1-Acyl-sn-glycerol-3-phosphate acyltransferase in maturing safflower seeds and its contribution to the non-random fatty acid distribution of triacylglycerol

A 20,000 X g particulate preparation isolated from maturing safflower seeds catalyzed the acylation of 1-acyl-sn-glycerol 3-phosphate with acyl-CoA to form phosphatidate. The specific activity of the reaction exceeded 200 nmol min-1 mg protein-1. Although this preparation was also capable of catalyzing the acylation of sn-glycerol 3-phosphate with acyl-CoA, the hydrolysis of phosphatidate, and the acylation of 1,2-diacylglycerol, phosphatidate was the only major product when the preparation was incubated with 1-acyl-glycerol-3-P and acyl-CoA. The enzyme responsible for this phosphatidate synthesis, 1-acyl-glycerol-3-P acyltransferase, showed a strict acyl-CoA specificity. The relative order of specificity for acyl-CoA was linoleoyl = oleoyl greater than palmitoleoyl greater than elaidoyl greater than cis-vaccenoyl greater than stearoyl = palmitoyl. This observation strongly suggests that the fatty acid composition of position 2 in phosphatidate synthesized in vivo primarily depends on both the acyl-CoA specificity of the 1-acyl-glycerol-3-P acyltransferase and the fatty acid composition of the acyl-CoA pool in the cell. Thus, the absence of saturated fatty acids at position 2 of safflower triacylglycerol may be explained in terms of the acyl-CoA specificity of the 1-acyl-glycerol-3-P acyltransferase. The fatty acid moiety esterified at position 1 of glycerol-3-P also affected the effectiveness of the reaction. The 1-acyl-glycerol-3-P acyltransferase utilized 1-acyl-glycerol-3-P molecular species in the following order of effectiveness: linoleoyl = oleoyl greater than palmitoyl. With a rise in incubation temperature, the initial rates of acylation with unsaturated acyl-CoA species increased more rapidly than those for saturated acyl-CoA species. A similar tendency was observed for saturated and unsaturated acyl acceptors. These data suggest that affinity of the acyltransferase for substrates may vary in response to changes in temperature, and that 1-acyl-glycerol-3-P acyltransferase may be involved in the alteration of the individual fatty acid compositions at positions 1 and 2 of glycerolipids in tissues grown at different temperatures. Based on these findings, further metabolism of 1-acyl-glycerol-3-P acyltransferase products could be the major factor determining the non-random distribution of fatty acids in safflower triacylglycerol.     

An enzyme in mung bean leaves that damages cell organelles which leads to loss of their matrix enzymes

An enzyme causing loss of a matrix enzyme (hydroxypyruvate reductase)from leaf peroxisomes was found in an extract of the primaryleaves of the mung bean and was purified 37-fold from the extract.The enzyme required calcium, magnesium, manganese or zinc ionfor its activity. Loss of matrix enzymes also was induced inmitochondria and chloroplasts. When etiolated seedlings of themung bean were illuminated, organelle-damaging activity in theprimary leaves increased markedly. (Received March 17, 1980; )     

Changes in mitochondrial activity during enlargement of mung bean root cells

A mitochondrial fraction was separately prepared from two differentregions of mung bean roots; from root-tips which contained mainlyimmature cells and from tissue, other than the root-tips, whichwas composed of mature and fully vacuolated cells. The malatedehydrogenase, fumarase and aconitase activities per cell didnot increase or did so only slightly during cell growth. Respiratoryactivity of both tissue sections and the crude mitochondrialfraction also seemed to increase slightly as cells matured.However, the cytochrome oxidase and succinate cytochrome c reductaseactivities per cell increased significantly during cell enlargement.There was no difference in the distribution-profiles of thecytochrome oxidase and malate dehydrogenase activities aftersucrose density gradient centrifugation, between mitochondrialfractions prepared from two regions of the. roots. The malatedehydrogenase activity per unit of cytochrome oxidase activityin purified mitochondria of immature cells was much higher thanthat of mature cells. The results suggest that enzymes in mitochondrialmatrix are mainly synthesized in immature cells or during celldivision. In contrast, enzymes in the cristae seem to be formedduring cell maturation, as well as being formed in immaturecells. (Received January 26, 1973; )     

Correspondence of minor subunits of plant mitochondrial F1ATPase to F1F0ATPase subunits of other organisms

In addition to two major alpha- and beta-subunits, the soluble oligomycin-insensitive F1ATPase purified from sweet potato root mitochondria contains four different minor subunits of gamma (Mr = 35,500), delta (Mr = 27,000), delta' (Mr = 23,000), and epsilon (Mr = 12,000) (Iwasaki, Y., and Asashi, T. (1983) Arch. Biochem. Biophys. 227, 164-173). Among these minor subunits, the delta-subunit specifically cross-reacted with an antibody against the delta-subunit of maize mitochondrial F1 which contains only three minor gamma-, delta- and epsilon-subunits like F1ATPases from other organisms, indicating that the delta'-subunit is an extra subunit of sweet potato F1 which is absent in the maize F1. All of the four minor subunits of sweet potato F1 were purified and their N-terminal amino acid sequences of 30-36 residues were determined. The N-terminal sequence of gamma-subunit was homologous to those of the gamma-subunits of bacterial F1 and mammalian mitochondrial F1. The N-terminal sequence of the delta-subunit was homologous to those of the delta-subunits of bacterial F1, chloroplast CF1, and oligomycin sensitivity conferring protein of bovine mitochondrial F1F0. A sequence homology was also observed between the sweet potato epsilon-subunit and the epsilon-subunit of bovine mitochondrial F1. The N-terminal sequence of the delta'-subunit did not show any significant sequence homology to known protein sequences. These subunit correspondences place plant mitochondrial F1 at an unique position in the evolution of F1ATPase.     

Pyrrolinone derivatives as a new class of P2X3 receptor antagonists Part 2: Discovery of orally bioavailable compounds

Some P2X3 receptor antagonists have been developed as new therapeutic drugs for pain. We discovered a novel chemotype of P2X3 receptor antagonists with a pyrrolinone skeleton. Because of SAR studies to improve bioavailability of lead compound 2, compound (R)-24 was identified, which showed an analgesic effect against neuropathic pain by oral administration. We constructed a human P2X3 homology model as a template for the zebrafish P2X4 receptor, which agreed with SAR studies of pyrrolinone derivatives.     

Toll-Like Receptor 4 +3725 G/C Polymorphism, Helicobacter pylori Seropositivity, and the Risk of Gastric Atrophy and Gastric Cancer in Japanese

Background:  Toll-like receptor 4 (TLR4) Asp299Gly and Thr399Ile polymorphisms were reported to be a risk factor of gastric carcinoma or its precursors in Caucasian and Indian population, but these polymorphisms are absent in Japanese. We investigated the associations of TLR4 +3725 G/C polymorphism, another functional polymorphism of TLR4 , with risk of gastric cancer and gastric atrophy in Japanese.
Materials and Methods:  Study subjects were 583 histologically diagnosed gastric cancer patients and age- and sex-matched 1592 control outpatients, who visited Aichi Cancer Center Hospital from 2001 to 2005. Serum anti- H. pylori IgG antibody and pepsinogens were measured to evaluate H. pylori infection and gastric atrophy, respectively. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by a logistic model.
Results:  Among the seropositive subjects, the age- and sex-adjusted OR of gastric atrophy was 1.17 (95%CI: 0.91–1.50) for G/C , 1.20 (95%CI: 0.76–1.89) for C/C , and 1.18 (95%CI: 0.93–1.49) for G/C + C/C relative to G/G genotype. The age- and sex-adjusted OR of severe gastric atrophy among H. pylori seropositive subjects was 1.43 (95%CI: 0.99–2.06) for G/C , 1.47 (95%CI: 0.76–2.88) for C/C , and 1.43 (95%CI: 1.01–2.04) for G/C + C/C . The OR of gastric cancer compared with gastric atrophy controls was not statistically significant.
Conclusion:  Our study found that TLR4 +3725 G/C polymorphism was a risk factor of severe gastric atrophy in H. pylori seropositive Japanese. Our results underscored the significance of the variations in host innate immunity due to TLR4 polymorphism as genetic predispositions to gastric precancerous lesions in Eastern Asian populations with the same backgrounds.     

Chemical and Immunochemical Detection of 8-Halogenated Deoxyguanosines at Early Stage Inflammation

Myeloperoxidase (MPO) generates reactive halogenating species that can modify DNA. The aim of this study was to investigate the formation of 8-halogenated 2′-deoxyguanosines (8- halo-dGs) during inflammatory events. 8-Bromo-2′-dG (8-BrdG) and 8-chloro-2′-dG (8-CldG) were generated by treatment of MPO with hydrogen peroxide at physiological concentrations of Cl⁻ and Br⁻. The formation of 8-halo-dGs with other oxidative stress biomarkers in lipopolysaccharide-treated rats was assessed by liquid chromatography tandem mass spectrometry and immunohistochemistry using a novel monoclonal antibody (mAb8B3) to 8-BrdG-conjugated keyhole limpet hemocyanin. The antibody recognized both 8-BrdG and 8-CldG. In the liver of lipopolysaccharide-treated rats, immunostaining for 8-halo-dGs, halogenated tyrosines, and MPO were increased at 8 h, whereas those of 8-oxo-2′-dG (8-OxodG) and 3-nitrotyrosine were increased at 24 h. Urinary excretion of both 8-CldG and 8-BrdG was also observed earlier than those of 8-OxodG and modified tyrosines (3-nitrotyrosine, 3-chlorotyrosine, and 3- bromotyrosine). Moreover, the levels of the 8-halo-dGs in urine from human diabetic patients were 8-fold higher than in healthy subjects (n = 10, healthy and diabetic, p < 0.0001), whereas there was a moderate difference in 8-OxodG between the two groups (p < 0.001). Interestingly, positive mAb8B3 antibody staining was observed in liver tissue from hepatocellular carcinoma patients but not in liver tissue from human cirrhosis patients. These data suggest that 8-halo-dGs may be potential biomarkers of early inflammation.     

Deletion of a chaperonin 60 beta gene leads to cell death in the Arabidopsis lesion initiation 1 mutant

Lesion mimic mutants develop spontaneous cell death without pathogen attack. Some of the genes defined by these mutations may function as regulators of cell death, whereas others may perturb cellular metabolism in a way that leads to cell death. To understand the molecular mechanism of cell death in lesion mimic mutants, we isolated a lesion initiation 1 (len1) mutant by a T-DNA tagging method. The len1 mutant develops lesions on its leaves and expresses systemic acquired resistance (SAR). LEN1 was identified to encode a chloroplast chaperonin 60 beta (Cpn60 beta), a homologue of bacterial GroEL. The recombinant LEN1 had molecular chaperone activity for suppressing protein aggregation in vitro. Moreover, len1 plants develop accelerated cell death to heat shock stress in comparison with wild-type plants. The chlorophyll a/b binding protein (CAB) was present in len1 plants at a lower level than in the wild-type plants. These results indicate that LEN1 functions as a molecular chaperone in chloroplasts and its deletion leads to cell death in Arabidopsis.     

Sarcolipin inhibits polymerization of phospholamban to induce superinhibition of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs)

Sarcolipin (SLN), a regulator of the sarco(endo)plasmic reticulum Ca(2+)-ATPase of fast-twitch skeletal muscle (SERCA1a), is also expressed in cardiac and slow-twitch skeletal muscles where phospholamban (PLN) and SERCA2a are expressed. Co-expression in HEK-293 cells of SLN tagged N-terminally with a FLAG epitope (NF-SLN), PLN, and SERCAs followed by measurement of the Ca(2+) dependence of Ca(2+) transport activity in isolated microsomal fractions showed that NF-SLN can reduce the apparent Ca(2+) affinity of both SERCA1a (DeltaK(Ca) = -0.22 +/- 0.01 pCa units) and SERCA2a (DeltaK(Ca) = -0.37 +/- 0.04 pCa units). When SERCA1a or SERCA2a were co-expressed with both NF-SLN and PLN, inhibition was synergistic, reducing DeltaK(Ca) by about -1.0 pCa units. Co-immunoprecipitation showed that NF-SLN increased the binding of PLN to SERCA, whereas PLN did not increase the binding of NF-SLN to SERCA. Elevated Ca(2+) dissociates both PLN and NF-SLN from their complexes with both SERCA1a and SERCA2a, but NF-SLN induced resistance to Ca(2+) dissociation of the PLN.SERCA complex. Co-immunoprecipitation of PLN and NF-SLN without SERCA showed that NF-SLN binds directly to PLN and that NF-SLN inhibits the formation of PLN pentamers. Thus the ability of NF-SLN to elevate the content of PLN monomers can account, at least in part, for the superinhibitory effects of NF-SLN in the presence of PLN.