Analysis of a primer-independent GTF-I from Streptococcus salivarius

Abstract A glucosyltransferase (GTF) gene, designated gtfL , from Streptococcus salivarius was cloned and expressed in Escherichia coli and its nucleotide sequence determined. The GTF-L enzyme catalysed the synthesis of water-insoluble glucan in a primer-independent manner. The nucleotide sequence and derived amino acid sequence of GTF-L were similar in size and domain structure to previously sequenced glucosyltransferases. However, a 464-bp region of high variability was identified which could be selectively amplified from strains of S. salivarius by the polymerase chain reaction and could therefore form the basis for species identification. No sequence-specific motifs related to the solubility and linkage of the glucan product or its need for a dextran primer could be ascertained.     

How does the G protein,Gi2, transduce mitogenic signals?

Serpentine receptors coupled to the heterotrimeric G protein, G_i2, are capable of stimulating DNA synthesis in a variety of cell types. A common feature of the G_i2-coupled stimulation of DNA synthesis is the activation of the mitogen-activated protein kinases (MAPKs). The regulation of MAPK activation by the G_i2-coupled thrombin and acetylcholine muscarinic M₂ receptors occurs by a sequential activation of a network of protein kinases. The MAPK kinase (MEK) which phosphorylates and activates MAPK is also activated by phosphorylation. MEK is phosphorylated and activated by either Raf or MEK kinase (MEKK). Thus, Raf and MEKK converge at MEK to regulate MAPK. G_i2-coupled receptors are capable of activating MEK and MAPK by Raf-dependent and Raf-independent mechanisms. Pertussis toxin catalyzed ADP-ribosylation of α_i2 inhibits both the Raf-dependent and-independent pathways activated by G_i2-coupled receptors. The Raf-dependent pathway involves Ras activation, while the Raf-independent activation of MEK and MAPK does not involve Ras. The Raf-independent activation of MEK and MAPK most likely involves the activation of MEKK. The vertebrate MEKK is homologous to the Ste11 and Byr2 protein kinases in the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The yeast Ste11 and Byr2 protein kinases are involved in signal transduction cascades initiated by pheromone receptors having a 7 membrane spanning serpentine structure coupled to G proteins. MEKK appears to be conserved in the regulation of G protein-coupled signal pathways in yeast and vertebrates. Raf represents a divergence in vertebrates from the yeast pheromone-responsive protein kinase system. Defining MEKK and Raf as a divergence in the MAPK regulatory network provides a mechanism for differential regulation of this system by G_i2-coupled receptors as well as other receptor systems, including the tyrosine kinases.     

Mapping of Murine Fibroblast Growth Factor Receptors Refines Regions of Homology between Mouse and Human Chromosomes

The genes for the fibroblast growth factor receptors Fgfr2, Fgfr3, and Fgfr4 have been mapped in the mouse using an interspecific backcross mapping panel. The Fgfr loci map to previously defined regions of homology between human and mouse chromosomes and provide additional information regarding human/mouse comparative mapping.     

The effect of pulsed microwaves on passive electrical properties and interspike intervals of snail neurons

The effects of pulsed microwaves (2.45 GHz, 10 μs, 100 pps, SAR: 81.5 kW/kg peak, 81.5 W/kg average) on membrane input resistance and action potential (AP) interval statistics were studied in spontaneously active ganglion neurons of land snails (Helix aspersa), at strictly constant temperature (20.8±.07°C worst case). Statistical comparison with sham-irradiated neurons revealed a significant increase in the mean input resistance of neurons exposed to pulsed microwaves (P ? .05 ). Pulsed microwaves had no visible effect on mean AP firing rate; this observation was confirmed by analysis of interspike intervals (ISIs). Using an integrator model for spontaneously active neurons, we found the net input current to be more variable in neurons exposed to pulsed microwaves. The mean input current was not affected. The standard deviation of ISIs and the autocorrelation of the input current were marginally affected, but these changes were not consistent across neurons. Although the observed effects were less obvious than those reported in other studies, they represent evidence of a direct interaction between neurons and pulsed microwaves, in the absence of macroscopic temperature changes. The data do not suggest a single, specific mechanism for such interaction. © 1993 Wiley-Liss, Inc.     

A 37-kilodalton glycoprotein from a baculovirus of Orgyia pseudotsugata is localized to cytoplasmic inclusion bodies.

The gene encoding a 37-kDa glycoprotein (gp37) of Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) was located and sequenced. gp37 of OpMNPV was found to have 62 and 37% amino acid sequence identity with gp37 of Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) and with a protein reported to be a component of occlusion bodies from Choristoneura biennis entomopoxvirus, respectively. The mRNA start site of the OpMNPV gp37 gene was mapped within a late promoter sequence (TTAAG). A TrpE fusion protein containing 55% of the OpMNPV gp37 gene amino acid sequence was used to generate a monospecific antiserum. Western immunoblot analysis of OpMNPV-infected Lymantria dispar cells detected gp37 beginning at 24 h postinfection. Immunoelectron microscopy indicated that the protein is concentrated in cytoplasmic inclusion bodies late in infection. In contrast to gp37 of AcMNPV which was present in the matrix of occlusion bodies, OpMNPV gp37 was not observed in this location. Neither OpMNPV nor AcMNPV gp37 was associated with the polyhedron envelope.     

The purification and immunocharacterisation of N-methylputrescine oxidase from transformed root cultures of Nicotiana tabacum L. cv SC58

The enzyme N-methylputrescine oxidase which catalyses the conversion of N-methylputrescine to N-methylpyrrolinium salt has been purified to homogeneity from transformed roots of Nicotiana tabacum L. cv SC58. The enzyme has an apparent sub-unit molecular weight of 53 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with gel-filtration studies, indicating that the native form is a dimer. The K _m of the enzyme for N-methylputrescine has been estimated to be 0.1 mM. Polyclonal antibodies raised to the purified protein recognise one product in an immunoblot of a crude extract of transformed root tissue and will immunoprecipitate N-methylputrescine oxidase activity from such an extract. The antibodies also show a high degree of specificity in immunoblots of crude extracts of transformed root cultures from a range of other solanaceous and non-solanaceous species but do not cross-react with a partially purified preparation of pea-seedling diamine oxidase.Abbreviations MPO N-methylputrescine oxidase - PVDF polyvinylidene difluoride - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We would like to thank members of the Plant Cell Biotechnology Group, Institute of Food Research, Norwich Laboratory, for their helpful discussions during the preparation of this paper.     

A matter of timing: System requirements for repair and their temporal dimensions

Research into repair within the circular economy (CE) typically focuses on technical aspects of design, policy, and markets, and often assumes simplified conditions for the user/owner and the product system to explain the barriers to scaling repair activities. However, factors occurring at pre-use stages of the product's life cycle can significantly influence whether, and to what extent, repair is viable or possible, that is, warranty duration, after-sale service provision, and access to necessities. The passing of time can directly and indirectly affect the ability, difficulty, and thus, the likelihood of repair activities being performed at each stage of the product's life cycle. Drawing from the literature and applying inductive systems-thinking tools, we propose a framework for considering the “System of Repairability.” We delineate how the passing of time (temporal dimensions) affects one's ‘‘ability to repair,’’ as a product progresses through different life cycle phases (i.e., breakdown vs. repair vs. disposal), and the point(s) at which the repair is considered or attempted (i.e., year of usage). By integrating life cycle and temporal (time-based) dimensions into a broad System of Repairability framework, we clarify relevant interconnections, iterations, sequences, and timing of decision points, stakeholders, and necessary conditions to facilitate an outcome of successful repair at the individual level, and thus intervention strategies for scaling repair within CE. We discuss how a policy mix can address the life cycle of products and the repair system more holistically. We conclude with a future outlook on how temporal dimensions can inform policy strategies and future research.     

The stimulatory effect of chronic lithium treatment on basal thyrotropin secretion in rats: In vivo antagonism by methylparaben

Chronic treatment of rats with lithium chloride was examined in order to determine its effect on hypothalamic monoamine and metabolite content, basal thyrotropin (TSH) secretion and thyroid function. The hypothalamic concentrations of noradrenaline (NA), dopamine (DA) and its metabolites, dihydroxyphenylacetic acid. (DOPAC) and homovanillic acid (HVA) in the lithium treated rats remained unaltered when compared to control levels. NA turnover and the NA metabolite, 3-methoxy-4-hydroxyphenylglycol (total MHPG), were significantly lower (p<0.01), whereas both serotonin (5-HT) and its metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), were significantly higher (p<0.01 and p<0.02, respectively) in the lithium treated rat hypothalami than in controls. Chronic lithium treatment significantly elevated basal TSH levels (p<0.05). This effect was antagonized by methylp-hydroxybenzoate (methylparaben, p<0.01), which did not itself affect basal TSH levels. Free serum T₃ and T₄ levels were not significantly affected by chronic lithium treatment, although T₄ tended to be slightly lower than control levels. The monoamine changes observed in the hypothalamus of lithium treated rats did not appear to account for the elevated TSH levels observed in these rats since NA activity which is generally regarded as stimulatory was decreased and 5-HT which has an inhibitory effect on TSH secretion, was increased. The elevated TSH levels may have been due to a reduced negative feedback inhibition of TSH release by the mildly reduced circulating T₄ levels caused by chronic lithium treatment. A further possibility is that the pituitary cGMP (and hence TSH) response to TRH may have been enhanced by chronic lithium treatment and methylparaben may have antagonized this effect.