The TraT lipoprotein as a vehicle for the transport of foreign antigenic determinants to the cell surface of Escherichia coli K12: structure–function relationships in the TraT protein

The TraT protein is a surface-exposed lipoprotein, specified by plasmids of the IncF group, that mediates serum resistance and surface exclusion. The structure and function of the TraT protein determined by plasmid R6-5 was probed by genetic insertion of a foreign antigenic determinant, the C3 epitope of polio virus, at residues 61, 125, 180, 200 or 216 of the protein. The chimaeric proteins were transported to the outer membrane and, in three cases, immunoassays with an anti-C3 monoclonal antibody indicated that the C3 epitope was exposed on the cell surface. Three of the hybrids, with insertions at residues 125, 180 and 200, assembled into the trypsin-resistant oligomeric form characteristic of the wild-type protein, which suggested that these regions are not involved in TraT subunit:subunit interactions. Additionally, the hybrid protein carrying the C3 epitope at position 180 functioned in a genetic suppression assay and retained partial surface-exclusion activity. Thus, its localization, folding and organization does not appear to be grossly altered from that of the wild-type protein. Applications of the protein for the transport of foreign antigenic determinants to the cell surface are discussed.     

EFFECT OF OLIGEMIA ON REGIONAL METABOLITE LEVELS IN CAT BRAIN

Abstract— Incomplete cerebral ischemia (oligemia) was produced in cat by carotid occlusion combined with arterial hypotension. Lowering arterial pressure to 50–60 Torr for 20 min caused marked alterations of the ATP, phosphocreatine, and lactate content of subcortical white matter. In contrast, metabolite levels in cerebral cortex and caudate nucleus were only moderately perturbed from control values. More severe oligemia resulted when arterial pressure was lowered to 30 Torr for 20 min following carotid occlusion. Metabolite levels in cortex, caudate nucleus, and white matter were greatly altered from control. In the gray matter there was regional heterogeneity of metabolic alteration, as evidenced from the pattern of NADH tissue fluorescence. The cortex contained micro-patches (0.1mm) of increased NADH, which frequently exhibited a columnar orientation.
These findings demonstrate two distinct types of cerebral inhomogeneity of metabolic failure with reduced blood flow; white matter fails before gray matter, and there is micro-heterogeneity of metabolic failure in the gray matter.     

Genetic Characterization of Accumulation of Polyhydroxyalkanoate from Styrene in Pseudomonas putida CA-3

Pseudomonas putida CA-3 is capable of accumulating medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when growing on the toxic pollutant styrene as the sole source of carbon and energy. In this study, we report on the molecular characterization of the metabolic pathways involved in this novel bioconversion. With a mini-Tn5 random mutagenesis approach, acetyl-coenzyme A (CoA) was identified as the end product of styrene metabolism in P. putida CA-3. Amplified flanking-region PCR was used to clone functionally expressed phenylacetyl-CoA catabolon genes upstream from the sty operon in P. putida CA-3, previously reported to generate acetyl-CoA moieties from the styrene catabolic intermediate, phenylacetyl-CoA. However, the essential involvement of a (non-phenylacetyl-CoA) catabolon-encoded 3-hydroxyacyl-CoA dehydrogenase is also reported. The link between de novo fatty acid synthesis and PHA monomer accumulation was investigated, and a functionally expressed 3-hydroxyacyl-acyl carrier protein-CoA transacylase (phaG) gene in P. putida CA-3 was identified. The deduced PhaG amino acid sequence shared >99% identity with a transacylase from P. putida KT2440, involved in 3-hydroxyacyl-CoA MCL-PHA monomer sequestration from de novo fatty acid synthesis under inorganic nutrient-limited conditions. Similarly, with P. putida CA-3, maximal phaG expression was observed only under nitrogen limitation, with concomitant PHA accumulation. Thus, β-oxidation and fatty acid de novo synthesis appear to converge in the generation of MCL-PHA monomers from styrene in P. putida CA-3. Cloning and functional characterization of the pha locus, responsible for PHA polymerization/depolymerization is also reported and the significance and future prospects of this novel bioconversion are discussed.     

Clostridial neurotoxins compromise the stability of a low energy SNARE complex mediating NSF activation of synaptic vesicle fusion.

A 20S complex composed of the cytosolic fusion proteins NSF and SNAP and the synaptosomal SNAP receptors (SNAREs) synaptobrevin, syntaxin and SNAP-25 is essential for synaptic vesicle exocytosis. Formation of this complex is thought to be regulated by synaptotagmin, the putative calcium sensor of neurotransmitter release. Here we have examined how different inhibitors of neurotransmitter release, e.g. clostridial neurotoxins and a synaptotagmin peptide, affect the properties of the 20S complex. Cleavage of synaptobrevin and SNAP-25 by the neurotoxic clostridial proteases tetanus toxin and botulinum toxin A had no effect on assembly and disassembly of the 20S complex; however, the stability of its SDS-resistant SNARE core was compromised. This SDS-resistant low energy conformation of the SNAREs constitutes the physiological target of NSF, as indicated by its ATP-dependent disassembly in the presence of SNAP and NSF. Synaptotagmin peptides caused inhibition of in vitro binding of this protein to the SNAREs, a result that is inconsistent with synaptotagmin's proposed role as a regulator of SNAP binding. Our data can be reconciled by the idea that NSF and SNAP generate synaptotagmin-containing intermediates in synaptic vesicle fusion, which catalyse neurotransmitter release.     

Lamb pregastric enzyme-catalysed hydrolysis of 4-nitrophenylalkanoates and monoacid triglycerides

Three lamb pregastric enzymes, isolated from the commercial extract from the tongue and epiglottal region of lamb, have been used to catalyze the hydrolysis of a series of 4-nitrophenylalkanoate esters (C2–C12) at 37°C, pH 7.2 and maximum activity was obtained against the decanoate ester in all cases. Burst kinetics were observed for activity of the principal lipase component against the decanoate ester. This enzyme was also used as a catalyst for the hydrolysis of monoacid triglycerides (C4:0 to C10:0) at 35°C, pH 6.5 and maximum activity was obtained against tributyrin (C4:0). A suggestion is made for orientation of ester substrates within the active site of the enzymes.     

Differences in the patterns of methylation in rat liver ribosomal ribonucleic acid after reaction in vivo with methyl methanesulphonate and NN-dimethylnitrosamine

1. rRNA was isolated from rat liver at short intervals after the intraperitoneal injection of [(14)C]methyl methanesulphonate (50mg/kg) or NN-di[(14)C]methylnitrosamine (2mg/kg). These doses were chosen to minimize the effects of toxicity. 2. The following methods of hydrolysis of [(14)C]methylated rRNA were employed: enzymic digestion to nucleosides at pH8; alkaline hydrolysis and conversion into nucleosides; acid hydrolysis to bases. 3. The methylation products were analysed by chromatography on columns of Dowex-50 (H(+) form) and Dowex-50 (NH(4) (+) form). 4. With both methylating agents the principal product of methylation was 7-methylguanine. Differences were obtained, however, in the molar proportions of the minor bases 3-methylcytosine, 1-methyladenine and 7-methyladenine. Methylation at the O-6 position of guanine was a significant feature of rRNA obtained from the NN-di[(14)C]methylnitrosamine-treated animals but was not detected in rRNA after treatment with [(14)C]methyl methanesulphonate.     

Combination of high-performance liquid chromatography and thin-layer chromatography separation of five adducted nucleotides isolated from liver resulting from intraperitoneal administration with 7H-dibenzo[c,g]carbazole to mice

7H-Dibenzo[c,g]carbazole, DBC, is a potent environmental liver carcinogen. Liver DNA from mice treated with DBC exhibited seven distinct DBC-DNA adducts as detected by ³²P-postlabeling using multidimensional TLC. To improve quantitation and chemically characterize the adducts, DNA samples were hydrlyzed, ³²P-postlabeled and the adducts were separated from the unadducted normal nucleotides on TLC using a D1 solvent, 0.65 M sodium phosphate (pH 6.8). Adducts were eluted from the TLC plates with 4.0 M pyridinium formate, concentrated, resuspended in 50% aqueous methanol and injected onto the HPLC; five individual adduct peaks were resolved and collected by this method. This approach will prove useful to decrease analysis time and improve chemical characterization of tightly clustered DNA adducts generated in vivo.