Multiple sequence alignment of protein families showing low sequence homology: a methodological approach using database pattern-matching discriminators for G-protein-linked receptors.

A multiple alignment has been constructed, containing 37 sequences from related families of membrane-bound receptors believed to share the same structural framework as rhodopsin. Sequence homology within families was high (occasionally greater than 90%), but homology between them was generally low (20% or less). Database pattern-scanning methods were therefore used to construct a set of discriminators to aid both the task of alignment and the identification of distantly related sequences showing similar rhodopsin-like transmembrane helices. The results indicate that these discriminators are uniquely able to identify each of the transmembrane helices without major cross-reaction with similar regions in unrelated integral membrane proteins. This ability engenders more accurate alignments of the sequences and facilitates structural analysis and model building of the receptors.     

Influence of dose and route of administration of bovine follicular fluid and the suppressive effect of purified bovine inhibin (Mr 31,000) on plasma FSH concentrations in ovariectomized ewes

Ovariectomized Merino ewes were used to develop an in-vivo bioassay for purified bovine inhibin of Mr 31,000. Various doses (0.25, 0.5, 1 or 2 ml) of bovine follicular fluid, given either by the intravenous (i.v.) or intracarotid route (i.c.) resulted in significant linear dose-related suppression of plasma FSH and interval to maximum suppression. Control ewes (1.0 ml steer plasma) showed no significant change in FSH over the same period. Doses of 470 and 2590 U of pure inhibin given i.v. caused a significant suppression of FSH in plasma in all ewes. The in-vivo potency estimate of the high dose (2760 U, 1420-4690 fiducial limits) agreed well with the in-vitro assay of potency. There were no significant changes observed in mean plasma LH after treatment with the higher dose of pure inhibin. There were no rebound effects of treatment with bovine follicular fluid or pure inhibin on FSH concentrations above that of controls. It is concluded that the form of bovine inhibin of Mr 31,000, which is believed to be the predominant circulating form, is biologically active when administered in vivo.     

Cardiovascular effects of training for a marathon run in unfit middle aged men.

The effects of a 30 week exercise programme on serum lipid values, blood pressure, and cardiac function were assessed in a group of sedentary men aged 35-50 training for their first marathon. Mean serum cholesterol concentration (n = 33) fell by 12% from 6.54 (SE 0.18) to 5.76 (0.15) mmol/l (mean fall 0.78 mmol/l; 95% confidence interval 0.52 to 1.04 mmol/l), serum triglyceride concentration (n = 33) by 22% from 1.56 (0.17) to 1.21 (0.09) mmol/l (mean fall 0.34 mmol/l; 95% confidence interval 0.12 to 0.56 mmol/l), and mean blood pressure (n = 27) by 10% from 102 (2) to 92 (2) mm Hg (mean fall 10 mm Hg; 95% confidence interval 7 to 13 mm Hg). These changes were not explained by changes in body composition. Peak exercise left ventricular end diastolic volume (n = 16) increased with training; as a result of this and an increased exercise left ventricular ejection fraction peak exercise cardiac output increased from 19.9 (1.2) to 23.1 (3.0) l/min (mean rise 3.2 l/min; 95% confidence interval 1.5 to 5.0 l/min). Maximum oxygen consumption increased from 33.9 (1.6) to 39.0 (1.3) ml/kg/min (mean rise 5.0 ml/kg/min; 95% confidence interval 1.8 to 8.2 ml/kg/min). This study showed favourable effects on coronary risk factors and cardiac function and supports the place of regular exercise in coronary prevention programmes.     

Ovine trophoblast protein-1 and human interferon alpha reduce prostaglandin synthesis by ovine endometrial cells

Ovine trophoblast protein-1 (oTP-1), a protein secreted by the sheep conceptus immediately prior to implantation has sequence homology with alpha interferon. We have previously shown that, in parallel with human alpha interferon (IFN), oTP-1 reduces the release of prostaglandins (PG) E and F2 alpha from cultured ovine endometrial cells. Here we have examined the time and dose dependence of these actions and the possible site of action of the peptides. The concentrations of oTP-1 and IFN required for 50% inhibition of PGE release were 92 pg/ml and 0.88 pg/ml and for PGF2 alpha release, 165 pg/ml and 1.12 pg/ml respectively. Significant effects on PG release were not measured before 12 h after addition of peptide to culture dishes. Following removal of the peptides, the cells released less PGs for a further 18 h but then recovered. A large increase in PG synthesis and release occurred from cells cultured with added serum or arachidonic acid (AA) and an interactive effect was demonstrated between them, AA having a greater stimulatory effect on PG released in the presence of serum. However, in all cases oTP-1 and IFN continued to attenuate prostaglandin release. We conclude that the IFNs act directly or indirectly on the prostaglandin synthase enzyme.     

Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

Variability and transport of suspended sediment,particulate and dissolved organic carbon in the tidal freshwater Hudson River

Measurements of suspended matter, particulate organic carbon and dissolved organic carbon were made over a three year period at stations spanning 150 km of the tidal freshwater Hudson River. Suspended matter concentrations varied from year-to-year and were not related to freshwater discharge. The increase in suspended matter with depth in vertical profiles suggests that, during medium to low flow conditions, resuspension of bottom sediments was as important a source of sediment as loadings from tributaries. Particulate organic carbon showed significant variability among stations, and both autochthonous primary production and detrital organic matter are contributing to POC standing stocks. Dissolved organic carbon represented over half of the total organic carbon in the water column and showed little variation among stations.Examining downstream changes in transport showed that there was significant production of both suspended matter and POC within the study reach during the ice-free season. Tributary loadings within the study reach do not appear to be the cause of these increases in downstream transport. Dissolved organic carbon behaved conservatively in that there was no evidence for net production or net consumption within the river.The spatial/temporal patterns and analyses of transport suggest that suspended matter and POC, but not DOC, were controlled to a significant extent by processes occurring within the river and were not simply related to loadings from outside.     

Purification, properties and cation activation of galactosyltransferase from lactating-rat mammary Golgi membranes

Galactosyltransferase was purified from Golgi membranes of lactating-rat mammary gland and studied with respect to its physical and enzymic (lactose synthetase) properties. The enzyme occurred in both monomeric (43-46 kDa) and apparently dimeric (90 kDa) forms. It was very unstable except in the presence of phospholipid, detergent, or cations binding to site 2. The amino acid composition and the N-terminal sequence closely resembled that of the human and bovine milk enzymes, particularly in respect to a Pro-Pro-Pro-Pro sequence. Kinetic studies demonstrated a high-affinity Mn2+-binding site (1) essential for activity, and a low-affinity Mn2+-binding site (2) that could also bind spermidine or clupeine. Mn2+ binding at site 2 raised Vmax fivefold. Spermidine binding at site 2 enhanced Mn2+ binding at site 1, and influenced binding of glucose. At physiological glucose concentration, clupeine or spermidine activated nearly as well as 15 mM MnCl2 and are regarded as models of a natural cation activator that remains to be isolated. Evidence is given for an essential histidine residue in the galactosyltransferase. It is proposed that site 1 Mn2+ participates directly in the reaction mechanism, whereas site 2 is a regulator site allosterically activated by a basic protein.     

Inhibin secretion by the sheep ovary

An in-vitro bioassay for inhibin based on FSH content or release by rat pituitary cells was validated for measuring inhibin activity in ovine plasma and lymph. Dose-dependent increases in inhibin activity were detected in peripheral plasma of 4 ovariectomized ewes 1 min after i.v. injections of ovine follicular fluid, and the half-life of inhibin in plasma for 2 ewes was 45 and 50 min, respectively. Inhibin was detected in ovarian lymph but not in ovarian or jugular venous plasma, even after treatment of ewes with PMSG to induce folliculogenesis. Destruction of visible follicles (greater than 0.5 mm diameter) on the ovaries of 4 PMSG-treated ewes by electrocautery was followed by a rapid and sustained decline in secretion of inhibin in ovarian lymph for up to 4 h. Ovarian lymph flow rates were either unchanged or slightly increased after cautery. Oestrogen concentrations in peripheral venous plasma declined within 15-30 min of cautery, but concentrations remained well above baseline. There was a significant decrease in peripheral progesterone concentrations in these same samples, but not until 2-3 h after cautery. FSH in peripheral plasma was depressed or non-detectable in PMSG-treated ewes and neither FSH nor LH concentrations in peripheral plasma were significantly altered up to 4 h after cautery of ovarian follicles. It is concluded that (a) antral follicles (greater than 0.5 mm) are the source of inhibin present in ovarian lymph, and (b) the ovarian lymphatic system is a route by which inhibin could reach the peripheral circulation, particularly in the luteal phase when ovarian lymph flow rates are high.     

ATP-sensitive inward rectifier and voltage- and calcium-activated K+ channels in cultured pancreatic islet cells

Summary K⁺ channels in cultured rat pancreatic islet cells have been studied using patch-clamp single-channel recording techniques in cell-attached and excised inside-out and outside-out membrane patches. Three different K⁺-selective channels have been found. Two inward rectifier K⁺ channels with slope conductances of about 4 and 17 pS recorded under quasi-physiological cation gradients (Na⁺ outside, K⁺ inside) and maximal conductances recorded in symmetrical K⁺-rich solutions of about 30 and 75 pS, respectively. A voltage- and calcium-activated K^– channel was recorded with a slope conductance of about 90 pS under the same conditions and a maximal conductance recorded in symmetrical K⁺-rich solutions of about 250 pS. Single-channel current recording in the cell-attached conformation revealed a continuous low level of activity in an apparently small number of both the inward rectifier K⁺ channels. But when membrane patches were excised from the intact cell a much larger number of inward rectifier K⁺ channels became transiently activated before showing an irreversible decline. In excised patches opening and closing of both the inward rectifier K⁺ channels were unaffected by voltage, internal Ca²⁺ or externally applied tetraethyl-ammonium (TEA) but the probability of opening of both inward rectifier K⁺ channels was reduced by internally applied 1–5mm adenosine-5-triphosphate (ATP). The large K⁺ channel was not operational in cell-attached membrane patches, but in excised patches it could be activated at negative membrane potentials by 10^–7 to 10^–6 m internal Ca²⁺ and blocked by 5–10mm external TEA.