Metabonomic investigations of aging and caloric restriction in a life-long dog study

Long-term restriction of energy intake without malnutrition is a robust intervention that has been shown to prolong life and delay age-related morbidity. A 1H NMR-based metabonomic strategy was used to monitor urinary metabolic profiles throughout the lifetimes of control-fed and diet-restricted dogs. Urinary metabolic trajectories were constructed for each dog, and metabolic variation was found to be predominantly influenced by age. Urinary excretion of creatinine increased with age, reaching a maximum between ages 5 and 9 years and declining thereafter. Excretion of mixed glycoproteins was noted at earlier ages, which may be a reflection of growth patterns. In addition, consistent metabolic variation related to diet was also characterized, and energy-associated metabolites, such as creatine, 1-methylnicotinamide, lactate, acetate, and succinate, were depleted in urine from diet-restricted dogs. Both aging and diet restriction altered activities of the gut microbiotia, manifested by variation of aromatic metabolites and aliphatic amine compounds. This analysis allowed the metabolic response to two different physiological processes to be monitored throughout the lifetime of the canine population and may form part of a strategy to monitor and reduce the impact of age related diseases in the dog, as well as providing more general insights into extension of longevity in higher mammals.     

Influence of 6-diazo-5-oxo-L-norleucine (DON), a glutamine analogue, on cartilaginous differentiation in mouse limb buds in vitro.

The antibiotic, 6-diazo-5-oxo-L-norleucine (DON), an analogue of L-glutamine, causes limb malformations in several species, including mice. This report shows that DON also interferes with differentiation of cartilaginous rudiments of mouse limb buds grown as organ cultures for 3 to 8 days in medium containing the teratogen. DON (5 mug/ml) inhibits growth of the explants and interferes with production of normal matrix by chondrocytes. The cartilage of DON-treated cultures exhibits a striking lack of matrix, compared with that of control explants which contains abundant metachromatic matrix. Differentiation of osteoblasts, and secretion of osteoid around the scapula and humerus are enhanced by DON. The direct effects of DON on growth and chondrogenesis, which can be prevented by addition of L-glutamine (1 mg/ml) to the medium, can be attributed to the known interference of DON in L-glutamine-dependent steps in metabolism. The possible relationships between these effects of DON in vitro and the malformations produced in vivo, are discussed.     

Virulence determinants, drug resistance and mobile genetic elements of Laribacter hongkongensis: a genome-wide analysis

Background

Laribacter hongkongensis is associated with community-acquired gastroenteritis and traveler's diarrhea. In this study, we performed an in-depth annotation of the genes in its genome related to the various steps in the infective process, drug resistance and mobile genetic elements.

Results

For acid and bile resistance, L. hongkongensis possessed a urease gene cassette, two arc gene clusters and bile salt efflux systems. For intestinal colonization, it possessed a putative adhesin of the autotransporter family homologous to those of diffusely adherent Escherichia coli (E. coli) and enterotoxigenic E. coli. To evade from host defense, it possessed superoxide dismutase and catalases. For lipopolysaccharide biosynthesis, it possessed the same set of genes that encode enzymes for synthesizing lipid A, two Kdo units and heptose units as E. coli, but different genes for its symmetrical acylation pattern, and nine genes for polysaccharide side chains biosynthesis. It contained a number of CDSs that encode putative cell surface acting (RTX toxin and hemolysins) and intracellular cytotoxins (patatin-like proteins) and enzymes for invasion (outer membrane phospholipase A). It contained a broad variety of antibiotic resistance-related genes, including genes related to β-lactam (n = 10) and multidrug efflux (n = 54). It also contained eight prophages, 17 other phage-related CDSs and 26 CDSs for transposases.

Conclusions

The L. hongkongensis genome possessed genes for acid and bile resistance, intestinal mucosa colonization, evasion of host defense and cytotoxicity and invasion. A broad variety of antibiotic resistance or multidrug resistance genes, a high number of prophages, other phage-related CDSs and CDSs for transposases, were also identified.     

Prevalence of human papilloma virus in esophageal carcinomas: a polymerase chain reaction-based study

Objectives: Human papilloma virus (HPV) has been repeatedly found in esophageal carcinoma tissues. However, detection rates of HPV DNA in these tumors have varied markedly. Differences in detection methods, sample types and geographic regions of the sample origin have been suggested as potential causes of this discrepancy. This study was undertaken to analyze the prevalence of HPV in esophageal carcinoma. Study Design: HPV L1 DNA was evaluated in a total of 49 esophageal carcinoma samples, including 44 cases of squamous cell carcinoma (SCC) and 5 cases of adenocarcinoma. Seventeen control samples of esophageal brushings were also analyzed. The HPV L1 fragment was detected using MY09/MY11 primers. Results: In test samples, 17/49 (34.7%) were positive for HPV L1 and, in comparison, none of the control samples were positive. HPV DNA was identified in 17/37 (46%) cases of non-keratinizing SCC and was not identified in any case of esophageal keratinizing SCC and adenocarcinoma. Conclusion: This study defines a significant association of HPV with esophageal non-keratinizing SCC. Our findings raise the possibility that HPV is involved in esophageal carcinogenesis, especially the non-keratinizing type of SCC. Further investigation with a larger sample size over broader geographic areas may be warranted.     

PDGF-Independent Activation of PDGF-β Receptors in NIH-3T3 Cells Transformed by c-met Protooncogene

We have previously reported that c-met protooncogene, a member of a new class of receptor tyrosine-kinase gene family, is transforming when overexpressed in NIH-3T3 cells. In this paper, we report that the c-met protooncogene-transformed cells proliferate in a serum- and growth factor-free medium and exhibit constitutive tyrosine phosphorylation of several cellular proteins including the met protooncogene-encoded p145 and p185. Further investigations revealed platelet-derived growth factor (PDGF)-independent phosphorylation of PDGF-β receptors in the transformed cells. Phosphoamino acid analysis revealed phosphorylation of PDGF receptors at tyrosine and serine residues. The PDGF receptor phosphorylation is unlikely to occur via autocrine production of PDGF since we could not detect PDGF activity both at the RNA level and at a functional protein level. Additionally, phospholipase C-γ (PLC-γ) a substrate of activated PDGF receptors, was found to be physically associated with PDGF receptors in the absence of PDGF stimulation in (transformed cells. Furthermore, PDGF receptors coimmunoprecipitated along with PLC-γ. Taken together, our results demonstrate a PDGF-independent phosphorylation and activation of PDGF-β receptor in NIH-3T3 cells transformed by c-met protooncogene.     

Initiator-promoter coupling of phospholipases D and A2 in platelets upon cholesterol incorporation.

Since phospholipases exist within a membrane lipid environment, it is not unreasonable to assume that cholesterol capable of changing the lipid environment can effect the coupling relationship among the signal transducing components. Our previous study showed that the 'molecular switch' through which membrane cholesterol modulates cyclic nucleotide levels and Na+/H+ exchange within human platelets is phospholipase A2. We demonstrate here that membrane cholesterol initiates the activation of phosphatidyl choline phospholipase D and phosphatidic acid thus generated promotes the activation of its phospholipase A2 in the presence of extraplatelet calcium. More important, inhibition of phospholipase D by zinc blocks the activation of phosphatidic acid phospholipase A2 in platelets upon cholesterol incorporation. Our result led us to postulate that membrane cholesterol induced initiation promotion coupling of phospholipases D and A2 in human platelets may be responsible for the hypersensitized state of platelets in hypercholesterolemic patients.     

Primary structure, physicochemical properties, and chemical modification of NAD(+)-dependent D-lactate dehydrogenase. Evidence for the presence of Arg-235, His-303, Tyr-101, and Trp-19 at or near the active site.

The NAD(+)-dependent D-lactate dehydrogenase was purified to apparent homogeneity from Lactobacillus bulgaricus and its complete amino acid sequence determined. Two gaps in the polypeptide chain (10 residues) were filled by the deduced amino acid sequence of the polymerase chain reaction amplified D-lactate dehydrogenase gene sequence. The enzyme is a dimer of identical subunits (specific activity 2800 +/- 100 units/min at 25 degrees C). Each subunit contains 332 amino acid residues; the calculated subunit M(r) being 36,831. Isoelectric focusing showed at least four protein bands between pH 4.0 and 4.7; the subunit M(r) of each subform is 36,000. The pH dependence of the kinetic parameters, Km, Vm, and kcat/Km, suggested an enzymic residue with a pKa value of about 7 to be involved in substrate binding as well as in the catalytic mechanism. Treatment of the enzyme with group-specific reagents 2,3-butanedione, diethylpyrocarbonate, tetranitromethane, or N-bromosuccinimide resulted in complete loss of enzyme activity. In each case, inactivation followed pseudo first-order kinetics. Inclusion of pyruvate and/or NADH reduced the inactivation rates manyfold, indicating the presence of arginine, histidine, tyrosine, and tryptophan residues at or near the active site. Spectral properties of chemically modified enzymes and analysis of kinetics of inactivation showed that the loss of enzyme activity was due to modification of a single arginine, histidine, tryptophan, or tyrosine residue. Peptide mapping in conjunction with peptide purification and amino acid sequence determination showed that Arg-235, His-303, Tyr-101, and Trp-19 were the sites of chemical modification. Arg-235 and His-303 are involved in the binding of 2-oxo acid substrate whereas other residues are involved in binding of the cofactor.