Killing of Fusobacterium nucleaturn,Porphyromonas gingivalis and Prevotella intermedia by protegrins

Protegrins are broad spectrum antibiotic peptides isolated from porcine leukocytes. In this study, we (i) examine the sensitivity of Gram-negative, anaerobic periodontal pathogens to synthetic protegrins; (ii) determine the relative potencies of protegrin congeners against these bacteria; and (iii) compare the potency of protegrins with other antibiotic peptides, including magainin MSI-78, tachyplesin I, cecropin P1, human defensins HNP-1-3, and clavanin A. Synthetic l - and d -enantiomers of protegrin 1 (PG-1 and D-PG-1, respectively), and L-enantiomers of protegrins 2, 3 and 5 (PG-2, PG-3 and PG-5) were tested against Fusobacteriurn nucleatum, and black-pigmented organisms including Porphyromonas gingivalis and Prevotella intermedia. Strains of both F. nucleatum and the black-pigmented organisms were sensitive to PG-1, and exhibited mean ED₉₉ of 2.2-2.3 μg/ml and 3.4-9.9 μg/ml, respectively. The D-form was statistically more potent than the L-form against these oral anaerobes, and although this difference in potency is unlikely to be of decisive therapeutic significance, the d -form may be of value given ability to resist microbial and host-derived proteases. PG-1 was more potent than magainin, tachyplesin, cecropin, defensins and clavanin under test conditions. Hypertonic saIt concentrations and heat-inactivated serum were found to be inhibitory to the bactericidal activity of PG-1. PG-1 was found to induce morphologic alterations in the ultrastructural appearance of F. nucleatum consistent with damage to the bacterial membranes. We conclude that protegrins may be useful antimicrobial agents in therapy against Gram-negative anaerobic bacteria believed to be involved in chronic, adult forms of periodontal infections.     

Mapping the diatom redox-sensitive proteome provides insight into response to nitrogen stress in the marine environment

Diatoms are ubiquitous marine photosynthetic eukaryotes responsible for approximately 20% of global photosynthesis. Little is known about the redox-based mechanisms that mediate diatom sensing and acclimation to environmental stress. Here we used a quantitative mass spectrometry-based approach to elucidate the redox-sensitive signaling network (redoxome) mediating the response of diatoms to oxidative stress. We quantified the degree of oxidation of 3,845 cysteines in the Phaeodactylum tricornutum proteome and identified approximately 300 redox-sensitive proteins. Intriguingly, we found redox-sensitive thiols in numerous enzymes composing the nitrogen assimilation pathway and the recently discovered diatom urea cycle. In agreement with this finding, the flux from nitrate into glutamine and glutamate, measured by the incorporation of ¹⁵N, was strongly inhibited under oxidative stress conditions. Furthermore, by targeting the redox-sensitive GFP sensor to various subcellular localizations, we mapped organelle-specific oxidation patterns in response to variations in nitrogen quota and quality. We propose that redox regulation of nitrogen metabolism allows rapid metabolic plasticity to ensure cellular homeostasis, and thus is essential for the ecological success of diatoms in the marine ecosystem.Aerobic organisms produce reactive oxygen species (ROS) as a byproduct of oxygen-based metabolic pathways, such as photosynthesis, photorespiration, and oxidative phosphorylation (1). Perturbations in oxygenic metabolism under various stress conditions can induce oxidative stress from overproduction of ROS (2, 3). Because ROS are highly reactive forms of oxygenic metabolites, critical mechanisms for ROS detoxification have evolved consisting of ROS-scavenging enzymes and small molecules, including glutathione (GSH) (4). As the most abundant low molecular weight thiol antioxidant, GSH has critical roles in maintaining a proper cellular thiol–disulfide balance and in detoxifying H₂O₂ via the ascorbate–GSH cycle (5).Although classically ROS were considered toxic metabolic byproducts that ultimately lead to cell death, it is now recognized that ROS act as central secondary messengers involved in compartmentalized signaling networks (1, 6–8). Modulation of various cell processes by ROS signaling is mediated largely by posttranslational thiol oxidation, whereby their physical structure and biochemical activity are modified upon oxidation (9). Thus, the redox states of these proteins possess crucial information needed for cell acclimation to stress conditions (10, 11). The emergence of advanced redox proteomic approaches, such as the OxICAT method (12), has created new opportunities to identify redox-sensitive proteins (e.g., redoxome) on the system level and to quantify their precise level of oxidation on exposure to environmental stress conditions.Marine photosynthetic microorganisms (phytoplankton) are the basis of marine food webs. Despite the fact that their biomass represents only approximately 0.2% of the photosynthetic biomass on earth, they are responsible for nearly 50% of the annual global carbon-based photosynthesis and greatly influence the global biogeochemical carbon cycle (13). This high ratio of productivity to biomass, reflected in high turnover rates, makes phytoplankton highly responsive to climate change. Phytoplankton can grow rapidly and form massive blooms that stretch over hundreds of kilometers in the oceans and are regulated by such environmental factors as nutrient availability and biotic interactions with grazers and viruses.Diatoms are a highly diverse clade of phytoplankton, responsible for roughly 20% of global primary productivity (14). Consequently, diatoms play a central role in the biogeochemical cycling of important nutrients, including carbon, nitrogen, and silica, which constitute part of their ornate cell wall. As members of the eukaryotic group known as stramenopiles (or heterokonts), diatoms are derived from a secondary endosymbiotic event involving red and green algae engulfed within an ancestral protest (15).The unique multilineage content of diatom genomes reveals a melting pot of biochemical characteristics that resemble bacterial, plant, and animal traits, including the integration of a complete urea cycle, fatty acid oxidation in the mitochondria, and plant C4-like related pathways (16, 17). During bloom succession, phytoplankton cells are subjected to diverse environmental stress conditions that lead to ROS production, such as allelopathic interactions (18), CO₂ availability (19, 20), UV exposure (21), iron limitation (22), and viral infection (23). Recently reported evidence suggests that diatoms possess a surveillance system based on the induction of ROS that have been implicated in response to various environmental stresses (22, 24). Nevertheless, very little is known about cell signaling processes in marine phytoplankton and their potential role in acclimation to rapid fluctuations in the chemophysical gradients in the marine environment (25).Using a mass spectrometry-based approach, we examined the diatom redoxome and quantified its degree of oxidation under oxidative stress conditions. The wealth of recently identified redox-sensitive proteins participating in various cellular functions suggests a fundamental role of redox regulation in diatom biology. We mapped the redox-sensitive enzymes into a metabolic network and evaluated their role in the adjustment of metabolic flux under variable environmental conditions. We further explored the redox sensitivity of the primary nitrogen-assimilating pathway and demonstrated the role of compartmentalized redox regulation in cells under nitrogen stress conditions using a redox-sensitive GFP sensor targeted to specific subcellular localizations.     

Inhibition of isolated Mycobacterium tuberculosis fatty acid synthase I by pyrazinamide analogs

An analog of pyrazinamide (PZA), 5-chloropyrazinamide (5-Cl-PZA), has previously been shown to inhibit mycobacterial fatty acid synthase I (FASI). FASI has been purified from a recombinant strain of M. smegmatis (M. smegmatis Deltafas1 attB::M. tuberculosis fas1). Following purification, FASI activity and inhibition were assessed spectrophotometrically by monitoring NADPH oxidation. The observed inhibition was both concentration and structure dependent, being affected by both substitution at the 5 position of the pyrazine nucleus and the nature of the ester or N-alkyl group. Under the conditions studied, both 5-Cl-PZA and PZA exhibited concentration and substrate dependence consistent with competitive inhibition of FASI with K(i)s of 55 to 59 microM and 2,567 to 2,627 microM, respectively. The results were validated utilizing a radiolabeled fatty acid synthesis assay. This assay showed that FASI was inhibited by PZA and pyrazinoic acid as well as by a series of PZA analogs.     

Sagittal spinopelvic parameters in children with achondroplasia: identification of 2 distinct groups

Imatinib mesylate has become the treatment of choice for gastrointestinal stromal tumors (GISTs) and has made a revolutionary impact on survival rates. Bone marrow necrosis is a very rare adverse event in malignant GIST. Bone metastases are also rarely encountered in the setting of this disease. The authors report on a patient with malignant GIST who developed a bone lesion, mimicking spinal metastasis on both MR imaging and FDG-PET/CT. Corpectomy and anterior fusion was performed, but the pathology report was consistent with bone marrow necrosis. Radiological and clinical similarities made the distinction between metastasis and bone marrow necrosis challenging for the treating physicians. Instead of radical surgical excision, more conservative methods such as percutaneous or endoscopic bone biopsies may be more useful for pathological confirmation, even though investigations such as MR imaging and FDG-PET/CT indicate metastatic disease.     

Unexpected pulmonary involvement in extrapulmonary tuberculosis patients

BACKGROUND: This study aimed to assess the utility of sputum examinations and chest radiographs (CXRs) in patients with extrapulmonary tuberculosis (XPTB) to detect pulmonary involvement of tuberculosis (TB). METHODS: We studied 72 XPTB patients who were managed through the TB Program, King County, WA, from January 2003 through November 2004. RESULTS: The two most common sites of XPTB were the lymph nodes (36 [50%]) and pleura (12 [17%]). Thirty-five of 72 XPTB patients (49%) had abnormal CXR findings. Sputum was not obtained from 15 patients despite sputum induction. Of the 57 patients from whom sputum was collected, 30 (53%) had abnormal CXR findings, 5 (9%) had sputum smears that were positive for acid-fast bacilli, and 12 (21%) had sputum cultures that were positive for Mycobacterium tuberculosis. Weight loss was significantly associated with positive sputum culture findings in a multivariate analysis (odds ratio, 4.3; 95% confidence interval, 1.01 to 18.72; p = 0.049). There was no significant difference in the occurrence of positive sputum culture results between patients with abnormal CXR findings and those with normal CXR findings (7 of 30 patients [23%] vs 5 of 27 patients [19%], respectively; p = 0.656). Of 24 HIV-negative XPTB patients with normal CXR findings, 2 patients (8%) had positive sputum culture findings. CONCLUSIONS: CXR results did not reliably differentiate XPTB patients with and without positive sputum culture findings. Some XPTB patients had positive sputum culture results despite normal CXR findings and negative HIV status. Weight loss in XPTB patients was associated with positive sputum culture results. Sputum examinations in XPTB patients, regardless of the CXR results, may identify potentially infectious cases of TB.     

Guanine polynucleotides are self‐antigens for human natural autoantibodies and are significantly reduced in the human genome

In the course of investigating anti‐DNA autoantibodies, we examined IgM and IgG antibodies to poly‐G and other oligonucleotides in the sera of healthy persons and those diagnosed with systemic lupus erythematosus (SLE), scleroderma (SSc), or pemphigus vulgaris (PV); we used an antigen microarray and informatic analysis. We now report that all of the 135 humans studied, irrespective of health or autoimmune disease, manifested relatively high amounts of IgG antibodies binding to the 20‐mer G oligonucleotide (G20); no participants entirely lacked this reactivity. IgG antibodies to homo‐nucleotides A20, C20 or T20 were present only in the sera of SLE patients who were positive for antibodies to dsDNA. The prevalence of anti‐G20 antibodies led us to survey human, mouse and Drosophila melanogaster (fruit fly) genomes for runs of T20 and G20 or more: runs of T20 appear > 170 000 times compared with only 93 runs of G20 or more in the human genome; of these runs, 40 were close to brain‐associated genes. Mouse and fruit fly genomes showed significantly lower T20/G20 ratios than did human genomes. Moreover, sera from both healthy and SLE mice contained relatively little or no anti‐G20 antibodies; so natural anti‐G20 antibodies appear to be characteristic of humans. These unexpected observations invite investigation of the immune functions of anti‐G20 antibodies in human health and disease and of runs of G20 in the human genome.