Aggregation-dependent turnover of flagellar adhesion molecules in chlamydomonas gametes

Previous studies on flagellar adhesion in chlamydomonas (Snell, W. and S. Roseman. 1979. J. Biol. Chem. 254:10820-10829.) have shown that as gametes adhere to flagella isolated from gametes of the opposite mating type, the adhsiveness of the added flagella but not of the gametes is lost. The studies reported here show that the addition of protein synthesis inhibitors (cycloheximide [CH] or anisomycin) to the medium of such cell- flagella mixtures causes the cells to lose their adhesiveness. This loss, however, occurs only after the cells have interacted with 4-8 flagella/cell and does not occur if the cells are kept in CH (7 h) without aggregating. The availability of an impotent (imp) mating type plus (MT(+)) mutant (provided by U.W. Goodenough), which adheres but is unable to undergo the fusion that normally follows adhesion, made it possible to determine whether a similar loss of adhesiveness occurs in mixtures of matting type minus (mt(-)) and imp mt(+) gametes. In the absence of inhibitor, mt(-) and imp mt(+) gametes adhered to each other (without fusing) for several hours; however, in the presence of CH or anisomycin, the gametes began to de-adhere 35 min after mixing, and, by 90 min, 100 percent of the cells were single again. This effect was reversible, and the rapid turnover of cells were single again. This effect was reversible, and the rapid turnover of molecules involved in adhesion occurred only during adhesion inasmuch as gametes pretreated for 4 h with CH were able to aggregate in CH for the same length of time as nonpretreated cells aggregated in CH. By the addition of CH at various times after the mt(-) and imp mt(+) gametes were mixed, measurements were made of the “pool size” of the molecules involved in adhesion. The pool reached a minimum after 25 min of aggregation, rapidly increased for the next 25 min, and then leveled off at the premixing level. These results suggest that flagellar adhesion in chlamydomonas causes modification of surface molecules (receptors, ligands), which brings about their inactivation and stimulates their replacement.     

A polysaccharide isolated from Cordyceps sinensis,a traditional Chinese medicine,protects PC12 cells against hydrogen peroxide-induced injury

Cordyceps sinensis, a well-known traditional Chinese medicine, possesses activities in anti-tumour, anti-oxidation and stimulating the immune system; however, the identity of active component(s) is not determined. By using anti-oxidation activity-guided fractionation, a polysaccharide of molecular weight approximately 210 kDa was isolated from cultured Cordyceps mycelia by ion-exchange and sizing chromatography. The isolated polysaccharide, having strong anti-oxidation activity, contains glucose, mannose and galactose in a ratio of 1 : 0.6 : 0.75. The pre-treatment of isolated polysaccharide on the cultured rat pheochromocytoma PC12 cells shows strong protective effect against hydrogen peroxide (H(2)O(2))-induced insult. Treatment of the cells with the isolated polysaccharide at 100 microg/ml prior to H(2)O(2) exposure significantly elevated the survival of PC12 cells in culture by over 60%. In parallel, the H(2)O(2)-induced production of malondialdehyde in cultured cells was markedly reduced by the polysaccharide treatment. Moreover, the pre-treatment of the isolated polysaccharide significantly attenuated the changes of glutathione peroxidase and superoxide dismutase activities in H(2)O(2)-treated cells in a dose-dependent manner. This is the first report in identifying a polysaccharide from Cordyceps, which protects against the free radical-induced neuronal cell toxicity.     

Chronotropism and blood flow patterns following teratogenic doses of catecholamines in 5-day-old chick embryos.

In vivo heart rates of 5-day-old chick embryos were recorded from electrodes placed in close proximity to the heart. L-epinephrine (4X10(-10) mole), 1-norepinephrine (1X10(-9)mole) and 1-isoproterenol (1.6X10(-10)mole) in 5 microliter of isotonic saline transiently accerlerated the mean heart rate by almost 9 percent. L-phenylephrine (2X10(-9)mole/5microliter) and the experimental procedure produced no appreciable effect. The positive chronotropic effect of the catecholamines was found to be highly significant (P less than 0.0005) as computed by Student's t test. However, no direct relationship could be established between the chronotropic response and the aortic arch anomalies produced. A prolonged reduction of blood flow in the primitive heart tube and the sixth aortic arch after administration of epinephrine and isoproterenol is apparently related to the induction of hypoplastic right pulmonary artery with absent or hypoplastic right ductus arteriosus.     

Vitamin K supplementation in postmenopausal women with osteopenia (ECKO trial): a randomized controlled trial

Background

Vitamin K has been widely promoted as a supplement for decreasing bone loss in postmenopausal women, but the long-term benefits and potential harms are unknown. This study was conducted to determine whether daily high-dose vitamin K1 supplementation safely reduces bone loss, bone turnover, and fractures.

Methods and Findings

This single-center study was designed as a 2-y randomized, placebo-controlled, double-blind trial, extended for earlier participants for up to an additional 2 y because of interest in long-term safety and fractures. A total of 440 postmenopausal women with osteopenia were randomized to either 5 mg of vitamin K1 or placebo daily. Primary outcomes were changes in BMD at the lumbar spine and total hip at 2 y. Secondary outcomes included changes in BMD at other sites and other time points, bone turnover markers, height, fractures, adverse effects, and health-related quality of life. This study has a power of 90% to detect 3% differences in BMD between the two groups. The women in this study were vitamin D replete, with a mean serum 25-hydroxyvitamin D level of 77 nmol/l at baseline. Over 2 y, BMD decreased by −1.28% and −1.22% (p = 0.84) (difference of −0.06%; 95% confidence interval [CI] −0.67% to 0.54%) at the lumbar spine and −0.69% and −0.88% (p = 0.51) (difference of 0.19%; 95% CI −0.37% to 0.75%) at the total hip in the vitamin K and placebo groups, respectively. There were no significant differences in changes in BMD at any site between the two groups over the 2- to 4-y period. Daily vitamin K1 supplementation increased serum vitamin K1 levels by 10-fold, and decreased the percentage of undercarboxylated osteocalcin and total osteocalcin levels (bone formation marker). However, C-telopeptide levels (bone resorption marker) were not significantly different between the two groups. Fewer women in the vitamin K group had clinical fractures (nine versus 20, p = 0.04) and fewer had cancers (three versus 12, p = 0.02). Vitamin K supplements were well-tolerated over the 4-y period. There were no significant differences in adverse effects or health-related quality of life between the two groups. The study was not powered to examine fractures or cancers, and their numbers were small.

Conclusions

Daily 5 mg of vitamin K1 supplementation for 2 to 4 y does not protect against age-related decline in BMD, but may protect against fractures and cancers in postmenopausal women with osteopenia. More studies are needed to further examine the effect of vitamin K on fractures and cancers. Trial registration: ClinicalTrials.gov (#{"type":"clinical-trial","attrs":{"text":"NCT00150969","term_id":"NCT00150969"}}NCT00150969) and Current Controlled Trials (#ISRCTN61708241)     

Chromatographic Evidence for the Presence of Multiple Tachykinins in the Bovine Adrenal Medulla

Among the mammalian tachykinins, substance P (SP) has been shown to be the most potent at modulating the response due to nicotinic acetylcholine receptor stimulation of bovine adrenal chromaffin cells. SP-like immunoreactivity has been detected in nerve terminals innervating the adrenal medulla; however, little is known of the presence of other tachykinins in this tissue. In this study, reverse-phase HPLC was used to fractionate peptides in bovine adrenal medullary extracts, and the fractions were analyzed by radioimmunoassay using antisera to SP or neurokinin A (NKA). The results show that both NKA- and SP-like immunoreactivities are present in the adrenal medulla. The presence of neurokinin B is also indicated. The presence of multiple tachykinins in this tissue raises questions as to their functions in the adrenal medulla.     

Ontogeny of adrenal VIP content and release from adrenocortical cells in the ovine fetus

The present study was designed to investigate the presence of VIP in fetal adrenals, to determine the changes in adrenal VIP content associated with maturation, and to explore the factors which regulate fetal adrenal VIP release. Adrenal glands from ovine fetuses at 70 to 140 days gestation were used. Adrenal VIP content, as measured by radioimmunoassay, were low at 70 and 80 days of gestation. This was followed by a rapid increase in VIP content from 80 to 110 days reaching a plateau between 110 and 130 days at levels comparable to that in the adult. A significant fall in adrenal VIP content occurred at 140 days, immediately prior to term. Release of VIP from fetal adrenocortical cells in vitro was significantly elevated by angiotensin II at 10(-5) M, while ACTH had no effect. Acetylcholine at 50 microM and high potassium stimulated fetal adrenal VIP release while norepinephrine did not. These results suggest that the VIP neuronal system in the ovine fetal adrenal matures between 80 and 110 days of gestation. Furthermore, the release of VIP from the fetal adrenocortical cells may be regulated by angiotensin II and cholinergic neurotransmitters.     

The MgADP-induced decrease of the SH1-SH2 fluorescence resonance energy transfer distance of myosin subfragment 1 occurs in two kinetic steps

The fluorescence resonance energy transfer distance between 5-[2-[iodoacetyl)amino)ethyl]aminoaphthalene-1-sulfonic acid covalently attached to the SH1 thiol of myosin subfragment 1 as the energy donor and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide attached to SH2 as the energy acceptor has been found to decrease by about 7 A in the presence of MgADP (Dalby, R. E., Weiel, J., and Yount, R. G. (1983) Biochemistry 22, 4696-4706; Cheung, H. C., Gonsoulin, F., and Garland, F. (1985) Biochim. Biophys. Acta 832, 52-62). Fluorescence stopped-flow experiments on the same system have yielded biphasic traces which are resolvable into a fast and slow component, k1 and k2, respectively. Results of experiments in which k1 and k2 were measured as a function of excess ADP concentration showed: 1) a nonlinear dependence of the apparent rate constants on [ADP]; 2) k1 is a factor of 10 faster than k2. These results are consistent with the 3-step mechanism previously proposed for nucleotide binding to myosin S1 (Garland, F., and Cheung, H. C. (1979) Biochemistry 18, 5281-5289). Kinetic experiments in which the anisotropy of the donor was monitored show this quantity to be unchanged over the course of the reaction. The biphasic decrease of donor intensity is assigned to an increase in energy transfer efficiency which, from the above results, is due to a decrease in donor-acceptor distance, occurring in two steps. The fast step is associated with a 4-5-A decrease of the donor-acceptor separation, while the slow step is associated with a further decrease of approximately 2 A.     

Solution conformations of DNAs containing binding sites of the catabolite gene activator protein and lac repressor protein: characterization by Raman spectroscopy

Raman spectra from three subfragments of the Escherichia coli lactose promoter region were obtained in 0.1 M NaCl. The three DNAs are 21, 40, and 62 bp in length. The 21 and 62 bp DNAs contain the binding site for the catabolite gene activator protein (CAP). The 40 bp DNA contains the binding site for the lac repressor. A quantitative analysis of Raman band characteristics indicates an overall B-type conformation for these gene regulatory sites. Bands which correspond to A-family (807 cm-1) and B-family (834 cm-1) deoxyribose phosphate vibrations have the same intensities as bands found in heterogeneous DNAs. The spectra of the 21 bp CAP site have, however, a small band at 867 cm-1 and several other small differences similar to some characteristics observed in C-DNA spectra. Several dG nucleosides in the CAP site appear to be altered from the conventional C2'-endo/anti conformation. At 45 degrees C, well below the melting region of these DNAs, small changes occur in the spectra of the 40 bp lac repressor site which are not observed in the other DNAs. A weak band occurs at 705 cm-1, and intensity changes are observed at 497, 682, and 792 cm-1. The changes suggest that the conformations of several dG nucleosides are altered and that a small region may exist with characteristics of an A-family backbone. This conformational change at 45 degrees C coincides with previous NMR observations indicating an enhanced imino proton exchange rate at a GTG sequence within the lac operator site.