakoid membranes.

Amino Acid Sequence↗

Superovulation in red deer (Cervus elaphus) and Père David's deer (Elaphurus davidianus), and fertilization rates following artificial insemination with Père David's deer semen.

Two comparative studies were undertaken using adult, female red and Père David's deer to examine the ovulatory response of these animals to a superovulation regimen and fertilization rates following inter- and intraspecific laparoscopic insemination. In Expt 1 six Père David's deer and 12 red deer hinds were treated during the breeding season with an intravaginal progesterone-impregnated controlled internal drug release device (CIDR) for 14 days, with 200 iu pregnant mares' serum gonadotrophin (PMSG) administered 72 h before the device was withdrawn and eight injections of ovine FSH given at 12 h intervals starting at the time of PMSG administration. Oestrous behaviour began one day after CIDR device withdrawal (Père David's deer: 24.00 +/- 2.32 h; red deer: 24.60 +/- 2.23 h). The duration of oestrus was greater in Père David's deer than in red deer (17.50 +/- 1.43 h and 8.25 +/- 3.25 h, respectively, P < 0.001). The peak LH surge of Père David's deer was 68.65 +/- 4.74 ng ml-1 occurring 29.00 +/- 2.41 h after removal of the CIDR devices. In comparison, the peak LH surge in red deer was 17.09 +/- 3.64 ng ml-1 (P < 0.001), occurring 24.00 +/- 0.00 h after CIDR device withdrawal. Pre-surge concentrations of LH were also greater (P < 0.001) in Père David's deer (1.37 +/- 0.11 ng ml-1) than in red deer hinds (0.41 +/- 0.02 ng ml-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Follicular development from ovarian xenografts in SCID mice.

Cortical slices of either cat or sheep ovaries were grafted under the renal capsules of ovariectomized SCID mice. The grafts became vascularized and were still surviving with large follicles present at autopsy up to nine months later. As developing follicles undergo atresia during the period of ischaemia after ovarian grafting, those found in long-term grafts at autopsy had presumably started to grow from the primordial stage after transplantation. Some follicles had reached a diameter of 3 mm with a normal antrum and appeared to be cytologically normal, but the latent period for the emergence of antral follicles was shorter in cat compared with sheep grafts. Oestradiol production from grafts, as indicated by vaginal cornification and plasma measurements collected at autopsy, was not constant and circulating concentrations varied among animals, and were sometimes far in excess of the normal physiological range of the host. The vaginal smears never presented cytological patterns like those of the normal mouse oestrous cycle, and ovulation had not occurred in any of the grafts. These results demonstrate that ovarian xenografts in SCID mice can serve as experimental models for investigating follicle development in species in which follicle growth in vitro and studies of the parent animal are impracticable.

Animals↗

Effects of recombinant bovine somatotrophin, insulin-like growth factor-I and insulin on bovine granulosa cell steroidogenesis in vitro.

Our previous studies have demonstrated that physiological concentrations of metabolic hormones, including recombinant bovine somatotrophin (BST), insulin-like growth factor-I (IGF-I) and insulin, can significantly stimulate the proliferation of bovine granulosa cells cultured under serum-free conditions. In this study we investigated the effects of these factors on bovine granulosa cell steroidogenesis using the same culture system. Bovine granulosa cells were obtained from antral follicles classified into three size classes: small, < 5 mm; medium-sized, 5-10 mm and large, > 10 mm in diameter. Whilst not affecting steroidogenesis by granulosa cells from small and medium-sized follicles, BST (10-1000 ng/ml) stimulated the secretion of both oestradiol and progesterone by granulosa cells from large follicles in a dose-dependent manner. Insulin (1-1000 ng/ml) and IGF-I (10-1000 ng/ml) stimulated the secretion of oestradiol and progesterone by granulosa cells from all three size categories of follicles in a dose-dependent manner. FSH (200 ng/ml) alone increased progesterone secretion by granulosa cells from all three size classes of follicles, but had no effect on oestradiol secretion by granulosa cells. Both IGF-I (200 ng/ml) and insulin (30 ng/ml) acted in synergy with FSH (200 ng/ml) to stimulate steroidogenesis by granulosa cells from all three size categories of follicles, but no such interaction was observed between BST (50 ng/ml) and FSH (200 ng/ml). In conclusion, BST, IGF-I and insulin significantly influence the steroidogenic activity of bovine granulosa cells cultured under serum-free conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

The Saskatchewan Clinical Stroke Prevention Project: design.

The Saskatchewan Clinical Stroke Prevention Project aims to examine the process and impact of incorporating enhanced stroke prevention into the routine clinical practice of family physicians. Twenty-four physicians in three practices in Saskatchewan are participating in a staged series of educational interventions over two years to enhance their management of smoking, transient ischemic attack/stroke, atrial fibrillation and hypertension. The components of each intervention include a seminar, printed materials, a one-to-one case discussion or "academic detailing," a self-documented chart audit and changes to the office system. Patients for whom this intervention is targeted are those 55 to 75 years of age presenting for a periodic health examination with one or more of the four main risk factors for stroke. Intervention will consist of counselling on relevant risk factors by the doctor and nurse teams, supported by appropriate patient education materials. Patient follow-up will be carried out according to clinical indications and will be done at least 3, 6, 12 and 24 months after entry into the study. Evaluation comprises measures of the process of implementing change in preventive practice as well as of the impact of such change. The former includes semi-structured interviews and focus groups with physicians, support staff and patients. The latter includes an assessment of knowledge, attitudes and charted practice before and after intervention.

Adult↗

Demonstration of two isoforms of the SERCA-2b type Ca2+,Mg(2+)-ATPase in pancreatic endoplasmic reticulum.

An antibody raised against a 12 amino acid peptide corresponding to the C-terminal sequence of the SERCA-2b Ca2+,Mg(2+)-ATPase precipitated Ca2+,Mg(2+)-ATPase activity from pancreatic rough ER. Thapsigargin and vanadate inhibited the activity with the same concentration-dependence as for native ER membranes. Partial purification of Ca2+,Mg(2+)-ATPase using Reactive Dye-agarose affinity chromatography resulted in activation of the enzyme, suggesting the presence of an endogenous inhibitor which was detached by binding to the Reactive Dye. Immunoblots and analysis of immunoprecipitated protein revealed two bands of molecular masses approx. 111 kDa and 97 kDa. It is concluded that pancreatic ER Ca2+,Mg(2+)-ATPase is of the SERCA-2b type and consists of two isoforms.

Amino Acid Sequence↗

The milk-alkali syndrome. A reversible form of acute renal failure.

The milk-alkali syndrome became rare with the advent of modern ulcer therapy with nonabsorbable antacids, histamine2 blockers, and sucralfate. An increased frequency of this syndrome seems likely with the growing popularity of the use of calcium carbonate as an antacid or as calcium supplementation to prevent osteoporosis. We treated five patients who had six episodes of the milk-alkali syndrome; four of these cases were diagnosed between 1990 and 1992. All patients were ingesting massive quantities of calcium and absorbable alkali and were unaware of the toxic effects of these compounds. All patients presented with the triad of hypercalcemia, metabolic alkalosis, and renal failure. All metabolic abnormalities were corrected, and renal function improved with appropriate supportive measures and cessation of calcium and alkali ingestion. In two patients, the renal failure was so severe that dialysis was necessary. In four patients, either the serum amino-terminal parathyroid hormone or 1,25-dihydroxycholecalciferol levels were appropriately decreased in response to hypercalcemia. The serum carboxy-terminal parathyroid hormone levels were increased because of renal failure. Since both physicians and patients are often unaware of the calcium and alkali content of many nonprescription medicines, the diagnosis of the milk-alkali syndrome, a reversible cause of renal failure, can be missed if a detailed history of such intake is not elicited. Measurement of the serum amino-terminal parathyroid hormone and 1,25-dihydroxycholecalciferol levels may help differentiate milk-alkali syndrome from primary hyperparathyroidism.

Acute Kidney Injury↗

Effect of recombinant bovine somatotropin on the superovulatory response to pregnant mare serum gonadotropin in heifers.

Treatment of mature heifers with recombinant bovine somatotropin (BST) increases the number of ovarian follicles of 2-5 mm in diameter. This study was carried out to investigate whether the increase in the number of small follicles induced by BST treatment can enhance the superovulatory response. In the first experiment, 24 heifers were assigned, in a randomized block design, to four treatment groups: 1) control; 2) BST; 3) pregnant mare serum gonadotropin (PMSG); 4) BST+PMSG. On Day 7 of the estrous cycle, animals in groups 2 and 4 received injections of 320 mg BST (in a sustained release formulation), while heifers in groups 1 and 3 received 10 ml saline. Five days later heifers in groups 3 and 4 were treated with a single dose of 2000 IU PMSG to induce superovulation. Embryos were recovered nonsurgically on Days 6-8 of the following cycle, and all heifers underwent laparoscopy on Day 9 to assess ovulation rate. In a second experiment, 24 heifers were assigned randomly to four treatment groups: 1) PMSG (1000 IU); 2) BST (320 mg) + PMSG (1000 IU); 3) PMSG (2000 IU); 4) BST (320 mg) + PMSG (2000 IU), and then treated as for experiment I. In both experiments, all heifers were scanned daily using real-time ultrasound from the day before BST injection until the day of ovulation to monitor dynamics of ovarian follicular growth and development, and blood samples were collected daily throughout the experimental period for measurement of peripheral progesterone, estradiol-17 beta, growth hormone (GH), and insulin-like growth factor-I (IGF-I).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

The effect of recombinant bovine somatotrophin on ovarian follicular growth and development in heifers.

The effects of recombinant bovine somatotrophin (BST) on the dynamics of ovarian follicular growth and development and peripheral insulin concentrations were investigated. Initially, studies were carried out in a population of Hereford x Friesian heifers to validate the ultrasound technique. In the first experiment, 12 heifers were injected daily with either 25 mg BST or vehicle for two oestrous cycles, and the effects on follicular dynamics and peripheral insulin were determined. In a second experiment, 12 heifers were given a single injection of 10 ml saline or 320 mg BST in a sustained-release formulation to examine the temporal relationships between growth hormone (GH), insulin-like growth factor-1 (IGF-1), insulin and the number of follicles. The validation studies demonstrated that small follicles (< 5 mm in diameter) could be clearly detected by real-time ultrasound, and that 75% (9 of 12) of heifers showed three waves of dominant follicle development during the oestrous cycle, whereas the remainder had only two waves. The changes in the numbers of follicles of the three size categories (< 5 mm, small; 5-10 mm, medium-sized and > 10 mm, large) also displayed a wave pattern similar to that of the dominant follicle, with a marked reduction in the number of subordinate follicles as the dominant follicle grew and reached its maximum size. In Expt 1, BST treatment increased the number of small follicles and caused a rise in peripheral insulin concentrations (P < 0.01) throughout the treatment period.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

The relationship between selection for pituitary responsiveness to gonadotrophin releasing hormone in sheep and differences in gonadotrophin subunit mRNAs.

Selection of the luteinizing hormone (LH) response to exogenous gonadotrophin-releasing hormone (GnRH) in sheep has resulted in the establishment of two lines (High and Low) with a fivefold difference in pituitary sensitivity to GnRH. The effect of selection on gonadotrophin gene expression in the presence or absence of an exogenous gonadotrophin-releasing hormone (GnRH) challenge in twenty-week-old ram lambs from both lines was examined. Before treatment with either GnRH or saline, LH and follicle-stimulating hormone (FSH) concentrations were significantly higher in the High line than in the Low line animals (LH and FSH: P < 0.01). One hour after either GnRH or saline, all animals were slaughtered. In the absence of a GnRH challenge, there were significantly higher concentrations of all three gonadotrophin subunit mRNAs in the High line compared with the Low line, corresponding to the higher basal concentrations of LH and FSH. When comparing treatments between the lines, following a GnRH challenge, LH beta subunit mRNA was significantly (P < 0.001) higher in both lines than before the GnRH, whereas there was no significant change in either alpha or FSH beta subunit mRNA. These results indicate that the differences in basal gonadotrophin secretion are related to differences in gonadotrophin subunit mRNAs with the High line animals having an inherently greater amount of all three gonadotrophin subunit mRNAs. Selection has not altered the differential amounts of gonadotrophin subunit mRNAs, since there is an overall increase in all three gonadotrophin subunits. GnRH appears to preferentially control LH beta mRNA in both High and Low line animals.

Animals↗