Plasma resistin concentration determined by common variants in the resistin gene and associated with metabolic traits in an aged Japanese population.

AIMS/HYPOTHESIS: Resistin is a cytokine derived from adipose tissue and is implicated in obesity-related insulin resistance and type 2 diabetes mellitus. Polymorphisms of the resistin gene (RETN) have been shown to affect the plasma resistin concentration. The aims of this study were to identify polymorphisms of RETN that influence plasma resistin concentration and to clarify the relation between plasma resistin level and metabolic disorders in an aged Japanese cohort. METHODS: The study participants comprised 3133 individuals recruited to a population-based prospective cohort study (KING study). Plasma resistin concentration, BMI, abdominal circumference, blood pressure, fasting plasma glucose and serum insulin concentrations, HbA(1c) content and serum lipid profile were measured in all participants. The HOMA index of insulin resistance (HOMA-IR) was also calculated. Eleven polymorphisms of RETN were genotyped. RESULTS: A combination of ANOVA and multiple linear regression analysis in screening and large-scale subsets of the study population revealed that plasma resistin concentration was significantly associated with rs34861192 and rs3745368 polymorphisms of RETN. Multiple linear regression analysis with adjustment for age and sex also showed that the plasma resistin level was significantly associated with serum concentrations of HDL-cholesterol, triacylglycerol and insulin, as well as with BMI. CONCLUSIONS/INTERPRETATION: Our results implicate the rs34861192 and rs3745368 polymorphisms of RETN as robust and independent determinants of plasma resistin concentration in the study population. In addition, plasma resistin level was associated with dyslipidaemia, serum insulin concentration and obesity. TRIAL REGISTRATION: ClinicalTrials.gov NCT00262691.

Honeycomb-like porous chitosan films prepared via phase transition of poly(N-isopropylacrylamide) during water evaporation under ambient conditions.

Honeycomb-like porous chitosan (CS) films are attractive tools for developing functional materials for filters, catalyses, adsorbents, biomaterials, etc. A simple method for fabricating honeycomb-like porous CS films without special reagents, facilities, and techniques would make them accessible. Here we introduce an easily available method for fabricating honeycomb-like CS films without a strong acid/base, toxic reagents, or special facilities/techniques. An aqueous solution containing CS and poly(N-isopropylacrylamide) (PNIPAm) was allowed to stand at 25 °C to evaporate water. After 3 days, CS-PNIPAm composite films with homogenously phase-separated PNIPAm particles were obtained. The PNIPAm particles were removed by immersion in methanol, and the resulting films dried under reduced pressure to become honeycomb-like porous CS films. The pore size could be varied in the range of 0.5-3.0 µm by altering the CS concentration and the molecular weight of CS where the pore size was reduced under conditions with stronger interaction between CS molecules. We reveal that the key to success with this system is the decrease of lower critical solution temperature (LCST) of PNIPAm with water evaporation. In addition, we confirmed the removed PNIPAm was recyclable in this system. Furthermore, we found this method was also applicable to alginate. The proposed facile method for fabricating honeycomb-like porous polymeric films could provide various functional porous materials.

Effects of levamisole on pathogenesis of Marek's disease.

Levamisole was administered to chickens previously inoculated with Merek's disease virus (MDV) or infected by contact, and the influence of the drug on the mortality rate of Merek's disease (MD) was examined. The chickens inoculated with MDV and then administered levamisole (3 mg/bird) began to die earlier than chickens not treated with levamisole. However, the chickens infected with MDV by contact and then treated with the levamisole had delayed deaths. When the capacity of peritoneal exudate cells (PEC) to inhibit MDV plaque formation on chicken kidney cell (CKC) cultures was examined, the inhibitory activity of PEC from chickens inoculated with MDV and treated with levamisole at 4 days or 2 months of age was much weaker than that of PEC from chickens of the same ages that were not treated with levamisole. This effect was also noted when levamisole was added to CKC cultures to examine the inhibitory activity of PEC from MDV-infected chickens not treated with levamisole. These results indicate that the administration of levamisole to MDV-inoculated chickens in doses used in the present experiment suppressed the macrophage restriction on MDV replication and hastened the death of chickens by MD during the early course of infection.

Inhibitory effects of macrophages against Marek's disease virus plaque formation in chicken kidney cell cultures.

Inhibition of plaque formation by Marek's disease virus (MDV) in chicken kidney cell cultures was investigated with the use of peritoneal exudate cells (PEC) from chickens. PEC from MDV-infected White Leghorn chickens inhibited the formation of MDV plaques, whereas the inhibitory effect of PEC from chickens vaccinated with herpesvirus of turkey (HVT) or PEC from normal chickens was very weak. However, PEC from either normal chickens or HVT-vaccinated chickens inhibited the MDV plaque formation in the presence of serum from MDV-infected chickens but not from normal or HVT-vaccinated chickens. The capacity of PEC to inhibit plaque formation was significantly reduced when PEC was treated with carrageenan but not with antithymus or antibursa cell serum. These results indicate that macrophages may have a role in protection against Marek's disease by reducing the number of MDV-infected cells and thereby decreasing the spread of the virus in vivo.

Detection of a new antigen associated with chicken thrombocytes on Marek's disease lymphoblastoid cell line.

A monoclonal antibody, 2C12, produced against MDCC-MSB1 cells, reacted with several cell lines derived from Marek's disease (MD), including MDCC-MSB1 cells, normal chicken thrombocytes, and MD tumor cells. It did not react with cell lines derived from avian leukosis or reticuloendotheliosis, cells from normal chicken thymus and bursa, chicken kidney cells and quail fibroblast cultures inoculated with MD virus, or with erythrocytes from 1-day-old and adult chickens, cow, sheep, and horse. Anti-thrombocyte serum prepared in a rabbit reacted with thrombocytes and MDCC-MSB1 cells. The specificities of antibody 2C12 and anti-thrombocyte serum against MDCC-MSB1 cells and thrombocytes were confirmed by cross-absorption, blocking, and double-membrane fluorescent antibody tests. The existence of a polypeptide with an apparent molecular weight of 103,000, common to both MDCC-MSB1 cells and thrombocytes, was demonstrated by Western blotting analyses.

Topographical analysis of tumor-associated antigens on bovine leukemia virus-induced bovine lymphosarcoma.

Tumor-associated antigens (TAAs) expressed on tumor cells from cattle with enzootic bovine leukosis were divided into three groups by using 13 monoclonal antibodies: common TAA; partially common TAA; and individually distinct TAA. TAA was extracted from tumor cells and purified by ion exchange chromatography on diethylaminoethyl-cellulose and isoelectric focusing. The common TAA, which was detected on all tumors tested, was eluted with 0.6 M KCl in ion exchange chromatography on diethylaminoethyl-cellulose, and the isoelectric point of the antigen was 6.8. The partially common TAA, which was detected on some (but not all) of the tumors tested, was eluted with 0.4 to 0.8 M KCl, and the isoelectric points of the antigen were 5.3, 5.8, and 6.4. The individually distinct TAA was present in the fractions eluted with 0.4 to 0.8 M KCl, and the isoelectric point of the antigen was 5.5. Results of competitive binding assay and Western blot analysis showed that the common TAA was a polypeptide with a molecular weight of 74,000; that it has at least two independent antigenic regions; that the partially common and individually distinct TAAs were a polypeptide with a molecular weight of 64,000; and that the antigenic determinants on the common TAA, partially common TAA, and individually distinct TAA existed independently from each other.

Monoclonal antibody to chicken fetal antigen on Marek's disease lymphoblastoid cell line MDCC-MSB1.

A monoclonal antibody (2H3) to chicken fetal antigen (CFA) expressed on the cell surface of the Marek's disease lymphoblastoid cell line MDCC-MSB1 was generated. 2H3 reacted specifically with various cells from chicken embryos, especially with fetal red blood cells, fetal bursa-derived lymphocytes, fetal liver cells, and embryo fibroblasts but not with fetal peripheral blood lymphocytes. 2H3 reacted also with all lymphoblastoid cell line cells tested: Marek's disease, avian leukosis, and reticuloendotheliosis line cells. On the basis of 2H3 specificity, CFA detected by 2H3 seemed to be similar to chicken fetal red blood cell antigen previously reported, but not to chicken alpha-fetoprotein. A transient increase of reactivities with 2H3 was observed in lymphocytes prepared from thymus, spleen, and bursa of chickens inoculated with Marek's disease virus or herpesvirus of turkeys from 7 to 21 days postinoculation. The average percentage of CFA-positive cells in lymphoid cells from Marek's disease virus-infected chickens at the peaks was higher than that of the cells in Marek's disease tumor cells from 146- and 156-day-old chickens in the field. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis revealed that the monoclonal 2H3 is recognized a Mr 50,000 oncodevelopmental cell surface antigen present on MDCC-MSB1 cells. The possible differences between CFA detected by 2H3 and chicken fetal red blood cell antigen expressed on normal chicken fetal erythrocytes are discussed.

Tumor-associated antigens on bovine leukemia virus-induced bovine lymphosarcoma identified by monoclonal antibodies.

Thirteen monoclonal antibodies directed against tumor cells from cattle from enzootic bovine leukosis (EBL) were obtained. They reacted with tumor cells but not with normal bovine cells or bovine leukemia virus antigens. According to the reactivities of these antibodies with 19 individual tumors, the 13 monoclonal antibodies can be divided into three groups. Antibodies of the first group reacted with all the EBLs tested; those of the second group reacted with several, but not all, of the EBLs tested; and those of the third group reacted only with homologous tumor cells. Therefore, tumor-associated antigen (TAA) on the EBL tumor may possess common TAA, partially common TAA, and individually distinct TAA. The TAAs were solubilized from the tumor cells by treatment with 0.2% sodium deoxycholate and partially purified by diethylaminoethyl-cellulose column chromatography. Eleven of the 13 monoclonal antibodies reacted with this soluble TAA. The monoclonal antibodies belonging to the first group inhibited in vitro the growth of the bovine lymphoid cell line derived from the EBL tumor.

Reduced myocardial sarcoplasmic reticulum Ca(2+)-ATPase mRNA expression and biphasic force-frequency relations in patients with hypertrophic cardiomyopathy.

BACKGROUND: The relationship between left ventricular (LV) contractile functional reserve and gene expression of Ca(2+)-handling proteins in patients with hypertrophic cardiomyopathy (HCM) remains to be clarified. METHODS AND RESULTS: We calculated the maximum first derivative of LV pressure (LV dP/dt(max)) and the LV pressure half-time (T(1/2)) during pacing in 14 patients with nonobstructive HCM (LV ejection fraction >55%) and 7 control subjects. Endomyocardial tissue was obtained, and mRNA levels of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2), ryanodine receptor-2, phospholamban, calsequestrin, and Na(+)/Ca(2+) exchanger were quantified by use of a real-time quantitative reverse transcription-polymerase chain reaction method. Group A consisted of 7 HCM patients who showed a progressive rise in the LV dP/dt(max) with increased heart rate. Group B consisted of 7 HCM patients in whom the heart rate-LV dP/dt(max) relation was biphasic at physiological pacing rates. Both the mean maximal wall thickness and the LV hypertrophy score in group B were greater than in group A (20+/-5 versus 15+/-3 mm and 7+/-1 versus 5+/-2 points, respectively). SERCA2 mRNA levels were significantly lower in group B (SERCA2/GAPDH ratio 0.34+/-0.15) compared with group A (0.72+/-0.27) and control subjects (0.85+/-0.47), whereas the mRNA expression of ryanodine receptor-2, phospholamban, calsequestrin, and Na(+)/Ca(2+) exchanger were similar in all groups. CONCLUSIONS: These results suggest that downregulation of SERCA2 mRNA, resulting in altered Ca(2+) handling, may contribute to impaired LV contractile reserve in HCM patients with severe hypertrophy, even in the absence of detectable baseline systolic dysfunction.

Impaired force-frequency relations in patients with hypertensive left ventricular hypertrophy. A possible physiological marker of the transition from physiological to pathological hypertrophy.

BACKGROUND: The extent to which force-frequency and relaxation-frequency relations (FFR and RFR, respectively) and exercise-induced adrenergic stimulation affect myocardial inotropic and lusitropic reserves has not been established in patients with left ventricular (LV) hypertrophy (LVH). METHODS AND RESULTS: We calculated the maximum first derivative of LV pressure (LV dP/dtmax) and the LV pressure half-time (T1/2) during pacing, exercise, and isoproterenol infusion in 17 patients with hypertensive LVH and 9 control subjects to investigate the influence of increases in heart rate (HR) and adrenergic stimulation on inotropic and lusitropic reserves. Group A consisted of 10 LVH patients who showed a progressive increase in the HR-LV dP/dtmax relation. Group B consisted of 7 LVH patients in whom the HR-dP/dtmax relation at physiological pacing rates was biphasic. The LV mass index was larger and the LV ejection fraction was smaller in group B than in group A (244+/-72 g/m2 versus 172+/-22 g/m2 and 55+/-18% versus 72+/-6%, respectively; both P<0.05). The increase in LV dP/dtmax was greater during exercise than pacing alone for similar increases in HR in all groups (P<0.05) (group A, 111+/-22% versus 25+/-14%; group B, 105+/-35% versus 14+/-10%; control, 111+/-24% versus 25+/-12%). T1/2 was shorter (P<0.05) during exercise than with pacing alone in all groups (group A, 41+/-6% versus 11+/-3%; group B, 38+/-9% versus 14+/-4%; control, 44+/-6% versus 12+/-5%). Isoproterenol infusion caused similar increases in LV dP/dtmax and similar decreases in T1/2 in all groups. CONCLUSIONS: The FFR was biphasic in patients with severe LVH irrespective of LV function but was preserved in patients with less severe LVH and control subjects. Importantly, the RFR and adrenergic control of both inotropic and lusitropic reserves were well preserved in all LVH patients. A biphasic FFR at physiological pacing rates may be one of the earliest markers of the transition from physiological adaptation to the pathological process in LVH patients.

Impaired response of left ventricular relaxation to exercise-induced adrenergic stimulation in patients with hypertrophic cardiomyopathy.

OBJECTIVES: We investigated the effect of adrenergic stimulation on left ventricular relaxation in patients with hypertrophic cardiomyopathy. BACKGROUND: Exercise-induced decreases in acceleration of left ventricular relaxation have been observed in patients with hypertrophic cardiomyopathy. However, data on sequential changes in left ventricular relaxation during exercise are limited. METHODS: We measured right (fluid filled) and left (high fidelity micromanometer) ventricular pressures during moderate supine ergometer exercise and during rapid right atrial pacing in four groups of patients: 9 with severe hypertrophic cardiomyopathy, 9 with moderate hypertrophic cardiomyopathy, 10 with hypertension and moderate hypertrophy and 5 control subjects. RESULTS: There was a curvilinear relation between the time constant of relaxation (tau) and heart rate in all groups during exercise. There was no difference in the slope of this relation between the two hypertrophic cardiomyopathy subgroups. Although the slope of this relation between tau and heart rate was steeper in the hypertensive than the moderate hypertrophic cardiomyopathy group (p < 0.001, analysis of covariance), the decrease in tau during right atrial pacing was similar in both groups. There were no significant differences in plasma levels of catecholamines at rest or at peak exercise among groups or in maximal heart rate during pacing. CONCLUSIONS: Pacing-induced changes in tau in hypertrophic cardiomyopathy were similar to those in hypertensive hypertrophy, but remarkable decrease in exercise-induced acceleration of tau were observed only in hypertrophic cardiomyopathy. Our results may indicate a depressed left ventricular relaxation response to exercise-induced adrenergic stimulation in hypertrophic cardiomyopathy.

Biphasic changes in left ventricular end-diastolic pressure during dynamic exercise in patients with nonobstructive hypertrophic cardiomyopathy.

OBJECTIVES: The aim of this study was to clarify the serial changes in left ventricular (LV) end-diastolic pressure (LVEDP) during dynamic exercise in patients with hypertrophic cardiomyopathy (HCM). BACKGROUND: Although HCM is characterized by impaired resting LV diastolic function, serial changes in LVEDP during exercise have not been characterized. METHODS: We simultaneously measured LV pressure and LV dimensions during symptom-limited supine bicycle exercise in 5 healthy individuals and 20 patients with HCM. Exercise thallium-201 scintigraphic studies were also performed. RESULTS: The LVEDP (baseline: 12 +/- 5 mm Hg) progressively increased to a maximum value at peak exercise (28 +/- 8 mm Hg) in 11 patients with HCM (group I). In the remaining nine patients with HCM (group II), changes in LVEDP during exercise were biphasic, with an initial progressive increase and a subsequent gradual decline up to peak exercise (14 +/- 4 mm Hg at baseline, 27 +/- 5 mm Hg at the critical heart rate, 16 +/- 3 mm Hg at peak exercise). Exercise-induced changes in LV dimensions and LV peak systolic pressures were similar in both groups. However, the maximum first derivative of LV pressure was greater and the LV pressure half-time was shorter in group II than in group I at a similar peak exercise heart rate. The biphasic changes in LVEDP disappeared by pretreatment with propranolol. The LV hypertrophy scores were higher in group I than in group II. Exercise thallium-201 images showed more severe perfusion defects in group I than in group II patients. CONCLUSIONS: The biphasic changes in LVEDP seen during exercise may be related to improved coronary microcirculation in response to beta-adrenergic stimulation in patients with mild to moderate HCM.

Facile preparation of surface N-halamine chitin nanofiber to endow antibacterial and antifungal activities.

N-halamine chitin nanofiber (NF) film was prepared by the reaction of chitin NF film with sodium hypochlorite solution to endow the film with antibacterial and antifungal activities. The amount of active chlorine content loaded on the chitin NF film depended on the sodium hypochlorite concentration and reaction time. FT-IR, UV-vis, XRD, and TG analyses showed that the N-H bond was substituted to the N-Cl bond and that the reaction took place at the chitin NF surface. After chlorination, the characteristic nanochitin morphology was maintained. Although the active chlorine content of the film gradually decreased by disassociation of the N-Cl bond, chlorine was rechargeable into chitin NF by another sodium hypochlorite solution treatment. The chlorinated chitin NF film showed strong efficacies against Gram-negative and -positive bacteria of Escherichia coli and Staphylococcus aureus, respectively. Moreover, the films showed 100% and 80% inhibition of spore germination when faced against Alternaria alternata and Penicillium digitatum fungi, respectively.

Molecular cloning of bovine leukemia virus DNA integrated into the bovine tumor cell genome.

The bovine leukemia virus (BLV) DNA harbored in the bovine tumor cell genome was cloned in lambda Charon 4A phage. Using either representative or 3' half-enriched BLV cDNA as a blot hybridization probe, clone lambda BLV-1 was shown to carry 9 kb of the BLV genome, flanked by cellular sequences at both ends. Restriction mapping with twelve endonucleases and hybridization of the DNA fragments to BLV cDNA representing a 3'-end portion of the viral genome revealed the presence and precise location of two long terminal repeats (LTRs) and virus-cell junctions. Thus, lambda BLV-1 appears to contain the complete BLV genome and flanking tumor cellular sequences. The restriction map of the cloned BLV proviral DNA closely resembles that previously reported for unintegrated linear proviral DNA, but differs significantly from that of the integrated provirus of another BLV isolate, the difference occurring preferentially in the putative gag and pol genes.

Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections.

We describe and discuss a method of protein extraction for Western blot analysis from formalin-fixed, paraffin-embedded tissue sections. From 5-mm2 50-micron-thick tissue sections, an abundance of proteins could be extracted by incubating the sections in lysis buffer containing 2% sodium dodecyl sulfate (SDS) at 100C for 20 min followed by incubation at 60C for 2 hr. Extracts yielded discernible protein bands ranging from 10 kD to 120 kD as identified by SDS-polyacrylamide gel electrophoresis (PAGE). Western blot analysis successfully detected membrane-bound protein such as E-cadherin, cytosolic protein such as beta-catenin, and nuclear proteins including proliferating cell nuclear antigen (PCNA), mutant-type p53, cyclin D1, cyclin E, and cyclin-dependent kinases (CDKs). With this technique, we could examine cyclin D1 and CDK2 expression in small adenomas compared with cancer tissues and normal mucosa. The simple method of protein extraction described here should make it possible to use large-scale archives of formalin-fixed, paraffin-embedded samples for Western blot analysis, and its application could lead to detailed analysis of protein expression. This new technique should yield valuable information for molecular biology.

Isolation and characterization of rainbow trout C-reactive protein.

An acute phase serum component, C-reactive protein (CRP), was isolated from the sera of rainbow trout (Salmo gairdneri). The isolation was based on its calcium-dependent binding affinity for pneumococcal C-polysaccharide (CPS) according to the isolation procedure of human C-reactive protein. In SDS-PAGE, the nonreduced CRP showed two subunits with molecular weights of 43,700 and 26,600, respectively, at a molar ratio of 1:1. The reduced CRP showed a single subunit of 26,600. The molecular weight of the native protein was estimated as 66,000 by native gradient PAGE and 81,400 by sedimentation equilibrium analysis using ultracentrifugation. The antigenic determinant on CPS-reactive site was destroyed by periodate oxidation, indicating that rainbow trout CRP is a glycoprotein. CRP levels in rainbow trout serum measured by the CPS-ELISA procedure showed that the rainbow trout CRP could behave as an acute phase reactant, following experimental infection with the fish pathogen Vibrio anguillarum.

Activation of trout macrophages and production of CRP after immunization with Vibrio anguillarum.

To examine the mechanism of the protection of rainbow trout (Salmo gairdneri) against Vibrio anguillarum in the early stage of immunization, the activation of macrophages and production of C-reactive protein (CRP) were investigated. Fish immunized with formalin-killed bacteria emulsified in Freund's complete adjuvant (FCA) resisted intraperitoneal challenge with living bacteria seven and ten days after immunization. The activation of macrophages was demonstrated by a significant increase of the chemiluminescent (CL) response and phagocytic activity. These fish also showed a significant increase of the CRP level in sera. Fish immunized with V. anguillarum alone or injected with FCA, however, did not resist the challenge. Though FCA itself increased CRP level and the sera enhanced phagocytic activity, increase of CL activity was weak. These results indicated that the increase of CL activity and opsonising effect of CRP on the phagocytosis of specifically activated macrophages concern to host defense in the early stage of infection.

PyNPase expression in human colon cancer.

Although colorectal cancer tissue is rich in pyrimidine nucleoside phosphorylase (PyNPase), there is no consensus as to whether cancer cells or stromal cells predominately express PyNPase. We micro-dissected OCT compound embedded frozen tissue sections into epithelial and stromal components and then analyzed the extracted samples separately. The PyNPase expression level was higher in stromal cells than in cancer cells and the difference increased with inflammation induced by the immunostimulator OK432. These results suggest that stromal cells are the major PyNPase source in colorectal cancer.

Cdk2/cdc2 expression in colon carcinogenesis and effects of cdk2/cdc2 inhibitor in colon cancer cells.

Cyclin dependent kinases propel the cell cycle in collaboration with cyclins. We have examined the expression of cdk2/cdc2 in adenoma and focal carcinoma in adenomatous tissue to explore their role in tumorigenesis of colorectum. Immunohistochemical study revealed that cdk2/cdc2 was overexpressed in a subsets of adenoma (14/50; 28.0%) but this overexpression was much more obvious in focal carcinoma (13/15; 86.7%). These results suggest that cdk2/cdc2 is remarkably upregulated together with a malignant change. In an effort to demonstrate a significant role for cdk2/cdc2 in colon cancer, we investigated growth and apoptosis with butyrolactone I, a specific inhibitor for cdk2/cdc2, using 4 colon carcinoma cell lines (HCT116, LoVo, HT29, Colo 320DM). Butyrolactone I inhibited proliferation of all colon carcinoma cell lines at 100 microM and it induced apoptosis in LoVo cell line with induction of p53. Our findings suggest that inhibition of cdk2/cdc2 may be a useful strategy against colon cancer.

Cloning and nucleotide sequence of the gene (citA) encoding a citrate carrier from Salmonella typhimurium.

A cryptic citrate transport gene (citA) from Salmonella typhimurium chromosome was cloned and its nucleotide sequence was determined. The cloned plasmid conferred citrate-utilizing ability on wild-type Escherichia coli, which cannot grow on citrate as the sole source of carbon. The resultant E. coli transformant was able to transport citrate. A 1,302-base-pair open reading frame with a preceding ribosomal binding site was found in the cloned DNA fragment. The 434-amino-acid protein that could be translated from this open reading frame is highly hydrophobic (69% nonpolar amino acid residues), consistent with the fact that the transport protein is an intrinsic membrane protein. The molecular weight of this protein was calculated to be 47,188. The gene sequence determined is highly homologous to those of Cit+ plasmid-mediated citrate transport gene, citA, from E. coli, the chromosomal citA gene from Citrobacter amalonaticus and the chromosomal cit+ gene from Klebsiella pneumoniae. The hydropathy profile of the deduced amino acid sequence suggests that this carrier has 12 hydrophobic segments, which may span the membrane lipid bilayer.