Phosphatidylcholine molecular species involved in Γ-linolenic acid biosynthesis in microsomes from borage seeds

Boraginaceae seeds are particularly rich in Γ -linolenic acid (6,9,12-octadecatrienoic acid, Γ -18:3). In microsomes, the analysis of phosphatidylcholine (PC) molecular species by HPLC led to identification of 15 different molecular species; among them 4 contained Γ -18:3, mostly at position 2 of sn -glycerol. Time courses of acylation and desaturation in PC molecular species were examined when [¹⁴C]oleoyl-CoA or [¹⁴C]linoleoyl-CoA was provided as substrates to isolated microsomes. With [¹⁴C]oleoyl-CoA or [¹⁴C]linoleoyl-CoA and in the absence of NADH, 3 main labelled PC molecular species were found: 18:2/[¹⁴C]18:1, 16:0/[¹⁴C]18:1 and 18:1/[¹⁴C]18:1. When NADH was present in the incubation medium, the fatty acids were progressively desaturated by the Δ12- and Δ6-desaturases successively (with [¹⁴C]oleoyl-CoA as precursor) or by the Δ6-desaturase alone (with [¹⁴C]linoleoyl-CoA as precursor). In both types of experiments, 7 final desaturation products in microsomes were evidenced; among them, 3 contained radioactive Γ -18:3, i.e . 18:2/[¹⁴C] Γ -18:3, 18:1/[¹⁴C] Γ -18:3 and 16:0/[¹⁴C] Γ -18:3. While the Δ12-desaturase had no specificity for position on the glycerol backbone, labelled Γ -linolenic acid was recovered exclusively in the sn -2 position.     

Computational and functional analysis of β-lactam resistance in Zymomonas mobilis

Zymomonas mobilis, a Gram-negative ethanologenic non-pathogenic bacterium, is reported to exhibit resistance to high concentrations of β-lactam antibiotics. In the present study, Z. mobilis was found to be resistant to I-IV generations of cephalosporins and carbapenems, i.e. narrow, broad and extended spectrum β-lactam antibiotics. We have analysed the genome of Z. mobilis (GenBank accession No.: NC 006526) harbouring multiple genes coding for β-lactamases (BLA), β-lactamase domain containing proteins (BDP) and penicillin binding proteins (PBP). The conserved domain database analysis of BDPs predicted them to be members of metallo β-lactamase superfamily. Further, class C specific multidomain AmpC (β-lactamase C) was found in the three β-lactamases. The β-lactam resistance determinants motifs, HXHXD, KXG, SXXK, SXN, and YXN are present in the BLAs, BDPs and PBPs of Z. mobilis. The predicted theoretical pI and aliphatic index values suggested their stability. One of the PBPs, PBP2, was predicted to share functional association with rod shape determining proteins (GenBank accession Nos. YP_162095 and YP_162091). Homology modelling of three dimensional structures of the β-lactam resistance determinants and further docking studies with penicillin and other β-lactam antibiotics indicated their substrate-specificity. Semi-quantitative PCR analysis indicated that the expression of all BLAs and one BDP are induced by penicillin. Disk diffusion assay, SDS-PAGE and zymogram analysis confirms the substrate specificity of the β-lactam resistance determinants. This study gives a broader picture of the β-lactam resistance determinants of a non-pathogenic ethanologenic Z. mobilis bacterium that could have implications in laboratories since it is routinely used in many research laboratories in the world for ethanol, fructooligosaccharides, levan production and has also been reported to be present in wine and beer as a spoilage organism.     

Effect of Down-Regulation of Ethylene Biosynthesis on Fruit Flavor Complex in Apple Fruit

The role of ethylene in regulating sugar, acid, texture and volatile components of fruit quality was investigated in transgenic apple fruit modified in their capacity to synthesize endogenous ethylene. Fruit obtained from plants silenced for either ACS (ACC synthase; ACC-1-aminocyclopropane-1-carboxylic acid) or ACO (ACC oxidase), key enzymes responsible for ethylene biosynthesis, expectedly showed reduced autocatalytic ethylene production. Ethylene suppressed fruits were significantly firmer than controls and displayed an increased shelf-life. No significant difference was observed in sugar or acid accumulation suggesting that sugar and acid composition and accumulation is not directly under ethylene control. Interestingly, a significant and dramatic suppression of the synthesis of volatile esters was observed in fruit silenced for ethylene. However, no significant suppression was observed for the aldehyde and alcohol precursors of these esters. Our results indicate that ethylene differentially regulates fruit quality components and the availability of these transgenic apple trees provides a unique resource to define the role of ethylene and other factors that regulate fruit development.     

Increased serum levels of interleukin-18 in patients with diabetic nephropathy

In the present study we measured interleukin-18 (IL-18) and tumour necrosis factor-alpha (TNF-alpha) levels by enzyme linked immunosorbent assay (ELISA) in sera from 65 diabetic [30 with type 1 insulin dependent diabetes mellitus (IDDM) and 35 with type 2 non-insulin dependent diabetes mellitus (NIDDM)] patients and 15 healthy volunteers, to investigate their associations with metabolic parameters and to elucidate their roles in the pathogenesis of diabetic complications especially diabetic nephropathy. Levels of IL-18 and TNF-alpha were significantly higher in both IDDM and NIDDM individuals as compared to the control group. Similarly, their levels in patients with diabetic nephropathy increased gradually according to the clinical stage of the disease, being highest in macroalbuminuric stage. Correlation analyses showed that the serum IL-18 and TNF-alpha concentration were positively correlated with each other and positively with fasting plasma glucose (FPG), 2h postprandial glucose, glycosylated hemoglobin (HbA1c), triglyceride, and urinary albumin levels and negative correlation between TNF-alpha and high density lipoprotein cholesterol (HDL-C) were also found in diabetic subjects. High serum levels of IL-18 and TNF-alpha suggested that they might play a role in the pathogenesis of DM and in the development of nephropathy in diabetic patients whether of type 1 or 2.     

Lipid-transfer proteins: Tools for manipulating membrane lipids

Like other eukaryotic cells, plant cells contain proteins able to bind or to transfer lipids. Since they are able to facilitate movements of various phospholipids between membranes and are also capable of binding fatty acids or acyl-CoAs, they have been termed lipid-transfer proteins (LTP). LTPs are basic proteins containing 90 to 95 residues (molecular mass 9 kDa), eight of them being cysteines found in conserved locations. These proteins have been used to manipulate in vitro the lipid composition of isolated membranes either from plant or mammalian sources. In addition to purified LTPs, recombinant LTPs produced by genes expressed in microorganisms can be used for this purpose. Several genes coding for these proteins have been characterized in various plants with different patterns of expression. However, it remains to be investigated whether these recombinant proteins behave functionally as LTPs. The use of purified or recombinant LTPs is promising for the study of the effect of lipid composition on membrane functional properties.     

Lymphatic Vessels Are Essential for the Removal of Cholesterol from Peripheral Tissues by SR-BI-Mediated Transport of HDL

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The actin bundling activity of ITPKA mainly accounts for its migration-promoting effect in lung cancer cells

Expression of Ins(1,4,5)P₃-kinase-A (ITPKA), the neuronal isoform of Ins(1,4,5)P₃-kinases, is up-regulated in many tumor types. In particular, in lung cancer cells this up-regulation is associated with bad prognosis and it has been shown that a high level of ITPKA increases migration and invasion of lung cancer cell lines. However, since ITPKA exhibits actin bundling and Ins(1,4,5)P₃-kinase activity, it was not clear which of these activities account for ITPKA-promoted migration and invasion of cancer cells. To address this issue, we inhibited endogenous actin bundling activity of ITPKA in lung cancer H1299 cells by overexpressing the dominant negative mutant ITPKA^L34P. Analysis of actin dynamics in filopodia as well as wound-healing migration revealed that ITPKA^L34P inhibited both processes. Moreover, the formation of invasive protrusions into collagen I was strongly blocked in cells overexpressing ITPKA^L34P. Furthermore, we found that ATP stimulation slightly but significantly (by 13%) increased migration of cells overexpressing ITPKA while under basal conditions up-regulation of ITPKA had no effect. In accordance with these results, overexpression of a catalytic inactive ITPKA mutant did not affect migration, and the Ins(1,4,5)P₃-kinase-inhibitor GNF362 reversed the stimulating effect of ITPKA overexpression on migration. In summary, we demonstrate that under basal conditions the actin bundling activity controls ITPKA-facilitated migration and invasion and in presence of ATP the Ins(1,4,5)P₃-kinase activity slightly enhances this effect.