Disulfide and secondary structures of recombinant human granulocyte colony stimulating factor

Molecular characteristics and secondary structures of recombinant methionyl human granulocyte colony stimulating factor produced by genetically engineered Escherichia coli are described. Limited radiolabeling of the protein with tritiated iodoacetate and determination of the labeled residue revealed that this recombinant protein contains only one free cysteine at position 17 which is not essential for activity. The free cysteine is inaccessible to modification unless the molecule is unfolded under denaturing conditions. The molecule forms two disulfide bridges which were assigned as Cys(36)-Cys(42) and Cys(64)-Cys(74) based on the results of isolation and characterization of disulfide-containing peptides obtained from a subtilisin digest of the intact protein. CD analyses and secondary structure prediction suggest that the molecule is abundant in alpha-helical structures.     

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)     

Separation of micro-organisms from meat and their rapid enumeration using a membrane filtration-epifluorescent microscopy technique

A new method of separating bacteria from beef mince has been developed, in which an alkaline protease, Alcalase 0.6 L, was used to degrade the meat proteins, leaving micro-organisms in suspension. The organisms were then counted, using a membrane filtration-epifluorescent microscopy technique. A correlation coefficient of 0.97 was obtained between this method and the standard plate count, indicating its suitability for use in quality control.     

Prenatal diagnosis of familial amyloidotic polyneuropathy: evidence for an early expression of the associated transthyretin methionine 30

Summary Transthyretin methionine 30 (TTR Met 30), which is associated with familial amyloidotic polyneuropathy, originates in a single base substitution (A for G) in the second exon of the TTR gene. This autosomal dominant disease can be diagnosed by RFLP analysis of NsiI-digested DNA. The amplification of DNA by PCR improves the diagnosis method, making it suitable for prenatal diagnosis. Using PCR-amplified DNA, prenatal diagnosis of two at-risk fetuses was performed. Control Met 30 and normal DNA (either genomic or produced by site directed mutagenesis) were processed in parallel. The diagnosis was made by hybridization with allele-specific oligonucleotide probes, and later confirmed by screening of the mutant protein in the amniotic fluid and, when possible, in the sera from the newborns. TTR Met 30 was detected in the amniotic fluid of a positive fetus whose father was the carrier of the mutation. This indicates that the mutant protein is expressed very early in development.     

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.     

The interaction of Trichomonas vaginalis and Tritrichomonas foetus with epithelial cells in vitro

The Madin-Darby canine kidney epithelial cell line (MDCK) was used as a model for trichomonad-host cell interaction. Two laboratory strains of the human parasite Trichomonas vaginalis and the cattle's parasite Tritrichomonas foetus or their supernatants from axenic cultures were allowed to interact with confluent epithelial cultures. The interaction process studied by transmission and scanning electron microscopy revealed that both parasites adhere to monolayers through flagella, cell body and particularly for T. foetus, through the posterior projection of the axostyle. A close contact region between the trichomonad's surface and MDCK cells was observed. A study of the involvement of trichomonad surface component in the interaction process indicated that cytochalasin B treated-parasites adhere much less to epithelial monolayers than untreated parasites. Colchicine treatment did not affect such adhesion. Treatment of the parasites with trypsin reduced the adhesion of trichomonads to monolayers but did not interfere with the cytopathic effect. In contrast, treatment of the parasites with neuraminidase did not interfere with their adhesion to epithelial cells and the monolayer destruction was further increased.     

In vivo inhibition of nitrogenase by hydroxylamine in Rhodospirillaceae Role of nitric oxide

Rhodobacter capsulatus strains E1F1 and B10 and Rhodobacter sphaeroides DSM 158 did not use hydroxylamine as nitrogen source for growth but metabolized it mainly through the glutamine synthetase reaction. Hydroxylamine had a high toxicity for cells growing either under phototrophic or dark-aerobic conditions. l-methionine-d,l-sulfoximine partially inhibited hydroxylamine uptake and increased the inhibition time of nitrogenase activity by this nitrogen compound. Nitric oxide was also a powerful inhibitor of nitrogenase in intact cells of R. capsulatus. Since low amounts of NO were produced from hydroxylamine, short-term inhibition of nitrogenase in the presence of this compound could be mediated in vivo by nitric oxide.Abbreviations GS glutamine synthetase - MSX l-methionine-d,l-sulfoximine - MTA mixed alkyltrimethylammonium bromide     

Infection of BALB/cByJ mice with the JHM strain of mouse hepatitis virus alters in vitro splenic T cell proliferation and cytokine production.

Earlier studies from this laboratory showed that infection of BALB/cByJ mice by a natural route with mouse hepatitis virus, strain JHM (MHV-JHM), results in functional splenic T cell suppression in vitro. This was evidenced by reduced concanavalin A-driven spleen cell proliferation and interleukin (IL)2 production measured after conventional intervals of cell culture (72 and 24 h, respectively). The purpose of the present work was to determine whether MHV-induced T cell dysfunction is kinetic or absolute and whether production of other T-cell derived cytokines is defective. Bioassays revealed that production of IL2, gamma interferon, and IL4 by spleen cells from acutely infected mice is suppressed and that some of the defects are kinetic as well as absolute. Proliferative responses of both CD4+ and CD8+ T cells were depressed, but neither cell type contained infectious virus. Cells that proliferated poorly in response to concanavalin A were fully capable of responding to specific virus stimulation. These results further emphasize the potential complications that MHV infection may pose to immunologic research using mice.