Determination of a new oral iron chelator, ICL670, and its iron complex in plasma by high-performance liquid chromatography and ultraviolet detection

ICL670 is a representative of a new class of orally active tridentate selective iron chelators. Two molecules of ICL670 are required to form a complete hexacoordinate chelate Fe–[ICL670]₂ with one ferric iron. A simple and rapid HPLC–UV method for the separate determination of ICL670 and Fe–[ICL670]₂ in the plasma of iron-overloaded patients is described. Plasma samples were prepared as rapidly as possible, the tubes being kept at 4°C. Plasma proteins were precipitated with methanol. The supernatant was diluted with water and placed on the refrigerated sample rack of an autosampler before injection. The chromatographic separations were achieved on an Alltima C₁₈ column using 0.05 M Na₂HPO₄ and 0.01 M tetrabutylammonium hydrogen sulfate–acetonitrile–methanol (41:9:50, v/v/v) as mobile phase. The analytes were detected at 295 nm. Calibration and quality control samples were prepared in normal human plasma. The mean accuracy (n=6) over the entire investigated concentration range 0.25–20 μg/ml ranged from 91 to 109% with a coefficient of variation (C.V.) from 4 to 8% for ICL670, and from 95 to 105% with a C.V. from 2 to 20% for the iron complex. The dissociation of the complex during analysis was shown to be marginal. The iron removal from plasma of iron-overloaded patients by free ICL670 during analysis was low. The in vitro iron transfer from the iron pools of iron-overloaded plasma onto ICL670 was shown to be a slow process.     

Enantioselective HPLC determination of E-6087, a new COX-2 inhibitor, in human plasma: Validation and pharmacokinetic application

E-6087 is a nonsteroidal anti-inflammatory compound that selectively inhibits cyclooxygenase-2. Because E-6087 has a chiral center, this compound is a racemic mixture of two stereoisomers, (+)-(R)-E-6087 (E-6231) and (-)-(S)-E-6087 (E-6232). A normal-phase liquid-chromatographic method for the enantioselective determination of E-6087 in human plasma was developed and validated. The samples were extracted using solid-phase extraction cartridges containing C(18) sorbent, and the extracts were redissolved in absolute ethanol and injected into the chromatographic system. The enantiomeric separation was achieved on a chiral stationary-phase column of derivatized amylose, and the enantiomers were quantified by fluorescence detection. The method was validated for drug concentrations ranging from 5 to 400 ng/ml for both enantiomers. No peaks interfering with the quantification of enantiomers were observed. The limit of quantification was 5 ng/ml, with precision expressed as a coefficient of variation lower than 10.6% and accuracy expressed as relative error lower than 12.2%. The utility of this method was demonstrated by analysis of plasma samples from healthy volunteers given an oral dose of rac-E-6087. Peak plasma levels of E-6231 were higher than levels obtained for E-6232. Results were consistent with those obtained with a conventional reversed-phase method used for determination of the racemic compound.     

Enantioselective determination of hydroxychloroquine and its major metabolites in urine and the observation of a reversal in the (+)/(−)-hydroxychloroquine ratio

A sequential achiral-chiral high-performance liquid chromatographic system has been developed for the quantitation in urine of the enantiomers of hydroxychloroquine (HCQ), and of its 3 major metabolites, desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), and bisdesethylchloroquine (BDCQ). HCQ and its metabolites were separated and quantified on a cyano-bonded phase, and the enantiomeric ratios were determined using a Chiral-AGP chiral stationary phase. The assay validation and application of this method to a preliminary study in a human volunteer are presented. In this subject, the initial 0-4 h urine contained the 2 HCQ enantiomers in a ratio of (+)-HCQ:(?)-HCQ of 3:2; by the 2,064 h of the study, this ratio had reversed to (+)-HCQ:(?)-HCQ of 3:7. © 1993 Wiley-Liss, Inc.     

肠道菌群在非甾体类抗炎药肠病中的作用及机制

非甾体类抗炎药(NSAIDs)是全球范围内广泛使用的解热镇痛抗炎药, 但存在明显的胃肠道不良反应, 可导致胃肠道溃疡、出血和穿孔等。目前, NSAIDs引起的肠病的发病率较高, 且作用机制尚不明确。其中, 肠黏膜屏障损伤在NSAIDs肠病发病机制中起着主要作用。近期研究表明肠道菌群与NSAIDs在机体内相互影响, 且肠道微生物对肠黏膜屏障功能的维持有重要作用。肠道菌群失衡逐渐成为NSAIDs肠病的病理生理机制之一。本文综述了肠道菌群与NSAIDs药物作用的密切关系, 并从肠黏膜屏障损伤和修复的角度阐述肠道菌群在NSAIDs肠病发生发展中的作用及机制, 为以肠道菌群为靶标治疗NSAIDs肠病提供理论基础。

     

Determination of the enantiomers of citalopram,its demethylated and propionic acid metabolites in human plasma by chiral HPLC

A stereoselective HPLC assay has been developed to analyze the enantiomers of citalopram and of its three main metabolites in plasma after their separation on a Chiracel OD column. Using a fluorescence detector, the limit of quantification in plasma samples was 15, 4, 5, and 2 ng/ml for the enantiomers of citalopram (CIT), desmethylcitalopram (DCIT), didesmethylcitalopram (DDCIT), and for the citalopram propionic acid derivative (CIT-PROP), respectively. Except for CIT, all metabolites were derivatized with achiral reagents. Identification of the enantiomers was realized with an optical rotation detector which showed that the enantiomers invert their rotation depending on the polarity and nature of the solvent. Under varying conditions, a racemization study has shown that the pure enantiomers of CIT and its demethylated metabolites are configurationally stable. Preliminary results obtained with five patients treated with CIT show a mean S/R ratio of 0.7 for both CIT and its active metabolite DCIT and of 3.6 for CIT-PROP in plasma. This suggests that the pharmacologically relevant (+)-(S)-isomers of CIT and DCIT could be preferentially and steroselectively metabolized to CIT-PROP. © 1995 Wiley-Liss, Inc.     

Efficient synthesis of apricoxib, CS-706, a selective cyclooxygenase-2 inhibitor, and evaluation of inhibition of prostaglandin E2 production in inflammatory breast cancer cells

An efficient synthesis of apricoxib (CS-706), a selective cyclooxygenase inhibitor, was developed using copper catalyzed homoallylic ketone formation from methyl 4-ethoxybenzoate followed by ozonolysis to an aldehyde, and condensation with sulfanilamide. This method provided multi-gram access of aprocoxib in good yield. Apricoxib exhibited potency equal to celecoxib at inhibition of prostaglandin E2 synthesis in two inflammatory breast cancer cell lines.     

Identification of HUHS190, a human naftopidil metabolite,as a novel anti-bladder cancer drug

We carried out structure-activity relationship study on anti-cancer effects of naftopidil (1) and its metabolites, resulted in identification of 1-(4-hydroxy-2-methoxyphenyl)piperazin-1-yl)-3-(naphthalen-1-yloxy) propan-2-ol (2, HUHS190), a major human metabolite of 1, which exhibited the most selective toxicities between against normal and cancer cells (Table 1). 2 was more hydrophilic compared to 1, was enough to be prepared in high concentration solution of more than 100 μM in saline for an intravesical instillation drug. Moreover, serum concentration of 2 was comparable to that of 1, an oral preparation drug, after oral administration at 32 mg/kg (Fig. 3). Both of 1 and 2 showed broad-spectrum anti-cancer activities in vitro, for example, 1 and 2 showed inhibitory activity IC₅₀ = 21.1 μM and 17.2 μM for DU145, human prostate cancer cells, respectively, and IC₅₀ = 18.5 μM and 10.5 μM for T24 cells, human bladder cancer cells. In this study, we estimated anticancer effects of 2 in a bladder cancer model after intravesical administration similar to clinical cases. A single intravesical administration of 2 exhibited the most potent inhibitory activities among the clinical drugs for bladder cancers, BCG and Pirarubicin, without obvious side effects and toxicity (Fig. 4). Thus, HUHS190 (2) can be effective for patients after post-TURBT therapy of bladder cancer without side effects, unlike the currently available clinical drugs.     

The influence of auranofin, a clinically established antiarthritic gold drug, on bone metabolism: analysis of its effects on human multipotent adipose-derived stem cells, taken as a model

Auranofin is a gold-based antiarthritic drug in clinical use for more that 25 years. However, in spite of a long established use, its specific effects on bone metabolism are still greatly controversial. We have analyzed in vitro the actions of auranofin on human multipotent adipose-derived stem (hMADS) cells, used as a model for bone metabolism, since these cells were reported to undergo osteogenesis both in vitro and in vivo. Cytotoxicity of auranofin on hMADS cells, differentiated into osteoblasts, was initially assessed. Thereafter, the consequences of exposure to nontoxic but clinically relevant auranofin concentrations were analyzed by monitoring the seleno-protein glutathione peroxidase 3 or alkaline phosphatase, a characteristic biomarker of osteogenesis. Notably, we found that chronic treatment with auranofin alters only weakly the levels of alkaline phosphatase, thus implying an overall modest effect on osteogenesis. In contrast, auranofin turned out to greatly affect glutathione peroxidase 3 activity. The possible medical implications of these findings are discussed.     

Preparation of matrine ethosome,its percutaneous permeation in vitro and anti-inflammatory activity in vivo in rats

The aim of this work was to evaluate the preparation of matrine ethosome and the percutaneous permeation in vitro and the anti-inflammatory activity in vivo in the rat skin. The matrine ethosomes were prepared by the ethanol injection-sonication method. The particle size of the ethosomes was measured by a laser particle-size analyzer, and the entrapment efficiency was detected by ultracentrifugation. The anti-inflammatory activity in vivo of the matrine formulations was determined by a reflection spectrophotometer. In this study, we found that the average particle size of matrine ethosomes was in the range of 50–200?nm with a narrow distribution, and the entrapment efficiency was in the range of 40–90%. Compared with other formulations, matrine ethosomes had the largest 24-hour accumulative permeation quantity (60.5%) and with no permeation lag time. Matrine ethosomes were able to make the induced erythema disappear more rapidly than the nonethosomes formulations of matrine. This study reveals that the average particle size of matrine ethosomes decreases with the increase of ethanol concentration and increases with the increase of phospholipid concentration, while the entrapment efficiency increases with the increase of the concentration of both ethanol and phospholipid. Matrine ethosomes can increase the percutaneous permeation of matrine in the experiment in vitro and improve the anti-inflammatory activity of matrine in vivo in rat skin.     

The enantioselective enrichment,metabolism, and toxicity of fenoxaprop-ethyl and its metabolites in zebrafish

To investigate the impacts of the widely used chiral herbicide fenoxaprop-ethyl (FE) on aquatic organisms, the enrichment, metabolism, acute toxicity, and oxidative stress of fenoxaprop-ethyl and its main metabolites fenoxaprop (FA), ethyl-2-(4-hydroxyphenoxy)propanoate (EHPP), 2-(4-hydroxyphenoxy)propanoic acid (HPPA), and 6-chloro-2,3-dihydrobenzoxazol-2-one (CDHB) in zebrafish were studied. The enantioselectivity of fenoxaprop-ethyl and its chiral metabolites was also determined. Fenoxaprop-ethyl quickly degraded in zebrafish by aquatic exposure. FA, HPPA, and CDHB were the main metabolites that were enriched in the zebrafish. In the metabolism experiment, the half-lives of the metabolites were 0.92–1.72 days in zebrafish. The R-enantiomers of FA and HPPA were preferentially enriched and metabolized with enantiomeric fractions (EFs) of 0.65–0.85. According to the 96-h acute toxicity, FA, HPPA, EHPP, and CDHB were less toxic to zebrafish than FE, following the order of FE > CDHB > EHPP > FA > HPPA. The S-enantiomers of FE, FA, CDHB, and EHPP were more toxic than the R-enantiomers. FE and its metabolites could significantly increase catalase (CAT) and superoxide dismutase (SOD) activity and the malondialdehyde (MDA) content in gill and liver tissues, indicating their oxidative stress, and these effects were also enantioselective. This work could supply more information for evaluating the risks of fenoxaprop-ethyl on aquatic organisms concerning their metabolites and enantiomers.     

Identification and quantitation of novel vitamin E metabolites, sulfated long-chain carboxychromanols, in human A549 cells and in rats

The metabolism of vitamin E involves oxidation of the phytyl chain to generate the terminal metabolite 7,8-dimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (CEHC) via intermediate formation of 13'-hydroxychromanol and long-chain carboxychromanols. Conjugated (including sulfated) metabolites were reported previously but were limited to CEHCs. Here, using electrospray and inductively coupled plasma mass spectrometry, we discovered that gamma-tocopherol (gamma-T) and delta-T were metabolized to sulfated 9'-, 11'-, and 13'-carboxychromanol (9'S, 11'S, and 13'S) in human A549 cells. To further study the metabolites, we developed a HPLC assay with fluorescence detection that simultaneously analyzes sulfated and nonconjugated intermediate metabolites. Using this assay, we found that sulfated metabolites were converted to nonconjugated carboxychromanols by sulfatase digestion. In cultured cells, approximately 45% long-chain carboxychromanols from gamma-T but only 10% from delta-T were sulfated. Upon supplementation with gamma-T, rats had increased tissue levels of 9'S, 11'S, and 13'S, 13'-hydroxychromanol, 13'-carboxychromanol, and gamma-CEHC. The plasma concentrations of combined sulfated long-chain metabolites were comparable to or exceeded those of CEHCs and increased proportionally with the supplement dosages of gamma-T. Our study identifies sulfated long-chain carboxychromanols as novel vitamin E metabolites and provides evidence that sulfation may occur parallel with beta-oxidation. In addition, the HPLC fluorescence assay is a useful tool for the investigation of vitamin E metabolism.     

Stereoselective disposition and chiral inversion of KE-298, a new antirheumatic drug,in rats

The present study was an attempt to elucidate the relationship between stereoselective pharmacokinetics and protein binding of KE-298 and its active metabolites, deacetyl-KE-298 (M-1) and S-methyl-KE-298 (M-2). Metabolic chiral inversion was also investigated. The levels of unchanged KE-298 in plasma after oral administration of (+)-(S)-KE-298 to rats were lower than those of (−)-(R)-KE-298, whereas the levels of M-1 and M-2 after administration of (+)-(S)-KE-298 were higher than after (−)-(R)-KE-298. In vitro, rat plasma protein binding of (+)-(S)-KE-298 was lower than that of (−)-(R)-KE-298. In contrast, the binding of (+)-(S)-M-1 and (+)-(S)-M-2 was higher than that of (−)-(R)-M-1 and (−)-(R)-M-2. Displacement studies revealed that the (+)-(S) and (−)-(R)-enantiomers of KE-298 and their metabolites bound to the warfarin binding site on rat serum albumin. These results suggest that the stereoselective plasma levels in KE-298 and its metabolites were closely related to enantiomeric differences in protein binding, attributed to quantitative differences in binding to albumin rather than to the different binding sites. Unidirectional chiral inversion was detected after oral administration of either (−)-(R)-KE-298 or (−)-(R)-M-2 to rats both yielding (+)-(S)-M-2. Chirality 9:22–28, 1997 © 1997 Wiley-Liss, Inc.     

Evacetrapib is a novel, potent, and selective inhibitor of cholesteryl ester transfer protein that elevates HDL cholesterol without inducing aldosterone or increasing blood pressure

Cholesteryl ester transfer protein (CETP) catalyses the exchange of cholesteryl ester and triglyceride between HDL and apoB containing lipoprotein particles. The role of CETP in modulating plasma HDL cholesterol levels in humans is well established and there have been significant efforts to develop CETP inhibitors to increase HDL cholesterol for the treatment of coronary artery disease. These efforts, however, have been hampered by the fact that most CETP inhibitors either have low potency or have undesirable side effects. In this study, we describe a novel benzazepine compound evacetrapib (LY2484595), which is a potent and selective inhibitor of CETP both in vitro and in vivo. Evacetrapib inhibited human recombinant CETP protein (5.5 nM IC(50)) and CETP activity in human plasma (36 nM IC(50)) in vitro. In double transgenic mice expressing human CETP and apoAI, evacetrapib exhibited an ex vivo CETP inhibition ED(50) of less than 5 mg/kg at 8 h post oral dose and significantly elevated HDL cholesterol. Importantly, no blood pressure elevation was observed in rats dosed with evacetrapib at high exposure multiples compared with the positive control, torcetrapib. In addition, in a human adrenal cortical carcinoma cell line (H295R cells), evacetrapib did not induce aldosterone or cortisol biosynthesis whereas torcetrapib dramatically induced aldosterone and cortisol biosynthesis. Our data indicate that evacetrapib is a potent and selective CETP inhibitor without torcetrapib-like off-target liabilities. Evacetrapib is currently in phase II clinical development.     

Survivability of a probiotic Lactobacillus casei in the gastrointestinal tract of healthy human volunteers and its impact on the faecal microflora

AIM: The aim of this study was to measure the gastrointestinal survival of Lactobacillus casei and its impact on the gut microflora in healthy human volunteers. METHODS AND RESULTS: Twenty healthy volunteers took part in a double-blind placebo-controlled probiotic feeding study (10 fed probiotic, 10 fed placebo). The probiotic was delivered in two 65 ml aliquots of fermented milk drink (FMD) daily for 21 days at a dose of 8.6 +/- 0.1 Log(10)Lact. casei CFU ml(-1) FMD. Faecal samples were collected before, during and after FMD or placebo consumption, and important groups of faecal bacteria enumerated by fluorescent in situ hybridization (FISH) using oligonucleotide probes targeting the 16S rRNA. The fed Lact. casei was enumerated using selective nutrient agar and colony identity confirmed by pulsed field gel electrophoresis. Seven days after ingestion of FMD, the Lact. casei was recovered from faecal samples taken from the active treatment group at 7.1 +/- 0.4 Log(10) CFU g(-1) faeces (mean +/- SD, n = 9) and numbers were maintained at this level until day 21. Lact. casei persisted in six volunteers until day 28 at 5.0 +/- 0.9 Log(10) CFU g(-1) faeces (mean +/- SD, n = 6). Numbers of faecal lactobacilli increased significantly upon FMD ingestion. In addition, the numbers of bifidobacteria were higher on days 7 and 21 than on days 0 and 28 in both FMD fed and placebo fed groups. Consumption of Lact. casei had little discernible effect on other bacterial groups enumerated. CONCLUSIONS: Daily consumption of FMD enabled a probiotic Lact. casei strain to be maintained in the gastrointestinal tract of volunteers at a stable relatively high population level during the probiotic feeding period. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has confirmed that this probiotic version of Lact. casei survives well within the human gastrointestinal tract.     

Cunninghamella blakesleeana-mediated biotransformation of a contraceptive drug,desogestrel, and anti-MDR-Staphylococcus aureus activity of its metabolites

Staphylococcus aureus is one of the most infectious agents among staphylococcal bacteria. Currently many strains of S. aureus have developed resistance against available antibiotics. Therefore, the treatment of infections caused by them is a major challenge. During current study, desogestrel (1), a contraceptive drug, was found to be a potent growth inhibitor of drug resistant strains of S. aureus. Therefore, in search of new and effective agents against multi-drug resistant S. aureus strains, whole-cell bio-catalytic conversion of desogestrel (1) by Cunninghamella blakesleeana ATCC 8688A at pH 7.0 and 25 °C was carried out, yielding three new metabolites, 13-ethyl-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-6β,15β,17β-triol (2), 13-ethyl-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-3β,6β,17β-triol (3), and 13-ethyl-11-methylene-18,19-dinor-17α-pregn-20-yn-3α,5α,6β,17β-tetraol (4), along with a known metabolite, 13-ethyl-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-6β,17β-dihydroxy-3-one (5). Among them, compounds 1–2 showed a potent activity against S. aureus EMRSA-17, S. aureus NCTC 13277 (MRSA-252), and S. aureus NCTC 13143, and clinically isolated Pakistani strain of S. aureus in an in vitro Microplate Alamar Blue Assay (MABA). Vancomycin was used as the standard drug in this assay. In addition, compound 1 also showed a significant activity against vancomycin-resistant S. aureus (VRSA) ATCC 700699. Compounds 1–5 were also evaluated against 3T3 normal cell line (mouse fibroblast) where they all were identified as non-cytotoxic. The present study thus provides new leads for the development of anti-bacterial drugs against MDR S. aureus.     

Analysis of eighteen antidepressants,four atypical antipsychotics and active metabolites in serum by liquid chromatography: a simple tool for therapeutic drug monitoring

Therapeutic drug monitoring necessitates efficient, fast and reliable analytical methods validated by external quality control. We therefore devised an isocratic reversed-phase HPLC method with ultraviolet detection and optimised this to quantify mirtazapine, reboxetine, moclobemide, venlafaxine, O-desmethylvenlafaxine, paroxetine, fluvoxamine, fluoxetine, norfluoxetine, sertraline, citalopram, amitriptyline, nortriptyline, imipramine, desipramine, doxepin, nordoxepin, clomipramine, norclomipramine, trimipramine, mianserine, maprotiline, normaprotiline, amisulpride, clozapine, norclozapine, quetiapine, risperidone and 9-OH-risperidone in human serum. After solid-phase extraction of the drugs and metabolites, the chromatographic separation was achieved on a Nucleosil 100-Protect 1 column with acetonitrile-potassium dihydrogenphosphate buffer as mobile phase. The method was validated for therapeutic and toxic serum ranges. A linear relationship (r>0.998) was obtained between the concentration and the detector signal. Recoveries were between 75 and 99% for the drugs and metabolites. The accuracy of the quality control samples, expressed as percent recovery, ranged from 91 to 118%; intra- and inter-assay-relative standard deviations were 0.9-10.2% and 0.9-9.7%, respectively. Additional external quality control is carried out since 3 years. This method is applicable to rapidly and effectively analyze serum or plasma samples for therapeutic drug monitoring of about 30 antidepressants and atypical antipsychotics.     

Febuxostat (TMX-67), a novel, non-purine, selective inhibitor of xanthine oxidase, is safe and decreases serum urate in healthy volunteers

In order to evaluate the safety, pharmacological properties, and urate-lowering efficacy of febuxostat, a non-purine, selective inhibitor of xanthine oxidase, a Phase 1, 2-week, multiple-dose, placebo-controlled, dose-escalation study was conducted in 154 healthy adults of both sexes. Daily febuxostat doses in the range 10 mg to 120 mg resulted in proportional mean serum urate reductions ranging from 25% to 70% and in proportional increases in maximum febuxostat plasma concentrations and area under plasma concentration versus time curves. Accompanying the hypouricemic effect were increases in serum xanthine concentrations, decreases in urinary uric acid excretion, and increases in urinary xanthine and hypoxanthine excretion, confirming inhibition of xanthine oxidase activity by febuxostat. Hepatic conjugation and oxidative metabolism were the major pathways of elimination of febuxostat from the body, and renal elimination did not appear to play a significant role. Although not uncommon, adverse events were mild and self-limited, and no deaths or serious adverse events were observed. Febuxostat is a safe and potent hypouricemic agent in healthy humans.     

Pharmacokinetics of the enantiomers of trans-tramadol and its active metabolite, trans-O-demethyltramadol, in healthy male and female chinese volunteers

By using a high-performance capillary electrophoresis method, the pharmacokinetics of the enantiomers of trans-tramadol (trans-T) and its active metabolite, trans-O-demethyltramadol (M1), was studied in healthy male and female Chinese volunteers after oral administration of 100 mg trans-T hydrochloride. The values of Cmax for the enantiomers of trans-T and M1, and AUC0- infinity for (-)-trans T, (+)-M1, and (-)-M1 were higher in females than in males. The values of V(d)/F for the enantiomers of trans-T and CLr for (+)-M1 were lower in females than in males. The value of t(1/2) for (-)-M1 was longer in females than in males. There were significant differences in pharmacokinetic parameters of the two enantiomers of trans-T or M1 both in males and in females. The (+)/(-)-enantiomeric ratios of t(max), V(d)/F for trans-T in males were significantly different from those in females and the (+)/(-)-enantiomeric ratios of pharmacokinetic parameters for M1 in males were similar to those in females. There are gender-related differences in the pharmacokinetics of the enantiomers of trans-T and M1 which may be due to the greater body weights for men and/or the higher CYP2D6 activity in women. The pharmacokinetics of trans-T and M1 is stereoselective in men and women. There is a gender-related difference in the stereoselectivity in pharmacokinetics of trans-T in human and the stereoselectivity in pharmacokinetics of M1 in men is similar to that in women. Chirality 16:112-118, 2004.     

Association of SNP41, SNP56 and a novel SNP in PDE4D gene with stroke and its subtypes

A genetic link of the carbon metabolism and DNA replication was recently reported for the representative of Gram-negative bacteria, Escherichia coli. Our studies showed that the viability of thermosensitive replication mutants at high temperature can be improved or fully recovered by deleting certain genes of central carbon metabolism (CCM). In order to improve our understanding of this phenomenon, in this study we analyzed the length and nucleoid distribution of suppressed thermosensitive replication mutants. The dysfunctions in the replication machinery generally lead to formation of elongated cells (termed filaments) that originate from an inhibition of cell division dependent on replication-stress, and to abnormal distribution and compaction of nucleoids. The results reported here provide evidence that deletion of the pta and ackA CCM genes significantly reduces observed cell length in the replication mutants dnaA46, dnaB8, dnaE486, dnaG(ts) and dnaN159. A weaker effect was shown in the tktB dnaE486 double mutant. The CCM enzyme dysfunction restored also the nucleoid shape and position in double mutants. The specificity of these effects was confirmed by overexpression of fully functional genes coding for relevant CCM enzymes, which caused the reversion to the initial filamentous and nucleoid phenotypes. These results indicate that CCM mutations can rescue (or reduce) the cell division defects resulting from various replication mutations. We thus suggest that the replication-metabolism connection may serve as a general mechanism affecting DNA duplication at various levels to adjust this process and the cell division to the status of cell physiology.