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PubMed2004

Community structure and metabolism through reconstruction of microbial genomes from the environment.

Microbial communities are vital in the functioning of all ecosystems; however, most microorganisms are uncultivated, and their roles in natural systems are unclear. Here, using random shotgun sequencing of DNA from a natural acidophilic biofilm, we report reconstruction of near-complete genomes of Leptospirillum group II and Ferroplasma type II, and partial recovery of three other genomes. This was possible because the biofilm was dominated by a small number of species populations and the frequency of genomic rearrangements and gene insertions or deletions was relatively low. Because each sequence read came from a different individual, we could determine that single-nucleotide polymorphisms are the predominant form of heterogeneity at the strain level. The Leptospirillum group II genome had remarkably few nucleotide polymorphisms, despite the existence of low-abundance variants. The Ferroplasma type II genome seems to be a composite from three ancestral strains that have undergone homologous recombination to form a large population of mosaic genomes. Analysis of the gene complement for each organism revealed the pathways for carbon and nitrogen fixation and energy generation, and provided insights into survival strategies in an extreme environment.

Archaea↗
PubMed2005

Microbial population dynamics at effluent treatment plants.

The requirements for treated wastewater are becoming increasingly more stringent, and therefore the improved efficiency of biological treatment processes is indispensable at industrial effluent treatment plants (ETPs). Microorganisms such as bacteria play an important role in the natural cycling of materials and particularly in the decomposition of organic wastes. The knowledge of the interactions among these microbial populations needs to be harnessed for optimum evaluation and functioning of effluent treatment plants. Modern molecular techniques have revolutionized the methods of assessing these microbial populations. The combination of the results of these microbial assessments along with the on-site parameters at ETPs would favor an efficient treatment. In this review, the various approaches and importance of correlating the microbial population dynamics and treatment of wastewater at industrial ETPs has been elaborated.

Bacteria↗
PubMed1969

The enzymic hydroxylation of protocollagen models.

1. Synthetic polymers of l-prolyl-l-prolylglycine of defined chain length, (Pro-Pro-Gly)(n), were found to be substrates for the enzyme protocollagen-proline hydroxylase, with optimum chain length n=5. Boiling the polymer (Pro-Pro-Gly)(15) increased its activity as a substrate but had no effect on (Pro-Pro-Gly)(5). 2. Protection of both or one of the N- and C-terminal groups made (Pro-Pro-Gly)(3) a better substrate, and collagenase digestion of hydroxylated tert.-pentyloxy-carbonyl-(Pro-Pro-Gly)(3) benzyl ester indicated that the central prolyl residues were the major points of hydroxylation. 3. The results suggest that the long-chain peptides are optimum substrates but that a triple-stranded structure is inhibitory for hydroxylation.

Animals↗
PubMed2001

Acholeplasma laidlawii up-regulates granulysin gene expression via transcription factor activator protein-1 in a human monocytic cell line, THP-1.

An antimicrobial protein granulysin is constitutively expressed in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. However, little is known about the precise regulatory mechanisms underlying granulysin gene expression. In this study, we examined the regulatory mechanisms underlying granulysin gene expression using a human monocytic cell line, THP-1, treated with Acholeplasma laidlawii. The level of granulysin mRNA expression in THP-1 cells was significantly augmented in response to stimulation with A. laidlawii. The transfection of reporter gene constructs into THP-1 cells indicated that DNA sequences between residues -329 and -239, relative to the transcriptional start site of the granulysin gene, are responsible for mediating gene induction. In addition, mutagenesis of a putative activator protein-1 (AP-1)-binding site between residues -277 and -271 in the granulysin promoter resulted in the reduction of granulysin promoter activity. Electrophoretic mobility shift assays (EMSA) demonstrated that nuclear extract prepared from A. laidlawii-treated THP-1 cells can generate specific binding to DNA oligonucleotides encompassing the AP-1-binding site, whereas unstimulated nuclear extract from the cells failed to do so. Furthermore, competition and supershift assays confirmed that A. laidlawii can induce the activation of AP-1. These results indicate that AP-1 dominantly participates in the regulation of inducible granulysin gene expression in THP-1 cells. Therefore, the finding of inducible granulysin gene expression by A. laidlawii suggests that inducible granulysin in macrophages may function as a protective weapon when microbial invasion occurs.

Acholeplasma laidlawii↗
PubMed2002

Binding of hemolin to bacterial lipopolysaccharide and lipoteichoic acid. An immunoglobulin superfamily member from insects as a pattern-recognition receptor.

Hemolin, a plasma protein from lepidopteran insects, is composed of four immunoglobulin domains. Its synthesis is induced by microbial challenge. We investigated the biological functions of hemolin in Manduca sexta. It was found to bind to the surface of bacteria and yeast, and caused these micro-organisms to aggregate. Hemolin was demonstrated to bind to lipopolysaccharide (LPS) from Gram-negative bacteria and to lipoteichoic acid from Gram-positive bacteria. Binding of hemolin to smooth-type forms of LPS was competed for efficiently by lipoteichoic acid and by rough mutant (Ra and Rc) forms of LPS, which differ in polysaccharide length. Binding of hemolin to LPS was partially inhibited by calcium and phosphate. Hemolin bound to the lipid A component of LPS, and this binding was completely blocked by free phosphate. Our results suggest that hemolin has two binding sites for LPS, one that interacts with the phosphate groups of lipid A and one that interacts with the O-specific antigen and the outer-core carbohydrates of LPS. The binding properties of M. sexta hemolin suggest that it functions as a pattern-recognition protein with broad specificity in the defense against micro-organisms.

Animals↗
PubMed2001

The efficacy and safety of two oral moxifloxacin regimens compared to oral clarithromycin in the treatment of community-acquired pneumonia.

An international multi-centre, randomized, prospective, double-blind study compared oral moxifloxacin (200 mg or 400 mg once daily for 10 days) with oral clarithromycin (500 mg, twice daily for 10 days) in the treatment of community-acquired pneumonia (CAP). The clinical success rate in the evaluable population at the primary efficacy assessment, 3-5 days after the end of study treatment, was 93.9% in patients treated with 200 mg moxifloxacin; 94.4%, with 400 mg moxifloxacin; and 94.3%, with clarithromycin. Clinical success rates were maintained at follow-up, 21-28 days after the end of treatment: 90.7% (200 mg moxifloxacin), 92.8% (400 mg moxifloxacin) and 92.2% (clarithromycin). The 95% confidence intervals indicated that all three treatment regimens were equally effective in treating CAP. At follow-up, the 400 mg moxifloxacin dose had a slightly higher observed cure rate than the 200 mg moxifloxacin dose, but this was not statistically significant. The most frequently isolated pathogens were Streptococcus pneumoniae (42%), Haemophilus influenzae (19%), Haemophilus parainfluenzae (10%), Moraxella catarrhalis (6%), Klebsiella pneumoniae (5%) and Staphylococcus aureus (4%). The bacteriological success rate (eradication and presumed eradication) was 72.5% (29/40) for 200 mg moxifloxacin, 78.7% (37/47) for 400 mg moxifloxacin and 70.7% (29/41) for clarithromycin. The adverse event profile was comparable between the three treatment groups. Most adverse events, possibly or probably related to the study drug, were generally mild or moderate in severity and mostly related to the digestive system: diarrhoea, nausea and abdominal pain in 200 mg moxifloxacin patients; diarrhoea, liver function abnormalities and nausea in 400 mg moxifloxacin patients and liver function abnormalities, diarrhoea, nausea and taste perversion in clarithromycin patients. Study drugs were discontinued because of adverse events in 7/229 (3%) patients treated with 200 mg moxifloxacin, 11/224 (5%) with moxifloxacin 400 mg and 11/222 (5%) with clarithromycin. In all assessments, moxifloxacin was at least as effective clinically, and as well tolerated as clarithromycin in the treatment of CAP. Bacteriological success rates in moxifloxacin-treated patients were greater than those of clarithromycin. Moxifloxacin, given once daily, is free of many drug-drug interactions and requires no dosage adjustments in most renal hepatic deficient patients.

Administration, Oral↗
PubMed2000

The hepatic microvascular responses to sepsis.

The liver is believed to play a major role in the initiation of multiorgan failure, the most lethal complication in the clinical course of sepsis. Microbes and their virulence factors enter the hepatic circulation where they first activate sinusoidal endothelial cells and Kupffer cells to produce proinflammatory mediators, including TNF-alpha, IL-1, IL-6, reactive oxygen metabolites, and eicosanoids. These mediators cause not only microbial killing, but also structural and functional liver damage concerning mainly the parenchymal cells. Leukocytes are targeted to the liver sinusoids by chemoattractants and, like platelets, tether to the sinusoidal endothelial cells, which are in a procoagulant state of inflammatory activation. Clogging of the sinusoids by these cells leads to a decrease of blood flow through the sinusoids, which is further aggravated by endothelin-1 effectuating the constriction of hepatic stellate cells in the sinusoids. In contrast, both nitric oxide (NO) and carbon monoxide (CO) act as antagonists of endothelin-1 by mediating relaxation of sinusoidal vessels. By maintaining an adequate sinusoidal perfusion, both NO and CO are hepatoprotective during the early, hyperdynamic phase of sepsis characterized by an increased cardiac output and moderate peripheral vasodilation. However, during the late, hypodynamic phase of sepsis, massive overproduction of NO by the inducible NO synthase leads to circulatory collapse, which inevitably includes breakdown of the liver circulation.

Endothelium, Vascular↗
PubMed2006

The role of decay-accelerating factor as a receptor for Helicobacter pylori and a mediator of gastric inflammation.

Persistent gastritis induced by Helicobacter pylori is the strongest known risk factor for peptic ulcer disease and distal gastric adenocarcinoma, a process for which adherence of H. pylori to gastric epithelial cells is critical. Decay-accelerating factor (DAF), a protein that protects epithelial cells from complement-mediated lysis, also functions as a receptor for several microbial pathogens. In this study, we investigated whether H. pylori utilizes DAF as a receptor and the role of DAF within H. pylori-infected gastric mucosa. In vitro studies showed that H. pylori adhered avidly to Chinese hamster ovary cells expressing human DAF but not to vector controls. In H. pylori, disruption of the virulence factors vacA, cagA, and cagE did not alter adherence, but deletion of DAF complement control protein (CCP) domains 1-4 or the heavily O-glycosylated serine-threonine-rich COOH-terminal domain reduced binding. In cultured gastric epithelial cells, H. pylori induced transcriptional up-regulation of DAF, and genetic deficiency of DAF attenuated the development of inflammation among H. pylori-infected mice. These results indicate that DAF may regulate H. pylori-epithelial cell interactions relevant to pathogenesis.

Animals↗
PubMed2005

The role of gut-associated lymphoid tissues and mucosal defence.

The newborn infant leaves a germ-free intrauterine environment to enter a contaminated extrauterine world and must have adequate intestinal defences to prevent the expression of clinical gastrointestinal disease states. Although the intestinal mucosal immune system is fully developed after a full-term birth, the actual protective function of the gut requires the microbial stimulation of initial bacterial colonization. Breast milk contains prebiotic oligosaccharides, like inulin-type fructans, which are not digested in the small intestine but enter the colon as intact large carbohydrates that are then fermented by the resident bacteria to produce SCFA. The nature of this fermentation and the consequent pH of the intestinal contents dictate proliferation of specific resident bacteria. For example, breast milk-fed infants with prebiotics present in breast milk produce an increased proliferation of bifidobacteria and lactobacilli (probiotics), whereas formula-fed infants produce more enterococci and enterobacteria. Probiotics, stimulated by prebiotic fermentation, are important to the development and sustainment of intestinal defences. For example, probiotics can stimulate the synthesis and secretion of polymeric IgA, the antibody that coats and protects mucosal surfaces against harmful bacterial invasion. In addition, appropriate colonization with probiotics helps to produce a balanced T helper cell response (Th1=Th2=Th3/Tr1) and prevent an imbalance (Th1>Th2 or Th2>Th1) contributing in part to clinical disease (Th2 imbalance contributes to atopic disease and Th1 imbalance contributes to Crohn's disease and Helicobacter pylori-induced gastritis). Furthermore, a series of pattern recognition receptors, toll-like receptors on gut lymphoid and epithelial cells that interact with bacterial molecular patterns (e.g. endotoxin (lipopolysaccharide), flagellin, etc.), help modulate intestinal innate immunity and an appropriate adaptive immune response. Animal and clinical studies have shown that inulin-type fructans will stimulate an increase in probiotics (commensal bacteria) and these bacteria have been shown to modulate the development and persistence of appropriate mucosal immune responses. However, additional studies are needed to show that prebiotics can directly or indirectly stimulate intestinal host defences. If this can be demonstrated, then prebiotics can be used as a dietary supplement to stimulate a balanced and an appropriately effective mucosal immune system in newborns and infants.

Animals↗
PubMed2000

Resolution of sick building syndrome in a high-security facility.

The main objective of this article is to serve as a case study for other industrial hygiene (IH) professionals' review as a "real world" effort in responding to a facility perceived as "sick" by its occupants. As many industrial hygienists do not have extensive backgrounds in evaluating microbial air contaminants or the mechanical function of building HVAC units, the overall intent is to provide "lessons learned" to IH generalists who may be asked to participate in indoor environmental quality (IEQ) surveys. In September 1994, a suspected case of "sick building syndrome" was investigated (with significant airborne fungal loads confirmed) at a communications center after numerous occupants reported upper respiratory disease and/or allergy-type symptoms. The setting was a two-story structure approximately 30 years old, with a normal occupancy load of 350 to 400 persons. In addition to continual structural modifications, the central HVAC air conditioning systems had poor maintenance histories. Inspection of HVAC components revealed visible fungal growth on air filters and air ducts and in cooling fan condensate drip pans. Fungal air samples were collected with an Anderson N6 air sampler and Sabouraund dextrose agar media. Over a study period of 23 months, three rounds of 26 air samples were collected for 5 minutes each at 28.3 liters/minute airflow. Cultures exhibited fungi such as Aspergillus, Penicillium, Alternaria, and Cladosporium. Certain strains of these fungi produce mycotoxins that may cause a variety of deleterious health effects such as those described by occupants. Initial 1994 airborne fungal concentrations ranged from 85 to 6157 colony forming units (CFUs) per cubic meter of sampled air (CFU/m3). Some investigators have reported fungal concentrations as low as 245 CFU/m3 associated with complaint sites in other buildings. Remediation efforts involved hiring a dedicated mechanic to implement a HVAC preventive maintenance program (including regular replacement of all HVAC air filters and cleaning of accessible components with water/bleach solution). Post-abatement January 1996 re-sampling revealed a significant drop in airborne fungal colonies up to 97 percent (range = 21 to 1092 CFUs/m3)--which also coincided with physicians at the local hospital sensing a qualitative reduction in patient visits from facility workers. To address seasonal bias, a final August 1996 air sample round revealed a range of 14 to 500 CFUs/m3. Of the 21 workspaces sampled in all three rounds, nine continued to show a decline in CFUs/m3 from September 1994 baseline counts. These results demonstrate the critical role of an ongoing HVAC maintenance program for reducing potential reservoirs of fungal organisms in indoor work environments. Building renovations (especially those involving major changes to building layout and usage) can adversely affect IEQ if plans do not include coordinated updates and regular preventive maintenance of HVAC systems. Eventual negative outcomes can be reduced occupant productivity and deleterious health effects.

Air Pollution, Indoor↗
PubMed1979

Augmentation of macrophage complement receptor function in vitro. I. Characterization of the cellular interactions required for the generation of a T-lymphocyte product that enhances macrophage complement receptor function.

The function of complement receptors of mouse peritoneal macrophages was converted in vitro from mediating only attachment of macrophage complement receptor function was achieved by treating freshly explanted macrophages with supernates from cultures containing T lymphocytes and appropriately triggered macrophages. Fc receptor-mediated phagocyctosis by macrophages was required for the production of active supernates, for neither ingestion via the cells' complement receptors nor ingestion via nonimmunologic means was a sufficient stimulus for the macrophages' participation in the generation of supernatant activity. Fc receptor-triggered macrophages interacted by a contact dependent, but histocompatibility independent, mechanism with T lymphocytes, thereby signalling the lymphocytes to elaborate the active product. The possible significance of enhanced macrophage complement receptor function in inflammation, host defense against microbial pathogens, immune complex disease, and neoplasia is discussed.

Animals↗
PubMed1992

Desialylation of lysosomal membrane glycoproteins by Trypanosoma cruzi: a role for the surface neuraminidase in facilitating parasite entry into the host cell cytoplasm.

Trypanosoma cruzi enters host cells via formation of an acidic vacuole which is subsequently disrupted, allowing the parasite access to the cytoplasm. We show that in an acid environment, release of the parasite surface neuraminidase is enhanced, and this release is likely mediated by a phosphatidylinositol-specific phospholipase C (PIPLC), since antibodies to a carbohydrate epitope (CRD) revealed in glycosylphosphatidylinositol (GPI)-anchored proteins after PIPLC cleavage remove the great majority of the soluble neuraminidase activity from culture supernatants. The neuraminidase is active at acidic pH, and is capable of desialylating known vacuolar constituents, i.e., lysosomal membrane glycoproteins. Parasite escape into the cytoplasm is significantly facilitated in terminal sialylation-defective mutant Lec 2 cells, and enzymatically desialylated membranes are more susceptible to lysis by a parasite hemolysin previously implicated in vacuole membrane rupture. These findings provide evidence that terminal sialylation on carbohydrate moieties contributes to maintaining lysosomal membrane integrity, and indicate a role for a protozoan-derived neuraminidase in facilitating parasite entry into host cells. These observations raise the possibility that other microbial neuraminidases may serve a similar function in acidic intracellular compartments.

Animals↗
PubMed1998

Sequence analysis of the cupin gene family in Synechocystis PCC6803.

The recently described cupin superfamily of proteins includes the germin and germinlike proteins, of which the cereal oxalate oxidase is the best characterized. This superfamily also includes seed storage proteins, in addition to several microbial enzymes and proteins with unknown function. All these proteins are characterized by the conservation of two central motifs, usually containing two or three histidine residues presumed to be involved with metal binding in the catalytic active site. The present study on the coding regions of Synechocystis PCC6803 identifies a previously unknown group of 12 related cupins, each containing the characteristic two-motif signature. This group comprises 11 single-domain proteins, ranging in length from 104 to 289 residues, and includes two phosphomannose isomerases and two epimerases involved in cell wall synthesis, a member of the pirin group of nuclear proteins, a possible transcriptional regulator, and a close relative of a cytochrome c551 from Rhodococcus. Additionally, there is a duplicated, two-domain protein that has close similarity to an oxalate decarboxylase from the fungus Collybia velutipes and that is a putative progenitor of the storage proteins of land plants.

Amino Acid Sequence↗
PubMed1983

Leukemic cells as probes for sequential functional differentiation of the human granulocyte.

The purpose of this study was to sequence the functional properties of the neutrophil during maturation, using leukemic cells as experimental probes. Twenty-nine cases of acute leukemia, derived from granulocytic precursors, were studied. The functional tests that were evaluated included phagocytosis, bactericidal activity, random locomotion, and chemotaxis. These functional properties were correlated with Fc receptors and the stimulated nitroblue tetrazolium dye reduction test. The results indicated that the various functions of the neutrophil are acquired sequentially rather than concurrently. It is postulated that the sequence of functional differentiation is: phagocytosis leads to microbial killing leads to random locomotion leads to chemotaxis.

Adolescent↗
PubMed2003

Galectin-8 modulates neutrophil function via interaction with integrin alphaM.

The members of the galectin family are associated with diverse cellular events, including immune response. We investigated the effects of galectin-8 on neutrophil function. Human galectin-8 induced firm and reversible adhesion of peripheral blood neutrophils but not eosinophils to a plastic surface in a lactose-sensitive manner. Other human galectins, galectins-1, -3, and -9, showed low or negligible effects on neutrophil adhesion. Confocal microscopy revealed actin bundle formation in the presence of galectin-8. Cytochalasins inhibited both actin assembly and cell adhesion induced by galectin-8. Affinity purification of galectin-interacting proteins from solubilized neutrophil membrane revealed that N-terminal carbohydrate recognition domain (CRD) of galectin-8 bound promatrix metalloproteinase-9 (proMMP-9), and C-terminal CRD bound integrin alphaM/CD11b and proMMP-9. A mutant galectin-8 lacking the carbohydrate-binding activity of N-terminal CRD (galectin-8R69H) retained adhesion-inducing activity, but inactivation of C-terminal CRD (galectin-8R233H) abolished the activity. MMP-3-mediated processing of proMMP-9 was accelerated by galectin-8, and this effect was inhibited by lactose. Galectins-1 and -3 did not affect the processing. Superoxide production, an essential event in bactericidal function of neutrophils, was stimulated by galectin-8 to an extent comparable to that induced by fMLP. Galectin-8R69H but not galectin-8R233H could stimulate superoxide production. Taken together, these results suggest that galectin-8 is a novel factor that modulates the neutrophil function related to transendothelial migration and microbial killing.

Actins↗
PubMed1987

Clinical efficacy and safety of sulbactam/ampicillin in patients suffering from chronic liver disease.

Sulbactam is a new beta-lactamase inhibitor with pharmacokinetic characteristics in humans similar to those of ampicillin. A total of 41 patients hospitalized in the Clinic of Infectious Diseases, University of Naples, for chronic liver diseases, were treated with sulbactam/ampicillin (ratio 1:2) for urinary, respiratory, biliary tract or soft tissue infections. Sulbactam/ampicillin was administered im or iv at a dosage of 3-9 g/day depending on the site and severity of the infection. All the patients treated with sulbactam/ampicillin had clinical signs and symptoms of infection, and all the organisms isolated were sensitive to sulbactam/ampicillin (MIC less than 16 mg/l). For both Gram-positive and Gram-negative bacteria the sulbactam/ampicillin MICs were much lower than the ampicillin MICs. In agreement with the favourable in-vitro results, we observed good therapeutic efficacy. 85% of the patients recovered or improved within a few days of therapy, with no clinical relapses, and in 81% of the infections the responsible bacteria were completely eradicated. We observed a low number of side effects (3/41 oral candidosis; 3/41 pain at the im injection site) and no change in the blood chemistry tests.

Adult↗
PubMed1989

Ticarcillin/clavulanate in severe infections in patients with varying renal function.

The therapeutic efficacy of ticarcillin/clavulanate was assessed in 71 patients with severe infections: 38 acute pyelonephritis, 16 septicaemia and 19 miscellaneous infections. The patients were classified according to their renal function in: Group A, normal (16 cases); B, mild renal impairment (RI) with creatinine clearance (Clcr) between 80 and 40 ml/min (18 cases); C, moderate RI with Clcr between 40 and 15 ml/min (12 cases); D, severe RI with (Clcr) between 15 and 5 ml/min (13 cases) and E, terminal with (Clcr) less than 5 ml/min (12 cases). A total of 105 microorganisms (48.6% resistant to ticarcillin): 31 Pseudomonas aeruginosa, 18 Escherichia coli, 21 other Enterobacteriaceae, 2 Haemophilus influenzae, 10 Bacteroides spp., 14 enterococci, 8 staphylococci and 1 streptococcus, were isolated. All except six Ps. aeruginosa were sensitive to ticarcillin/clavulanate, using 75:10 microgram discs. Bacteriological eradication was obtained in 97% of the cases on the third day and at the end of treatment, and in 82% of the cases after one month. In all the assessable cases, the clinical symptoms disappeared on the third day except in one patient who developed a resistant strain (Klebsiella oxytoca). The wide range of bacteria assessed and the clinical-bacteriological success rates demonstrated that the ticarcillin/clavulanate combination had an efficacy/safety profile that could be considered excellent. Tolerance was good and side effects were not observed. This study confirms the practical efficacy of the recommended dosages derived from our previous kinetic studies in RI.

Adult↗