Unique sequences in region VI of the flagellin gene of Salmonella typhi

The H1 (now renamed fliC; lino et al., 1988) alleles specifying antigenically different Salmonella flagellins are identical at their ends but differ greatly towards the middle, where there are two hypervariable segments (regions IV and VI). The flagellar antigen, d, of Salmonella typhi, is found also as phase-1 antigen in many other Salmonella species. We cloned the H1-d gene of a strain of S. typhi and determined the nucleotide sequence of its two hypervariable regions. Comparison with gene H1-d of Salmonella muenchen showed substantial differences in region VI: four scattered amino acid differences and ten adjacent amino acids in the inferred S. typhi sequence, all of which differ from the corresponding nine amino acids in the S. muenchen sequence. The results of polymerase chain reaction amplification indicated the presence of the S. typhi version in all of 18 additional S. typhi strains and the presence of the S. muenchen version in all four non-S. typhi species with flagellar antigen d. The difference in amino acid sequence in segment VI may be responsible for the minor serological differences between antigens d of S. typhi and antigen d of S. muenchen.     

Prokaryotic and eukaryotic pyridoxal-dependent decarboxylases are homologous

Summary A database search has revealed significant and extensive sequence similarities among prokaryotic and eukaryotic pyridoxal phosphate (PLP)-dependent decarboxylases, includingDrosophila glutamic acid decarboxylase (GAD) and bacterial histidine decarboxylase (HDC). Based on these findings, the sequences of seven PLP-dependent decarboxylases from five different organisms have been aligned to derive a consensus sequence for this family of enzymes. In addition, quantitative methods have been employed to calculate the relative evolutionary distances between pairs of the decarboxylases comprising this family. The multiple sequence analysis together with the quantitative results strongly suggest an ancient and common origin for all PLP-dependent decarboxylases. This analysis also indicates that prokaryotic and eukaryotic HDC activities evolved independently. Finally, a sensitive search algorithm (PROFILE) was unable to detect additional members of this decarboxylase family in protein sequence databases.     

High cooperativity, specificity, and multiplicity in the protein kinase C-lipid interaction

The number of phosphatidylserine molecules involved in activating protein kinase C was determined in a mixed micelle system where one monomer of protein kinase C binds to one detergent:lipid micelle of fixed composition. Unusually high cooperativity, specificity, and multiplicity in the protein kinase C-phospholipid interaction are demonstrated by examining the lipid dependence of enzymatic activity. The rates of autophosphorylation and substrate (histone) phosphorylation are specifically regulated by the phosphatidylserine content of the micelles. Hill coefficients of 8-11 were calculated for phosphatidylserine-dependent stimulation of enzyme activity, with a maximum occurring in micelles containing greater than or equal to 12 phosphatidylserine molecules. The high specificity that exists is illustrated by the fact that phosphatidylethanolamine and phosphatidylglycerol, but not phosphatidylcholine or phosphatidic acid, can replace only some of the phosphatidylserine molecules. We propose that Ca2+ and acidic phospholipids cause the protein to undergo a conformation change revealing multiple phosphatidylserine binding sites and resulting in the highly cooperative and specific interaction of protein kinase C with phosphatidylserine. Consistent with this, the proteolytic sensitivity of protein kinase C increases approximately 10-fold in the presence of phosphatidylserine and Ca2+ compared to Ca2+ alone. The high degree of cooperativity and specificity may provide a sensitive method for the physiological regulation of protein kinase C by phospholipid.     

Isolated iron-molybdenum cofactor of nitrogenase exists in multiple forms in its oxidized and semi-reduced states

Electrochemical and EPR spectroscopic experiments demonstrate that the isolated iron-molybdenum cofactor from the molybdenum-iron protein of nitrogenase from Azotobacter vinelandii exists in multiple forms in both its oxidized and semi-reduced states. The particular forms found in either oxidation state appear to be a function of the acid/base status of the solvent, N-methylformamide. In "alkaline" N-methylformamide, a single, detectable form of iron-molybdenum cofactor is observed for both oxidized and semi-reduced states. The semi-reduced form, termed R(s-r), is the one previously recognized with an S = 3/2 EPR spectrum with apparent g values of 4.6, 3.4, 2.0. Its oxidized counterpart, termed B(ox), is characterized electrochemically by a differential pulse voltammetric reduction peak at -0.37 V versus the normal hydrogen electrode. In "acidic" solvent, two distinct, previously unrecognized redox pairs of iron-molybdenum cofactor forms exist. The two semi-reduced forms, N(s-r) and W(s-r), are characterized by EPR spectra with g = 4.5, 3.6, 2.0 and g = 4.9, 3.1, 1.9, respectively. Their oxidized counterparts, A(ox) and C(ox), have differential pulse voltammetric reduction peaks at -0.32 and -0.43 V versus the normal hydrogen electrode, respectively. Manipulations of either the isolation protocol or the sample conditions affects both the type and distribution of forms present. Each form likely corresponds to a biologically significant state of the cofactor cluster within the protein.     

Identification of IL-1 receptors on human monocytes

The expression and functional analysis of IL-1 beta R on human monocytes were investigated. Binding of 125I-IL-1 to human monocytes was found to be specific and saturable. Scatchard plot analysis revealed a single class of receptors with a binding constant of 600 pM and a receptor density of approximately 100 binding sites per cell. At 37 degrees C 54% of the labeled ligand was internalized over 2 h of incubation. Addition of 0.2% sodium azide to the cells reduced ligand internalization to 9% of total bound. Cross-linking studies revealed that the IL-1R in human monocytes had a Mr of 80 kDa. The addition of IL-1 to monocytes caused changes in membrane Ag expression as assessed by flow cytometric analysis. The results of this study identify IL-1 receptors on monocytes and suggest that IL-1 may act as an effector molecule for monocytes by enhancing expression of Ag correlated with cell differentiation and immune function.     

Activation of recA protein: The salt-induced structural transition

Purified recA protein is induced by high salt concentrations to hydrolyse ATP even in the absence of DNA. By small angle neutron scattering we show that this salt activation results from a structural transition of the protein filament in the presence of ATPγS from the inactive, compact form (a helical polymer of pitch 70 Å and cross-sectional radius of gyration R_c 40 Å) to the open form (a helical filament of pitch 95 Å and R_c 35 Å, which are the same structural parameters as in the ATPase active complex with DNA and ATP), without detectable change in the degree of association. We conclude that activation of recA is due to the same structural change whether induced by the binding of DNA or by salt. Indeed, the other enzymatic activity of recA, the proteolytic cleavage of the lexA repressor, is found to be inducible by the same salt concentrations as those of the structural transition.     

Glutathione amide and its perthiol in anaerobic sulfur bacteria.

Chromatium species produced the novel biological thiol glutathione amide, gamma-L-glutamyl-L-cysteinylglycine amide (GASH), when grown photoheterotrophically. GASH was largely converted to the corresponding perthiol during photoautotrophic growth on sulfide, suggesting that GASH may have a function in anaerobic sulfide metabolism. This unprecedented form of glutathione metabolism was probably present in anaerobic ancestors of modern cyanobacteria and purple bacteria.     

Characterisation of Human 5-Hydroxytryptamine2A and 5-Hydroxytryptamine2C Receptors Expressed in the Human Neuroblastoma Cell Line SH-SY5Y: Comparative Stimulation by Hallucinogenic Drugs

Abstract: Stable transfection of the human neuroblastoma cell line SH-SY5Y with the human 5-hydroxytryptamine_2A (5-HT_2A) or 5-HT_2C receptor cDNA produced cell lines demonstrating ligand affinities that correlated closely with those for the corresponding endogenous receptors in human frontal cortex and choroid plexus, respectively. Stimulation of the recombinant receptors by 5-HT induced phosphoinositide hydrolysis with higher potency but lower efficacy at the 5-HT_2C receptor (pEC₅₀ = 7.80 ± 0.06) compared with the 5-HT_2A receptor (pEC₅₀ = 7.30 ± 0.08). Activation of the 5-HT_2A receptor caused a transient fourfold increase in intracellular Ca²⁺ concentration. Whole-cell recordings of cells clamped at ?50 mV demonstrated a small inward current (2 pA) in response to 10 µM 5-HT for both receptors. There were no differences in potency or efficacy of phosphoinositide hydrolysis among four hallucinogenic [d-lysergic acid diethylamide (LSD), 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI), 5-methoxy-N,N-dimethyltryptamine, and mescaline] and three nonhallucinogenic drugs (m-chlorophenylpiperazine, quipazine, and ergotamine). Comparison of equipotent doses producing 20% of the maximal response induced by 5-HT revealed selective activation of the 5-HT_2A receptor by LSD and to a lesser degree by DOI, mescaline, and ergotamine. Quipazine and 5-methoxy-N,N-dimethyltryptamine were relatively nonselective, whereas m-chlorophenylpiperazine selectively activated the 5-HT_2C receptor. It is unlikely therefore that hallucinosis is mediated primarily by activity at the 5-HT_2C receptor, whereas activity at the 5-HT_2A receptor may represent an important but not unique mechanism associated with hallucinogenic drug action.     

DNA methylation and Dc8-GUS transgene expression in carrot (Daucus carota L.)

Summary DNA methylation has been associated with gene activity in differentiating and developing plant tissues. The objective of this study was to determine the involvement of methylation in the expression of a gene transferred into carrot (Daucus carota L.) tissues by particle bombardment. Expression of the Dc8-GUS gene construct in response to treatments with 5-azacytidine (S-azaC) and to in vitro methylation by methylases was investigated by histochemical assay of GUS activity. The 5-azaC treatment increased the frequency of Dc8-driven GUS expression in both calli and somatic embryos. The increase occurred with treatment either to E. coli containing the plasmid insert or to the carrot tissues before bombardment. GUS expression, increased by the 5-azaC treatment, was enhanced by ABA treatment of both calli and somatic embryos and was more prominent in the latter. Increased digestion of the 5-azaC-treated plasmid DNA with EcoRII suggested that demethylation had occurred. In vitro methylation of Dc8-GUS by methylases generally resulted in a lower frequency of GUS expression. SssI methylase completely inhibited GUS expression. The level of GUS expression was correlated with the extent of methylation of the plasmid.Abbreviations ABA Abscisic Acid - 5-azaC 5-azacytidine - GUS -glucuronidase - Dc8 carrot promoter