Expression of fatty acid binding proteins inhibits lipid accumulation and alters toxicity in L cell fibroblasts

High levels of saturated,branched-chain fatty acids are deleterious to cells and animals,resulting in lipid accumulation and cytotoxicity. Although fatty acidbinding proteins (FABPs) are thought to be protective, this hypothesishas not previously been examined. Phytanic acid (branched chain,16-carbon backbone) induced lipid accumulation in L cell fibroblastssimilar to that observed with palmitic acid (unbranched,C₁₆): triacylglycerol free fatty acid > cholesterol > cholesteryl ester phospholipid. Althoughexpression of sterol carrier protein (SCP)-2, SCP-x, or liver FABP(L-FABP) in transfected L cells reduced [³H]phytanic aciduptake (57-87%) and lipid accumulation (21-27%), nevertheless [³H]phytanic acid oxidation was inhibited(74-100%) and phytanic acid toxicity was enhanced in the orderL-FABP SCP-x > SCP-2. These effects differed markedly fromthose of [³H]palmitic acid, whose uptake, oxidation, andinduction of lipid accumulation were not reduced by L-FABP, SCP-2, orSCP-x expression. Furthermore, these proteins did not enhance thecytotoxicity of palmitic acid. In summary, intracellular FABPs reducelipid accumulation induced by high levels of branched-chain but notstraight-chain saturated fatty acids. These beneficial effects wereoffset by inhibition of branched-chain fatty acid oxidation thatcorrelated with the enhanced toxicity of high levels of branched-chainfatty acid.

     

Contribution of chiral HPLC in tandem with polarimetric detection in the determination of absolute configuration by chemical interconversion method: Example in 1-(thi)oxothiazolinyl-3-(thi)oxothiazolinyl toluene atropisomer series

We report the determination of the absolute configuration of eight stereoisomers in the series of chiral 1-(thi)oxothiazolinyl-3-(thi)oxothiazolinyl toluene atropisomers 1-3, from the known absolute configuration of one stereoisomer, determined by X-ray crystallography. The method uses the affiliation between signs of rotation of polarised light during chemical transformations which preserve the absolute configuration and also during rotation around a single pivot bond producing a compound of known configuration. The use of chiral HPLC in tandem with a chirality detector gives a decisive advantage since such correlation can be performed on a mixture of a very limited quantity of compounds, without tedious purification steps. The method shown as an example in this article, which uses chiral HPLC with chirality detection, may prove useful in many other cases where the determination of the absolute configuration is necessary and where a chemical interconversion method can be used on a microscale.     

Relationship between chromosomal changes complexity and disease aggressiveness in myeloid and lymphoid disorders

In this paper are presented four cases, with unusual chromosomal abnormalities, identified at the first presentation, among over 100 patients with myeloid and lymphoid acute and chronic leukemias cytogenetically investigated. The complexity and nature of cytogenetic abnormalities was in direct relationship with the disease evolution. The first case, a 22 years old man with acute lymphoblastic leukemia type L3, exhibited many structural changes in bone marrow cells with diploid number of chromosomes: del(3)(q26); del (5)(p13); t(8;14) (q24;q32); del(9)(p11q11);inv(15)(p12qter). The second case, a 62 years old woman, diagnosed as poorly differentiated acute leukemia, refractory to treatment, showed hiperdiploidy (48–54 chromosomes) and 3–4 markers derived from chromosomes 5 and 12. The third case, a young man of 27 years old, diagnosed as acute myeloid leukemia, apart of Philadelphia chromosome, presented trisomy 16, both in diploid and aneuploid cells. None of these three patients did respond to any medical therapy. Their rapid death was a powerful proof of the correlation between the complexity of genome changes and disease aggressiveness. In the fourth case, a constitutional translocation t(3;5)(q26.3;q21) identified in a 72 years old woman with essential thrombocythemia, appeared not to be involved in the etiology of the disease. In this case, the treatment with hydroxyurea was successful and the disease evolution was favourable. In conclusion, we appreciate that in the three cases of myeloid and lymphoid leukemias it was a direct relationship between the complexity of genomic changes and the aggressiveness of the disease.     

Mechanism of the E. coli tau processivity switch during lagging-strand synthesis

The E. coli replication machinery employs a beta clamp that tethers the polymerase to DNA, thus ensuring high processivity. The replicase also contains a processivity switch that dissociates the polymerase from its beta clamp. The switch requires the tau subunit of the clamp loader and is regulated by different DNA structures. At a primed site, the switch is "off." When the replicase reaches the downstream primer to form a nick, the switch is flipped, and tau ejects the polymerase from beta. This switch has high fidelity for completed synthesis, remaining "off" until just prior to incorporation of the last nucleotide and turning "on" only after addition of the last dNTP. These actions of tau are confined to its C-terminal region, which is located outside the clamp loading apparatus. Thus, this highly processive replication machine has evolved a mechanism to specifically counteract processivity at a defined time in the lagging-strand cycle.     

Complete Genome Sequence of φHSIC, a Pseudotemperate Marine Phage of Listonella pelagia

The genome for the marine pseudotemperate member of the Siphoviridae HSIC has been sequenced using a combination of linker amplification library construction, restriction digest library construction, and primer walking. HSIC enters into a pseudolysogenic relationship with its host, Listonella pelagia, characterized by sigmoidal growth curves producing >10⁹ cells/ml and >10¹¹ phage/ml. The genome (37,966 bp; G+C content, 44%) contained 47 putative open reading frames (ORFs), 17 of which had significant BLASTP hits in GenBank, including a β subunit of DNA polymerase III, a helicase, a helicase-like subunit of a resolvasome complex, a terminase, a tail tape measure protein, several phage-like structural proteins, and 1 ORF that may assist in host pathogenicity (an ADP ribosyltransferase). The genome was circularly permuted, with no physical ends detected by sequencing or restriction enzyme digestion analysis, and lacked a cos site. This evidence is consistent with a headful packaging mechanism similar to that of Salmonella phage P22 and Shigella phage Sf6. Because none of the phage-like ORFs were closely related to any existing phage sequences in GenBank (i.e., none more than 62% identical and most <25% identical at the amino acid level), HSIC is unique among phages that have been sequenced to date. These results further emphasize the need to sequence phages from the marine environment, perhaps the largest reservoir of untapped genetic information.     

Reversible inhibitors of lambda integrase-mediated recombination efficiently trap Holliday junction intermediates and form the basis of a novel assay for junction resolution

The bacteriophage lambda integrase catalyzes four site-specific recombination pathways with distinct protein and DNA requirements and nucleoprotein intermediates. Some of these intermediates are very transient and difficult to obtain in significant amounts, due to the high efficiency and processivity of integrase, the lack of requirements for external energy factors or metal ions, and the highly reversible nature of each of the intermediates. We have previously used mixture-based combinatorial libraries to identify hexapeptides that trap 40-60% of recombination substrates at the Holliday junction stage of the reaction. These inhibitors discriminate between the four pathways, blocking one of them (bent-L recombination) more severely than the others and blocking the excision pathway least. We presume that these differences reflect specific conformational differences of the nucleoprotein intermediates in each pathway. We have now identified new inhibitors of the excision pathway. One of these, WRWYCR, is over 50-fold more potent at inhibiting excision than the previously identified peptides. This peptide stably traps Holliday junction complexes in all recombination pathways mediated by integrase as well as Cre. This finding and other data presented indicate that the peptide's target is a common feature shared by the Holliday junction complexes assembled by tyrosine recombinases. We have taken advantage of reversible inhibition by the active peptides to develop a new assay for Holliday junction resolution. This assay is particularly useful for determining junction resolution rates in cases where complexes directly assembled on junction substrates undergo little or no catalysis.     

Relationship of eNOS gene variants to diseases that have in common an endothelial cell dysfunction

The endothelial cell (EC) dysfunction is a common characteristic of various pathologies that include atherosclerosis, hypertension, and Fabry's disease. Aware of the role of eNO and ACE in EC dysfunction, we questioned whether polymorphism of eNOS and/or ACE gene may be a common denominator in these pathologies. Patients with CHD (108), HT (109), Fabry's disease (37) and healthy subjects (control, 141) were genotyped for the eNOSG894T by RFLP-PCR technique and for eNOS4b/a, and ACEI/D polymorphisms by PCR amplification. The results of these studies were statistically evaluated. Compared to controls, the frequency of the eNOSG894T (T allele) was higher in CHD (P=0.03) and Fabry (P=0.01), while the eNOS4b/a (a allele) in CHD (P=0.01) and HT patients (P=0.01). The proportion of the ACEI/D was similar in all subjects. In CHD patients at "low risk" of atherogenic factors, the frequency of the T and a alleles of eNOS gene was high (P=0.03 and 0.02, respectively). Carriers of the T allele of eNOSG894T were over-represented (P=0.04) in Fabry subgroup with renal failure. Compared to women, the eNOS894T alleles were more frequent (P=0.03) in men with CHD and HT, whereas ACE I/D in men (P=0.03) with HT. These findings suggest: (i) the frequency of eNOSG894T and/or eNOS4b/a is significantly associated with coronary dysfunction; (ii) eNOS4b/a confers a relatively high risk of hypertension in subjects with atherogenic risk factors; (iii) the frequency of eNOSG894T is high in Fabry hemizygotes with renal complications. Therefore, eNOS gene polymorphism represent a frequent risk factor for vascular abnormalities in CHD, HT and Fabry's disease, afflictions which have in common, the endothelial dysfunction.     

Novel fluo-4 analogs for fluorescent calcium measurements

We report new fluorescent calcium indicators based on fluo-4. Attachment of a carboxamide or methylenecarboxamide moiety to the BAPTA chelator portion of fluo-4 allowed for the attachment of dextrans, protein-reactive moieties, and biotin. In particular, a high affinity fluo-4 dextran conjugate was prepared and shown to be functional in brain slices. All new probes were characterized spectroscopically and exhibited large fluorescence increases upon calcium-binding. The biotinylated version of fluo-4 formed a persistent streptavidin complex which still responded to increasing calcium concentrations with a large fluorescence increase.