Telomere structure in Euplotes crassus: characterization of DNA-protein interactions and isolation of a telomere-binding protein.

The nucleoprotein structure of telomeres from Euplotes crassus was studied by using nuclease and chemical footprinting. The macronuclear telomeres were found to exist as DNA-protein complexes that are resistant to micrococcal nuclease digestion. Each complex encompassed 85 to 130 base pairs of macronuclear DNA and appeared to consist of two structural domains that are characterized by dissimilar DNA-protein interactions. Dimethyl sulfate footprinting demonstrated that very sequence-specific and salt-stable interactions occur in the most terminal region of each complex. DNase I footprinting indicated that DNA in the region 30 to 120 base-pairs from the 5' end lies on a protein surface; the interactions in this region of the complex are unlikely to be sequence specific. A 50-kilodalton telomere-binding protein was isolated. Binding of this protein protected telomeric DNA from BAL 31 digestion and gave rise to many of the sequence-specific DNA-protein interactions that were observed in vivo. The telomeric complexes from E. crassus were very similar in overall structure to the complexes found at Oxytricha telomeres. However, telomeric complexes from the two ciliates showed significant differences in internal organization. The telomeric DNA, the telomere-binding proteins, and the resultant DNA-protein interactions were all somewhat different. The telomere-binding proteins from the two ciliates were found to be less closely conserved than might have been expected. It appears that the proteins are tailored to match their cognate telomeric DNA.     

The unfolding and attempted refolding of mitochondrial aspartate aminotransferase from pig heart.

The role of prostaglandins in the regulation of muscle protein breakdown is controversial. We examined the influence of arachidonic acid (5 microM), prostaglandin E2 (PGE2) (2.8 microM) and the prostaglandin-synthesis inhibitor indomethacin (3 microM) on total and myofibrillar protein breakdown in rat extensor digitorum longus and soleus muscles incubated under different conditions in vitro. In other experiments, the effects of indomethacin, administered in vivo to septic rats (3 mg/kg, injected subcutaneously twice after induction of sepsis by caecal ligation and puncture) on plasma levels and muscle release of PGE2 and on total and myofibrillar protein breakdown rates were determined. Total and myofibrillar proteolysis was assessed by measuring production by incubated muscles of tyrosine and 3-methylhistidine respectively. Arachidonic acid or PGE2 added during incubation of muscles from normal rats did not affect total or myofibrillar protein degradation under a variety of different conditions in vitro. Indomethacin inhibited muscle PGE2 production by incubated muscles from septic rats, but did not lower proteolytic rates. Administration in vivo of indomethacin did not affect total or myofibrillar muscle protein breakdown, despite effective plasma levels of indomethacin with decreased plasma PGE2 levels and inhibition of muscle PGE2 release. The present results suggest that protein breakdown in skeletal muscle of normal or septic rats is not regulated by PGE2 or other prostaglandins.     

Studies on the growth of the fetal sheep. Effects of surgical reduction in placental size, or experimental manipulation of uterine blood flow on plasma sulphation promoting activity and on the concentration of insulin-like growth factors I and II

The effect of long- and short-term manipulations of uterine blood flow on fetal plasma levels of IGF-I and -II have been studied in sheep at days 125-139 of pregnancy and compared with those in near term rats and guinea pig. The primary objective is to show that both long- and short-term reduction of uterine blood flow is associated with increase in the fetal plasma concentration of IGF-II while that of IGF-I falls. In the pregnant sheep long-term depression of utero-placental blood flow was caused by surgical reduction in placental mass (carunclectomy) prior to conception. This reduced fetal weight to 2.42 +/- 0.49 kg (SD) compared with 3.41 +/- 0.46 in controls; the respective values for uterine blood flow being 1694 +/- 558 and 913 +/- 324 ml/min respectively. This was associated with a fall in fetal plasma IGF-I concentration from 22.6 +/- 3.4 ng/ml to 14.9 +/- 1.31 ng/ml and a rise in IGF-II from 1952 +/- 284 ng/ml to 3360 +/- 914 ng/ml respectively. Similar changes in the plasma concentrations of IGF peptides were observed in fetal rats and guinea pigs in response to uterine artery ligation. Short-term reduction (60 min) of the uterine blood flow was caused either by compression of the common uterine artery to depress flow from 1491 +/- 375 to 648 +/- 216 ml/min or through intraarterial infusion of adrenaline at 35 ug/min to lower flow from 1628 +/- 339 to 1195 +/- 128 ml/min. Such falls in uterine blood flow had no significant effect on fetal plasma IGF-I levels but increased IGF-II levels by 30 to 60%.     

Early-blocked sporulation mutations alter expression of enzymes under carbon control in Bacillus subtilis

Summary The physiological roles of the gene subset defined by early-blocked sporulation mutations (spo0) and their second-site suppressor alleles (rvtA11 and crsA47) remain cryptic for both vegetative and sporulating Bacillus subtilis cells. To test the hypothesis that spo0 gene products affect global regulation, we assayed the levels of carbon- and nitrogen-sensitive enzymes in wild-type and spo0 strains grown in a defined minimal medium containing various carbon and nitrogen sources. All the spo0 mutations (except spo0J) affected both histidase and arabinose isomerase levels in an unexpected way: levels of both carbon-sensitive enzymes were two- to six-fold higher in spo0 strains compared to wild type, when cells were grown on the derepressing carbon sources arabinose or maltose. There was no difference in enzyme levels with glucose-grown cells, nor was there a significant difference in levels of the carbonindependent enzymes glutamine synthetase and glucose-6-phosphate dehydrogenase. This effect was not due to a slower growth rate for the spo0 mutants on the poor carbon and nitrogen sources used. The levels of carbon-sensitive enzymes were not simply correlated with sporulation ability in genetically suppressed spo0 mutants, but the rvtA and crsA suppressors each had such marked effects on wild-type growth and enzyme levels that these results were difficult to interpret. We conclude that directly or indirectly the spo0 mutations, although blocking the sporulation process, increase levels of carbon-sensitive enzymes, possibly at the level of gene expression.     

Chromoplast-Specific Proteins in Capsicum annuum

Chromoplasts are a common differentiation state of plastids in which the photosynthetic apparatus is absent and carotenoids accumulate to high levels. As a first step toward the isolation of chromoplast-specific genes, we have examined plastids of the bell pepper, Capsicum annuum L., for the presence of chromoplast-specific proteins. Intact chromoplasts were isolated from mature fruits of C. annuum var Emerald Giant, Golden Cal Wonder, and DNAP VS-12 by differential centrifugation followed by isopycnic sedimentation in gradients of silica sols. The plastids were then fractionated into soluble and membrane components and the proteins analyzed by one- and two-dimensional gel electrophoresis using isoelectric focusing, sodium dodecyl sulfate, and sodium dodecyl sulfate-urea gels. Two polypeptides with M_r of 35,000 and 58,000 accumulate to high levels in membrane fractions of chromoplasts of var Emerald Giant. These polypeptides are either not detectable or barely detectable in chloroplasts from immature fruits. Both polypeptides have been purified to near homogeneity. Yellow chromoplasts from var Golden Cal Wonder and red chromoplasts from var DNAP VS-12 contained the 35-kilodalton polypeptide, but not the 58-kilodalton species.     

Developmental toxicity evaluation of Bendectin in CD rats

Bendectin, composed of doxylamine succinate and pyridoxine HCl (1:1), is an antinauseant previously prescribed for nausea and vomiting during pregnancy. The present study examined the maternal and developmental effects of Bendectin (0, 200, 500, or 800 mg/kg/day, po) administered to timed-pregnant CD rats (36-41/group) during organogenesis (gestational days [gd] 6-15). At death (gd 20), all live fetuses were examined for external, visceral, and skeletal abnormalities. At 500 and 800 mg/kg/day, maternal toxicity included reduced food consumption during treatment and for the gestation period, increased water consumption in the posttreatment period, reduced weight gain during treatment, and sedation; water consumption was reduced during treatment and for the gestation period, and maternal mortality (17.1%) was observed only at the high dose. Developmental toxicity included reduced prenatal viability (800 mg/kg/day) and reduced fetal body weight/litter (500 and 800 mg/kg/day). In addition, reduced ossification of metacarpals (800 mg/kg/day), phalanges of the forelimbs (500 and 800 mg/kg/day), and of caudal vertebral centra (all doses) was observed. No increase in percent malformed live fetuses/litter was observed. The proportion of litters with one or more malformed fetuses was higher than vehicle controls only at 800 mg/kg/day, with short 13th rib (to which the test species is predisposed) as the predominant observation. By contrast, a positive control agent (nitrofen, 50 mg/kg/day, po, 14 dams) produced 85% malformed fetuses/litter with the predominant malformation being diaphragmatic hernia. In conclusion, the incidence of litters with one or more malformed fetuses was increased only at a dose of Bendectin which produced maternal mortality (17.1%) and other indices of maternal and developmental toxicity (see Discussion).     

Indomethacin, but not dazoxiben, reduced lung fluid filtration after E. coli infusion

Goats were divided into three groups and given infusions of live Escherichia coli bacteria. Group I received no treatment, group II was treated with indomethacin (a cyclooxygenase inhibitor), and group III with dazoxiben (a thromboxane synthase inhibitor). Double indicator-dilution extravascular lung water (EVLW) in group I was significantly different from the treated groups. There was an early increase in EVLW in group I and group III but not in group II animals. At 6 h EVLW's in group I, group II, and group III were 100, 45, and 30% above base line, respectively. Lymph flow (QL) and lymph-to-plasma protein ratio (L/P) was not statistically different between groups. Estimated total fluid filtration [QL + d(EVLW)/dt] in group I and III was markedly elevated between 0 and 1.5-2 h after E. coli infusion. Cardiac output (QT) decreased to 40% of base line in group I, and it decreased slightly in group II because of the indomethacin but did not decrease after E. coli. QT decreased in group III but recovered more rapidly than group I. Mean pulmonary arterial pressure increased more rapidly in group I and reached a higher peak than either treated group. At 6 h these groups had similar pulmonary arterial and pulmonary arterial wedge pressures. We conclude that 1) indomethacin but not dazoxiben blocks the early increase in total fluid filtration after bacterial infusion, 2) dazoxiben does not prevent the increased endothelial permeability resulting from infusion of live bacteria, and 3) indomethacin may somewhat ameliorate the endothelial permeability change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plant wound compounds from oviposition scars used in host discrimination by a stem-galling sawfly

The stem-galling sawfly Euura lasiolepisuses one or more plant wound compounds resulting from oviposition scars as cues in host discrimination (avoiding sites occupied by conspecifics). Four experiments were conducted to test hypotheses about how Euurapartitions resources. Experiment 1 demonstrated that Euuraavoids ovipositing on nodes with scars from previous ovipositions. Experiment 2 showed no evidence that the sawfly uses oviposition-deterring pheromones and indicated there is a time lag following oviposition before the oviposition scar becomes a deterrent. Experiment 3 showed that sawflies avoid artificially formed scars, demonstrating that a plant cue alone can lead to host discrimination. Experiment 4 showed that visual or tactile cues are not necessary for host discrimination and indicated that a plant wound compound functions as an oviposition deterrent. Both experimental results and field surveys showed that Euuraoviposition scars were more uniformly distributed than expected if sawflies were ignoring previous ovipositions.     

Field test of resource regulation by the bud-galling sawfly, Euura mucronata, on Salix cinerea

The attack by the bud-galling sawfly, Euura mucronata , on its willow host was simulated by removing every other living bud from previous year's shoots in a natural stand of Salix cinerea . The impact of simulated attack on the growth of the willow and the subsequent attack by E. mucronata was estimated after one growing season. Experimental bud-killing resulted in a growth reaction in S. cinerea characterized by significant increase in the length of new shoots produced by the remaining buds Also the number of buds and E. mucronata galls per shoot were significantly higher on bud-removal branches as compared with control branches. The overall survival of E. mucronata larvae was better on long shoots than on short shoots. The result shows that sawfly attack increases the amount of high quality resources for the subsequent generation of the same sawfly as predicted by "resource regulation hypothesis". We conclude that these highly specialized insect herbivores have adapted to utilize and to maintain the willow's juvenile traits. Rapid regrowth of willows after damage may have originally arisen as an adaptation in response to other, less specific pressures such as mammal browsing or snow and ice damage.     

A prospective study of maternal and fetal outcome in acute Lassa fever infection during pregnancy.

Several viral infections have been reported to result in more severe disease in pregnant than non-pregnant women, but the relative risks have not been well characterised. This has now been done for Lassa fever in a prospective study of 68 pregnant and 79 non-pregnant women who were admitted to hospital in Sierra Leone with confirmed Lassa fever. Lassa fever was the main cause of maternal mortality in the hospital, accounting for 25% of maternal deaths. Twelve of 40 patients in the third trimester died, compared with two of 28 in the first two trimesters and 10 of 79 non-pregnant women. The odds ratio for death in the third trimester compared with the first two trimesters was 5.57 (95% confidence intervals 1.02 to 30.26). The condition of the mother improved rapidly after evacuation of the uterus, whether by spontaneous abortion, evacuation of retained products of conception, or normal delivery; 10 of 26 women without uterine evacuation died, but only four of 39 women with evacuation died (p = 0.0016). The odds ratio for death with pregnancy intact was 5.47 (95% confidence interval 1.35 to 22.16). Fetal and neonatal loss was 87%. The risk of death from Lassa fever in the third trimester is significantly higher than that in the first two trimesters and higher than that for non-pregnant women, but evacuation of the uterus can significantly improve the mother''s chance of survival.