PRIMARY STRUCTURE OF ALLATOSTATIN 4, A NEVROPEPTIDE INHIBITOR OF JUVENILE HORMONE SYNTHESIS

Abstract Isolation and identification of allatostatin 4 (AST 4) from crude brain extracts of female Diploptera punctata are described. Synthetic analogues of allatostatin 4 truncated from the N-terminal were evaluated to gain some knowledge of structure-activity correlation. AST 4 reversibly inhibits the biosynthesis of JH in vitro . AST 4 has the following sequence: Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH₂. This neurohormone (AST 4) has no sequence similarity with any known neuropeptide from other organisms. Synthetic AST 4 as well as truncation fragments, including two with the five and six amino acids terminal residues deleted showed in vitro activity distinguishable from that of the native allatostatin.     

DNA synthesis precedes gliotoxin-induced apoptosis

The toxin gliotoxin induces apoptosis or programmed cell death in a variety of immune cells including thymocytes. Apoptosis induced by gliotoxin in thymocytes is unaffected by protein synthesis inhibitors nor is it associated with early changes in intracellular calcium levels (Beaver and Waring, 1994). This work shows that the cell lines P815 and WEHI7 and murine thymocytes when treated with gliotoxin show an early incorporation of tritiated thymidine over the concentration range which causes apoptosis. Proliferating cell nuclear antigen (PCNA), a marker for S phase, is elevated in cells following gliotoxin treatment and S phase DNA content is increased. Thymidine incorporation is inhibited by hydroxyurea, an inhibitor of replicative DNA synthesis not repair. Free radical scavangers have no effect on apoptosis induced by gliotoxin in thymocytes. Hydrogen peroxide-treated cells showed no enhanced thymidine incorporation and no apoptosis. Thus oxidative stress does not appear to be a factor in gliotoxin-induced apoptosis. Thymocytes treated with gliotoxin show increased phosphorylation of a 16.3 kDa protein, and apoptosis is inhibited by the tyrosine kinase inhibitor genistein, which also inhibited the increased thymidine incorporation in P815 cells. We conclude that one mechanism by which gliotoxin can cause apoptosis may be the induction of inappropriate entry of cells into the cell cycle followed by death.     

Membrane filtration method for bacteriological testing of water: enhanced colony visualization and stability on purification of phenol red indicator

Commercially-available phenol red indicator, purified by adsorption chromatography was incorporated into lauryl sulphate broth (LSB) used in the membrane filtration method for the detection of Escherichia coli and other coliform bacteria. Relative to LSB containing the impure dye or its major contaminant, the purified phenol red provided clear visualization of discrete yellow colonies observed against a white background. The colonies remained stable for at least 24 h at 25°C under standard laboratory lighting conditions. This simple procedure will enhance the detection of coliforms in samples.     

Analysis of inhibitors of bacteriophage T4 DNA polymerase.

Bacteriophage T4 DNA polymerase was inhibited by butylphenyl nucleotides, aphidicolin and pyrophosphate analogs, but with lower sensitivities than other members of the B family DNA polymerases. The nucleotides N2-(p-n-butylphenyl)dGTP (BuPdGTP) and 2-(p-n-butylanilino)dATP (BuAdATP) inhibited T4 DNA polymerase with competitive Ki values of 0.82 and 0.54 microM with respect to dGTP and dATP, respectively. The same compounds were more potent inhibitors in truncated assays lacking the competitor dNTP, displaying apparent Ki values of 0.001 and 0.0016 microM, respectively. BuPdGTP was a substrate for T4 DNA polymerase, and the resulting 3'-BuPdG-primer:template was bound strongly by the enzyme. Each of the non-substrate derivatives, BuPdGDP and BuPdGMPCH2PP, inhibited T4 DNA polymerase with similar potencies in both the truncated and variable competitor assays. These results indicate that BuPdGTP inhibits T4 DNA polymerase by distinct mechanisms depending upon the assay conditions. Reversible competitive inhibition predominates in the presence of dGTP, and incorporation in the absence of dGTP leads to potent inhibition by the modified primer:template. The implications of these findings for the use of these inhibitors in the study of B family DNA polymerases is discussed.     

The Subunit Composition of a GABAA/Benzodiazepine Receptor from Rat Cerebellum

Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABA_A receptors that show diazepam (or clonazepam)-insensitive [³H]Ro 15-4513 binding has been determined by immunoprecipitation with subunit-specific antibodies. These receptors have α₆, α₁, γ_2S, γ_2L, and β₂ or β₃ subunits colocalizing in the same receptor complex.     

Mapping of two new markers within the smallest interval harboring the spinal muscular atrophy locus by family and radiation hybrid analysis

The locus responsible for the childhood-onset proximal spinal muscular atrophies (SMA) has recently been mapped to an area of 2–3 Mb in the region q12–13.3 of chromosome 5. We have used a series of radiation hybrids (RHs) containing distinct parts of the SMA region as defined by reference markers. A cosmid library was constructed from one RH. Thirteen clones were isolated and five of these were mapped within the SMA region. Both RH mapping and fluorescence in situ hybridization analysis showed that two clones map in the region between loci D5S125 and D5S351. One of the cosmids contains expressed sequences. Polymorphic dinucleotide repeats were identified in both clones and used for segregation analysis of key recombinant SMA families. One recombination between the SMA locus and the new marker 9Ic (D5S685) indicates that 9Ic is probably the closest distal marker. The absence of recombination between the SMA locus and marker Fc (D5S684) suggests that Fc is located close to the disease gene. These new loci should refine linkage analysis in SMA family studies and may facilitate the isolation of the disease gene.     

A simple and efficient method for DNA extraction from grapevine cultivars andVitis species

A quick, simple, and reliable method for the extraction of DNA from grapevine species, hybrids, andAmpelopsis brevipedunculata (Vitaceae) has been developed. This method, based on that of Doyle and Doyle (1990), is a CTBA-based extraction procedure modified by the use of NaCl to remove polysaccharides and PVP to eliminate polyphenols during DNA purification. The method has also been used successfully for extraction of total DNA from other fruit species such as apple (Malus domestica), apricot (Prunus armeniaca), cherry (Prunus avium), peach (Prunus persica), plum (Prunus domestica), and raspberry (Rubus idaeus). DNA yield from this procedure is high (up to 1 mg/g of leaf tissue). DNA is completely digestible with restriction endonucleases and amplifiable in the polymerase chain reaction (PCR), indicating freedom from common contaminating compounds.     

Evidence for the role of glycoprotein G of respiratory syncytial virus in binding of Neisseria meningitidis to HEp-2 cells

Abstract Viral glycoproteins G and F are expressed on the surface of cells infected with respiratory syncytial virus (RSV). We investigated the role of these proteins in the previously reported enhanced binding of Neisseria meningitidis to RSV-infected HEp-2 cells. Virus particles attached to bacteria were detected by immunofluorescence with flow cytometry. Binding of FITC-labelled bacteria to RSV-infected cells was significantly inhibited by monoclonal antibody against glycoprotein G. Unlabelled bacteria interfered with binding of the anti-G monoclonal antibody to these cells. These interactions were not found with a monoclonal antibody against glycoprotein F. We propose that glycoprotein G of RSV expressed on the surface of infected cells might act as an additional receptor for meningococci.     

A fed batch surface culture process for the production of citric acid with reuse of Aspergillus niger mycelia

Citric acid production in surface culture process with reuse of mycelia for more than one batch of media has been reported in this work. A strategy for replacing media and thus increasing productivity in subsequent batches is discussed. An explanation for the decrease in citric acid accumulation beyond the second batch is suggested on the basis of oxygen limitation.