, 2008; Qualls et al., 2010; Murray & Wynn, 2011). Expression

of Arg1 by M2 ABC294640 manufacturer macrophages is required for the suppression of T cell proliferation (Pesce et al., 2009), although the corresponding studies in humans have yet to be performed. Moreover, experiments to test T-cell proliferation regulation by Arg1 in Mtb infection need further investigation. In M1 macrophages that are involved in Mtb infection, Arg1 expression and activity is an important mechanism by which Mtb regulates macrophage function by suppressing NO production (El Kasmi et al., 2008; Qualls et al., 2010). Additional studies are necessary to determine whether Arg1 expression by macrophages in human lungs of patients with TB facilitates or not pathogen survival. In humans, it has been reported that Arg1 is released by polymorphonuclear granulocytes and accumulate extracellularly inducing suppression of T-cell proliferation, cytokine synthesis, and also leads to CD3-chain down-regulation without altering T-cell viability (Munder et al., 2006). Besides regulating NO production, these Arg1-dependent events may also play a role in human Mtb infection. In addition, our results demonstrated that, iNOS is also expressed within macrophages associated with granulomas in human TB

lung samples. Interestingly, the number of Arg1-positive cells was higher than the iNOS-positive cells (Fig. 1h). Coexpression of Arg1 and iNOS in mycobacteria-infected cells has been documented, and indeed, competition Oxymatrine between iNOS and arginase for arginine this website has been suggested to contribute to the outcome of infection, because coexpression of Arg1 and iNOS alters the arginine balance such that NO production cannot be maximal (Modolell et al., 1995; Chang et al., 1998; Mills, 2001). Studies have demonstrated

that the expression of host Arg2 may also be up-regulated in macrophages infected by several intracellular pathogens such as Trypanosoma cruzi, Trypanosoma brucei, and Helicobacter pylori (Das et al., 2010). We have found that Arg2 expression is rarely observed in TB lungs, suggesting that Arg2 is not up-regulated in the Mtb-infected human lungs. Whether Arg2 is up-regulated in other tissues (e.g. lymph nodes and spleen) during TB infection remains to be investigated. Type II pneumocytes are specialized cells responsible for the secretion of surfactants such as SP-A, a lipoprotein complex that reduces the surface tension at the air–liquid interface of the lung, which in turn enables any fluid to be converted into droplets that can be rapidly removed. Type II pneumocytes also possess some phagocytic properties (Bermudez & Goodman, 1996; Sato et al., 2002). Mtb multiplies within human type II cell line in vitro, leading to pro-inflammatory citokyne production, which directly influences macrophage function (Sato et al., 2002).

Infection is a leading complication of immunosuppressive treatment and an important cause of mortality in Asian LN patients.[65] Management of patients would need to take into consideration infections that are prevalent or endemic in some Asian countries, such

as hepatitis Belnacasan mw B and tuberculosis, since prophylaxis or pre-emptive treatment may be indicated.[66, 67] The risk of infective complications influences the dose of immunosuppressive agents. The starting dose of prednisolone is usually in the range of 0.8–1 mg/kg body weight daily for the initial treatment of severe proliferative LN. The use of pulse methylprednisolone varies, but many would use intravenous pulse methylprednisolone at 0.5–1 g daily for three days in patients who show extensive crescents on renal biopsy or rapidly progressive renal impairment. Also there is variation on the rate of corticosteroid dose tapering. The target dose of MMF for induction therapy in severe LN is mostly in the range of 1.5–2 g/day, and a high tolerance to MMF is generally observed. The choice and duration of MMF treatment are often dependent on financial Tanespimycin cell line considerations, since MMF or mycophenolic acid sodium is either a self-financed item or second-line (to CYC) immunosuppressive agent in many Asian countries, although health insurance reimbursement is available in some countries with specified criteria. In this context, quality-of-life scores were higher

during MMF treatment compared with scores associated with CYC induction in patients who had experience with both treatments, while the treatment cost associated with MMF could be partially offset by savings from the reduced incidence of complications.[34, isothipendyl 68] Also, the data from a recent

report showed that in patients who had been treated with prednisolone and MMF as continuous induction-maintenance immunosuppression the risk of disease flare was lower when MMF was given for at least 24 months compared with shorter treatment durations[35] In general, treatment is guided by disease severity, which is informed by histological data indicating the class, severity, and reversibility of nephritis, and clinical data which include the change in proteinuria, renal function, serology and extra-renal lupus manifestations. A summary of the consensus recommendations by ALNN members is presented (Table 3). Initial (Induction) immunosuppression in the form of combination therapy with corticosteroids (e.g. prednisolone 0.8 mg/kg/day) and either MMF or CYC (Level 1b) Pulse corticosteroid (e.g. methylprednisolone 0.5 to 1.0 g/day for 3 days) advisable when renal biopsy shows crescentic involvement >10% or evidence of deteriorating renal function. (Level 5) Tapering of corticosteroids to begin after 2 weeks except in patients with no sign of improvement, aiming to reach <20 mg/day after 3 months and ≤7.5 mg/day after 6 months.

The role of FcRn includes the maintenance of serum IgG and albumin levels and the delivery of antigen in the form of immune complexes to degradative compartments within cells. The FcRn–IgG interaction is strictly pH-dependent, with a maximum at pH 6, and becomes undetectable as near neutral pH is approached, a feature that is essential for efficient transport. IgG transport between the blood and

CH5424802 mw interstitial compartments may proceed by convection through paracellular pores in the vascular endothelium, or via FcRn-mediated transcytosis across vascular endosomal cells. Because of the redundancy of the transport systems, high-dose IVIG may help to block FcRn resulting in the enhanced clearance of pathogenic autoantibodies, but will never be able to block it completely, as

indicated in several experimental studies to date [42]. Although improving the binding of IgG to FcRn in vitro generally translates to an improved serum IgG half-life in vivo, this is not always the case. Recombinant therapeutics genetically engineered to contain IgG fragments with the CH2–CH3 domain that binds to FcRn can have significantly prolonged half-life due to protection of catabolism through FcRn binding. However, increased binding affinity to the FcRn does not appear to be proportional to the half-life extension. For example, when comparing variants of Herceptin antibody (an ERBB2-specific human IgG1 against mammary tumour cells) with a threefold Lenvatinib concentration increase in FcRn binding at acidic pH and another variant with a 12-fold increased binding at acidic pH and also enhanced binding at more neutral pH,

both antibodies exhibited similar half-lives when tested in a humanized FcRn transgenic mouse model [43]. Increased binding may enhance degradation of IgG under neutral tuclazepam conditions. Clearly, there is an obvious need to have a better understanding of FcRn in the exact regulation of IgG-mediated responses and half-life in vivo. Research in immunoglobulin therapy with IVIG or SCIG has shown that therapy targets and treatment options evolve in parallel. Achieving good clinical outcomes to enable a state of health as found in immunocompetent individuals is achievable with the use of 0·4–0·6 g/kg/month for many patients with PI, although some patients may require higher doses. For patients with autoimmune neuropathies, an empirically derived starting dose of 2 g/kg is used frequently in the acute setting as in Guillain–Barré syndrome. For maintenance treatment, evidence from a recent randomized placebo-controlled trial in chronic inflammatory demyelinating neuropathy suggests that a dose of 1 g/kg every 3 weeks is sufficient to maintain strength [44]. Indications for review of immunoglobulin doses in patients with PI and autoimmune neuropathies are summarized in Table 5.

CNV of

NKG2C was assessed by a PCR method based on the approach used by Miyashita et al. [43], with modifications. Briefly, homo- and heterozygosity for the NKG2C gene and its deletion were determined by a single Cytoskeletal Signaling inhibitor PCR that yields amplicons of different lengths in each genotype. This is achieved by means of two primer pairs recognizing sequence motifs specific for, either NKG2C, or the gene arrangement resulting from its deletion [60]. In one child participating in the study this analysis could not be performed. Comparisons of categorical variables among study groups were performed using the chi-square or Fisher exact test, as appropriate. Continuous variables were compared using the Mann–Whitney U test. A p value <0.05 was considered statistically significant.

Spearman’s rank correlation coefficient was used to evaluate the association between continuous variables. Multivariate analysis was carried out using linear regression analysis; the dependent variables were subjected to logarithmic transformation prior to inclusion in the regression model. This work was supported by grants from Plan Nacional de I+D (SAF2010–22153-C03), and EU SUDOE program (SOE2/P1/E341). A.M. is supported by Asociación Española Contra el Cáncer (AECC). We thank Gemma Heredia and María Cañizares for their excellent technical assistance, and Joan Vila for his expert advice in statistical analysis. We are grateful to patients and their families for generously accepting to participate in this study. The authors declare no financial or commercial conflict Napabucasin in vitro of interest. Disclaimer: Supplementary materials have been peer-reviewed but not copyedited. Figure 1. Variable NKG2C surface expression intensity in NK cells. Table 1. Clinical findings at birth and NK-cell immunophenotype in children with symptomatic congenital infection. Table 2. Summary of multivariate linear regression analysis. “
“To target gestational diabetes mellitus (GDM) by means of temporal variation Ribonucleotide reductase in pregnancy-associated plasma protein A (PAPP-A) and soluble human leukocyte antigen-G (sHLA-G). Retrospective analysis of PAPP-A and sHLA-G blood levels in historical samples of 112 GDM and 112 controls,

drawn at first trimester, and prospective study in 18 GDM and 105 controls collected in triplicate along the pregnancy. Six hundred and sixty-five samples were analyzed. Gestational diabetes mellitus had significantly lower first-trimester PAPP-A concentrations than controls (2343 ± 1519 versus 2996 ± 1955 mU/mL, in retrospective brunch and 2490.57 ± 1828.52 versus 3240.84 ± 1930.69 mU/L in prospective one, P < 0.001). First-trimester sHLA-G level was significantly lower in GDM than in controls (52.88 ± 59.69 versus 66.81 ± 50.14 ng/mL, P < 0.001) and increased during gestation in diabetic women showing an opposite trend with respect to the controls. PAPP-A and sHLA-G are independent markers of GDM. Quantitative variations during pregnancy help to early unravel the onset of GDM.

The authors concluded that combining tamsulosin and 10 mg of propiverine for 12 weeks provides added benefit for these kinds of men. Tamsulosin plus propiverine

10 and 20 mg patients had significantly increased PVR. However, adverse events were few. The authors believe that propiverine 10 mg may inhibit predominantly actions of non-neuronal acetylcholine released from urothelium contributing to the pathophysiology of OAB and produce significant results. Tamsulosin and solifenacin INCB024360 chemical structure 2.5 mg combination therapy was conducted in the ASSIST study. The primary endpoint was mean change in urgency episodes evaluated using 3-day bladder diary. It was statistically significantly reduced in tamsulosin plus solifenacin 5 mg compared with tamsulosin plus placebo. However, urgency episodes per 24 h were also reduced in tamsulosin plus solifenacin 2.5 mg, but the change CH5424802 cell line was not statistically significant. A statistically significant reduction of OABSS urgency score was shown in both the tamsulosin plus solifenacin 2.5

and 5 mg group compared with tamsulosin plus placebo group. The authors suggested that combination therapy of alpha-blocker plus antimuscarinic may decrease the dose of antimuscarinic to avoid the risk of adverse event in the future.25 Most men with LUTS have both storage and voiding symptoms. This suggests that BPO and DO may coexist. OAB occurs in 50–75% of men with BPO. It is expected that combination therapy with an alpha-blocker and an anticholinergic agent in patients with OAB and BPO could significantly

alleviate symptoms and improve QoL. There are still some concerns because this approach could aggravate voiding symptoms, increase the risk of acute urinary retention, or increase adverse effects. The definition of low dose is not yet known. However, it can be expected there will be some benefits and very mild or no adverse effects in low-dose combination therapy. There is a very small number of clinical reports about low-dose combination therapy. Thalidomide Good randomized controlled trials are needed to proving the effect of this approach. No conflict of interest have been declared by the authors. “
“Objective: Pressure-flow study is a method used to evaluate the degree of bladder outlet obstruction and the strength of detrusor contractility during voiding. However, whether or not the operation for benign prostate hyperplasia should be avoided in detrusor underactivity patients remains controversial. To address this, we performed a retrospective analysis of our pressure-flow study data for benign prostate hyperplasia patients. We especially focused on the backgrounds of patients with weak detrusor contractility. Methods: Patients (n = 288; average age, 71.5 years) who underwent pressure-flow study to evaluate operative indications between February 2001 and April 2010 were included in this study. We analyzed the relationships between background factors and detrusor contraction strength according to Schäfer’s nomogram.

The agarose layer was removed and the number of plaques was measured. Overnight cultures were MG-132 datasheet adjusted to the desired concentration of bacteria in PBS by OD600 nm. Hartley guinea pigs were inoculated in the conjunctival sac with 1 × 109 CFU of S. flexneri 2457T, 2457Tsen and M4243A strains. Guinea pigs were examined daily for 5 days, and their inflammatory responses were graded according to the standard Sereny scale (Cersini et al., 2003). Culture medium from triplicate wells of T84 or HEp-2 cell monolayers

infected in triplicate with S. flexneri wild-type strain 2457T, 2457TΔsen, M4243A and 2457TΔsen transformed with pBAD vector, pSen and pJS26 plasmids were evaluated in duplicate by enzyme-linked immunosorbent assay for IL-8 as described (Harrington et al., 2005). Whole-cell RNA was isolated from a 10-mL sample of the bacterial cultures of wild-type Shigella strain 2457T using the Trizol method according to the manufacturer’s protocols (Invitrogen). RNA was treated with RNase-free DNase I to eliminate the contaminating

DNA using the RNeasy kit (Qiagen). cDNA was synthesized from 1 μg of bacterial RNA using random hexamer primers NVP-AUY922 molecular weight and the Thermoscript RT enzyme (Invitrogen). The PCR reaction was performed using 2 μL of cDNA with the primers C1 (CGCAATAAAATATGAGAATGCAG), P1 (GGGCTGCTCTATCGCTGTAA), P2 (GGGGACAAACCACATCAATC) and S1 (GGCAATTGTTTTGAGTGCAA). Statistical significance between means was analyzed using the unpaired Student t-test with a threshold of P<0.05. Values are expressed as means ± SEs of the mean of three experiments. Several Shigella virulence factors reach their cellular targets by

injection into the eukaryotic cell via the T3SS injectosome. Buchrieser et al. (2000) suggested that ShET-2 could function as a T3SS effector protein, based on the similarity of ShET-2 (which they called OspD3) to another protein (OspD1) that was shown to be secreted by this secretion system. To investigate the possible secretion of ShET-2 by the T3SS, S. flexneri wild-type strain 2457T strain was transformed with pSen (Table 1), which encodes a full-length ShET-2-coding gene (sen gene) fused to a histidine hexamer (His6) at its C-terminus. Figure 1 shows that recombinant ShET-2 protein is secreted in the presence of CR, which induces secretion of type III effectors in Shigella (Bahrani et al., 1997), whereas no secretion of ShET-2 protein was observed when cells were Methane monooxygenase incubated without CR dye (data not shown). As a control for leakage of cytoplasmic proteins, we found no increase in the presence of the protein GroEL in these supernatants. The pSen plasmid was also transformed into S. flexneri harboring mutations in virF or virB (defective in expression of the complete T3SS and Ipa invasins), spa47 (defective in injectosome assembly) or mxiM (defective in the T3SS-associated ATPase). When these mutants were incubated with CR, neither ShET-2 nor the positive control protein IpaB were found in the supernatant fraction (Fig. 1).

Furthermore, repeated sequences from the same individual can vary in copy number in different organs and tissues [16]. The general mechanisms

that lead to changes in copy number include homologous recombination and non-homologous repair mechanisms [17]. Changes in copy number might alter the expression levels of genes included in the CNVR. For example, the salivary amylase gene, AMY1, shows CNV in human populations, and the amount of salivary amylase is directly proportional to the copy number of AMY1[18]. More importantly, CNVs shape tissue transcriptomes on a global scale [19]. Additional copies of genes also provide redundancy that allows some copies to evolve new or modified functions while other copies maintain the original function. CNVs can represent benign polymorphic variations or convey clinical phenotypes by mechanisms such as altered gene dosage and gene disruption. CNV Dabrafenib molecular weight can be responsible for sporadic birth defects [20], other sporadic traits, Mendelian diseases and complex traits including autism, schizophrenia, epilepsy, Parkinson

disease, Alzheimer disease, human immunodeficiency virus (HIV) infection and mental retardation [21–23]. Interestingly, the set of genes that vary in copy number seems to be enriched for genes involved in olfaction, immunity and secreted proteins [24]. The following diseases are associated with CNVs of the immune genes: (i) CNVs of FCGR3B and FCGR2C (encoding different Fcγ receptors) have been associated with a range of autoimmune diseases, including check details systemic lupus erythematosus (SLE), polyangiitis, Wegener’s granulomatosis and idiopathic thrombocytopenic purpura [25–27]. (ii) CNVs of the complement genes CFHR1 and CFHR3, which belong to the complement factor H protein family, have been associated with age-related macular degeneration and atypical haemolytic-uraemic syndrome [28–30]. Complement C4 gene copy number has been related directly

with systemic lupus erythematosus (SLE) [31]. (iii) On chromosome 8, a unit of seven β-defensin genes, which encode anti-microbial peptides with other diverse functions such as chemokine activity [32], has variability in its copy number [33]: low copy number has been associated with Crohn’s disease [34,35], and high copy number with predisposition to psoriasis [36]. (iv) In Pembrolizumab mouse this review, we will examine one of the most striking examples of CNV in the human genome, the chemokine genes CCL3L and CCL4L. Chemokines are a large superfamily of small structurally related cytokines that regulate cell trafficking of various types of leucocytes to areas of injury, and play key roles in both inflammatory and homeostatic processes. Chemokines are classified into four families based on the arrangement of the first two cysteines of the typically conserved four cysteines: CXC, CC, C and CX3C (where X is any amino acid) [37].

281 ATYPICAL PRESENTATION OF ANTI-GLOMERULAR BASEMENT MEMBRANE DISEASE WITH CO-EXISTING IgA NEPHROPATHY A LECAMWASAM1, A SKENE2, D LEE1, L MCMAHON1 1Department of Renal Medicine, Eastern Health, Melbourne, Victoria; 2Department of Anatomical Pathology, Austin Health,

Melbourne, Victoria, Australia Background: We report a case of atypical presentation of anti-glomerular basement membrane (anti-GBM) Small molecule library datasheet disease co-existing with IgA nephropathy. Case Report: A 56-year-old Caucasian normotensive man presented with prodromal symptoms for a month. Kidney function deteriorated over 3 weeks with serum creatinine from 134 to 194 μmol/L, while it was normal 14 months prior. Urine microscopy revealed microscopic haematuria but no red cell casts, and spot urine protein-to-creatinine ratio was 0.057 mg/mmol. Anti-GBM antibody titre was 57 units/mL (<20), and anti-neutrophil cytoplasmic antibody was negative. Urgent treatment was commenced consisting of intravenous methylprednisolone, oral cyclophosphamide and plasmapheresis.

Renal biopsy showed 20% crescents. Immunohistochemical studies (IHC) were performed as there was inadequate renal cortex for immunofluorescence check details (IF) studies. IHC showed mesangial IgA deposits and weak IgG but no observable linear staining, favouring IgA nephropathy

with occasional crescents, and plasmapheresis was ceased. His kidney function worsened, and a second renal biopsy was performed 5 days later showing 41% crescents. Repeat IHC studies identified no IgG deposits and weak mesangial IgA staining. Interestingly, IF studies revealed patchy but linear IgG and mesangial IgA staining consistent with anti-GBM disease with mild IgA nephropathy. Plasmapheresis PTK6 was reinstituted followed by undetectable circulating anti-GBM antibody, normalisation of kidney function, proteinuria and haematuria at 5 months follow-up. Conclusions: Our case reinforces the importance of strong clinical suspicion for atypical presentation of anti-GBM disease in the context of acute kidney injury and circulating anti-GBM antibody, as early initiation of treatment is paramount for favourable outcomes. Co-existing glomerulonephritis, prodromal symptoms and less rapid deterioration in kidney function are not uncommon. Linear IgG deposits may be more sensitive by IF compared to IHC.

pneumoniae (13). Moreover cathelicidins, such as CRAMP and defensins, constitute two important families of antimicrobial peptides (4). The evidence indicates

that cathelicidins are also likely to possess anti-mycoplasmal Alpelisib price activity. In the present study, we examined the antimicrobial activity of CRAMP against M. pneumoniae and the expression of CRAMP in BALF of M. pneumoniae-infected mice. To this end, we developed a sandwich ELISA to quantitate CRAMP levels. CRAMP was found to exert antimicrobial activity in vitro against M. pneumoniae. High concentrations of CRAMP were detected in BALF of M. pneumoniae-infected mice. Neutrophils in BALF showed a fair amount of CRAMP in their cytoplasm and M. pneumoniae caused the release

of CRAMP buy Erlotinib from neutrophils. Thus, our results suggest that CRAMP plays a critical role in protection against M. pneumoniae infection in a murine model. Mycoplasma pneumoniae FH and M. pneumoniae M129, originally clinical isolates, were cultured in PPLO medium (Becton Dickinson, Sparks, MD, USA) as described previously (14). These strains were centrifuged for 10 min at 20,000 g and washed with PBS twice. Then the cells were suspended to a concentration of 1 × 108 CFU/mL in PBS and subsequently used for antimicrobial assays and infection of mice. Cathelin-related antimicrobial peptide (C-terminus peptide) was chemically synthesized by Bex (Tokyo, Japan). The amino acid sequence is as follows; GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE. Rabbit anti-CRAMP Ab was prepared by immunizing rabbits with KLH-conjugated CRAMP peptide emulsified in complete Freund’s adjuvant. Repeated boosts were carried out every two weeks four times. Then sera were obtained and a MAbTrap Kit (Amersham Biosciences, Uppsala, Sweden) was used to isolate the IgG fraction. Mycoplasma pneumoniae strains prepared as above were diluted to 2 × 105 mL in 10 mM SPB (pH 7.4) containing 0.03% Luria-Bertani broth. The strains were harvested from an exponential phase culture. A 25-μL aliquot of M. pneumoniae

was incubated with 25 μL of CRAMP at various concentrations for 3 hr at 37°C dipyridamole as previously described (13). The mixture of M. pneumoniae and CRAMP was serially diluted 10-fold with SPB and plated on PPLO agar plates. Mycoplasmal colonies were enumerated the following day. BALB/c mice (5 weeks old) (Kyudo, Tosu, Saga, Japan) were intranasally infected with 50 μL of M. pneumoniae M129 (5 × 107 CFU) in PBS. After 24 hr, 1 mL of PBS was injected into the bronchial tracts of the mice and BALF obtained from them as previously described (15). After centrifugation of BALF at 400 g for 5 min, the supernatants were used for measurement of CRAMP concentration, whereas the cells of the pellets were used for detection of intracellular CRAMP antigens. All experimental procedures on animals were reviewed and approved by the Kurume University School of Medicine Institutional Animal Care and Use Committee.

In order to complement

the null mhuA allele, the full-length mhuA gene was amplified by PCR with the primer pair A1 and A4, and the resulting amplicon was ligated into the XbaI site of a broad-host-range plasmid, pRK415. The resulting hybrid plasmid, pRK415-mhuA, was transformed into E. coliβ2155, and crossed with the ΔiucDΔmhuA strain. Tetracycline-resistant colonies were selected, and plasmid transfer confirmed by PCR and restriction enzyme analysis of the extracted plasmid. Two kinds of DNA fragments containing upstream regions of the mhuA gene were amplified by PCR with primer pairs Xba-I-P (5′-ctagtctagaACGGAACCGCAGACATGGTGTTG-3′), or Xba-I-P2 (5′-ctagtctagaTTTGATAACTCAAGGAGCTAGGAGC-3′) and Sph-I-P (5′-acatgcatgcTACAACAATTGCACTAGCGAGC-3′) Y-27632 chemical structure CP-690550 in vivo (the small italic letter sequences in Xba-I-P and Xba-I-P2, and Sph-I-P are polylinkers with XbaI and SphI sites, respectively). PCR fragments digested with

SphI-XbaI were ligated into the same enzyme sites of pAA224 (17), and the resulting promoter-lacZ reporter plasmids, termed pVMB2 and pVMB3, were then individually transformed into E. coli WAM131 (15). The degree of expression of the promoter-fused lacZ gene in E. coli WAM131 cells harboring the respective reporter plasmids was estimated by β-galactosidase activity measured by the method of Miller (22), after they had been grown at 37oC in +Fe or −Fe (with DPD) medium for 20 hr and 12 hr, respectively. Nucleotide sequence data for the V. mimicus mhuA and mhuB genes have been deposited in the EMBL/GenBank/DDBJ databases under the accession number AB048382. In order to eliminate background growth resulting from aerobactin-mediated iron uptake in the −Fe medium, a ΔiucD mutant incapable of synthesizing aerobactin was first constructed from V. mimicus 7PT and then 4-Aminobutyrate aminotransferase used to assess whether this species can utilize heme and hemoglobin as iron sources. The deletion

in the iucD gene was confirmed by PCR analysis of the ΔiucD chromosomal DNA with the primer pair D5 and D6, which revealed an amplicon of the expected size (ca. 2.8-kb) (Fig. 1a). In the growth assay, the ΔiucD strain showed no growth in the −Fe medium, while the addition of hemin at 10 μM or hemoglobin at 2.5 μM to the same medium restored growth to a degree comparable to that found in the +Fe medium (Fig. 1b) These data clearly indicate that V. mimicus can utilize heme and hemoglobin as iron sources. The ORF in the 5121-bp cloned region together with relevant inserts in the constructed plasmids are shown in Figure 2. Fur-box-containing gene fragments from V. mimicus 7PT (10, 21) were isolated through application of FURTA system (14). One of the FURTA-positive clones, termed pVM3, possessed two partial ORF, whose deduced amino acid sequences were significantly homologous to the V. cholerae HutA and VCA0575 proteins involved in heme and hemoglobin utilization (11, 23). To clone surrounding regions of these partial ORF, V.