Effect of oxaloacetate and phosphorylation on ATP-citrate lyase activity

ATP-citrate lyase (CL) catalyzes the conversion of citrate and CoA to oxaloacetate (OA) and acetyl-CoA. As the coupled malic dehydrogenase (MDH) assay is not able either to study the effect of oxaloacetate (OA) on CL activity or to measure accurately CL activity in biological samples, a new assay was developed. The CL-citrate coupled CAT assay measures the amount of acetyl-CoA formed by transferring radiolabeled acetyl-CoA synthesized from [14C]citrate to chloramphenicol with chloramphenicol acetyltransferase (CAT). Employing this assay, the rate of increase in acetyl-CoA synthesis from citrate is linear with respect to added CL. Kinetic values for ATP, CoA and citrate are similar to those obtained using the MDH assay. The effect of CL phosphorylation on enzyme activity was determined. CL phosphorylated by cAMP-dependent protein kinase or by this kinase and glycogen synthase kinase-3 (GSK-3) decreases the apparent Vmax without changing the apparent Km. The effect of OA, a product of the enzyme reaction, on CL activity was also determined. Computational analysis of the data obtained without added OA and at three concentrations of OA indicate that the apparent Km for the substrate is not altered even though the apparent Vmax is decreased. The effect of OA on the activity of phosphorylated enzyme was also determined. OA decreases the apparent Vmax of the phosphorylated enzyme to the same extent as in control CL. This assay is able to measure CL activity in cytosol from 3T3-L1 adipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temporal relationships and IL-2 dependency of prolactin-induced lymphocyte proliferation

Recombinant preparations of human prolactin (hPRL) and interleukin 2 (hIL-2) as well as monoclonal antibodies to these growth factors were used to study the synergistic interaction of PRL and IL-2 in Nb2 rat lymphoma lactogen-dependent cells. It was shown that IL-2 stimulated Nb2 cell proliferation in lactogen-free culture medium. Experiments with short-term exposure to growth factor demonstrated that PRL was required only during the initial 12 h of incubation while IL-2 was mitogenic regardless of the time it was added. Antibody to IL-2 partially inhibited both PRL- and IL-2-induced proliferation whereas antibody to PRL significantly decreased PRL but not IL-2-induced proliferation. These findings suggest that the complete mitogenic effect of PRL on Nb2 cells requires stimulation of IL-2 production.     

Amylase thermolability in body fluids

The thermolability of amylase was measured in saliva, pancreatic juice, urine, adult and neonatal sera. The mean percentage thermolability from these fluids was 100%, 99%, 87%, 44% and 23% respectively. In patients with acute pancreatitis and mumps the amylase was 84% and 83% thermolabile during the acute phase. On resolution of the pancreatitis this dropped towards normal. Patients with a pancreatic pseudocyst showed a high mean percentage thermolability (82%). These results could suggest that a component of amylase in human serum is not of pancreatic or salivary origin. In addition, this simple technique may be helpful in the diagnosis of pancreatic pseudocyst.     

Association of abnormal uterine discharge with new intramammary infection in the early postpartum period in multiparous dairy cows

The association of abnormal uterine discharge with the development of intramammary infection (IMI) was studied in 62 multiparous Holstein cows during the nonlactating period and from lactation days 3 through 30. Duplicate milk samples were obtained from each mammary gland at approximately day 30 of the nonlactating period. Milk samples for bacteriologic culture also were obtained from each gland from all cows at the end of the previous lactation, at parturition, and on a minimum of 7 additional dates during the first 30 days of lactation. Beginning after parturition and continuing once weekly for 4 weeks, each cow was examined, using a vaginal speculum to visually estimate the quantity of abnormal uterine discharge in the vagina. Additionally, uterine swab specimens were obtained for aerobic bacteriologic culture. Cows were allotted to groups on the basis of the maximal amount of abnormal uterine discharge observed at any 1 of the 4 examinations. Cows in group 1 had normal discharge or < 30 ml of abnormal discharge; in group 2, > or = to 30 ml of abnormal discharge, observed only on examination by vaginal speculum; and in group 3, > or = to 30 ml of abnormal discharge visible externally. A difference was not detected in the development of new IMI in the nonlactating period between cows that subsequently developed uterine discharge and those that did not. Although significant differences were not found, a tendency for lactating cows with abnormal uterine discharge to be at increased risk for developing new IMI was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

De novo protein synthesis by bovine uterine tube (oviduct) epithelial cells changes during co-culture with bull spermatozoa

Polypeptides secreted by uterine tube epithelial cells (UTEC) may facilitate sperm cell capacitation in vivo. This experiment evaluated the effect of sperm-UTEC co-culture on de novo protein synthesis by epithelial cells of the tubal isthmus. Comparisons of the patterns of proteins secreted into medium were made between four culture groups incubated for 24 h in the presence of 35S-methionine: group 1, sperm cells alone; group 2, control UTEC monolayers; group 3, UTEC co-cultured with sperm cells; and group 4, UTEC partitioned by a diffusible membrane from sperm cells during culture. Two-dimensional PAGE followed by fluorography was used to analyze conditioned medium containing secreted proteins from each group. The experiment was replicated four times. Sperm cells alone secreted no detectable proteins, whereas control UTEC monolayers produced a wide array of polypeptides. Sperm cells attached to UTEC in co-culture within minutes, and the resultant protein profile for these UTEC differed markedly from that of the control UTEC. Several new proteins were seen only from co-cultured cells, whereas other protein groups that were present with UTEC alone were absent in the co-culture medium of group 3. The protein pattern expressed by UTEC partitioned from sperm cells (group 4) was intermediate between that of the group 2 controls and that of co-cultured UTEC (group 3). In summary, the attachment of sperm cells to the UTEC during co-culture changed the types and quantities of proteins secreted into the conditioned medium as compared to those of control UTEC monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

A serological survey for bovine immunodeficiency-like virus in Ontario dairy cattle and associations between test results, production records and management practices

The release of waste anesthetic gases (WAG) in hospital operating rooms (ORs) was evaluated to determine if staff exposure to nitrous oxide exceeded the American Conference of Governmental Industrial Hygienists (ACGIH) Threshold Limit Value (TLV) and investigate possible correlations between symptoms and nitrous oxide exposure. The monitoring strategy consisted of nitrous oxide measurements by personal monitoring of the anesthetists and scrub nurses, and area monitoring at the exhaust grills. In addition, room ventilation rates and carbon dioxide concentrations were measured. Self-administered questionnaires were given to both the operating room personnel and staff on control wards. Nitrous oxide levels exceeded the current TLV of 50 ppm in 4 of 12 ORs. Anesthetists typically received the highest nitrous oxide exposure. There was a strong correlation (r2 = 0.90) for nitrous oxide dosimetry results between anesthetists and scrub nurses, and a fair correlation (r2 = 0.35) between area monitoring results and the anesthetists' personal exposures. Carbon dioxide levels commonly exceeded 1000 ppm on control wards. A correlation between reported symptoms and nitrous oxide exposure was not demonstrated. Reported symptoms more closely correlated with carbon dioxide levels. Nitrous oxide dosimetry of the anesthetists appears to be the only accurate strategy for monitoring human exposure to WAG in an operating room.     

Different roles of tumour necrosis factor alpha and interleukin 1 in murine streptococcal cell wall arthritis

In this study two different aspects of tumour necrosis factor alpha (TNF-alpha) and interleukin 1 (IL-1) in locally induced murine streptococcal cell wall arthritis (SCW) were investigated. First, the kinetics and interdependence of TNF-alpha and IL-1 release; and second; their involvement in inflammation and cartilage destruction. Kinetic studies showed that the TNF-alpha peak level preceded the IL-1 peak level. However, in vivo neutralization of TNF-alpha did not result in decreased IL-1 bioactivity or immunoreactivity, suggesting that there is no dominant TNF-alpha-dependent IL-1 release in this model. Inflammation was studied by measuring knee joint swelling and inflammatory cell influx. Impact on cartilage was studied by measuring chondrocyte proteoglycan synthesis and cartilage proteoglycan depletion. The role of TNF-alpha in these phenomena was investigated using anti-TNF-alpha antibodies and tumour necrosis factor binding protein (TNFbp). Similarly, the role of IL-1 was studied using anti-IL-1 antibodies or IL-1 receptor antagonist (IL-1Ra). Anti-TNF-alpha treatment significantly reduced joint swelling, whereas this effect was not found by using anti-IL-1 or IL-1Ra. In contrast, neutralization of IL-1, but not TNF-alpha, resulted in a significant decrease of chondrocyte proteoglycan synthesis inhibition. Moreover, histology revealed that anti-IL-1 treatment reduced cartilage proteoglycan depletion and inflammatory cell influx. Combined anti-TNF-alpha/anti-IL-1 treatment significantly suppressed both inflammation and cartilage damage. However, the impact on these separate parameters did not exceed the effects of either anti-TNF-alpha or anti-TNF-1. It can be concluded that both TNF-alpha and IL-1 exert specific activities in SCW arthritis. The involvement of TNF-alpha in this model is limited to joint swelling, whereas IL-1 plays a dominant role in cartilage destruction and inflammatory cell influx.     

Castanospermine, an oligosaccharide processing inhibitor, reduces membrane expression of adhesion molecules and prolongs heart allograft survival in rats

The clinical characteristics and natural history of 55 cases with antenatally diagnosed fetal uropathy were investigated. Percutaneous aspiration of the fetal pelvic or vesical urine was performed to decompress progressive unilateral hydronephrosis in 2 cases and to evaluate renal function in another 2 cases of bilateral hydronephrosis. As the postnatal diagnosis, upper urinary tract dilatation (hydronephrosis or hydronephroureter, 33 cases) and renal dysplasia (15 cases) made up 87% of all cases. A combination of hydronephrosis in one kidney and renal dysplasia in the other was also found in another 2 cases. Among 35 cases with upper urinary tract dilatation, 27 cases demonstrated pelviureteric junction stenosis and surgical intervention was necessary in 15 cases. In 17 cases with renal dysplasia, spontaneous regression was observed in only 3 cases and surgical intervention by means of percutaneous nephrostomy and nephrectomy was performed in 4 and 6 cases, respectively.