Abstract-Nitric oxide (NO) derived from endothelial cells is profoundly related to the maintenance of physiological vascular tone. Impairment of endothelial NO generation brought about by gene polymorphism is considered the major deterioration factor for progressive renal disease, including diabetic nephropathy. The present study aimed to elucidate the Glu298Asp polymorphism of endothelial NO synthase (eNOS) in patients with end-stage renal disease (ESRD) and its role as a predisposing factor for cardiovascular complications. Glu298Asp in exon 7 of the eNOS gene was determined by polymerase chain reaction, followed by restriction fragment length polymorphism analysis, in ESRD patients (nϭ185) and compared with that of unrelated healthy individuals (nϭ304). Key Words: polymorphism Ⅲ polymerase chain reaction Ⅲ nitric oxide synthase Ⅲ diabetes mellitus T he endothelial isoform of nitric oxide (NO) synthase (eNOS, NOSIII) is a constitutively expressed 135 kDa protein predominantly associated with the particulated specific structures in the plasmalemmal membrane, caveolae, of vascular endothelial cells. 1,2 NO is produced from L-arginine and diffuses to vascular smooth muscle cells, where it increases the concentration of cGMP by stimulating soluble guanylate cyclase, leading to vascular relaxation. It also inhibits platelet and/or leukocyte adhesion to vascular endothelium. Therefore, the impairment of eNOS expression has been considered a primary factor for diseases such as hypertension, coronary artery disease, thromboembolic diseases, and atherosclerosis. Indeed, knocking out the gene encoding eNOS in mice resulted in significant hypertension, and aortic rings from these animals studied ex vivo displayed no relaxation in response to acetylcholine. 3 Patients with endstage renal disease (ESRD) derived from diabetes mellitus (DM) nephropathy have a higher prevalence of cardiovascular complications than those with non-DM ESRD, and this limits their 5-year survival rate to less than 50%. Therefore, polymorphism in eNOS is considered one of the major predisposing factors for endothelial dysfunction.The gene polymorphisms of eNOS have been detected at 4b/4a variable number of tandem repeats in intron 4, Glu298Asp in exon 7, CA repeat in intron 13, A27 to C (A to C nucleotide conversion) in intron 18, and G10 to T in intron 23. A part of candidate variations (4b/4a tandem repeats in intron 4) has been investigated in glomerulonephritis and/or ESRD patients, but no conclusive results have been obtained so far. Recently, the G/T polymorphism in exon 7 coding for Glu298Asp was detected and reported to have a remarkable association with coronary spasm, acute myocardial infarction, 4 and hypertension. 5 To date, this is the only known eNOS polymorphism associated with an altered protein sequence, though recent expression studies have demonstrated no functional difference between 298Glu and 298Asp despite the accumulating clinical evidence. 6,7

The cls gene of Escherichia coli is responsible for the synthesis of a major membrane phospholipid, cardiolipin, and has been proposed to encode cardiolipin synthase. This gene cloned on a pBR322 derivative was disrupted by either insertion of or replacement with a kanamycin-resistant gene followed by exchange with the homologous chromosomal region. The proper genomic disruptions were confirmed by Southern blot hybridization and a transductional linkage analysis. Both types of disruptants had essentially the same properties; cardiolipin synthase activity was not detectable, but the strains grew well, although their growth rates and final culture densities were lower than those of the corresponding wild-type strains and strains with the classical cls-l mutation. A disruptant harboring a plasmid that carried the intact cls gene grew normally.The results indicate that the cls gene and probably the cardiolipin synthase are dispensable for E. coli but may confer growth or survival advantages. Low but definite levels of cardiolipin were synthesized by all the disruptants. Cardiolipin content of the cis mutants depended on the dosage of the pss gene,-and attempts to transfer a null allele of the cls gene into a pss-l mutant were unsuccessful. We point out the possibilities of minor cardiolipin formation by phosphatidylserine synthase and of the essential nature of cardiolipin for the survival of E. coli cells.Cardiolipin, one of the major phospholipids of Escherichia coli, is unique in its structure among membrane lipids (i.e., tetraacyl structure with two phosphate groups) and has been postulated to play specific roles in membrane functions. It is synthesized from two molecules of phosphatidylglycerol by cardiolipin synthase (4, 20), which differs from its eucaryotic counterpart that utilizes CDP-diacylglycerol and phosphatidylglycerol as substrates (21). The gene cls is responsible for cardiolipin formation, and several lines of evidence indicate, though not definitely, that it is the structural gene for cardiolipin synthase (10).An E. coli mutation (cls-J) that results in a defective cardiolipin synthase has been isolated (14), and the cells with this mutation have been shown to display only minor growth phenotypes, despite decreased levels of cardiolipin (14, 18). Therefore, it has been uncertain whether the cls gene and cardiolipin are essential in E. coli cells or whether the residual low levels of cardiolipin observed maintain membrane functions that are dependent on this particular phospholipid.To determine whether the cls gene is essential and to analyze the physiological roles, if any, of this gene, an attempt to obtain a null mutation of this gene seemed most useful. This paper describes the construction, by genetic manipulations, of such mutants and their characteristic properties. The results show that the cls gene is not essential. Cardiolipin was formed at low levels even in the null cls mutants. We also present results that favor a hypothesis of secondary cardiolipin formation by phosphatidylserine synt...

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The mechanism that assures the balanced synthesis of zwitterionic (phosphatidylethanolamine) and acidic phospholipids (phosphatidylglycerol and cardiolipin) in Escherichia coli has been examined by genetically manipulating the two enzymes at the biosynthetic branch point, i.e., phosphatidylglycerophosphate synthase, encoded by pgsA, and phosphatidylserine synthase, encoded by pssA. A mutant in which the most part of the pssA gene was replaced with a drug resistance gene lacked phosphatidylserine synthase and phosphatidylethanolamine and required divalent metal ions for growth, as did a previously reported insertion-inactivated pssA mutant. When this mutant harbored a plasmid containing a Bacillus subtilis gene that encodes membrane-bound phosphatidylserine synthase, the phosphatidylethanolamine content was dependent on its activity, in contrast to that with the soluble E. coli counterpart. A defective mutation, pgsA3, caused reductions not only in acidic-phospholipid synthesis but also in phosphatidylethanolamine synthesis, despite the normal level of phosphatidylserine synthase activity. These results, together with previous observations, indicate that phosphatidylserine synthesis requires the membrane-associated form of phosphatidylserine synthase, which is related to the membrane-levels of acidic phospholipids, thus yielding balanced compositions of zwitterionic and acidic phospholipids.

Monocytic myeloid-derived suppressor cells (mMDSCs) are a class of immunosuppressive immune cells with prognostic value in many solid tumors. It is reported that the proportion of mMDSCs in the peripheral blood can be a predictive marker for response to cancer immunotherapy. In this study, we performed a correlation analysis of the proportion of mMDSCs in freshly-drawn peripheral blood, levels of plasma proteins, and demographic factors in colorectal cancer (CRC) patients, to find factors that could be used to predict mMDSC proportions. Freshly-drawn mMDSCs were measured using flow cytometry on peripheral blood mononuclear cells (PBMCs) from healthy donors (n = 24) and CRC patients (n = 78). The plasma concentrations of 29 different cytokines, chemokines, growth factors, and enzymes were measured using a multiplex assay or enzyme-linked immunosorbent assay. Correlation analysis to find mMDSC-associated factors was conducted using univariate and multivariate models. In univariate correlation analysis, there were no plasma proteins that were associated with mMDSC proportions in CRC patients. In multivariate analysis, considering all variables including age, sex, and plasma proteins, levels of inducible nitric acid synthase (iNOS) (p = 0.013) and platelet-derived growth factor (PDGF)-BB (p = 0.035) were associated with mMDSC proportion in PBMCs (mMDSC proportion [%] = 0.2929 − 0.2389 * PDGF-BB + 0.3582 * iNOS) (p < 0.005, r = 0.32). Measuring the plasma concentrations of iNOS and PDGF-BB may be useful in predicting the proportion of mMDSCs in CRC patients’ peripheral blood. Further research is required to establish and validate these predictive factors. Data registration Patient data were registered in an anonymization system at Tsukuba Clinical Research & Development Organization (T-CReDO).

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CP-230821 is a novel, potent LDL receptor gene transcriptional upregulator which decreases total plasma cholesterol level. Interestingly, this plasma LDL decrease does not alter hepatic lipid contents. A series of experiments was undertaken to study the molecular biology of this phenomenon. Twelve hours after CP-230821 treatment, the transcriptional activity and mRNA level of the LDL receptor gene in HepG2 cells were increased by 264% and 426%, respectively. Although treatment with the HMG-CoA reductase inhibitor compactin also increased LDL receptor gene transcription and mRNA, CP-230821 did not increase the level of HMG-CoA reductase gene transcription or mRNA. These results indicate that LDL receptor gene activity may play an important role in the decrease of plasma LDL level. These results further suggest that the LDL receptor gene and the HMG-CoA reductase gene are not strictly coordinately controlled.