Neuronal calcium channel antagonists. Discrimination between calcium channel subtypes using omega-conotoxin from Conus magus venom

The omega-conotoxins from the venom of fish-hunting cone snails are probably the most useful of presently available ligands for neuronal Ca channels from vertebrates. Two of these peptide toxins, omega-conotoxins MVIIA and MVIIB from the venom of Conus magus, were purified. The amino acid sequences show significant differences from omega-conotoxins from Conus geographus. Total synthesis of omega-conotoxin MVIIA was achieved, and biologically active radiolabeled toxin was produced by iodination. Although omega-conotoxins from C. geographus (GVIA) and C. magus (MVIIA) appear to compete for the same sites in mammalian brain, in amphibian brain the high-affinity binding of omega-conotoxin MVIIA has narrower specificity. In this system, it is demonstrated that a combination of two omega-conotoxins can be used for biochemically defining receptor subtypes and suggested that these correspond to subtypes of neuronal Ca2+ channels.     

Differential response to environmental alcohol among second-chromosome arrangements in experimental populations of Drosophila buzzatii

Drosophila buzzatii feeds and breeds on the decaying cladodes and fruits of several species of Opuntia (prickly pear) which contain significant levels of ethanol and isopropanol. The potential influence of these two alcohols on the inversion polymorphism of the second and fourth chromosomes was investigated in ten experimental populations with different amounts of alcohol (either ethanol or isopropanol) added to the culture medium. All populations were started with the offspring of 29 wild females collected at Adeje (Tenerife, Canary Islands) and their genetic composition was monitored for about two years (more than 30 generations). Consistent changes in the frequency of most second- and fourth-chromosome arrangements occurred in all populations including those without alcohol (control). The comparison of inversion frequency through the various treatments revealed a significant influence of the alcohol on the frequency changes of the four second-chromosome arrangements. Moreover, this influence was of a different type for every one of them, sometimes with opposite effects between alcohols and/or concentrations. These results indicate genetic differentiation among second-chromosome arrangements with regard to alcohol and suggest that the alcohol heterogeneity found in the species' trophic niche may play an important role in the maintenance of this polymorphism and also in the recent historical changes in the frequency of some arrangements associated with colonization.This paper is number X of the series The evolutionary history of Drosophila buzzatii.     

The effect of glucose-6-phosphate isomerase genotype onin vitro specific activity andin vivo flux inMytilus edulis

Four samples of the musselMytilus edulis were taken between 1984 and 1987 from Stony Brook, New York, and used to study the glucose-6-phosphate isomerase (GPI) polymorphism in this species.In vitro specific activity andin vivo flux measured in the same animals were found to be significantly correlated. A significant effect of GPI genotype on flux was observed in one of the samples; overall, significant evidence of effect of genotype on enzyme activity was also obtained. GPI activities of common genotypes tend to deviate less from the population mean than those of rare (frequency less than 5%) genotypes. This suggests the possibility that rare GPI genotypes are rare as a consequence of having biochemical properties that deviate from an optimum level and, therefore, having a lower fitness. In support of this hypothesis, we found in one of our samples that shell length is a concave function of GPI activity with an intermediate optimum activity level. The financial support provided to P.J.N.S. by the Luso-American Educational Commission (Fulbright Program), the Instituto Nacional de Investigacao Científica (Portugal), and the Faculdade de Ciências da Universidade de Lisboa during several stages of this research is gratefully acknowledged. Financial support from the Ministerio de Educatión y Ciencia (Spain) in the form of a postdoctoral Fulbright/MEC fellowship to M.S. is also gratefully acknowledged. Research was supported by National Science Foundation Grant BSR-8415060 to R.K.K. This is contribution No. 736 from the Program in Ecology and Evolution, State University of New York at Stony Brook. On leave from Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, Campo Grande C2, Lisboa, Portugal.     

NMR studies of DNA (R+)n.(Y-)n.(Y+)n triple helices in solution: imino and amino proton markers of T.A.T and C.G.C+ base-triple formation

High-resolution exchangeable proton two-dimensional NMR spectra have been recorded on 11-mer DNA triple helices containing one oligopurine (R)n and two oligopyrimidine (Y)n strands at acidic pH and elevated temperatures. Our two-dimensional nuclear Overhauser effect studies have focused on an 11-mer triplex where the third oligopyrimidine strand is parallel to the oligopurine strand. The observed distance connectivities establish that the third oligopyrimidine strand resides in the major groove with the triplex stabilized through formation of T.A.T and C.G.C+ base triples. The T.A.T base triple can be monitored by imino protons of the thymidines involved in Watson-Crick (13.65-14.25 ppm) and Hoogsteen (12.9-13.55 ppm) pairing, as well as the amino protons of adenosine (7.4-7.7 ppm). The amino protons of the protonated (8.5-10.0 ppm) and unprotonated (6.5-8.3 ppm) cytidines in the C.G.C+ base triple provide distinct markers as do the imino protons of the guanosine (12.6-13.3 ppm) and the protonated cytidine (14.5-16.0 ppm). The upfield chemical shift of the adenosine H8 protons (7.1-7.3 ppm) establishes that the oligopurine strand adopts an A-helical base stacking conformation in the 11-mer triplex. These results demonstrate that oligonucleotide triple helices can be readily monitored by NMR at the individual base-triple level with distinct markers differentiating between Watson-Crick and Hoogsteen pairing. Excellent exchangeable proton spectra have also been recorded for (R+)n.(Y-)n.(Y+)n 7-mer triple helices with the shorter length permitting spectra to be recorded at ambient temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prevention of insulin resistance by environmental manipulation as young rats mature

This study was initiated in an attempt to see if the insulin resistance associated with maturation in young rats could be prevented by environmental manipulation. Consequently, seven week-old rats were either housed in standard laboratory cages and fed a calorie-restricted diet or placed individually in exercise wheel cages and allowed to eat chow ad lib. A control group of rats was housed in standard laboratory cages from seven weeks to five months of age, and also allowed to eat chow ad lib. When studied at five months of age, the chow-fed rats weighed more (624 +/- 8 g) than either the calorie restricted (479 +/- 9 g) or exercise trained (485 +/- 13 g) rats. Insulin action was compared in the three groups by assessing the steady-state serum glucose (SSSG) and insulin (SSSI) concentrations achieved during a continuous intravenous infusion of glucose and exogenous insulin. The results of these studies indicated that SSSG concentration was significantly higher (P less than 0.001) in chow-fed rats than in the two experimental groups. Since SSSI concentrations were the same in all three groups, lower SSSG concentrations in calorie-restricted and exercise trained rats indicates that insulin-stimulated glucose uptake was preserved in these two groups as compared to the chow-fed population. In an attempt to understand why exercise training and calorie restriction prevented the development of insulin resistance, muscle glycogen synthase activity and muscle capillary density were compared in the three groups of five month-old rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

The cytotaxonomy ofEmilia spp. (Asteraceae: Senecioneae) occurring in Brazil

Analysis of several populations in a large part of the distribution area of the genusEmilia in Brazil has revealed only two species: the diploidE. sonchifolia and the tetraploidE. fosbergii. The more widely reportedE. coccinea was not found. They show a karyotype constancy in morphology and chromosome number (2n = 10 and 2n = 20, respectively), C-banding pattern and number of secondary constrictions. Some indications were found thatE. fosbergii may be an allopolyploid and that its ancestors had different genome sizes.     

Neurosecretory and trophic action on fetal rat neuroblasts induced by an amino acid mixture

The effects of a synthetically obtained mixture of amino acids (FACE) were investigated on the trophic and neurosecretory activity of in vitro cultures of fetal rat neuronal cells. The addition of 10(-6) M FACE to the culture medium significantly increased cell DNA content. Secretions of IR-SRIF, IR-VIP, and IR-GRF were also augmented in different proportions by the presence of FACE. Time studies demonstrated that IR-SRIF was significantly increased after 48 (P less than 0.05) and 72 (P less than 0.01) hr of exposure to FACE, and IR-VIP secretion was potentiated after only 24 hr of culture. Dose-response experiments with 10(-7) to 10(-4) M FACE indicated that concentrations of 10(-5) and 10(-4) M significantly increased both somatostatin released to the medium and cell content of IR-SRIF. FACE concentrations as low as 10(-10) M augmented the secretion of IR-GRF, and there was a dose-response correlation between 10(-10) and 10(-5) M FACE. The release and cell content of IR-VIP were also increased by FACE, with a dose-response relation at concentrations of 10(-9) to 10(-6) M. It can thus be concluded that FACE has a powerful effect on the multiplication and survival of fetal cerebrocortical cells and is also an important potentiator of IR-SRIF, IR-VIP, and IR-GRF secretion.     

Differentiation of myoblasts and CNS cells grown either separately or as co-cultures on microcarriers

Dispersed neuronal and muscular elements from fetal or neonatal origin, can organize and mature in culture when grown on positively charged cylindrical microcarriers (MCS), to a stage which simulatein vivo maturation. Cells arrange themselves on the MCS to form aggregates which remain floating in the nutrient medium. In such a tridimensional organization, the neuronal tissue is capable of regenerating a network of nerve fibers which establish synapse interconnections and undergo myelination. Oligodendrocytes organize on MCS in a tridimensional pattern and produce extensive myelin-like membranes. Myoblasts in MC-cultures fuse into polynucleated myotubes which become striated and contract spontaneously. Creatine kinase and acetylcholine receptor (AChR) are formed during myogenesis in similar quantities in MC-cultures and in monolayers. When both neuronal and muscle tissues are prepared from the same fetus (autologous nerve-muscle co-cultures) and are cultured on MCS, they interconnect to form neuro-muscular junctions. Cells from both tissues, exhibit better differentiation, for longer periods in MC-cultures than they do in monolayers. The floating functional entities are easy to sample and can be harvested for ultrastructural, immunocytochemical and biochemical analysis. In addition, MC-cultures can be used as a good tool for the study of acute and chronic exposures to toxicological agents, as well as for implantation into demyelinated, injured or dystrophic tissues. In this case the MCS in the implanted entities will serve as identifiable markers.     

Molecular analysis of yellow fever virus 17DD vaccine strain.

The Oswaldo Cruz Foundation produces most of the yellow fever (YF) vaccine prepared worldwide. As part of a broader approach to determine the genetic variability in YF 17D seeds and vaccines and its relevance to viral attenuation the 17DD virus was purified directly from chick embryo homogenates which is the source of virus used for vaccination of millions of people in Brazil and other countries for half a century. Neutralization and hemagglutination tests showed that the purified virus is similar to the original stock. Furthermore, radioimmune precipitation of 35S-methionine-labeled viral proteins using mouse hyperimmune ascitic fluid revealed identical patterns for the purified 17DD virus and the YF 17D-204 strain except for the 17DD E protein which migrated slower on SDS-PAGE. This difference is likely to be due to N-linked glycosylation. Finally, comparison by northern blot hybridization of virion RNAs of purified 17DD with two other strains of YF 17D virus revealed only genome-sized molecules for all three viruses. These observations suggest that the vaccine phenotype is primarily associated with the accumulation of mutations.