Hepcidin is the key regulator of systemic iron homeostasis. The iron-sensing mechanisms and the role of intracellular iron in modulating hepatic hepcidin secretion are unclear. Therefore, we created a novel cell line, recombinant-TfR1 HepG2, expressing iron-response-element-independent TFRC mRNA to promote cellular iron-overload and examined the effect of excess holotransferrin (5g/L) on cell-surface TfR1, iron content, hepcidin secretion and mRNA expressions of TFRC, HAMP, SLC40A1, HFE and TFR2. Results showed that the recombinant cells exceeded levels of cell-surface TfR1 in wild-type cells under basal (2.8-fold; p<0.03) and holotransferrin-supplemented conditions for 24h and 48h (4.4- and 7.5-fold, respectively; p<0.01). Also, these cells showed higher intracellular iron content than wild-type cells under basal (3-fold; p<0.03) and holotransferrin-supplemented conditions (6.6-fold at 4h; p<0.01). However, hepcidin secretion was not higher than wild-type cells. Moreover, holotransferrin treatment to recombinant cells did not elevate HAMP responses compared to untreated or wild-type cells. In conclusion, increased intracellular iron content in recombinant cells did not increase hepcidin responses compared to wild-type cells, resembling hemochromatosis. Furthermore, TFR2 expression altered within 4h of treatment, while HFE expression altered later at 24h and 48h, suggesting that TFR2 may function prior to HFE in HAMP regulation.

There are few descriptions of young adults with self-reported hemochromatosis or iron overload (H/IO). We analyzed initial screening data in 7,343 HEmochromatosis and IRon Overload Screening (HEIRS) Study participants ages 25-29 years, including race/ethnicity and health information; transferrin saturation (TS) and ferritin (SF) measurements; and HFE C282Y and H63D genotypes. We used denaturing high-pressure liquid chromatography and sequencing to detect mutations in HJV, TFR2, HAMP, SLC40A1, and FTL. Fifty-one participants reported previous H/IO; 23 (45%) reported medical conditions associated with H/IO. Prevalences of reports of arthritis, diabetes, liver disease or liver cancer, heart failure, fertility problems or impotence, and blood relatives with H/IO were significantly greater in participants with previous H/IO reports than in those without. Only 7.8% of the 51 participants with previous H/IO reports had elevated TS; 13.7% had elevated SF. Only one participant had C282Y homozygosity. Three participants aged 25-29 years were heterozygous for potentially deleterious mutations in HFE2, TFR2, and HAMP promoter, respectively. Prevalences of self-reported conditions, screening iron phenotypes, and C282Y homozygosity were similar in 1,165 participants aged 30 years or greater who reported previous H/IO. We conclude that persons who report previous H/IO diagnoses in screening programs are unlikely to have H/IO phenotypes or genotypes. Previous H/IO reports in some participants could be explained by treatment that induced iron depletion before initial screening, misdiagnosis, or participant misunderstanding of their physician or the initial screening questionnaire.

Taz is a chimeric receptor consisting of the periplasmic, transmembrane and most of the HAMP linker domains of the Escherichia coli aspartate receptor (Tar(Ec)) and the cytoplasmic signalling domain of the E. coli osmosensor EnvZ. Aspartate is one of several attractant ligands normally sensed by Tar and it interacts with Taz to induce OmpR-dependent transcription from the ompC promoter--albeit with reduced sensitivity relative to the chemotactic response it evokes via Tar. By combining Taz with a reporter system that expresses green fluorescent protein (GFP) from the ompC promoter, we were able to examine the interaction of Taz with all 20 natural amino acids. Some amino acids (Leu, Met, Val and Ser) reduced GFP expression, which in the case of leucine is likely attributed to a direct effect on the receptor, rather than an indirect effect through the leucine responsive protein (Lrp). Surprisingly, amino acids like Met and Ser--which are also attractants for Tar--'inhibited' Taz. Moreover, Taz exhibits a higher sensitivity to Leu compared with Asp, which is the inverse of Tar. Our results show the exquisite sensitivity of chemotactic receptors. Small conformational changes induced by making the chimera may have changed the way it responds to different amino acids.

BACKGROUND

X-linked sideroblastic anemia (XLSA) is associated with iron overload and mutations in ALAS2, which encodes 5-aminolevulinate synthase. There are few reports of XLSA in persons of sub-Saharan African descent.

METHODS

A 47-year-old African-American man had microcytic anemia, elevated iron measures, cardiomyopathy, hepatic cirrhosis, diabetes mellitus, a history of cocaine use and hepatitis C. We amplified and directly sequenced his genomic DNA to detect mutations of SLC40A1, HFE, TFR2, HAMP, HJV and ALAS2.

RESULTS

The subject's transferrin saturation was 100% and his serum ferritin was 2,960 ng/ml. An MRI scan revealed diffusely decreased T(2) signals of the heart, liver and pancreas. Transjugular right endomyocardial and liver biopsy specimens revealed marked iron deposition in cardiac myocytes and hepatocytes, and cirrhosis. He died of progressive cardiomyopathy. He was hemizygous for ALAS2 R452S (exon 9; c.1354C->>A) and heterozygous for SLC40A1 R561G (exon 8; c.1681A->>G). He did not have coding region mutations in HFE, TFR2, HAMP or HJV.

CONCLUSIONS

ALAS2 R452S largely explains this patient's microcytic anemia and multi-organ iron overload and dysfunction. SLC40A1 R561G may have increased his iron absorption and overload further. Acquired factors, especially cocaine use and hepatitis C, may have contributed to his clinical phenotype.

The Escherichia coli Tsr protein contains a periplasmic serine-binding domain that transmits ligand occupancy information to a cytoplasmic kinase-control domain to regulate the cell's flagellar motors. The Tsr input and output domains communicate through conformational changes transmitted through a transmembrane helix (TM2), a five-residue control cable helix at the membrane-cytoplasm interface, and a four-helix HAMP bundle. Changes in serine occupancy are known to promote TM2 piston displacements in one subunit of the Tsr homodimer. We explored how such piston motions might be relayed through the control cable to reach the input AS1 helix of HAMP by constructing and characterizing mutant receptors that had one-residue insertions or deletions in the TM2-control cable segment of Tsr. TM2 deletions caused kinase-off output shifts; TM2 insertions caused kinase-on shifts. In contrast, control cable deletions caused kinase-on output, whereas insertions at the TM2-control cable junction caused kinase-off output. These findings rule out direct mechanical transmission of TM2 conformational changes to HAMP. Instead, we suggest that the Tsr control cable transmits input signals to HAMP by modulating the intensity of structural clashes between out-of-register TM2 and AS1 helices. Inward displacement of TM2 might alter the sidechain environment of control cable residues at the membrane core-headgroup interface, causing a break in the control cable helix to attenuate the register mismatch and enhance HAMP packing stability, leading to a kinase-off output response. This helix-clutch model offers a new perspective on the mechanism of transmembrane signaling in chemoreceptors.

Dysregulated intracellular iron homeostasis has been found not only in rodent and human hepatocellular carcinomas but also in several preneoplastic pathological states associated with hepatocarcinogenesis; however, the precise underlying mechanisms of metabolic iron disturbances in preneoplastic liver and the role of these disturbances remain unexplored. In the present study, using an in vivo model of rat hepatocarcinogenesis induced by 2-acetylaminofluorene, we found extensive alterations in cellular iron metabolism at preneoplastic stages of liver carcinogenesis. These were characterized by a substantial decrease in the levels of cytoplasmic non-heme iron in foci of initiated hepatocytes and altered expression of the major genes responsible for the proper maintenance of intracellular iron homeostasis. Gene expression analysis revealed that the decreased intracellular levels of iron in preneoplastic foci might be attributed to increased iron export from the cells, driven by upregulation of ferroportin (Fpn1), the only known non-heme iron exporter. Likewise, increased Fpn1 gene expression was found in vitro in TRL1215 rat liver cells with an acquired malignant phenotype, suggesting that upregulation of Fpn1 might be a specific feature of neoplastically transformed cells. Other changes observed in vivo included the downregulation of hepcidin (Hamp) gene, a key regulator of Fpn1, and this was accompanied by decreased levels of CCAAT/enhancer binding proteins alpha and beta, especially at the Hamp promoter. In conclusion, our results demonstrate the significance of altered intracellular iron metabolism in the progression of liver carcinogenesis and suggest that correction of these alterations could possibly affect liver cancer development.

The effects of changes in macrophage iron status, induced by single or multiple iron injections, iron depletion or pregnancy, on both immune function and mRNA expression of genes involved in iron influx and egress have been evaluated. Macrophages isolated from iron deficient rats, or pregnant rats at day 21 of gestation, either supplemented with a single dose of iron dextran, 10 mg, at the commencement of pregnancy, or not, showed significant increases of macrophage ferroportin mRNA expression, which was paralleled by significant decreases in hepatic Hamp mRNA expression. IRP activity in macrophages was not significantly altered by iron status or the inducement of pregnancy +/- a single iron supplement. Macrophage immune function was significantly altered by iron supplementation and pregnancy. Iron supplementation, alone or combined with pregnancy, increased the activities of both NADPH oxidase and nuclear factor kappa B (NFkappaB). In contrast, the imposition of pregnancy reduced the ability of these parameters to respond to an inflammatory stimuli. Increasing iron status, if only marginally, will reduce the ability of macrophages to mount a sustained response to inflammation as well as altering iron homeostatic mechanisms.

OBJECTIVE

To determine the molecular basis of a mild hemochromatosis phenotype in a man of Scottish-Irish descent.

METHODS

We sequenced genomic DNA to detect mutations of HFE, SLC40A1, TFR2, HAMP, and HFE2. RNA isolated from blood mononuclear cells was used to make cDNA. RT-PCR was performed to amplify ferroportin from cDNA, and amplified products were visualized by electrophoresis and sequenced.

RESULTS

The proband was heterozygous for the novel mutation c.1402G->>A (predicted G468S) in exon 7 of the ferroportin gene (SLC40A1). Located in the last nucleotide before the splice junction, this mutation results in aberrant splicing to a cryptic upstream splice site located at nt 990 within the same exon. This causes truncation of ferroportin after glycine 330 and the addition of 4 irrelevant amino acids before terminating. The truncated ferroportin protein, missing its C-terminal 241 amino acids, would lack all structural motifs beyond transmembrane region 7. The patient was also heterozygous for the common HFE H63D polymorphism, but did not have coding region mutations in TFR2, HAMP, or HFE2.

CONCLUSIONS

We conclude that this patient represents a unique example of hemochromatosis due to a single base-pair mutation of SLC40A1 that results in aberrant splicing and truncation of ferroportin.

Hepcidin, an iron regulatory peptide, plays a central role in the maintenance of systemic iron homeostasis by inducing the internalization and degradation of the iron exporter, ferroportin. Hepcidin expression in the liver is regulated in response to several stimuli including iron status, erythropoietic activity, hypoxia and inflammation. Hepcidin expression has been shown to be reduced in phenylhydrazine-treated mice, a mouse model of acute hemolysis. In this mouse model, hepcidin suppression was associated with increased expression of molecules involved in iron transport and recycling. The present study aims to explore whether the response to phenylhydrazine treatment is affected by hepcidin deficiency and/or the subsequently altered iron metabolism. Hepcidin1 knockout (Hamp(-/-)) and wild type mice were treated with phenylhydrazine or saline and parameters of iron homeostasis were determined 3 days after the treatment. In wild type mice, phenylhydrazine administration resulted in significantly reduced serum iron, increased tissue non-heme iron levels and suppressed hepcidin expression. The treatment was also associated with increases in membrane ferroportin protein levels and spleen heme oxygenase 1 mRNA expression. In addition, trends toward increased mRNA expression of duodenal iron transporters were also observed. In contrast, serum iron and tissue non-heme iron levels in Hamp(-/-) mice were unaffected by the treatment. Moreover, the effects of phenylhydrazine on the expression of ferroportin and duodenal iron transporters were not observed in Hamp(-/-) mice. Interestingly, mRNA levels of molecules involved in splenic heme uptake and degradation were significantly induced by Hamp disruption. In summary, our study demonstrates that the response to phenylhydrazine-induced hemolysis differs between wild type and Hamp(-/-) mice. This observation may be caused by the absence of hepcidin per se or the altered iron homeostasis induced by the lack of hepcidin in these mice.

Nik1 orthologs or group III hybrid histidine kinases (HHK) are ubiquitous signaling molecules in fungal pathogens. Besides osmosensing, they are also involved in hyphal morphogenesis, virulence, and conidiation. They are important molecular targets for antifungal agents. Nik1 orthologs contain a varying number of HAMP domain repeats (poly-HAMP) in the N-terminal region. Poly-HAMP plays a crucial role in their function. So far, the role of HAMP domains in their function has been studied only in a few Nik1 orthologs. In this paper, we describe the functional characterization of a Nik1 ortholog (ClNik1p) from Candida lusitaniae, an emerging and important fungal pathogen. We show that ClNik1p acts as a bona fide osmosensor and negatively regulates the downstream HOG pathway in Saccharomyces cerevisiae. Our data suggests a differential role of the HAMP domains in the functionality of ClNik1p. The HAMP domains H1, H2, H3 and H5 are essential for kinase activity, and H4 domain has a regulatory role. Among the HAMP like linker domains, only H4b was crucial for the activity of ClNik1p.

Because dietary fats affect the regulation and use of body iron, we hypothesized that iron regulatory and transport genes may be affected by dietary fat. A model of early-stage I to II, nonalcoholic fatty liver was used in which rats were fed standard (35% energy from fat) or high-fat (71% energy from fat) liquid diets with normal iron content (STD/HF groups). In addition, intraperitoneal injections of iron dextran were given to iron-loaded (STD+/HF+ groups) and iron-deficient diets to STD-/HF- groups. Plasma osmolality, hemoglobin level, and mean corpuscular hemoglobin concentration were increased in all STD diet groups compared with all HF diet groups. Plasma iron and transferrin saturation were affected by an interaction between dietary fat and iron. They were high in the STD group (normal iron) compared with their respective HF group. Similarly, this group also showed a 4-fold increase in the messenger RNA expression of the hepatic hemochromatosis gene. Spleen iron was high in the iron-loaded STD+ group compared with all other groups. Hepatic iron and messenger RNA expression of peroxisome proliferator-activated receptor-γ, CCAAT/enhancer binding protein α, interleukin-6, and iron transport genes (transferrin receptor 2, divalent metal transporter 1 iron-responsive element, and divalent metal transporter 1 non-iron-responsive element) were increased, whereas tumor necrosis factor α was decreased in the HF diet groups. The expression of iron regulatory gene HAMP was not increased in the HF diet groups. Iron regulatory and transport genes involved in cellular and systemic iron homeostasis may be affected by the macronutrient composition of the diet.

Hepcidin, a circulatory antimicrobial peptide, is involved in iron homeostasis, inflammation, infection and metabolic signals. Humans express one hepcidin gene, <em>HAMP</em> but mice express two hepcidin genes, <em>Hamp</em>1 and <em>Hamp</em>2. Consecutive gene targeting events were performed to produce transgenic mice expressing conditional alleles of either <em>Hamp</em>1 or both <em>Hamp</em>1 and <em>Hamp</em>2 (<em>Hamp</em>1/2). The deletion of <em>Hamp</em>1 alleles elevated <em>Hamp</em>2 expression, particularly in males, which was reduced by endotoxin treatment. The tissue levels of iron and other biometals were quantified by inductively coupled mass spectrometry. The ubiquitous or liver-specific deletion of <em>Hamp</em>1 alleles yielded similar quantitative changes in iron levels in the liver, duodenum, spleen, kidney, heart and brain. The introduction of <em>Hamp</em>2 null allele did not exacerbate the iron-related phenotype of <em>Hamp</em>1 null allele. Besides iron, <em>Hamp</em>1 null allele significantly elevated the levels of selenium in the liver, manganese in the liver and duodenum, and copper in the brain. Mice with conditional <em>Hamp</em> alleles will be useful to determine the tissue-specific regulation and functions of <em>Hamp</em>1 and <em>Hamp</em>2 in biometal homeostasis and other biological processes.

Halobacterial pharaonis phoborhodopsin [ppR, also called Natronomonas pharaonis sensory rhodopsin II (NpSRII)] is a phototaxis protein which transmits a light signal to the cytoplasm through its transducer protein (pHtrII). pHtrII, a two-transmembrane protein that interacts with ppR, belongs to the group of methyl-accepting chemotaxis proteins (MCPs). Several mutation studies have indicated that the linker region connecting the transmembrane and methylation regions is necessary for signal transduction. However, the three-dimensional (3D) structure of an MCP linker region has yet to be reported, and hence, details concerning the signal transduction mechanism remain unknown. Here the structure of the pHtrII linker region was investigated biochemically and biophysically. Following limited proteolysis, only one trypsin resistant fragment in the pHtrII linker region was identified. This fragment forms a homodimer with a Kd value of 115 microM. The 3D structure of this fragment was determined by solution NMR, and only one alpha-helix was found between two HAMP domains of the linker region. This alpha-helix was significantly stabilized within transmembrane protein pHtrII as revealed by CW-EPR. The presence of Af1503 HAMP domain-like structures in the linker region was supported by CD, NMR, and ELDOR data. The alpha-helix determined here presumably works as a mechanical joint between two HAMP domains in the linker region to transfer the photoactivated conformational change downstream.

Plant responses to biotic stress involve non-self perception, signaling, and altered defense phenotypes. During attack, defoliating insects deposit gland secretions (GS) and complex foregut derived oral secretions (OS) that include GS and combined products of plant, insect, and microbial interactions. GS-derived and OS-derived biochemicals that trigger defense are termed Herbivore Associated Molecular Patterns (HAMPs) while those that promote susceptibility are termed effectors. These functions are highly context and species specific. The magnitude and direction of plant responses are orchestrated by the interaction of damage, OS/GS components, predicted receptor-ligand interactions, ion fluxes, protein kinase signaling cascades, phytohormone interactions, transcription factor activation, altered translation, and defense biosynthesis. Unlike plant-pathogen recognition, a remaining challenge is the discovery of plant receptors for defoliator-derived HAMPs.

Hereditary hemochromatosis, one of the commonest genetic disorder in Caucasians, is mainly associated to homozygosity for the C282Y mutation in the HFE gene, which is highly prevalent (allele frequency up to near 10% in Northern Europe) and easily detectable through a widely available "first level" molecular test. However, in certain geographical regions like the Mediterranean area, up to 30% of patients with a HH phenotype has a negative or non-diagnostic (i.e. simple heterozygosity) test, because of a known heterogeneity involving at least four other genes (HAMP, HJV, TFR2, and SLC40A1). Mutations in such genes are generally rare/private, making the diagnosis of atypical HH essentially a matter of exclusion in clinical practice (from here the term of "non-HFE" HH), unless cumbersome traditional sequencing is applied. We developed a Next Generation Sequencing (NGS)-based test targeting the five HH genes, and applied it to patients with clinically relevant iron overload (IO) and a non-diagnostic first level genetic test. We identified several mutations, some of which were novel (i.e. HFE W163X, HAMP R59X, and TFR2 D555N) and allowed molecular reclassification of "non-HFE" HH clinical diagnosis, particularly in some highly selected IO patients without concurring acquired risk factors. This NGS-based "second level" genetic test may represent a useful tool for molecular diagnosis of HH in patients in whom HH phenotype remains unexplained after the search of common HFE mutations.

BACKGROUND

The growing number of monitoring devices, combined with suboptimal patient monitoring and alarm management strategies, has increased "alarm fatigue," which have led to serious consequences. Most reported alarm man- agement approaches have focused on the critical care setting. Since 2007 Dartmouth-Hitchcock (Lebanon, New Hamp- shire) has developed a generalizable and effective design, implementation, and performance evaluation approach to alarm systems for continuous monitoring in general care settings (that is, patient surveillance monitoring).

METHODS

In late 2007, a patient surveillance monitoring system was piloted on the basis of a structured design and implementation approach in a 36-bed orthopedics unit. Beginning in early 2009, it was expanded to cover more than 200 inpatient beds in all medicine and surgical units, except for psychiatry and labor and delivery.

RESULTS

Improvements in clinical outcomes (reduction of unplanned transfers by 50% and reduction of rescue events by more than 60% in 2008) and approximately two alarms per patient per 12-hour nursing shift in the original pilot unit have been sustained across most D-H general care units in spite of increasing patient acuity and unit occupancy. Sample analysis of pager notifications indicates that more than 85% of all alarm conditions are resolved within 30 seconds and that more than 99% are resolved before escalation is triggered.

CONCLUSIONS

The D-H surveillance monitoring system employs several important, generalizable features to manage alarms in a general care setting: alarm delays, static thresholds set appropriately for the prevalence of events in this setting, directed alarm annunciation, and policy-driven customization of thresholds to allow clinicians to respond to needs of individual patients. The systematic approach to design, implementation, and performance management has been key to the success of the system.

OBJECTIVE

Hepcidin is a key regulator of systemic iron metabolism and its expression is modulated by hepatitis C virus (HCV) infection. Suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 act as negative regulators of the Jak/signal transducers and activators of transcription signaling pathway. In this study, we investigated how HCV infection modulates SOCS-1 and SOCS-3 production and how these SOCS proteins affect hepcidin production.

METHODS

The effects of SOCS-1 and SOCS-3 on hepcidin production were investigated using a complete genome, HCV replicon system.

RESULTS

Unexpectedly, basal expression levels of hepcidin (HAMP) mRNA and the bioactive form of hepcidin protein, hepcidin-25, were significantly higher in replicon cells. Regardless of HCV infection, STAT3 was activated in response to interleukin-6 (IL-6), but this activation was greater in replicon cells than in cured cells. Basal expression of the SOCS-3 protein was enhanced, but basal expression of SOCS-1 protein was reduced, in replicon cells. Expression of SOCS-3 increased dramatically in response to IL-6 stimulation but expression of SOCS-1 was not induced by IL-6. Interestingly, silencing of SOCS-1 and SOCS-3 gene expression enhanced STAT3 activation and HAMP gene expression. In addition, overexpression of SOCS-1 protein strongly suppressed STAT3 activation and HAMP gene expression.

CONCLUSIONS

This in vitro study shows that SOCS-3 expression was enhanced but SOCS-1 expression was reduced by HCV infection. The upregulation of hepcidin induced by IL-6 was found to be negatively regulated by SOCS-1 and SOCS-3. The modulation of SOCS1 and SOCS3 in HCV-infected hepatocytes may explain, at least in part, the relative shortage of hepcidin production in CH-C.

Plant defense against herbivores is modulated by herbivore-associated molecular patterns (HAMPs) from oral secretions (OS) and/or saliva of insects. Furthermore, feeding wounds initiate plant self-damage responses modulated by danger-associated molecular patterns (DAMPs) such as immune defense-promoting plant elicitor peptides (Peps). While temporal and spatial co-existence of both patterns during herbivory implies a possibility of their close interaction, the molecular mechanisms remain undetermined. Here we report that exogenous application of rice (Oryza sativa) peptides (OsPeps) can elicit multiple defense responses in rice cell cultures. Specific activation of OsPROPEP3 gene transcripts in rice leaves by wounding and OS treatments further suggests a possible involvement of the OsPep3 peptide in rice-herbivore interactions. Correspondingly, we found that simultaneous application of OsPep3 and Mythimna loreyi OS significantly amplifies an array of defense responses in rice cells, including mitogen-activated protein kinase activation, and generation of defense-related hormones and metabolites. The induction of OsPROPEP3/4 by OsPep3 points to a positive auto-feedback loop in OsPep signaling which may contribute to additional enhancement of defense signal(s). Finally, the overexpression of the OsPep receptor OsPEPR1 increases the sensitivity of rice plants not only to the cognate OsPeps but also to OS signals. Our findings collectively suggest that HAMP-DAMP signal integration provides a critical step in the amplification of defense signaling in plants.

Gastric cancer is one of the most common malignancies with high cancer-associated mortality rate globally. Hepcidin is the peptide hormone, which is critically important in the regulation of systemic iron homeostasis. Cumulating evidence has reported that the disturbed local expression of hepcidin may serve as a predictive biomarker in assessing the clinical outcomes in a range of cancer types. However, the expression profile of hepcidin in human gastric cancer is remains to be investigated. In the present retrospective study, using archived paraffin-embedded tissue blocks, the local production of hepcidin by immunohistochemical analysis was detected, and then its correlation with clinicopathological characteristics in human gastric cancer was evaluated. In parallel, using western blotting, quantitative reverse transcription polymerase chain reaction and chromatin immunoprecipitation assay, the local status of Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling in response to inflammatory stimuli mediated by interleukin (IL)-6, which in turn regulates transcriptional activity of hepcidin gene (HAMP) was also assessed. The results indicated that, the local production of hepcidin was significantly elevated in tumor tissues compared with adjacent non-tumor tissues, and was tightly correlated with increasing tumor stages according to the tumor node metastasis (TNM) classification. In addition, JAK/STAT3 signaling and the STAT3 binding affinity to the HAMP gene promoter were significantly enhanced, in parallel with an increased expression of hepcidin, in tumor tissues compared with adjacent non-tumor tissues. Collectively, the present study indicated that local expression of hepcidin in gastric cancer tumor tissues was positively correlated with increasing tumor stages, which may be closely associated with the upregulation of IL-6-mediated JAK/STAT3 signaling in human gastric cancer.

Botrytis cinerea is a filamentous phytopathogen with a high risk of developing resistance to fungicides. The phenylpyrrole fungicide fludioxonil has been reported to have excellent activity against B. cinerea and increasingly has been applied to control gray mold in China. In this study, molecular and biochemical characteristics of laboratory and field mutants of B. cinerea resistant to fludioxonil has been investigated. During 2012 to 2014, B. cinerea isolates collected from Jiangsu and Shandong Provinces in China were tested in vitro for sensitivity to fungicides commonly used to suppress gray mold of cucumber and tomato. Among the 75 isolates collected from cucumber in 2013, two were highly resistant (HR) to fludioxonil. Of the 308 isolates collected from tomato in 2014, four were fludioxonil-HR. This was the first time that B. cinerea isolates HR to fludioxonil had been detected in the field. Six fludioxonil-resistant mutants were obtained in the laboratory by selection on fungicide-amended media. These mutants exhibited stable resistance to fludioxonil, as indicated by resistance factor values that ranged from 34.38 to >10,000. In comparison with fludioxonil-sensitive isolates of B. cinerea, all field and laboratory mutants showed reduced fitness, as defined by mycelial growth, sporulation, virulence, and sensitivity to osmotic stress. When treated with fludioxonil at 1 μg/ml, sensitive isolates showed increased glycerol contents in mycelium and expression levels of Bchog1, while levels in field and laboratory HR mutants increased only slightly. Sequences of the Bos1 gene of field and laboratory fludioxonil-HR mutants showed that mutations in field mutants were located in the histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase (HAMP) domains of the N-terminal region, whereas mutations in the laboratory mutants were distributed in HAMP domains or in the HATPase_c domain of the C-terminal region. These results will enhance our understanding of the resistance mechanism of B. cinerea to fludioxonil.

The complex of two membrane proteins, sensory rhodopsin II (NpSRII) with its cognate transducer (NpHtrII), mediates negative phototaxis in halobacteria N. pharaonis. Upon light activation NpSRII triggers a signal transduction chain homologous to the two-component system in eubacterial chemotaxis. Here we report on crystal structures of the ground and active M-state of the complex in the space group I212121. We demonstrate that the relative orientation of symmetrical parts of the dimer is parallel ("U"-shaped) contrary to the gusset-like ("V"-shaped) form of the previously reported structures of the NpSRII/NpHtrII complex in the space group P21212, although the structures of the monomers taken individually are nearly the same. Computer modeling of the HAMP domain in the obtained "V"- and "U"-shaped structures revealed that only the "U"-shaped conformation allows for tight interactions of the receptor with the HAMP domain. This is in line with existing data and supports biological relevance of the "U" shape in the ground state. We suggest that the "V"-shaped structure may correspond to the active state of the complex and transition from the "U" to the "V"-shape of the receptor-transducer complex can be involved in signal transduction from the receptor to the signaling domain of NpHtrII.

Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays. Understanding stimulus detection and transmission at the molecular level requires precise structural characterisation of the array building block known as a core signalling unit. Here we introduce an Escherichia coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays. We use cryo-electron tomography and subtomogram averaging to provide a three-dimensional map of a complete core signalling unit, with visible densities corresponding to the HAMP and periplasmic domains. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields a molecular model of the transmembrane core signalling unit and enables spatial localisation of its individual domains. Our work thus offers a solid structural basis for the interpretation of a wide range of existing data and the design of further experiments to elucidate signalling mechanisms within the core signalling unit and larger array.

OBJECTIVE

Iron overload (IO) is a life-threatening complication of chronic hemolytic disorders such as β-thalassemia. IO results in severe cellular oxidative damage, leading to organ failure. Peroxiredoxin-2 (Prx2), a typical 2-cysteine-(Cys)-peroxiredoxin, is an important component of the cytoprotective system, but its response to IO is still to be fully defined.

RESULTS

We studied the effects of IO on Prx2-knockout mice (Prx2-/-). The absence of Prx2 enhanced toxicity due to IO on erythropoiesis. We found that IO failed to induce the typical hepcidin (Hamp) upregulation in Prx2-/- mice due to its failure to activate the signal transducer and activator of transcription-3 (STAT3) with intact Jak2 signaling. In Prx2-/- mice, the loss of Hamp response was also observed after administration of a single dose of oral iron. When lipopolysaccharide (LPS) was used to explore IL6-STAT3 activation in Prx2-/- mice, STAT3 activation and Hamp upregulation were once again defective. Treatment with PEP-fusion-recombinant-Prx2 (PEP Prx2) significantly increased STAT3 activation with upregulation of Hamp expression in both IO- and LPS-exposed Prx2-/- mice. We also confirmed the beneficial effects of PEP Prx2 on Hamp expression through STAT3 activation in β-thalassemic mice.

METHODS

We propose that Prx2 plays a key role in responding to cytotoxicity of IO, directly targeting STAT3-transcriptional factor in a Jak2-independent fashion and regulating Hamp in response to canonical stimuli.

CONCLUSIONS

Collectively, our data highlight a novel role of Prx2 in iron homeostasis. Prx2 is a key cytoprotector against IO that is induced either by iron supplementation or due to chronic hemolysis as in β-thalassemia. Prx2 is required to support STAT3 transcriptional activity and regulation of Hamp expression. Antioxid. Redox Signal. 28, 1-14.

Hepcidin is an antimicrobial peptide hormone that plays a central role in the metabolism of iron and its expression in the liver can be induced through two major pathways: the inflammatory pathway, mainly via IL-6; and the iron-sensing pathway, mediated by BMP-6. GATA-proteins are group of evolutionary conserved transcriptional regulators that bind to the consensus motif-WGATAR-in the promoter region. In hepatoma cells, GATA-proteins 4 and 6 in conjunction with the co-factor friend of GATA (FOG) were shown to modulate the transcription of HAMP. However, it is unclear as to which of the GATA-proteins drive the expression of HAMP in vivo. In this study, using in vitro and in vivo approaches, we investigated the relevance of GATA and FOG proteins in the expression of hepcidin following treatment with IL-6 and BMP-6. We found that treatment of Huh7 cells with either IL-6 or BMP-6 increased the HAMP promoter activity. The HAMP promoter activity following treatment with IL-6 or BMP-6 was further increased by co-transfection of the promoter with GATA proteins 4 and 6. However, co-transfection of the HAMP promoter with FOG proteins 1 or 2 repressed the promoter response to treatments with either IL-6 or BMP-6. The effects of both GATA and FOG proteins on the promoter activity in response to IL-6 or BMP-6 treatment were abrogated by mutation of the GATA response element-TTATCT-in the HAMP promoter region -103/-98. In vivo, treatment of mice with lipopolysaccharide led to a transient increase of Gata-6 expression in the liver that was positively correlated with the expression of hepcidin. Our results indicate that during inflammation GATA-6 is up-regulated in concert with hepcidin while GATA-4 and FOG (1 and 2) are repressed.