Benzopyrazine-Based Small Molecule Inhibitors As Trypanocidal and Leishmanicidal Agents: Green Synthesis, In Vitro, and In Silico Evaluations.

World Health Organization (WHO) identified twenty tropical disease categories as neglected tropical diseases (NTDs). Chagas' disease (also known as American trypanosomiasis) and leishmaniasis are two major classes of NTDs. The total number of mortality, morbidity, and disability attributed each year due to these two categories of diseases in magnitudes is much higher than the so-called elite diseases like cancer, diabetes, AIDS, cardiovascular and neurodegenerative diseases. Impoverished communities around the world are the major victim of NTDs. The development of new and novel drugs in the battle against Chagas' disease and leishmaniasis is highly anticipated. An easy and straightforward on-water green access to synthesize benzopyrazines is reported. This ultrasound-assisted procedure does not require any catalyst/support/additive/hazardous solvents and maintains a high atom economy. A series of eleven benzopyrazines has been synthesized, and most of the synthesized compounds possess the drug-likeness following Lipinski's "Rule of 5". Benzopyrazines 3 and 4 demonstrated moderate leishmanicidal activity against L. mexicana (M378) strain. The selective lead compound 1 showed good leishmanicidal, and trypanocidal activities (in vitro) against both L. mexicana (M378) and T. cruzi (NINOA) strains compared to the standard controls. The in vitro trypanocidal and leishmanicidal activities of the lead compound 1 have been validated by molecular docking studies against four biomolecular drug targets viz. T. cruzi histidyl-tRNA synthetase, T. cruzi trans-sialidase, leishmanial rRNA A-site, and leishmania major N-myristoyl transferase.

Dual Stain With SATB2 and CK20/Villin Is Useful to Distinguish Colorectal Carcinomas From Other Tumors.

OBJECTIVES: Small sample size limits the number of immunostains that may be attempted in colorectal carcinoma (CRC) biopsy specimens. We investigated the utility of dual stain with special AT-rich sequence binding protein 2 (SATB2) or caudal-type homeobox 2 (CDX2) and cytokeratin 20 (CK20) or villin in identifying CRC. METHODS: Tissue microarrays with 222 CRCs and 375 other carcinomas were built. Dual stain was performed pairing nuclear stains CDX2 or SATB2 with CK20 or villin. RESULTS: All four single stains showed excellent sensitivity (93%-99%) but variable specificity (56%-88%) for CRC. All four dual stains also showed excellent sensitivity (90%-96%) while much improved specificity (88%-98%) compared with single stains. SATB2 dual stain (with CK20 or villin) showed a higher specificity than CDX2 dual stain (with CK20 or villin) with a comparable sensitivity. CONCLUSIONS: SATB2 dual stain shows the greatest potential clinical utility in identifying CRC and is superior to CDX2 dual stain. More important, SATB2 dual stain could be helpful for specimens with limited tissues or those having a nonclassic staining pattern.

Dual Immunostain With SATB2 and CK20 Differentiates Appendiceal Mucinous Neoplasms From Ovarian Mucinous Neoplasms.

OBJECTIVES: Determination of the primary site of origin for mucinous neoplasms identified in the peritoneal and/or pelvic cavities may be challenging, with major differential diagnoses including appendiceal mucinous neoplasm (AMN) and ovarian mucinous neoplasm (OMN). Special AT-rich sequence binding protein 2 (SATB2) has been shown to be highly selectively expressed in the lower gastrointestinal tract, including the appendix. METHODS: We investigated the utility of a dual stain (DS) with SATB2 or caudal type homeobox 2 (CDX2) and cytokeratin 20 (CK20) or villin in distinguishing AMNs from OMNs. Tissue microarrays with 40 AMNs and 18 OMNs were stained with SATB2 or CDX2 paired with either CK20 or villin. RESULTS: SATB2 single stain showed a good sensitivity of 83% and the highest specificity of 78% for AMNs over OMNs among all four stains. DS with SATB2 and villin showed an identical sensitivity of 78% but specificity increased to 94%, while DS with SATB2 and CK20 showed a sensitivity of 80% and a specificity of 100%. In contrast, DS with CDX2 and CK20/villin showed slightly higher sensitivity but much lower specificity. CONCLUSIONS: DS with SATB2/CK20 shows the greatest potential clinical utility in distinguishing AMNs from OMNs and is superior to DS with CDX2/CK20. Importantly, DS could be helpful for specimens with limited tissues.

Mixed Adeno-neuroendocrine Carcinoma: An Aggressive Clinical Entity.

BACKGROUND: Mixed adeno-neuroendocrine carcinoma (MANEC) is a rare pathologic diagnosis recently defined by the World Health Organization in 2010. Due to poor understanding of MANEC as a clinical entity, there is significant variation in the management of these patients. The purpose of our study was to characterize MANEC to develop evidence-based treatment strategies. METHODS: The Ohio State University patient database was queried for the diagnosis of MANEC and 46 patients were identified. For comparison, the database also was queried for goblet cell carcinoid (GCC) of the appendix, signet ring cell carcinoma, and carcinoid/neuroendocrine tumor of the appendix. Charts were then retrospectively reviewed for clinicopathologic characteristics, patient treatment, and survival data. RESULTS: The mean age of diagnosis of MANEC was 54 years. Eighty-seven percent of MANEC arose from the appendix, with 28 % of patients undergoing appendectomy and 35 % undergoing right hemicolectomy as their index operation. Immunohistochemical staining was positive for chromogranin (82 %), synaptophysin (97 %), and CD56 (67 %). Sixty-seven percent of patients presented with stage IV disease and 41 % had nodal metastases. Overall survival was 4.1 years, which was statistically significantly different (p ≤ 0.05) compared with carcinoid tumors (13.4 years), GCC (15.4 years), and signet ring carcinoma (2.2 years). CONCLUSIONS: MANEC is a more aggressive clinical entity than both GCC of the appendix and carcinoid/neuroendocrine tumors of the appendix. Based on these findings, we recommend patients with MANEC tumors undergo aggressive multidisciplinary cancer management and close surveillance.

Appendiceal Neuroendocrine, Goblet and Signet-Ring Cell Tumors: A Spectrum of Diseases with Different Patterns of Presentation and Outcome.

PURPOSE: Appendiceal tumors are a heterogeneous group of diseases that include typical neuroendocrine tumors (TNET), goblet cell carcinoids (GCC), and atypical GCC. Atypical GCC are classified into signet-ring cell cancers (SRCC) and poorly differentiated appendiceal adenocarcinoids. The prognosis and management of these diseases is unclear because there are no prospective studies. The aim of this study is to assess the characteristics and outcome of appendiceal TNET, GCC, and SRCC patients. MATERIALS AND METHODS: Appendiceal TNET, GCC, and SRCC patients diagnosed between 1973 and 2011 were identified in the Surveillance Epidemiology and End Results (SEER) database. Demographics, type of surgery, and clinicopathologic characteristics were collected. Survival functions were estimated by the Kaplan-Meier method, and log-rank test was used to assess the difference in overall survival (OS) among the three histologies. RESULTS: The SEER database yielded 1,021 TNET patients, 1,582 with GCC, and 534 SRCC patients. TNET presented at a younger age (p < 0.001). Patients with SRCC presented with advanced stage disease (p < 0.001). The median OS (mOS) for GCC and TNET patients was not reached; mOS for SRCC was 24 months. Multivariate analysis stratified for stage revealed significantly longer survival for TNET and GCC than SRCC (p < 0.001). CONCLUSION: This is the largest report to date for appendiceal neuroendocrine tumor patients, suggesting a spectrum of diseases with different characteristics and outcomes. In this report, we present a treatment approach for this complex spectrum of disease, based on the experience of Ohio State and Emory Universities investigators.

The impact of tumor deposits on colonic adenocarcinoma AJCC TNM staging and outcome.

The definition of tumor deposits (TDs) in colonic adenocarcinoma has been modified in different editions of the AJCC/TNM staging system. Studies have shown that the presence of TD is associated with advanced tumor growth and poor prognosis. Most of these data were obtained in patients with simultaneous lymph node (LN) metastases. Reports focusing on the impact of TD in patients without LN metastasis are limited. We retrospectively restaged all right-sided colonic adenocarcinomas over a 10-year period using criteria from the fifth, sixth, and seventh AJCC edition. We compared the number of tumor nodule interpreted as LN and TD in each edition and evaluated the stage migration caused by TD definition change. We then assessed clinical significance of TD in the AJCC seventh edition by comparing 5-year overall survival of N1c patients versus other N category (N0, N1, N2) patients with similar T and M status. We showed that the average number of tumor nodules interpreted as LNs per case and the number of cases with positive LNs were significantly decreased with the seventh edition compared with fifth/sixth; however, numbers of cases with TDs and <12 LNs were significantly increased with the seventh edition compared with fifth/sixth. These changes, however, resulted in minimal effects on the final stage grouping. Our survival analysis showed that N1c patients had significantly worse survival compared with N0 patients. Although not statistically significant, the hazard ratios indicated that the N1c group might have worse survival than the N1 group and better survival than the N2 group. Therefore, we conclude that TDs predict patient outcome at least similarly to positive LNs.

Collagen immunostains can distinguish capsular fibrous tissue from septal fibrosis and may help stage liver fibrosis.

Core-needle biopsy remains essential for diagnosis of cirrhosis; however, evaluation of fibrosis in such biopsies is often challenging due to the fragmented nature of cirrhotic liver specimens. It is also common to see portions of liver capsules present in the biopsy which adds to the diagnostic challenge. The distinction between capsular/subcapsular fibrous tissue and septal fibrosis is critical to avoid potential overstaging of liver fibrosis. We compared the differential immunostaining in liver capsular and septal areas for collagens III, IV, V, VI, vitronectin, laminin, Orcein, and Trichrome in 15 whole sections of explanted cirrhotic livers and 5 simulated liver biopsies. Collagens III, IV, V, VI, Trichrome, and Orcein show distinct staining patterns in capsular fibrous tissue and septal fibrosis. Collagen IV shows strong diffuse septal staining and consistently weak to negative capsular staining. Collagens III and VI stain similar to IV for septal fibrosis, whereas collagen V, Trichrome, and Orcein show strong staining in both areas. Collagen IV, possibly with III or VI in addition to the routine Trichrome and hematoxylin and eosin stain, is useful in differentiating capsular fibrous tissue from septal fibrosis on challenging and fragmented liver biopsies.

Colonic manifestations of PTEN hamartoma tumor syndrome: case series and systematic review.

AIM: To investigate our clinical experience with the colonic manifestations of phosphatase and tensin homolog on chromosome ten (PTEN) hamartoma tumor syndrome (PHTS) and to perform a systematic literature review regarding the same. METHODS: This study was approved by the appropriate institutional review board prior to initiation. A clinical genetics database was searched for patients with PHTS or a component syndrome that received gastrointestinal endoscopy or pathology interpretation at our center. These patient's records were retrospectively reviewed for clinical characteristics (including family history and genetic testing), endoscopy results and pathology findings. We also performed a systematic review of the literature for case series of PHTS or component syndromes that reported gastrointestinal manifestations and investigations published after consensus diagnostic criteria were established in 1996. These results were compiled and reported. RESULTS: Eight patients from our institution met initial inclusion criteria. Of these, 5 patients underwent 4.2 colonoscopies at mean age 45.8 ± 10.8 years. All were found to have colon polyps during their clinical course and polyp histology included adenoma, hyperplastic, ganglioneuroma and juvenile. No malignant lesions were identified. Two had multiple histologic types. One patient underwent colectomy due to innumerable polyps and concern for future malignant potential. Systematic literature review of PHTS patients undergoing endoscopy revealed 107 patients receiving colonoscopy at mean age 37.4 years. Colon polyps were noted in 92.5% and multiple colon polyp histologies were reported in 53.6%. Common polyp histologies included hyperplastic (43.6%), adenoma (40.4%), hamartoma (38.3%), ganglioneuroma (33%) and inflammatory (24.5%) polyps. Twelve (11.2%) patients had colorectal cancer at mean age 46.7 years (range 35-62). Clinical outcomes secondary to colon polyposis and malignancy were not commonly reported. CONCLUSION: PHTS has a high prevalence of colon polyposis with multiple histologic types. It should be considered a mixed polyposis syndrome. Systematic review found an increased prevalence of colorectal cancer and we recommend initiating colonoscopy for colorectal cancer surveillance at age 35 years.

Different collagen types show distinct rates of increase from early to late stages of hepatitis C-related liver fibrosis.

During progression from normal liver to cirrhosis, total collagen increases nearly 10-fold with an abnormal increase in fibril-forming collagen and other extracellular matrix molecules. However, little is known regarding the changes each collagen type undergoes during fibrogenesis. We assessed the different collagen types by immunohistochemistry at various stages of hepatitis C-related liver fibrosis in core biopsies and compared changes in each with trichrome stain to better understand fibrogenesis. The possible utility in staging fibrosis was investigated. We found collagens III, IV, V, VI, vitronectin, and trichrome all showed statistically significant increases from early to late stages of fibrosis, but with temporal and quantitative differences. During the transition from early to late fibrosis, trichrome (stains primarily collagen I) and collagen IV showed the steepest increase and appear to be the most useful discriminators between early and late stages of fibrosis. Collagens V and VI have strong reactivity even in stage 1, which may be helpful in identifying early fibrosis when trichrome is weak or negative. Collagen III and vitronectin showed the most gradual increase. Interestingly, collagen V also showed increased staining in areas around inflammation/edema, which may overestimate established fibrosis as compared with trichrome.

Debating deposits: an interobserver variability study of lymph nodes and pericolonic tumor deposits in colonic adenocarcinoma.

CONTEXT: The American Joint Committee on Cancer's Cancer Staging Manual 7th edition defines pericolonic tumor deposits (TDs) as discrete tumor foci in pericolic fat showing no evidence of residual lymph node (LN). This definition relies on subjective features rather than size (5th edition) or shape (6th edition) and introduced the category N1c. Although typically straightforward, metastases are encountered for which the distinction between LNs and TDs is unclear. For data to be meaningful, agreement on distinguishing features between positive LNs and TDs is needed. OBJECTIVES: To assess agreement among gastrointestinal pathologists evaluating difficult metastases and to report the distinguishing features they found helpful. DESIGN: Twenty-five tumor metastases from right-sided colonic adenocarcinomas were selected in which the distinction between positive LNs and TDs was challenging. Virtual slides were reviewed by 7 gastrointestinal pathologists. A list of features potentially helpful in differentiating positive LNs and TDs was ranked for usefulness by each pathologist. Every metastasis was diagnosed as positive LN or TD. For each case diagnosed as positive LN, reviewers were asked to list every feature used in diagnosis. RESULTS: Complete agreement was found for 11 of 25 metastases, 5 positive LNs and 6 TDs (κ statistic, 0.48; 95% confidence interval, 0.28-0.67). Top-ranked features included round shape, peripheral lymphocyte rim, peripheral lymphoid follicles, possible subcapsular sinus, residual LN in surrounding fibroadipose tissue, and thick capsule. The top used features were similar among reviewers. CONCLUSIONS: Significant agreement on positive LNs and TDs in difficult colonic adenocarcinoma metastases was found among evaluators, but inconsistency remains. Round shape, peripheral lymphocyte rim, peripheral lymphoid follicles, possible subcapsular sinus, residual LN in surrounding fibroadipose tissue, and thick capsule were most often used to aid in diagnosis.

Fhit delocalizes annexin a4 from plasma membrane to cytosol and sensitizes lung cancer cells to paclitaxel.

Fhit protein is lost or reduced in a large fraction of human tumors, and its restoration triggers apoptosis and suppresses tumor formation or progression in preclinical models. Here, we describe the identification of candidate Fhit-interacting proteins with cytosolic and plasma membrane localization. Among these, Annexin 4 (ANXA4) was validated by co-immunoprecipitation and confocal microscopy as a partner of this novel Fhit protein complex. Here we report that overexpression of Fhit prevents Annexin A4 translocation from cytosol to plasma membrane in A549 lung cancer cells treated with paclitaxel. Moreover, paclitaxel administration in combination with AdFHIT acts synergistically to increase the apoptotic rate of tumor cells both in vitro and in vivo experiments.

Osteopontin expression is associated with improved survival in patients with pancreatic adenocarcinoma.

BACKGROUND: Osteopontin (OPN) is a secreted protein of the extracellular matrix. It has been used as a marker for tumor aggressiveness and correlated with clinical outcomes in several solid tumors, such as liver, lung, and breast. We determined the OPN expression and its influence on survival in patients with resected pancreatic adenocarcinoma. METHODS: Tissue microarrays were constructed from 245 resected pancreatic adenocarcinomas. Immunohistochemical staining for OPN was undertaken and compared to normal pancreas (n = 12). OPN expression was then correlated with patient demographics, tumor size, grade, node, and margin status. Survival curves were created by the Kaplan-Meier method and compared by log rank analysis. RESULTS: In total, 181 (74 %) of pancreatic adenocarcinoma tissues expressed OPN compared to 7 (58 %) of normal controls (p = 0.004). Expression was observed predominantly in the cytoplasm of the tumor cells. The median and 2 year overall survival was longer when OPN was expressed (17.1 vs. 11.6 months, and 38 vs. 24 %, respectively, p = 0.04). Multivariate analysis showed OPN expression and T stage to be independent predictors of overall survival, while other histopathologic factors such as tumor grade, tumor size, and nodal status were not. CONCLUSIONS: These results suggest that the presence of OPN expression in pancreatic adenocarcinoma may have a protective effect independent of tumor stage. This emphasizes the importance of the interaction between pancreatic cancer cells and their stromal elements.

The spectrum of hematologic malignancies involving the pancreas: potential clinical mimics of pancreatic adenocarcinoma.

Hematologic malignancies often involve the pancreas, causing potential diagnostic pitfalls and, rarely, potentially avoidable surgical resection. We review the spectrum of hematologic malignancies involving the pancreas and describe features useful in preoperative distinction from adenocarcinoma. Archived clinical, pathologic, and radiologic data (1965 to present) for hematologic malignancies involving the pancreas were reviewed and compared with the data for 157 surgically resected pancreatic adenocarcinomas. Of 42 cases, 27 (64%) were clinically "suspicious" for hematologic malignancies. Of the remaining 15 cases, 4 patients underwent resection for presumed pancreatic adenocarcinoma. Isolated pancreatic masses proved most difficult to identify clinically. Significant factors in distinguishing hematologic malignancies from adenocarcinoma included history of hematologic malignancy, young age, large tumor size, low CA19-9 level, B symptoms, and lack of jaundice or diabetes mellitus. Various hematologic malignancies involve the pancreas, most commonly diffuse large B-cell lymphoma. Pancreatic masses are usually correctly identified clinically. Preoperative and operative sampling is strongly recommended when hematologic malignancies cannot be excluded.

Serrated lesions of the appendix: a morphologic and immunohistochemical appraisal.

We performed a histologic and immunohistochemical assessment of 53 noninvasive appendiceal epithelial proliferations, appropriating terminology and using markers shown useful in differentiating serrated colorectal polyps. These were classified as hyperplastic polyp (HP), sessile serrated adenoma (SSA), mixed serrated and adenomatous lesion (MSAL), mucinous cystadenoma (MCA), or conventional adenoma (CAD). Immunohistochemical analysis for cytokeratin (CK) 20, Ki-67, MUC6, and beta-catenin was performed. Diagnoses were as follows: HP, 6; SSA, 12; HP vs SSA, 3; MSAL, 16; MCA, 14; and CAD, 2. All HPs showed expanded (beyond surface) CK20 and expanded or normal (base) Ki-67; 1 was MUC6+. Most SSAs and MSALs were CK20-expanded or expanded with random expression in deep crypts (Ex/I) and Ki-67-expanded, Ex/I (expanded with asymmetry), or normal. All SSAs and 8 of 16 MSALs were MUC6+. CADs were CK20-Ex/I, Ki-67-Ex, and MUC6-; 1 showed nuclear beta-catenin expression. Serrated appendiceal lesions can be categorized using colorectal terminology. MUC6 is associated with SSA morphologic features. Similar immunohistochemical patterns in SSA and MSAL suggest a link between these lesions.

Utilization of lactoferrin-bound and transferrin-bound iron by Campylobacter jejuni.

Campylobacter jejuni NCTC 11168 was capable of growth to levels comparable with FeSO4 in defined iron-limited medium (minimal essential medium alpha [MEMalpha]) containing ferrilactoferrin, ferritransferrin, or ferri-ovotransferrin. Iron was internalized in a contact-dependent manner, with 94% of cell-associated radioactivity from either 55Fe-loaded transferrin or lactoferrin associated with the soluble cell fraction. Partitioning the iron source away from bacteria significantly decreased cellular growth. Excess cold transferrin or lactoferrin in cultures containing 55Fe-loaded transferrin or lactoferrin resulted in reduced levels of 55Fe uptake. Growth of C. jejuni in the presence of ferri- and an excess of apoprotein reduced overall levels of growth. Following incubation of cells in the presence of ferrilactoferrin, lactoferrin became associated with the cell surface; binding levels were higher after growth under iron limitation. A strain carrying a mutation in the cj0178 gene from the iron uptake system Cj0173c-Cj0178 demonstrated significantly reduced growth promotion in the presence of ferrilactoferrin in MEMalpha compared to wild type but was not affected in the presence of heme. Moreover, this mutant acquired less 55Fe than wild type when incubated with 55Fe-loaded protein and bound less lactoferrin. Complementation restored the wild-type phenotype when cells were grown with ferrilactoferrin. A mutant in the ABC transporter system permease gene (cj0174c) showed a small but significant growth reduction. The cj0176c-cj0177 intergenic region contains two separate Fur-regulated iron-repressible promoters. This is the first demonstration that C. jejuni is capable of acquiring iron from members of the transferrin protein family, and our data indicate a role for Cj0178 in this process.

Regulation of denitrification genes in Neisseria meningitidis by nitric oxide and the repressor NsrR.

The human pathogen Neisseria meningitidis is capable of growth using the denitrification of nitrite to nitrous oxide under microaerobic conditions. This process is catalyzed by two reductases: nitrite reductase (encoded by aniA) and nitric oxide (NO) reductase (encoded by norB). Here, we show that in N. meningitidis MC58 norB is regulated by nitric oxide via the product of gene NMB0437 which encodes NsrR. NsrR is a repressor in the absence of NO, but norB expression is derepressed by NO in an NsrR-dependent manner. nsrR-deficient mutants grow by denitrification more rapidly than wild-type N. meningitidis, and this is coincident with the upregulation of both NO reductase and nitrite reductase even under aerobic conditions in the absence of nitrite or NO. The NsrR-dependent repression of aniA (unlike that of norB) is not lifted in the presence of NO. The role of NsrR in the control of expression of aniA is linked to the function of the anaerobic activator protein FNR: analysis of nsrR and fnr single and nsrR fnr double mutants carrying an aniA promoter lacZ fusion indicates that the role of NsrR is to prevent FNR-dependent aniA expression under aerobic conditions, indicating that FNR in N. meningitidis retains considerable activity aerobically.

Growth of benign and malignant schwannoma xenografts in severe combined immunodeficiency mice.

OBJECTIVES: Models for the development of new treatment options in vestibular schwannoma (VS) treatment are lacking. The purpose of this study is to establish a quantifiable human VS xenograft model in mice. STUDY DESIGN AND METHODS: Both rat malignant schwannoma cells (KE-F11 and RT4) and human malignant schwannoma (HMS-97) cells were implanted near the sciatic nerve in the thigh of severe combined immunodeficiency (SCID) mice. Additionally, human benign VS specimens were implanted in another set of SCID mice. Three-dimensional tumor volumes were calculated from magnetic resonance images over the next 6 months. RESULTS: Mice implanted with malignant schwannoma cells developed visible tumors within 2 weeks. Imaging using a 4.7-tesla magnetic resonance imaging and immunohistopathologic examination identified solid tumors in all KE-F11 and HMS-97 xenografts, whereas RT4 xenografts consistently developed cystic schwannomas. VS xenografts demonstrated variability in their growth rates similar to human VS. The majority of VS xenografts did not grow but persisted throughout the study, whereas two of 15 xenografts grew significantly. Histopathologic examination and immunohistochemistry confirmed that VS xenografts retained their original microscopic and immunohistochemical characteristics after prolonged implantation. CONCLUSIONS: This study describes the first animal model for cystic schwannomas. Also, we demonstrate the use of high-field magnetic resonance imaging to quantify VS xenograft growth over time. The VS xenografts represent a model complimentary to Nf2 transgenic and knockout mice for translational VS research.

Heme utilization in Campylobacter jejuni.

A putative iron- and Fur-regulated hemin uptake gene cluster, composed of the transport genes chuABCD and a putative heme oxygenase gene (Cj1613c), has been identified in Campylobacter jejuni NCTC 11168. Mutation of chuA or Cj1613c leads to an inability to grow in the presence of hemin or hemoglobin as a sole source of iron. Mutation of chuB, -C, or -D only partially attenuates growth where hemin is the sole iron source, suggesting that an additional inner membrane (IM) ABC (ATP-binding cassette) transport system(s) for heme is present in C. jejuni. Genotyping experiments revealed that Cj1613c is highly conserved in 32 clinical isolates. One strain did not possess chuC, though it was still capable of using hemin/hemoglobin as a sole iron source, supporting the hypothesis that additional IM transport genes are present. In two other strains, sequence variations within the gene cluster were apparent and may account for an observed negative heme utilization phenotype. Analysis of promoter activity within the Cj1613c-chuA intergenic spacer region revealed chuABCD and Cj1613c are expressed from separate iron-repressed promoters and that this region also specifically binds purified recombinant Fur(Cj) in gel retardation studies. Absorbance spectroscopy of purified recombinant His(6)-Cj1613c revealed a 1:1 heme:His(6)-Cj1613c binding ratio. The complex was oxidatively degraded in the presence of ascorbic acid as the electron donor, indicating that the Cj1613c gene product functions as a heme oxygenase. In conclusion, we confirm the involvement of Cj1613c and ChuABCD in heme/hemoglobin utilization in C. jejuni.

The pathogen Neisseria meningitidis requires oxygen, but supplements growth by denitrification. Nitrite, nitric oxide and oxygen control respiratory flux at genetic and metabolic levels.

The human pathogen Neisseria meningitidis is the major causative agent of bacterial meningitis. The organism is usually treated as a strict aerobe and is cultured under fully aerobic conditions in the laboratory. We demonstrate here that although N. meningitidis fails to grow under strictly anaerobic conditions, under oxygen limitation the bacterium expresses a denitrification pathway (reduction of nitrite to nitrous oxide via nitric oxide) and that this pathway supplements growth. The expression of the gene aniA, which encodes nitrite reductase, is regulated by oxygen depletion and nitrite availability via transcriptional regulator FNR and two-component sensor-regulator NarQ/NarP respectively. Completion of the two-step denitrification pathway requires nitric oxide (NO) reduction, which proceeds after NO has accumulated during batch growth under oxygen-limited conditions. During periods of NO accumulation both nitrite and NO reduction are observed aerobically, indicating N. meningitidis can act as an aerobic denitrifier. However, under steady-state conditions in which NO is maintained at a low concentration, oxygen respiration is favoured over denitrification. NO inhibits oxidase activity in N. meningitidis with an apparent Ki NO = 380 nM measured in intact cells. The high respiratory flux to nitrite after microaerobic growth and the finding that accumulation of the denitrification intermediate NO inhibits oxygen respiration support the view that denitrification is a pathway of major importance in N. meningitidis.