Role of the DinB Homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis

The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS) across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phase-dependent manner, with dinB2 levels 12- to 40-fold higher than those of dinB1. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the β-clamp, consistent with its canonical C-terminal β-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M. tuberculosis to compounds that form N²-dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.The emergence and global spread of multi- and extensively drug-resistant strains of Mycobacterium tuberculosis have further complicated the already daunting challenge of controlling tuberculosis (TB) (15). The mechanisms that underlie the evolution of drug resistance in M. tuberculosis by chromosomal mutagenesis and their association with the conditions that tubercle bacilli encounter during the course of infection are poorly understood (6). It has been postulated that hypoxia, low pH, nutrient deprivation, and nitrosative and oxidative stress impose environmental and host immune-mediated DNA-damaging insults on infecting bacilli (64). In addition, the observed importance of excision repair pathways for the growth and survival of M. tuberculosis in murine models of infection (13, 55) and the upregulation of M. tuberculosis genes involved in DNA repair and modification in pulmonary TB in humans provide compelling evidence that the in vivo environment is DNA damaging (51).Damage tolerance constitutes an integral component of an organism''s response to genotoxic stress, preventing collapse of the replication fork at persisting, replication-blocking lesions through the engagement of specialized DNA polymerases that are able to catalyze translesion synthesis (TLS) across the sites of damage (19, 21, 60). Most TLS polymerases belong to the Y family, which comprises a wide range of structurally related proteins present in bacteria, archaea, and eukaryotes (44). Of these, the DinB subfamily of Y family polymerases, whose founder member is Escherichia coli Pol IV (63), is conserved among all domains of life (44). The association of Y family polymerases with inducible mutagenesis has implicated these enzymes in the adaptation of bacteria to environmental stress (17, 20, 39, 54, 58, 59, 66). Their key properties are exemplified in E. coli Pol IV: the polymerase catalyzes efficient and accurate TLS across certain N²-dG adducts (27, 28, 34, 40, 45, 67) and has been implicated in the tolerance of alkylation damage (4); furthermore, overexpression of Pol IV significantly increases mutation rates in E. coli (reviewed in references 21 and 26), and dinB is the only SOS-regulated gene required at induced levels for stress-induced mutagenesis in this organism (20). Furthermore, overproduction of E. coli Pol IV inhibits replication fork progression through replacement of the replicative polymerase to form an alternate replisome in which Pol IV modulates the rate of unwinding of the DnaB helicase (25) and also reduces colony-forming ability (61).The M. tuberculosis genome encodes two Y family polymerase homologs belonging to the DinB subfamily, designated herein as DinB1 (DinX, encoded by Rv1537) and DinB2 (DinP, encoded by Rv3056), as well as a third, distantly related homolog encoded by Rv3394c (see Fig. S1 in the supplemental material) (9). On the basis of sequence similarity with their counterparts from E. coli (63) and Pseudomonas aeruginosa (54), including the complete conservation of key acidic residues essential for catalysis, DinB1 and DinB2 may be functional DNA polymerases (see Fig. S1). In contrast, Rv3394c lacks these residues and as such is unlikely to have polymerase activity (see Fig. S1). Unlike most Y family polymerase-encoding genes investigated with other bacteria (17, 26, 54, 58), dinB1 and dinB2 expression in M. tuberculosis is not dependent on RecA, the SOS response, or the presence of DNA damage (5, 7, 52). That these genes are regulated by other mechanisms and so may serve distinct roles in DNA metabolism in M. tuberculosis is suggested by the observation that dinB1 is differentially expressed in pulmonary TB (51) and is a member of the SigH regulon (30), whereas expression of dinB2 is induced following exposure to novobiocin (5).In this study, we adopted a genetic approach to investigate the function of dinB1 and dinB2 in M. tuberculosis. Mutants with altered complements or expression levels of dinB1 and/or dinB2 were analyzed in vitro and in vivo under conditions predicted to be phenotypically revealing based on DinB function established with other model organisms. The lack of discernible phenotypes in any of the assays employed suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.     

Abdominal wall and labial edema presenting in a girl with Henoch-Schönlein purpura: a case report

Introduction

We report a rare case of gastrointestinal perforation following dacarbazine infusion for metastatic melanoma. The condition is attributed to a responding malignant melanoma in the gastrointestinal tract.

Case presentation

A 52-year-old Caucasian man presented with abdominal pain and distension, malaise, night sweats, dysphagia and early satiety. A computed tomography scan showed massive ascites, lymphadenopathy and liver lesions suspect for metastases. An upper gastrointestinal endoscopy was performed and revealed multiple dark lesions of 5 mm to 10 mm in his stomach and duodenum. When his skin was re-examined, an irregular pigmented lesion over the left clavicle measuring 15 mm × 8 mm with partial depigmentation was found. Histological examination of a duodenal lesion was consistent with a diagnosis of metastatic melanoma. The patient deteriorated and his level of lactate dehydrogenase rapidly increased. The patient was started on systemic treatment with dacarbazine 800 mg/m² every three weeks and he was discharged one day after the first dose. On the sixth day he was readmitted with severe abdominal pain. A chest X-ray showed the presence of free intraperitoneal air that was consistent with gastrointestinal perforation. His lactate dehydrogenase level had fallen from 6969U/L to 1827U/L, supporting the conclusion that the response of gastrointestinal metastases to dacarbazine had resulted in the perforation of the patient's bowel wall. A laparotomy was discussed with the patient and his family but he decided to go home with symptomatic treatment. He died 11 days later.

Conclusion

Melanoma can originate in, as well as metastasize to, the gastrointestinal tract. Gastrointestinal perforations due to responding tumors are a well-known complication of systemic treatment of gastrointestinal lymphomas. However, as the response rate of metastatic melanoma to dacarbazine is only 10% to 20%, and responses are usually only partial, perforation due to treatment response in metastatic melanoma is rare. Medical oncologists should be aware of the risk of bowel perforation after starting cytotoxic chemotherapy on patients with gastrointestinal metastases.     

Manipulating cellular transport and immune responses: dynamic interactions between intracellular Salmonella enterica and its host cells

Intracellular survival and replication within eukaryotic host cells is of central importance for the pathogenesis of infections caused by Salmonella enterica. Intracellular Salmonella translocates a set of effector proteins by means of a type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI2) that manipulates normal host-cell functions. Intracellular survival and replication is linked to the function of the SPI2-T3SS, but recent observations show that many additional cellular functions are targeted by this virulence system. In this review, we focus on the recent observations on the interference of intracellular Salmonella with functions of the innate and adaptive immune system and the modification of endocytic and exocytic cellular transport. The common molecular basis of the different SPI2-dependent phenotypes could be the interference with cellular transport along microtubules.     

Integrin shedding as a mechanism of cellular adaptation during cardiac growth

Integrin-mediated cell-extracellular matrix (ECM) interactions are essential for multiple cellular processes; however, little is known regarding integrin turnover during these events. Recent studies have demonstrated shedding of cell surface molecules and suggested this as a potential mechanism for integrin turnover. Confocal microscopy of mouse hearts under different physiological conditions demonstrated the presence of beta(1)-integrin-immunoreactive material in the interstitium. Culture media from neonatal rat cardiac myocytes and fibroblasts contained a 55-kDa fragment of beta(1)-integrin. Attachment to ECM components, response to phorbol 12-myristate 13-acetate stimulation, and matrix metalloproteinase inhibition assays demonstrated that fibroblasts responded differently to the fragment compared with myocytes. The beta(1)-integrin fragment stimulated myocyte attachment to collagen and the fragment itself bound a variety of ECM proteins. These studies indicate that as myocytes and fibroblasts change size and shape, cellular contacts with the ECM are altered, resulting in the liberation of a beta(1)-integrin fragment from the cell surface. Integrin shedding may represent a novel mechanism of rapidly modifying cell-ECM contacts during various cellular processes.     

An arabinogalactan protein associated with secondary cell wall formation in differentiating xylem of loblolly pine

Arabinogalactan proteins (AGPs) are abundant plant proteoglycans implicated in plant growth and development. Here, we report the genetic characterization, partial purification and immunolocalization of a classical AGP (PtaAGP6, accession number AF101785) in loblolly pine (Pinus taeda L.). A PtaAGP6 full-length cDNA clone was expressed in bacteria. PtaAGP6 resembles tomato LeAGP-1 and Arabidopsis AtAGP17-19 in that they all possess a subdomain composed of basic amino acids. The accessibility of this domain in the glycoprotein makes it possible to label the PtaAGP6 epitopes on the cell surface or in the cell wall with polyclonal antibodies raised against this subdomain. The antibodies recognize the peptide of the basic subdomain and bind to the intact protein molecule. A soluble protein-containing fraction was purified from the differentiating xylem of pine trees by using -glucosyl Yariv reagent (-glcY) and was recognized by antibodies against the basic subdomain. Immunolocalization studies showed that the PtaAGP6 epitopes are restricted to a file of cells that just precede secondary cell wall thickening, suggesting roles in xylem differentiation and wood formation. The location of apparent labeling of the PtaAGP6 epitopes is separated from the location of lignin deposition. Multiple single nucleotide polymorphisms (SNPs) were detected in EST variants. Denaturing HPLC analysis of PCR products suggests that PtaAGP6 is encoded by a single gene. Mobility variation in denaturing gel electrophoresis was used to map PtaAGP6 SNPs to a site on linkage group 5.     

aph-1 and pen-2 are required for Notch pathway signaling,gamma-secretase cleavage of betaAPP,and presenilin protein accumulation

Presenilins are components of the gamma-secretase protein complex that mediates intramembranous cleavage of betaAPP and Notch proteins. A C. elegans genetic screen revealed two genes, aph-1 and pen-2, encoding multipass transmembrane proteins, that interact strongly with sel-12/presenilin and aph-2/nicastrin. Human aph-1 and pen-2 partially rescue the C. elegans mutant phenotypes, demonstrating conserved functions. The human genes must be provided together to rescue the mutant phenotypes, and the inclusion of presenilin-1 improves rescue, suggesting that they interact closely with each other and with presenilin. RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. aph-1 and pen-2, like nicastrin, are required for the activity and accumulation of gamma-secretase.     

Chemistry,distribution, and metabolism of tomato carotenoids and their impact on human health

Recent epidemiological studies have suggested that the consumption of tomatoes and tomato-based food products reduce the risk of prostate cancer in humans. This protective effect has been attributed to carotenoids, which are one of the major classes of phytochemicals in this fruit. The most abundant carotenoid in tomato is lycopene, followed by phytoene, phytofluene, zeta-carotene, gamma-carotene, beta-carotene, neurosporene, and lutein. The distribution of lycopene and related carotenoids in tomatoes and tomato-based food products has been determined by extraction and high-performance liquid chromatography-UV/Visible photodiode array detection. Detailed qualitative and quantitative analysis of human serum, milk, and organs, particularly prostate, have revealed the presence of all the aforementioned carotenoids in biologically significant concentrations. Two oxidative metabolites of lycopene, 2,6-cyclolycopene-1,5-diols A and B, which are only present in tomatoes in extremely low concentrations, have been isolated and identified in human serum, milk, organs (liver, lung, breast, liver, prostate, colon) and skin. Carotenoids may also play an important role in the prevention of age-related macular degeneration, cataracts, and other blinding disorders. Among 25 dietary carotenoids and nine metabolites routinely found in human serum, mainly (3R,3'R,6'R)-lutein, (3R,3'R)-zeaxanthin, lycopene, and their metabolites were detected in ocular tissues. In this review we identified and quantified the complete spectrum of carotenoids from pooled human retinal pigment epithelium, ciliary body, iris, lens, and in the uveal tract and in other tissues of the human eye to gain a better insight into the metabolic pathways of ocular carotenoids. Although (3R,3'R,6'R)-lutein, (3R,3'R)-zeaxanthin, and their metabolites constitute the major carotenoids in human ocular tissues, lycopene and a wide range of dietary carotenoids have been detected in high concentrations in ciliary body and retinal pigment epithelium. The possible role of lycopene and other dietary carotenoids in the prevention of age-related macular degeneration and other eye diseases is discussed.