Org Lett, 2004 Sep 2, 6(18), 3043 - 6 Synthesis of LuxS inhibitors targeting bacterial cell-cell communication; Alfaro JF et al.; {reaction: see text} Quorum sensing is a process by which bacteria sense cell density . This cell-cell communication process is mediated by autoinducers . A cross-species messenger, autoinducer-2 (AI-2) is produced from S-ribosyl-L-homocysteine by the LuxS enzyme . A proposed mechanism for LuxS is an aldose-ketose isomerization of S-ribosylhomocysteine followed by a beta-elimination . We report here the synthesis of two substrate analogues, S-anhydroribosyl-L-homocysteine and S-homoribosyl-L-cysteine, which prevent the initial and final step of the mechanism, respectively. Biotechnol Bioeng, 2004 Sep 20, 87(6), 687 - 94 Single nucleotide polymorphism detection in aldehyde dehydrogenase 2 (ALDH2) gene using bacterial magnetic particles based on dissociation curve analysis; Maruyama K et al.; Single nucleotide polymorphism (SNP) detection for aldehyde dehydrogenase 2 (ALDH2) gene based on DNA thermal dissociation curve analysis was successfully demonstrated using an automated system with bacterial magnetic particles (BMPs) by developing a new method for avoiding light scattering caused by nanometer-size particles when using commercially available fluorescent dyes such as FITC, Cy3, and Cy5 as labeling chromophores . Biotin-labeled PCR products in ALDH2, two allele-specific probes (Cy3-labeled detection probe for ALDH2*1 and Cy5-labeled detection probe for ALDH2*2), streptavidin-immobilized BMPs (SA-BMPs) were simultaneously mixed . The mixture was denatured at 70 degrees C for 3 min, cooled slowly to 25 degrees C, and incubated for 10 min, allowing the DNA duplex to form between Cy3- or Cy5-labeled detection probes and biotin-labeled PCR products on SA-BMPs . Then duplex DNA-BMP complex was heated to 58 degrees C, a temperature determined by dissociation curve analysis and a dissociated single-base mismatched detection probe was removed at the same temperature under precise control . Furthermore, fluorescence signal from the detection probe was liberated into the supernatant from completely matched duplex DNA-BMP complex by heating to 80 degrees C and measured . In the homozygote target DNA (ALDH2*1/*1 and ALDH2*2/*2), the fluorescence signals from single-base mismatched were decreased to background level, indicating that mismatched hybridization was efficiently removed by the washing process . In the heterozygote target DNA (ALDH2*1/*2), each fluorescence signals was at a similar level . Therefore, three genotypes of SNP in ALDH2 gene were detected using the automated detection system with BMPs . Med Sci Monit, 2004 Sep, 10(9), PI99 - PI103 Epub 2004 Aug 20. Efficacy of a topical suspension of bacterial antigens for the management of recurrent eczema in children; Mora R et al.; BACKGROUND: The aim of this study was to compare the efficacy and safety of a topical suspension of bacterial antigens for the management of recurrent eczema in children . MATERIAL/METHODS: Eighty pediatric patients, aged between 2 and 6 years, with a history of recurrent external auditory eczema, were enrolled in this study . Congenital immune deficiencies, treatment with corticosteroids, immunostimulant or immunosuppressive agents in the past 6 months, and bronchopulmonary disease were considered as exclusion criteria . The patients were randomized to receive topical suspension of bacterial antigens (Lantigen B) (Group A) or placebo (physiological solution) (Group B) . The efficacy parameters were evaluated before, at the end of, and one year after the beginning of the therapy . All the children underwent clinical examinations, blood test, skin prick tests (SPT), and clinical evaluation using the clinical score proposed by Rajka and Langeland, considering the intensity, extent, and course of lesions . RESULTS: In Group A, using the statistical Wilcoxon test, we saw an improvement in the clinical items measured . It is important to consider that the patients were allowed to receive concomitant medications to treat acute episodes, which may have partially contributed to the positive results obtained . CONCLUSIONS: A topical suspension of bacterial antigens was significantly more effective than placebo in the treatment of recurrent eczema in children. BJOG, 2004 Sep, 111(9), 982 - 8 Randomised treatment trial of bacterial vaginosis to prevent post-abortion complication; Miller L et al.; OBJECTIVE: To evaluate the efficacy of metronidazole to reduce post-abortion complications among women with bacterial vaginosis . DESIGN: A randomised, double-blind placebo-controlled trial . SETTING: An American outpatient abortion facility between April 1999 and June 2000 . SAMPLE: Women presenting for surgical abortion were screened for bacterial vaginosis using a pH and amines card test . METHODS: Women positive for elevated pH and amines on a self-collected vaginal discharge sample were randomised to 1000 mg oral metronidazole before abortion followed by 500 mg twice daily or placebo . All randomised women were also dispensed 100 mg doxycycline to take twice daily for seven days . MAIN OUTCOME MEASURES: Data were collected by phone, daily diary or visit and scored from 0 to 7 for post-abortion complications . Intention-to-treat analyses were completed prior to unblinding . RESULTS: Of 1764 women screened by card test, 638 (36%) were positive . Of these, 393 were randomised . Follow up data were available for 253 (64%) of these women . A complication score of 3 or more occurred in 21% of women assigned to metronidazole, compared with 19% in those assigned placebo (RR 1.1, 95% CI 0.7-1.9) . Among 153 women with Gram stain confirmation for bacterial vaginosis, there was a similar lack of benefit with treatment (RR 1.6, 95% CI 0.9-3.0) . No individual symptom or sign was statistically different between groups even for second trimester procedures . CONCLUSION: Among women undergoing abortion and diagnosed with bacterial vaginosis, oral metronidazole, in conjunction with doxycycline, did not reduce post-abortion complications. J Am Chem Soc, 2004 Sep 1, 126(34), 10598 - 602 Presentation and detection of azide functionality in bacterial cell surface proteins; Link AJ et al.; An improved protocol for copper-catalyzed triazole formation on the bacterial cell surface is described . Addition of highly pure CuBr to cells treated with azidohomoalanine (2) leads to ca . 10-fold more extensive cell surface labeling than previously observed . This highly active catalyst allows detection of the methionine analogues azidoalanine (1), azidonorvaline (3), and azidonorleucine (4) in cell surface proteins . Azidoalanine was previously believed to be silent with regard to the cellular protein synthesis machinery . J Biol Chem, 2004 Oct 29, 279(44), 46008 - 13 Epub 2004 Aug 23. A unifying model for the role of polyamines in bacterial cell growth, the polyamine modulon; Yoshida M et al.; We reported previously that the synthesis of specific proteins such as OppA, Cya, and RpoS (sigma(38)), which are important for cell growth and viability, is stimulated by polyamines at the level of translation . In this study we found that the synthesis of FecI and Fis was also stimulated by polyamines at the level of translation . The FecI and Fis proteins enhance the expression of mRNAs that are involved in iron uptake and energy metabolism and the expression of rRNA and some tRNAs . The Shine-Dalgarno (SD) sequence of their mRNAs was not obvious or was not located at the usual position . When the SD sequences were created at the normal position on these mRNAs, protein synthesis was no longer influenced by polyamines . Thus, the common characteristic of these mRNAs was to have a weak or ineffective SD sequence . We propose that a group of genes whose expression is enhanced by polyamines at the level of translation be referred to as a "polyamine modulon." By DNA microarray, we found that 309 of 2,742 mRNA species were upregulated by polyamines . Among the 309 up-regulated genes, transcriptional enhancement of at least 58 genes might be attributable to increased levels of the transcription factors Cya, RpoS, FecI, and Fis, which are all organized in the polyamine modulon . This unifying molecular mechanism is proposed to underlie the physiological role of polyamines in controlling the growth of Escherichia coli. Biophys J, 2004 Nov, 87(5), 3137 - 47 Epub 2004 Aug 23. Permeation properties of an engineered bacterial OmpF porin containing the EEEE-locus of Ca2+ channels; Miedema H et al.; The selectivity filter of the bacterial porin OmpF carries a small net charge close to -1 e and is therefore only slightly cation-selective . Calcium channels, on the other hand, contain four negatively charged glutamates, the EEEE-locus, and are among the most selective cation channels known . We aimed to turn the essentially nonselective OmpF into a Ca2+-selective channel . To that end, two additional glutamates (R42E and R132E) were introduced in the OmpF constriction zone that already contains D113 and E117 . Mutant OmpF containing this DEEE-locus has a high Ca2+ over Cl- selectivity and a Na+ current with a strongly increased sensitivity to 1 mM Ca2+ . The charge/space competition model, initially applied to the L-type Ca2+ channel, identifies the fixed charge and filter volume as key determinants of ion selectivity, with the precise atomic arrangement having only second-order effects . By implication, the reproduction of fixed charge and filter volume should transform two channels into channels of similar selectivity, even if the two belong to entirely different ion channel families, as is the case for OmpF and the L-type Ca2+ channel . The results presented here fit quite well in the framework of charge/space competition theory. Biophys J, 2004 Nov, 87(5), 3010 - 22 Epub 2004 Aug 23. Circular dichroism of carotenoids in bacterial light-harvesting complexes: experiments and modeling; Georgakopoulou S et al.; In this work we investigate the origin and characteristics of the circular dichroism (CD) spectrum of rhodopin glucoside and lycopene in the light-harvesting 2 complex of Rhodopseudomonas acidophila and Rhodospirillum molischianum, respectively . We successfully model their absorption and CD spectra based on the high-resolution structures . We assume that these spectra originate from seven interacting transition dipole moments: the first corresponds to the 0-0 transition of the carotenoid, whereas the remaining six represent higher vibronic components of the S2 state . From the absorption spectra we get an estimate of the Franck-Condon factors of these transitions . Furthermore, we investigate the broadening mechanisms that lead to the final shape of the spectra and get an insight into the interaction energy between carotenoids . Finally, we examine the consequences of rotations of the carotenoid transition dipole moment and of deformations in the light-harvesting 2 complex rings . Comparison of the modeled carotenoid spectra with modeled spectra of the bacteriochlorophyll QY region leads to a refinement of the modeling procedure and an improvement of all calculated results . We therefore propose that the combined carotenoid and bacteriochlorophyll CD can be used as an accurate reflection of the overall structure of the light-harvesting complexes. FEMS Immunol Med Microbiol, 2004 Sep 1, 42(1), 139 - 45 Interleukin 1-beta responses to bacterial toxins and sudden infant death syndrome; Moscovis SM et al.; We tested the hypothesis that significantly higher IL-1beta responses to toxic shock syndrome toxin (TSST) noted for parents of sudden infant death syndrome (SIDS) infants might be due in part to genetic factors such as the IL-1beta (C-511T) and IL-1RN (T+2018C) single nucleotide polymorphisms (SNP) . The first objective was to assess the distribution of these polymorphisms among SIDS infants, parents of SIDS infants and controls, and two ethnic groups: Aboriginal Australians who have a high incidence of SIDS; and Bangladeshis who in Britain have a low incidence of SIDS compared with Europeans . The second objective was to assess IL-1beta responses to endotoxin and toxic shock syndrome toxin (TSST) from leukocytes of smokers and non-smokers in relation to these polymorphisms . There were major differences in the distributions of the IL-1beta (C-511T) SNP between Europeans and Bangladeshis (p=0.00) and between Europeans and Aboriginal Australians (p=0.00); however, they were similar for the Bangladeshi and Aboriginal Australian subjects . The allele frequency distribution of the IL-1RN (T+2018C) SNP for the Aboriginal Australians was statistically different from the European group (p=0.00), but it was not different from the Bangladeshi group (p=0.09) . Compared with controls of European origin, there were no significant differences in the distribution of these polymorphisms among SIDS infants or parents of SIDS infants . For the IL-1beta (C-511T) SNP, the highest IL-1beta responses to endotoxin were obtained with leukocytes of non-smokers with the heterozygous CT genotype . Smokers had significantly lower levels of IL-1beta in response to endotoxin (p=0.01) and these differences were significant for donors with the wild type CC (p=0.00) and CT (p=0.03) genotypes . Similar patterns were observed for IL-1beta responses to TSST, but the differences were not significant . For the IL-1RN (T+2018C) SNP, the highest IL-1beta responses to endotoxin were obtained with leukocytes from non-smoker donors with the wildtype TT genotype and significantly lower responses were found with leukocytes from donors with the TC genotype (p=0.02) . The responses of smokers were lower but the differences were significant only for donors with the TT genotype (p=0.00) . Similar patterns were observed for IL-1beta responses to TSST, but the differences were not significant . IL-1beta responses to both endotoxin and TSST were increased for the small number of smokers with the TT genotype of the IL-1beta (C-511T) SNP . The TT genotype of the IL-1beta (C-511T) was found predominantly among Aboriginal Australian and Bangladeshi individuals but only a small proportion of Europeans . Smokers with the AA genotype of the IL-10 (G-1082A) SNP which is found predominantly among these two groups had significantly lower levels of IL-10 responses . If cigarette smoke enhances pro-inflammatory responses and reduces anti-inflammatory responses in individuals with these genotypes, this might partly explain the increased susceptibility of Aboriginal Australian infants to infections and SIDS. FEMS Immunol Med Microbiol, 2004 Sep 1, 42(1), 42 - 7 Common bacterial toxins and physiological vulnerability to sudden infant death: the role of deleterious genetic mutations; Morris JA; The common bacterial toxin hypothesis of sudden infant death syndrome (SIDS) is consistent with the epidemiological features of the condition including the age distribution, seasonal incidence, association with prone sleeping and with exposure to tobacco smoke . The hypothesis is supported by experimental evidence but there are two barriers to its acceptance: the speed of onset does not fit with conventional concepts of an infective process; furthermore, the hypothesis appears to offer a single explanation for what is regarded as a multifactorial disease . Concepts from information theory are used to explore these objections . Complex physiological systems process information and need a high level of redundancy to minimise error . Models show that deleterious mutations in such a system will interact synergistically . Environmental perturbations are most likely to cause failure (sudden death) in systems with several mutations . Models also indicate that mutation rates will pose a limit to the size of the functioning genome and, therefore, increased complexity in evolution depends on using old genes in new combinations rather than the chance appearance of new genes . The idea that we share our genes with the rest of creation (same genes but different combinations) leads to the following conjecture: for every receptor controlling the flow of information across a cell membrane there will be a bacterially coded molecule that can switch it off or on . Based on this premise, bacterial toxaemia could cause sudden death, merely the time it takes for a molecule to associate with or dissociate from its receptor . Regardless of the number of physiological systems involved in SIDS, the age distribution will have a unimodal peak corresponding to the age range during which infant serum IgG reaches its nadir . In this way, the two barriers to the common bacterial toxin hypothesis can be overcome: one explanation but multiple bacteria and toxins acting with variable speed on multiple target systems. Clin Lab Med, 2004 Sep, 24(3), 627 - 49, vi Emerging intracellular bacterial infections; Katz B et al.; Human infections by intracellular bacteria have been recognized for many years, but much of what we know about the pathogenesis of these diseases and their etiologic organisms has emerged within the past few years as a result of improved molecular-based means for their detection and classification . New insights concerning the epidemiology and pathogenesis of intracellular bacterial infections and methods for the detection of Chlamydophila pneumoniae, Ehrlichia chaffeensis, Anaplasma phagocytophilum, and Rickettsia species have made an impact on how we view them as agents of human disease . Emerging evidence suggesting a possible intracellular existence for another organism, Mycoplasma pneumoniae, may explain how this organism interacts with the host to induce chronic inflammatory conditions of the respiratory tract. Biochem J, 2004 Dec 1, 384(Pt 2), 385 - 90 Efficient production of active chicken avidin using a bacterial signal peptide in Escherichia coli; Hytonen VP et al.; Chicken avidin is a highly popular tool with countless applications in the life sciences . In the present study, an efficient method for producing avidin protein in the periplasmic space of Escherichia coli in the active form is described . Avidin was produced by replacing the native signal sequence of the protein with a bacterial OmpA secretion signal . The yield after a single 2-iminobiotin-agarose affinity purification step was approx . 10 mg/l of virtually pure avidin . Purified avidin had 3.7 free biotin-binding sites per tetramer and showed the same biotin-binding affinity and thermal stability as egg-white avidin . Avidin crystallized under various conditions, which will enable X-ray crystallographic studies . Avidin produced in E . coli lacks the carbohydrate chains of chicken avidin and the absence of glycosylation should decrease the non-specific binding that avidin exhibits towards many materials {Rosebrough and Hartley (1996) J . Nucl . Med . 37, 1380-1384} . The present method provides a feasible and inexpensive alternative for the production of recombinant avidin, avidin mutants and avidin fusion proteins for novel avidin-biotin technology applications. Clin Exp Immunol, 2004 Sep, 137(3), 460 - 8 Correlation of virulence, lung pathology, bacterial load and delayed type hypersensitivity responses after infection with different Mycobacterium tuberculosis genotypes in a BALB/c mouse model; Dormans J et al.; One of the most intriguing aspects of tuberculosis is that the outcome of an infection with M . tuberculosis (TB) is highly variable between individuals . The possibility of differences in virulence between M . tuberculosis strains or genotypes has only recently been studied . There is evidence of multifactorial genetic predisposition in humans that influences the susceptibility to tuberculosis . A better understanding of differences in virulence between M . tuberculosis genotypes could be important with regard to the efforts at TB control and the development of improved antituberculosis vaccines . Survival, lung pathology, bacterial load and delayed type hypersensitivity (DTH) responses of BALB/c mice after intratracheal infection with any of 19 different M . tuberculosis complex strains of 11 major genotype families were studied . The results indicate that among genetically different M . tuberculosis strains a very broad response was present with respect to virulence, pathology, bacterial load and DTH . 'Low'-responders were the H37Rv, Canetti, Beijing-1 strains, while Beijing-2,3, Africa-2 and Somalia-2 strains were 'high'-responders . A severe pathological response correlates with a high mortality and a high CFU counts in lungs, but poorly with the degree of the DTH response. Pediatr Res, 2004 Nov, 56(5), 804 - 9 Epub 2004 Aug 19. Evaluation of IL-8-concentrations in plasma and lysed EDTA-blood in healthy neonates and those with suspected early onset bacterial infection; Orlikowsky TW et al.; Plasma IL-8 is a diagnostic parameter of early-onset bacterial infection (EOBI) in neonates but has a short half-life . The detergent-lysed whole-blood (DLWB) IL-8 consists of both extracellular and cell-bound IL-8 . The objective of this study was to investigate kinetics of plasma and DLWB IL-8 in healthy newborns and those with suspected EOBI and to test the hypothesis that determination of DLWB IL-8 results in higher sensitivity for EOBI detection . Sixty-one neonates with clinical and serologic signs of EOBI composed the study group; 188 neonates with risk factors but without EOBI served as control subjects . IL-8 concentrations were determined in plasma and DLWB . In the control group, DLWB IL-8 concentrations were 280-fold higher (9599 pg/mL; SD 4433) up to 24 h post partum than corresponding plasma levels (34.2 pg/mL; SD 18.1) . The sensitivity of DLWB versus plasma IL-8 for EOBI was 0.97 versus 0.71 after 6 h and 0.70 versus 0.32 after 24 h . Corresponding values for specificity were 0.95 versus 0.90 after 6 h and 0.92 versus 0.99 after 24 h . After 24 h, the negative predictive value for DLWB versus plasma IL-8 was 0.80 versus 0.66 . DLWB IL-8 showed a higher sensitivity for EOBI within 6 h after first clinical suspicion than plasma IL-8 . It also remained elevated longer . Our results suggest that DLWB IL-8 results in a higher sensitivity for EOBI. Mol Biol Evol, 2004 Nov, 21(11), 2172 - 81 Epub 2004 Aug 18. The evolutionary fate of nonfunctional DNA in the bacterial endosymbiont Buchnera aphidicola; Gomez-Valero L et al.; Reduction of the genome size in endosymbiotic bacteria is the main feature linked to the adaptation to a host-associated lifestyle . We have analyzed the fate of the nonfunctional DNA in Buchnera aphidicola, the primary endosymbiont of aphids . At least 164 gene losses took place during the recent evolution of three B . aphidicola strains, symbionts of the aphids Acyrthosiphon pisum (BAp), Schizaphis graminum (BSg), and Baizongia pistacia (BBp) . A typical pattern starts with the inactivation of a gene, which produces a pseudogene, and is followed by the progressive loss of its DNA . Our results show that during the period from the separation of the Aphidinae and Pemphiginae lineages (86-164 MYA) to the divergence of BAp and BSg (50-70 MYA) the half-life of a pseudogene was 23.9 Myr . For the remaining periods of evolution, the ranges of values obtained for this parameter are of the same order of magnitude . These results have revealed that a gene inactivated during B . aphidicola evolution requires 40-60 Myr to become almost completely disintegrated . Moreover, we have shown a positive correlation between the decrease in the GC content and the DNA loss for these nonfunctional DNA regions . When gene losses are classified, based on the detection of a pseudogene or otherwise of an absent gene in the modern B . aphidicola genomes, we have observed a drastic reduction of DNA length in the latter versus the former relative to the functional gene . Finally, we have also detected a slight reduction in size of the intergenic regions in the three B . aphidicola strains, when they are compared with the size of the close relative Escherichia coli. JAMA, 2004 Aug 18, 292(7), 852 - 8 Small intestinal bacterial overgrowth: a framework for understanding irritable bowel syndrome; Lin HC; CONTEXT: Irritable bowel syndrome (IBS), which affects 11% to 14% of the population, is a puzzling condition with multiple models of pathophysiology including altered motility, visceral hypersensitivity, abnormal brain-gut interaction, autonomic dysfunction, and immune activation . Although no conceptual framework accounts for all the symptoms and observations in IBS, a unifying explanation may exist since 92% of these patients share the symptom of bloating regardless of their predominant complaint . EVIDENCE ACQUISITION: Ovid MEDLINE was searched through May 2004 for relevant English-language articles beginning with those related to bloating, gas, and IBS . Bibliographies of pertinent articles and books were also scanned for additional suitable citations . EVIDENCE SYNTHESIS: The possibility that small intestinal bacterial overgrowth (SIBO) may explain bloating in IBS is supported by greater total hydrogen excretion after lactulose ingestion, a correlation between the pattern of bowel movement and the type of excreted gas, a prevalence of abnormal lactulose breath test in 84% of IBS patients, and a 75% improvement of IBS symptoms after eradication of SIBO . Altered gastrointestinal motility and sensation, changed activity of the central nervous system, and increased sympathetic drive and immune activation may be understood as consequences of the host response to SIBO . CONCLUSIONS: The gastrointestinal and immune effects of SIBO provide a possible unifying framework for understanding frequent observations in IBS, including postprandial bloating and distension, altered motility, visceral hypersensitivity, abnormal brain-gut interaction, autonomic dysfunction, and immune activation. Indian J Environ Health, 2003 Jul, 45(3), 209 - 12 Effect of holding time and temperature on bacterial counts; Ahammed MM; Water samples of different pollution levels were collected from four different sources, viz., a canal, an open dug well, a deep tube well, and a drinking water tap, stored at refrigerator (4-5 degrees C) and ambient (28-30 degrees C) temperatures, and analysed at 0, 6, 12, 24 and 48 hours in order to find the effect of holding time and temperature on bacterial counts . The samples were analysed for total coliforms and for heterotrophic plate counts (HPC) . Results of the study indicate significant reduction in the coliform counts in all the water samples tested at both ambient and refrigerator temperatures . However, the rate of decline was much less in refrigerated samples compared to that in samples stored at ambient temperature . Further, the rate of decline was dependent on the source of water . After 48 hours of storage at ambient temperature, canal water samples showed the sharpest decline of 82% in coliform counts, and water samples from open dug well showed the least decline of 51% . While HPCs of the samples held at refrigerator temperature did not show significant changes with time, those samples held at ambient temperature increased upon storage . Up to 12 hours of storage, there was no significant changes in bacterial counts in any type of water at both the temperatures, which suggests that water samples can be stored at ambient temperatures for 12 hours without significantly affecting the coliform counts. Anesteziol Reanimatol, 2004 May-Jun, (3), 43 - 5 {Prevention and treatment of bacterial infection by the recombinant human granulocytic colony-stimulating factor in newborns}; Structure of alanine dehydrogenase from Archaeoglobus: active site analysis and relation to bacterial cyclodeaminases and mammalian mu crystallin; Biotechnology Division of the National Institute of Standards and Technology, Gaithersburg, MD 20899-8312, USA . travis.gallagher@nist.gov The hyperthermophilic archaeon Archaeoglobus fulgidus contains an L-Ala dehydrogenase (AlaDH, EC 1.4.1.1) that is not homologous to known bacterial dehydrogenases and appears to represent a previously unrecognized archaeal group of NAD-dependent dehydrogenases . The gene (Genbank; TIGR AF1665) was annotated initially as an ornithine cyclodeaminase (OCD) on the basis of strong homology with the mu crystallin/OCD protein family . We report the structure of the NAD-bound AF1665 AlaDH (AF-AlaDH) at 2.3 A in a C2 crystal form with the 70 kDa dimer in the asymmetric unit, as the first structural representative of this family . Consistent with its lack of homology to bacterial AlaDH proteins, which are mostly hexameric, the archaeal dimer has a novel structure . Although both types of AlaDH enzyme include a Rossmann-type NAD-binding domain, the arrangement of strands in the C-terminal half of this domain is novel, and the other (catalytic) domain in the archaeal protein has a new fold . The active site presents a cluster of conserved Arg and Lys side-chains over the pro-R face of the cofactor . In addition, the best ordered of the 338 water molecules in the structure is positioned well for mechanistic interaction . The overall structure and active site are compared with other dehydrogenases, including the AlaDH from Phormidium lapideum . Implications for the catalytic mechanism and for the structures of homologs are considered . The archaeal AlaDH represents an ancient and previously undescribed subclass of Rossmann-fold proteins that includes bacterial ornithine and lysine cyclodeaminases, marsupial lens proteins and, in man, a thyroid hormone-binding protein that exhibits 30% sequence identity with AF1665. Trends Genet, 2004 Sep, 20(9), 413 - 6 GroEL and the maintenance of bacterial endosymbiosis; Fares MA et al.; Many eukaryotic organisms have symbiotic associations with obligate intracellular bacteria . The clonal transmission of endosymbionts between host generations should lead to the irreversible fixation of slightly deleterious mutations in their non-recombinant genome by genetic drift . However, the stability of endosymbiosis indicates that some mechanism is involved in the amelioration of the effects of these mutations . We propose that the chaperone GroEL was involved in the acquisition of an endosymbiotic lifestyle not only by means of its over-production, as proposed by Moran, but also by its adaptive evolution mediated by positive selection to improve the interaction with the unstable endosymbiont proteome. J Biotechnol, 2004 Sep 9, 112(3), 313 - 22 Bacterial expression of biologically active recombinant musarmin 1 from bulbs of Muscari armeniacum L . and Miller; Antolin P et al.; Musarmins are type 1 ribosome-inactivating proteins with N-glycosidase activity on the 28 S rRNA that are present in bulbs of Muscari armeniacum L . and Miller at rather low concentrations . In the present work, a cDNA fragment coding for musarmin 1 was sub-cloned and expressed in Escherichia coli . The recombinant protein (rMU1) was synthesised as a polypeptide of 295 amino acids that was delivered to the periplasm and processed . Recombinant musarmin 1 present in the periplam has two forms: insoluble with a molecular mass of 29,423 and soluble with a molecular mass of 29,117 because of a small proteolytic shortening with respect to the insoluble one, presumably in the C-terminal . The yield of protein homogeneous by polyacrylamide gel electrophoresis was 23mgl-1 of bacterial culture . The recombinant musarmin 1 forms isolated from both the soluble and the insoluble (upon refolding) fractions retained full translational inhibitory and 28 S rRNA N-glycosidase activities as compared with the native protein . The recombinant protein displayed great stability towards trypsin, collagenase, rat plasma and rat liver protein extract, but was sensitive to the action of papain and proteinase K . The easy availability and full activity of the recombinant musarmin 1 makes it a good candidate for the preparation of immunotoxins for targeted therapy and for the construction of transgenic plants expressing it as antipathogenic agent. Scand J Infect Dis, 2004, 36(6-7), 523 - 4 Chron's disease, rare association with selective IgA immunodeficiency, and development of life-threatening bacterial infections; Manfredi R et al.; Life-threatening necrotizing fasciitis and relapsing Lemierre syndrome associated with Fusobacterium necrophorum septicaemia occurred in young adults with a moderate Chron's disease and a missed profound IgA deficiency . This unexpected association of a chronic bowel inflammatory syndrome with prominent IgA abnormalities and severe bacterial infection deserves careful attention by physicians faced with young patients with Chron's disease. Clin Infect Dis, 2004 Jul 15, 39(2), 206 - 17 Epub 2004 Jul 02. Serum procalcitonin and C-reactive protein levels as markers of bacterial infection: a systematic review and meta-analysis; Simon L et al.; A meta-analysis was performed to evaluate the accuracy of determination of procalcitonin (PCT) and C-reactive protein (CRP) levels for the diagnosis of bacterial infection . The analysis included published studies that evaluated these markers for the diagnosis of bacterial infections in hospitalized patients . PCT level was more sensitive (88% {95% confidence interval {CI}, 80%-93%} vs . 75% {95% CI, 62%-84%}) and more specific (81% {95% CI, 67%-90%} vs . 67% {95% CI, 56%-77%}) than CRP level for differentiating bacterial from noninfective causes of inflammation . The Q value for PCT markers was higher (0.82 vs . 0.73) . The sensitivity for differentiating bacterial from viral infections was also higher for PCT markers (92% {95% CI, 86%-95%} vs . 86% {95% CI, 65%-95%}); the specificities were comparable (73% {95% CI, 42%-91%} vs . 70% {95% CI, 19%-96%}) . The Q value was higher for PCT markers (0.89 vs . 0.83) . PCT markers also had a higher positive likelihood ratio and lower negative likelihood ratio than did CRP markers in both groups . On the basis of this analysis, the diagnostic accuracy of PCT markers was higher than that of CRP markers among patients hospitalized for suspected bacterial infections. J Perinatol, 2004 Dec, 24(12), 735 - 42 Prospective validation of a perinatal bacterial vaginosis screening risk score; Pastore LM et al.; OBJECTIVE: Prospectively validate an antenatal bacterial vaginosis (BV) risk score at two public health department obstetrics clinics . STUDY DESIGN: Women (n=409) entering prenatal care received a BV risk score (range 0 to 16) at their first visit and at 24 to 28 weeks' gestation . BV was measured with Gram-stained vaginal smears . Normal discharge was used as a surrogate for being asymptomatic . RESULTS: Approximately half of the women scored > or =3 at each assessment . In total 29% had true BV at the first assessment (13% at the second assessment) . The BV risk score (> or =3) had 91% sensitivity and 63% specificity, and the BV risk score (> or =5) had 88% sensitivity and 76% specificity . Among true cases, 42% were asymptomatic, of which 77% had risk scores > or =3 . All symptomatic BV cases had risk scores > or =3 . CONCLUSION: In practice, the risk score identified both asymptomatic and symptomatic cases . Asymptomatic women are of particular interest because they are not screened for BV under current practice guidelines. Environ Microbiol, 2004 Sep, 6(9), 921 - 7 Targeting modular polyketide synthases with iteratively acting acyltransferases from metagenomes of uncultured bacterial consortia; Piel J et al.; Bacterial type I polyketide synthases (PKSs) produce a wide range of biomedically important secondary metabolites . These enzymes possess a modular structure that can be genetically re-engineered to yield novel drug candidates not found in nature . Recently, we have reported the putative pederin PKS from an uncultured bacterial symbiont of Paederus fuscipes beetles . It belongs to an architecturally unusual PKS group, the members of which contain iteratively acting acyltransferases that are not integrated into the PKS modules but are encoded by isolated genes . As these systems are rare, often contain additional unusual features and are of smaller size than regular PKSs, the development of a method for the targeted isolation of new group members would be of great interest . Here, we present a phylogenetic approach to identify these systems rapidly in highly complex metagenomic DNA samples . To demonstrate its practical value, we located two pederin-type PKS systems putatively involved in the biosynthesis of antitumour polyketides in the metagenomic DNA of beetles, sponges and their uncultivated bacterial symbionts. Dev Dyn, 2004 Sep, 231(1), 199 - 203 Creation of a transgenic mouse for hair-cell gene targeting by using a modified bacterial artificial chromosome containing Prestin; Tian Y et al.; We made a transgenic mouse that expresses Cre recombinase activity in inner ear hair cells by using a modified bacterial artificial chromosome containing Prestin . Cre recombinase activity was restricted to inner and outer hair cells, a subset of vestibular hair cells, spiral and vestibular ganglia in the inner ear, and a subset of cells in the testis, epididymis, and ear bone . This mouse will be useful for hair-cell-specific gene targeting. Mol Cell, 2004 Aug 13, 15(3), 389 - 98 Exchange of regions between bacterial poly(A) polymerase and the CCA-adding enzyme generates altered specificities; Betat H et al.; Bacterial poly(A) polymerases (PAP) and tRNA nucleotidyltransferases are highly similar in sequence but display different activities: whereas tRNA nucleotidyltransferase catalyzes the addition of CCA to 3' ends of tRNAs, PAP adds poly(A) tails to a variety of transcripts . Using domain substitution experiments, we show that these enzymes follow a modular concept: exchange of N- and C-terminal regions leads to chimeric enzymes with unexpected activities, indicating that tRNA nucleotidyltransferase carries an "anchor domain" in the C-terminal section that restricts polymerization to three nucleotides . A 27 amino acid region was identified that determines whether poly(A) or CCA is synthesized by the enzyme chimeras . Sequence alignments suggest that the catalytic cores of both enzymes carry identical components involved in nucleotide recognition and incorporation . This seems to be the prerequisite for the observed reprogramming of the catalytic center of PAP to incorporate a sequence of defined length and composition instead of long stretches of A residues. J Gastroenterol Hepatol, 2004 Sep, 19(9), 988 - 93 Hypergastrinemia after Helicobacter pylori infection is associated with bacterial load and related inflammation of the oxyntic corpus mucosa; Chuang CH et al.; BACKGROUND AND AIM: Helicobacter pylori infection causes hypergastrinemia . This study aimed to determine the association between serum gastrin and the severity of H . pylori-related gastric histology . METHODS: A total of 458 dyspeptic patients were included in this study after the absence of gastric malignancy was confirmed using endoscopy . The gastric specimens of each patient were collected from the antrum and corpus for the analysis of H . pylori-related histology changes by updated Sydney's system . Before endoscopy, the fasting blood samples were collected for gastrin analysis . RESULTS: The H . pylori-infected patients had higher gastrin levels than those without infection (P = 0.01) . Gastrin levels were related to H . pylori density and acute and chronic inflammation scores in the corpus mucosa (P < 0.05), but not in the antral mucosa (P = NS) . Gastrin levels were also not related to the presence of gastric atrophy . Multivariate regression showed that the gastrin level was only related to acute corpus inflammation . However, in the patients without infection, the gastrin level was also associated with acute corpus inflammation . Nevertheless, the patients with denser H . pylori infection were more likely to have acute corpus gastritis than those with lighter H . pylori infection, and thus presented with higher gastrin levels (P < 0.05) . CONCLUSIONS: The increased level of gastrin of serum after H . pylori infection was associated with acute inflammation in the gastric corpus mucosa, but not in the antral mucosa . Denser H . pylori infection causes more severe corpus gastritis and thus may lead to a higher fasting level of gastrin of serum. J Environ Biol, 2004 Jan, 25(1), 93 - 8 Toxicological evaluation of bacterial decolourised anaerobically treated distillery effluent with common duckweed (Lemna minor); Chandra R et al.; Duckweed (Lemna minor) a small vascular plant, grows rapidly, is sensitive to a wide variety of toxicants and is easy to culture . A method is described that measures duckweed frond growth, chlorophyll, protein and biomass content as indicator of growth inhibition . The physico-chemical analysis of anaerobically treated distillery effluent revealed high BOD (28,000 mg/l), COD (52,400 mg/l) and dark brown colour (180,000 Co . Pt.) . This effluent showed high toxicity to Lemna minor after 96 h of exposure in laboratory condition . EC50 of the fronds for chlorophyll, protein and biomass was found to be 25%, however, the bacterial decolourised effluent showed reduction of BOD (87.50%), COD (84.50%) and colour (76%) . Further the toxicity evaluation with Lemna minor showed toxicity reduction up to 63% for all tested parameters . The EC50 noted for chlorophyll, protein and biomass was 100% concentration of decolourised effluent. Toxicon, 2004 Sep 1, 44(3), 243 - 9 No evidence for an endosymbiotic bacterial origin of tetrodotoxin in the newt Taricha granulosa; Lehman EM et al.; Tetrodotoxin (TTX) is a potent neurotoxin which is known to occur in numerous taxa, including newts . The origin of TTX is unknown, but production by symbiotic bacteria is suspected for some groups . Using PCR primers that specifically amplify 16S rRNA genes of bacteria, we examined tissues from rough-skin newts, Taricha granulosa, for the presence of bacteria which may produce TTX . No amplification of bacterial DNA was seen in samples taken from skin, liver, gonads or oviposited eggs-tissues known to contain TTX . Amplification of bacterial DNA was seen only in samples taken from newt intestines, a tissue with low concentrations of TTX . These results indicate that symbiotic bacteria are unlikely to be the source of TTX in newts. Acta Crystallogr D Biol Crystallogr, 1997 Mar, 53(Pt 2), 213 - 6 Crystallization and preliminary X-ray crystallographic and electron microscopic study of a bacterial DNA helicase (RSF1010 RepA); Roleke D; Helicases are ATP-driven enzymes essential for DNA unwinding . The broad host range plasmid RSFI010 harbours a gene (repA) encoding for one of the smallest known oligomeric helicases, RepA, a homo-hexamer with 30 kDa subunits . Electron micrographs indicate that the overall shape of RepA resembles a hexagon with globular monomers at the corners, diameter 140 A, and a central channel . Below pH 6, the molecules aggregate into tubular structures . The enzyme has been purified and crystallized using the hanging-drop vapour-diffusion method with polyethyleneglycol monomethylether as precipitating agent . The crystals exhibit the monoclinic space group P2(1) with unit-cell parameters a = 105.8, b = 180.3, c = 115.4 A, beta = 95.2 degrees, and diffract to 3.5 A resolution using rotating-anode Cu Kalpha radiation . Assuming two 180 kDa molecules per asymmetric unit, the volume per unit weight is V(m) = 3.06 A Da(-1), equivalent to a solvent content of 60% . A self-rotation search indicates that the sixfold axis of the hexamer is parallel to the ac plane and inclined at about 2 degrees to the c axis . The two hexamers are oriented head-to-head with point-group symmetry D(6). Commun Agric Appl Biol Sci, 2003, 68(2 Pt A), 141 - 5 Growth enhancement of ETBE-degrading bacterial consortium with various carbon sources; Bekri M et al.; In this study, we evaluated Ethyl tert-Butyl Ether (ETBE)-degrading consortia growths in the presence of diverse carbon sources (alcohols, alkanes, ether compounds and carbohydrates) . In a second step, we studied the consortium ability to maintain its ETBE degradation activity after growing on these carbon sources in presence or in absence of ETBE . The results indicate that the bacterial growth of ETBE-degrading consortia is enhanced three times more with addition of ethanol than with ETBE alone, while maintaining its ability to degrade ETBE . The bacterial yield growth rate was 0.504 d(-1) when growing on ETBE alone, 1.728 d(-1) on both ETBE and ethanol and 2.856 d(-1) on ethanol alone . Both ETBE and ethanol are completely degraded at 8.33 mg L(-1) h(-1) and 18.55 mg L(-1) h(-1) respectively for an initial OD of 0.4 . The frequency of ethanol addition, as growth co-substrate, was studied to preserve the ETBE-degrading capacity of the consortium, and to observe the stability of the genetic character of the ether degradation. Pediatr Infect Dis J, 2004 Aug, 23(8), 719 - 25 Topically applied sunflower seed oil prevents invasive bacterial infections in preterm infants in Egypt: a randomized, controlled clinical trial; Darmstadt GL et al.; BACKGROUND: Because the therapeutic options for managing infections in neonates in developing countries are often limited, innovative approaches to preventing infections are needed . Topical therapy with skin barrier-enhancing products may be an effective strategy for improving neonatal outcomes, particularly among preterm, low birth weight infants whose skin barrier is temporarily but critically compromised as a result of immaturity . METHODS: We tested the impact of topical application of sunflower seed oil 3 times daily to preterm infants <34 weeks gestational age at the Kasr El-Aini neonatal intensive care unit at Cairo University on skin condition, rates of nosocomial infections and mortality . RESULTS: Treatment with sunflower seed oil (n = 51) resulted in a significant improvement in skin condition (P = 0.037) and a highly significant reduction in the incidence of nosocomial infections (adjusted incidence ratio, 0.46; 95% confidence interval, 0.26-0.81; P = 0.007) compared with infants not receiving topical prophylaxis (n = 52) . There were no reported adverse events as a result of topical therapy . CONCLUSIONS: Given the low cost (approximately .20 dollars for a course of therapy) and technologic simplicity of the intervention and the effect size observed in this study, a clinical trial with increased numbers of subjects is indicated to evaluate the potential of topical therapy to reduce infections and save newborn lives in developing countries. Appl Environ Microbiol, 2004 Aug, 70(8), 4921 - 9 "Candidatus Endobugula glebosa," a specific bacterial symbiont of the marine bryozoan Bugula simplex; Lim GE et al.; The bryozoans Bugula neritina and Bugula simplex harbor bacteria in the pallial sinuses of their larvae as seen by electron microscopy . In B . neritina, the bacterial symbiont has been characterized as a gamma-proteobacterium, "Candidatus Endobugula sertula." "Candidatus E . sertula" has been implicated as the source of the bryostatins, polyketides that provide chemical defense to the host and are also being tested for use in human cancer treatments . In this study, the bacterial symbiont in B . simplex larvae was identified by 16S rRNA-targeted PCR and sequencing as a gamma-proteobacterium closely related to and forming a monophyletic group with "Candidatus E . sertula." In a fluorescence in situ hybridization, a 16S ribosomal DNA probe specific to the B . simplex symbiont hybridized to long rod-shaped bacteria in the pallial sinus of a B . simplex larva . The taxonomic status "Candidatus Endobugula glebosa" is proposed for the B . simplex larval symbiont . Degenerate polyketide synthase (PKS) primers amplified a gene fragment from B . simplex that closely matched a PKS gene fragment from the bryostatin PKS cluster . PCR surveys show that the symbiont and this PKS gene fragment are consistently and uniquely associated with B . simplex . Bryostatin activity assays and chemical analyses of B . simplex extracts reveal the presence of compounds similar to bryostatins . Taken together, these findings demonstrate a symbiosis in B . simplex that is similar and evolutionarily related to that in B . neritina. Appl Environ Microbiol, 2004 Aug, 70(8), 4848 - 54 Influence of humic substances on bacterial and viral dynamics in freshwaters; Anesio AM et al.; Bacterial and viral abundances were measured in 24 lakes with dissolved organic carbon (DOC) concentrations ranging from 3 to 19 mg of C liter(-1) . In addition, a laboratory experiment was performed to test the effects of different sources of carbon (i.e., glucose and fulvic acids) and nutrients on the dynamics of viruses and bacteria . In the lake survey, no correlation was found between virus abundance and DOC concentration, yet there was a significant positive correlation between bacterial abundance and DOC concentration . A negative correlation was found between the virus-to-bacteria ratio and DOC level . These results are in agreement with our findings in the laboratory, where virus counts were significantly lower in treatments with fulvic acid additions than in a control (mean, 67.4% +/- 6.5% of the control) . Virus counts did not differ significantly among the control and treatments with glucose, indicating that it was the type of organic carbon and not quantity which had an impact on viruses . Results from this study suggest that the way viruses control bacterial assemblages in humic lakes is different from the mechanism in clear water systems. Appl Environ Microbiol, 2004 Aug, 70(8), 4582 - 7 Enhanced arsenic accumulation in engineered bacterial cells expressing ArsR; Kostal J et al.; The metalloregulatory protein ArsR, which offers high affinity and selectivity toward arsenite, was overexpressed in Escherichia coli in an attempt to increase the bioaccumulation of arsenic . Overproduction of ArsR resulted in elevated levels of arsenite bioaccumulation but also a severe reduction in cell growth . Incorporation of an elastin-like polypeptide as the fusion partner to ArsR (ELP153AR) improved cell growth by twofold without compromising the ability to accumulate arsenite . Resting cells overexpressing ELP153AR accumulated 5- and 60-fold-higher levels of arsenate and arsenite than control cells without ArsR overexpression . Conversely, no significant improvement in Cd(2+) or Zn(2+) accumulation was observed, validating the specificity of ArsR . The high affinity of ArsR allowed 100% removal of 50 ppb of arsenite from contaminated water with these engineered cells, providing a technology useful to comply with the newly approved U.S . Environmental Protection Agency limit of 10 ppb . These results open up the possibility of using cells overexpressing ArsR as an inexpensive, high-affinity ligand for arsenic removal from contaminated drinking and ground water. Mikrobiyol Bul, 2004 Jan-Apr, 38(1-2), 27 - 32 {Determination of serologic markers against bacterial atypical pneumonia agents in pneumonia patients}; Pinar A et al.; Approximately one third of all community acquired pneumonia cases are caused by Legionella pneumophila, Mycoplasma pneumoniae and Chlamydophila pneumoniae (previously, Chlamydia pneumoniae) which are known as bacterial atypical pneumonia agents . Serological tests are used commonly for laboratory diagnosis of these agents . The aim of this study was to evaluate the causative role of bacterial atypical pneumonia agents in clinically diagnosed pneumonia patients . Acute and convalescent serum samples were collected from a total of 65 clinically diagnosed adult pneumonia patients in order to evaluate IgM and IgG positivities against L . pneumophila, M . pneumoniae and C . pneumoniae . IgM and IgG were evaluated by enzyme immunoassay (ELISA) for L . pneumophila and M . pneumoniae, and by indirect fluorescent antibody (IFA) method for C . pneumoniae . In acute serum samples, 4 (6.2%) M . pneumoniae IgM positivity in addition to 3 (4.6%) L . pneumophila IgG, 3 (4.6%) M . pneumoniae IgG and 62 (95.4%) C . pneumoniae IgG positivity were detected . In convelescent serum samples, 3 (4.6%) L . pneumophila, 1 (1.5%) M . pneumoniae, 3 (4.6%) C . pneumoniae IgM positivity and 4 (6.2%) L . pneumophila with 1 (1.5%) M . pneumoniae IgG positivity were detected in addition to acute sample positivities . According to these serological data, totally 16 (24.6%) of the patients were infected by bacterial atypical pneumonia agents . These results show that bacterial atypical pneumonia agents are important etiological factors for community acquired pneumonia. Rev Mal Respir, 2004 Jun, 21(3 Pt 1), 539 - 47 {Tobacco smoke and risk of bacterial infection}; Trosini-Desert V et al.; INTRODUCTION: Tobacco smoke is a proven risk factor for bacterial infection . STATE OF THE ART: In adults without COPD, smoking is associated with a significant increase in the relative risk (RR) of pneumonia (RR=2.97; 95% CI 1.52-5.81), S pneumoniae pneumonia (RR=2.50; 95% IC 1.50-5.10), Legionella infection (RR=3.75; 95% CI 2.17-6.17) . Smoking has clearly been shown to be associated with an increased risk of tuberculosis (RR=2.60; 95% CI 2,20-3,20), and also with increased incidence of post-operative infections . In young children whose parents smoke, passive exposure to tobacco smoke is associated with an increased relative risk of seasonal infections (RR=1.7; CI 95% 1.55-1.91) and recurrent otitis media (RR=1.48; 95% CI 1.08-2.04) . Passive smoking also increases risk of pneumonia in adults (RR=2.5; CI 95% 1.2-5.1) . Plausible explanations of the increased risk of infection in active or passive smokers include increased bacterial adherence, decrease of lung and nasal clearance, and changes in the immune response . CONCLUSIONS: Exposure to tobacco smoke approximately doubles the risk of infection . This increased burden of infection has significant healthcare cost implications . Each infectious episode in an individual should prompt an attempt at smoking cessation. Cytometry, 2004 Aug, 60A(2), 165 - 72 Green fluorescent protein-propidium iodide (GFP-PI) based assay for flow cytometric measurement of bacterial viability; Lehtinen J et al.; BACKGROUND: Several staining protocols have been developed for flow cytometric analysis of bacterial viability . One promising method is dual staining with the LIVE/DEAD BacLight bacterial viability kit . In this procedure, cells are treated with two different DNA-binding dyes (SYTO9 and PI), and viability is estimated according to the proportion of bound stain . SYTO9 diffuses through the intact cell membrane and binds cellular DNA, while PI binds DNA of damaged cells only . This dual-staining method allows effective separation between viable and dead cells, which is far more difficult to achieve with single staining . Although SYTO9-PI dual staining is practical for various bacterial viability analyses, the method has a number of disadvantages . Specifically, the passage of SYTO9 through the cell membrane is a slow process, which is significantly accelerated when the integrity of the cell membrane is disrupted . As a result, SYTO9 binding to DNA is considerably enhanced . PI competes for binding sites with SYTO9 and may displace the bound dye . These properties diminish the reliability of the LIVE/DEAD viability kit . In this study, we investigate an alternative method for measuring bacterial viability using a combination of green fluorescent protein (GFP) and PI, with a view to improving data reliability . METHODS: Recombinant Escherichia coli cells with a plasmid containing the gene for jellyfish GFP were stained with PI, and green and red fluorescence were measured by FCM . For comparison, cells containing the plasmid from which gfp was removed were stained with SYTO9 and PI, and analyzed by FCM . Viability was estimated according to the proportion of green and red fluorescence . In addition, bioluminescence and plate counting (other methods to assess viability) were used as reference procedures . RESULTS: SYTO9-PI dual staining of bacterial cells revealed three different cell populations: living, compromised, and dead cells . These cell populations were more distinct when the GFP-PI combination was used instead of dual staining . No differences in sensitivity were observed between the two methods . However, substitution of SYTO9 with GFP accelerated the procedure . Bioluminescence and plate counting results were in agreement with flow cytometric viability data . CONCLUSIONS: In bacterial viability analyses, the GFP-PI combination provided better distinction between current viability stages of E . coli cells than SYTO9-PI dual staining . Additionally, the overall procedure was more rapid . No marked differences in sensitivity were observed. Emerg Radiol, 2002 Dec, 9(6), 317 - 9 Epub 2002 Nov 30. Bacterial aortitis resulting in rapid development of infective "mycotic" aneurysm; Shu KE et al.; Aortitis and aneurysms with an infective etiology, though uncommon, must be recognized at imaging so that early medical and surgical intervention can improve survival . We report a case in which serial CT scans illustrate the short-interval development of an infective "mycotic" aneurysm resulting from bacterial aortitis. Nucleic Acids Res, 2004 Aug 02, 32(13), 4071 - 80 Print 2004. Cleavage of deoxyoxanosine-containing oligodeoxyribonucleotides by bacterial endonuclease V; Hitchcock TM et al.; Oxanine (O) is a deamination product derived from guanine with the nitrogen at the N1 position substituted by oxygen . Cytosine, thymine, adenine, guanine as well as oxanine itself can be incorporated by Klenow Fragment to pair with oxanine in a DNA template with similar efficiency, indicating that oxanine in DNA may cause various mutations . As a nucleotide, deoxyoxanosine may substitute for deoxyguanosine to complete a primer extension reaction . Endonuclease V, an enzyme known for its enzymatic activity on uridine-, inosine- and xanthosine-containing DNA, can cleave oxanosine-containing DNA at the second phosphodiester bond 3' to the lesion . Mg2+ or Mn2+, and to a small extent Co2+ or Ni2+, support the oxanosine-containing DNA cleavage activity . All four oxanosine-containing base pairs (A/O, T/O, C/O and G/O) were cleaved with similar efficiency . The cleavage of double-stranded oxanosine-containing DNA was approximately 6-fold less efficient than that of double-stranded inosine-containing DNA . Single-stranded oxanosine-containing DNA was cleaved with a lower efficiency as compared with double-stranded oxanosine-containing DNA . A metal ion enhances the binding of endonuclease V to double-stranded and single-stranded oxanosine-containing DNA 6- and 4-fold, respectively . Hypothetic models of oxanine-containing base pairs and deaminated base recognition mechanism are presented. Microbiology, 2004 Aug, 150(Pt 8), 2497 - 502 Cyclic di-GMP as a bacterial second messenger; D'Argenio DA et al.; Environmental signals trigger changes in the bacterial cell surface, including changes in exopolysaccharides and proteinaceous appendages that ultimately favour bacterial persistence and proliferation . Such adaptations are regulated in diverse bacteria by proteins with GGDEF and EAL domains . These proteins are predicted to regulate cell surface adhesiveness by controlling the level of a second messenger, the cyclic dinucleotide c-di-GMP . Genetic evidence suggests that the GGDEF domain acts as a nucleotide cyclase for c-di-GMP synthesis while the EAL domain is a good candidate for the opposing activity, a phosphodiesterase for c-di-GMP degradation. Trends Biochem Sci, 2004 Aug, 29(8), 392 - 5 BTLCP proteins: a novel family of bacterial transglutaminase-like cysteine proteinases; Ginalski K et al.; Using sequence similarity searches and top-of-the-range fold-recognition methods, we have identified a novel family of bacterial transglutaminase-like cysteine proteinases (BTLCPs) with an invariant Cys-His-Asp catalytic triad and a predicted N-terminal signal sequence . This family of previously uncharacterized hypothetical proteins encompasses sequences of unknown function from DUF920 (in the Pfam database) and COG3672 . BTLCPs are predicted to possess the papain-like cysteine proteinase fold and catalyze post-translational protein modification through transamidase, acetylase or hydrolase activity . Inspection of neighboring genes encoding BTLCPs suggests a link between this predicted activity and a type-I secretion system resembling ATP-binding cassette exporters of toxins and proteases involved in bacterial pathogenicity. Genome, 2004 Aug, 47(4), 764 - 8 Segmental duplications within the Glycine max genome revealed by fluorescence in situ hybridization of bacterial artificial chromosomes; Pagel J et al.; Soybean (Glycine max L . Merr.) is presumed to be an ancient polyploid based on chromosome number and multiple RFLP fragments in genetic mapping . Direct cytogenetic observation of duplicated regions within the soybean genome has not heretofore been reported . Employing fluorescence in situ hybridization (FISH) of genetically anchored bacterial artificial chromosomes (BACs) in soybean, we were able to observe that the distal ends of molecular linkage group E had duplicated regions on linkage groups A2 and B2 . Further, using fiber-FISH, it was possible to measure the molecular size and organization of one of the duplicated regions . As FISH did not require repetitive DNA for blocking fluorescence signals, we assume that the 200-kb genome region is relatively low in repetitive sequences . This observation, along with the observation that the BACs are located in distal euchromatin regions, has implications for genome structure/evolution and the approach used to sequence the soybean genome. Am J Obstet Gynecol, 2004 Jun, 190(6), 1582 - 7; discussion 1587-9 Fetal fibronectin and bacterial vaginosis are associated with preterm birth in women who are symptomatic for preterm labor; Stevens AO et al.; OBJECTIVE: The purpose of this study was to codify the relationship between bacterial vaginosis/fetal fibronectin and preterm labor/birth . STUDY DESIGN: In this prospective study, 185 women who were symptomatic for preterm labor were assessed for bacterial vaginosis and fetal fibronectin . RESULTS: These women comprised 4 groups: group A (n=23 women; +bacterial vaginosis/+fetal fibronectin); group B (n=31 women; -bacterial vaginosis/+fetal fibronectin); group C (n=47 women; +bacterial vaginosis/-fetal fibronectin); and group D (n=84 women; -bacterial vaginosis/-fetal fibronectin) . The time interval from gestational age at testing until delivery was significantly shorter for groups A and B versus groups C and D (P < or =.05 and P <.001, respectively) . Similarly, delivery at <32 weeks of gestation was increased in group B (26%) compared with groups A (9%), C (2%), and D (5%; P <.009; odds ratio, 165.90; 95% CI, 30.02, 916.08) . CONCLUSION: Women who are symptomatic for preterm labor should be considered for fetal fibronectin and bacterial vaginosis testing. Am J Obstet Gynecol, 2004 Jun, 190(6), 1504 - 8; discussion 3A A polymorphism in the promoter region of TNF and bacterial vaginosis: preliminary evidence of gene-environment interaction in the etiology of spontaneous preterm birth; Macones GA et al.; OBJECTIVE: The rarer of 2 alleles of a polymorphism in the promoter of the tumor necrosis factor alpha gene (TNF) has been associated with spontaneous preterm birth following preterm premature rupture of the fetal membranes in some populations . The aim of this study was to assess if the presence of symptomatic bacterial vaginosis amplifies the risk of spontaneous preterm birth in those with a "susceptible" TNF genotype . STUDY DESIGN: A case-control study was performed at our institution . Cases (n=125) were defined as women who delivered before 37 weeks as a result of ruptured membranes or preterm labor, while control subjects (n=250) were defined as women who delivered after 37 weeks . DNA was collected from maternal blood and analyzed for the TNF genotype . Information on symptomatic bacterial vaginosis and other risk factors for preterm birth was obtained by review of the antenatal record . Multiple logistic regression was also used to test the interaction between bacterial vaginosis, the TNF genotype, and preterm birth . RESULTS: Maternal carriers of the rarer allele (TNF-2) were at a significantly increased risk of spontaneous preterm birth {odds ratio (OR) 2.7, 95% CI 1.7-4.5} . The association between TNF-2 and preterm birth was modified by the presence of bacterial vaginosis, such that those with a "susceptible" genotype and bacterial vaginosis had increased odds of preterm birth compared with those who did not (OR 6.1, 95% CI 1.9-21.0) . CONCLUSION: This study provides preliminary evidence that an interaction between genetic susceptibilities (ie, TNF-2 carriers) and environmental factors (ie, bacterial vaginosis) is associated with an increased risk of spontaneous preterm birth. Biochim Biophys Acta, 2004 Jul 23, 1658(1-2), 50 - 7 Probing light-induced conformational transitions in bacterial photosynthetic reaction centers embedded in trehalose-water amorphous matrices; Francia F et al.; The coupling between electron transfer and protein dynamics has been studied in photosynthetic reaction centers (RC) from Rhodobacter sphaeroides by embedding the protein into room temperature solid trehalose-water matrices . Electron transfer kinetics from the primary quinone acceptor (Q(A)(-)) to the photoxidized donor (P(+)) were measured as a function of the duration of photoexcitation from 20 ns (laser flash) to more than 1 min . Decreasing the water content of the matrix down to approximately 5x10(3) water molecules per RC causes a reversible four-times acceleration of P(+)Q(A)(-) recombination after the laser pulse . By comparing the broadly distributed kinetics observed under these conditions with the ones measured in glycerol-water mixtures at cryogenic temperatures, we conclude that RC relaxation from the dark-adapted to the light-adapted state and thermal fluctuations among conformational substates are hindered in the room temperature matrix over the time scale of tens of milliseconds . When the duration of photoexcitation is increased from a few milliseconds to the second time scale, recombination kinetics of P(+)Q(A)(-) slows down progressively and becomes less distributed, indicating that even in the driest matrices, during continuous illumination, the RC is gaining a limited conformational freedom that results in partial stabilization of P(+)Q(A)(-) . This behavior is consistent with a tight structural and dynamical coupling between the protein surface and the trehalose-water matrix. Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1151 - 6 Identification of the bacterial endosymbionts of the marine ciliate Euplotes magnicirratus (Ciliophora, Hypotrichia) and proposal of 'Candidatus Devosia euplotis'; Vannini C et al.; This paper reports the identification of bacterial endosymbionts that inhabit the cytoplasm of the marine ciliated protozoon Euplotes magnicirratus . Ultrastructural and full-cycle rRNA approaches were used to reveal the identity of these bacteria . Based on analysis of 16S rRNA gene sequences, evolutionary trees were constructed; these placed the endosymbiont in the genus Devosia in the alpha-Proteobacteria . The validity of this finding was also shown by fluorescence in situ hybridization with a Devosia-specific oligonucleotide probe . Differences at the 16S rRNA gene level (which allowed the construction of a species-specific oligonucleotide probe) and the peculiar habitat indicate that the endosymbiont represents a novel species . As its cultivation has not been successful to date, the provisional name 'Candidatus Devosia euplotis' is proposed . The species- and group-specific probes designed in this study could represent convenient tools for the detection of 'Candidatus Devosia euplotis' and Devosia-like bacteria in the environment. Heart Vessels, 2004 Jul, 19(4), 179 - 82 Mid-term results of mitral valve repair for complicated active bacterial endocarditis in high-risk patients; Cebi N et al.; Mitral valve repair in endocarditis achieves a competent valve and prevents septic embolization and acute left ventricular failure, in which operative mortality could be increased . Early and mid-term results were examined to establish whether emergency mitral valve repair offers an advantage in complicated active endocarditis . Ten patients with complicated active native valve endocarditis underwent mitral valve repair . The mean age was 45.8 +/- 18.5 years; two patients were female (20%) . All patients had severe mitral regurgitation, which combined in one patient with mitral valve stenosis . New York Heart Association (NYHA) functional class was IV in all patients . The macroscopically infected tissue with vegetation in all patients was excised . Multiple techniques were required to achieve valve competence . There was one (10%) hospital death in a patient with persistent congestive heart failure, and a reoperation in another (10%) after 2 years . Mean follow-up was 32.1 +/- 12.7 months (range 1-45 months) and was complete . There were no late deaths, recurrent endocarditis, or thromboembolic events . Seven patients (77.7%) were in NYHA functional class I, and two (22.2%) were in class II . Mitral valve repair in complicated active bacterial endocarditis limited to leaflet tissues has a low operative mortality and valve-related morbidity, with promising mid-term survival in high-risk patients. Res Vet Sci, 2004 Dec, 77(3), 189 - 95 Detection of bacterial DNA in synovial fluid from horses with infectious synovitis; Pille F et al.; Standard culturing techniques are often unrewarding in confirming diagnosis of synovial infection in the equine patient . Several human studies report the use of sensitive polymerase chain reaction (PCR) techniques for the detection of bacterial involvement in acute synovitis . However, successful extraction of bacterial DNA directly from clinical samples from horses without prior culture has not been reported yet . The goal of this study was to develop a sensitive and reliable method for molecular detection and identification of bacterial species in synovial fluid from horses with infectious synovitis . Synovial fluid samples from 6 horses with culture confirmed synovial infection were used for broad range 16S rRNA gene PCR . Synovial aspirates of 2 healthy horses were used as negative controls . Following extraction and purification of synovial fluid DNA, all samples were processed by touchdown PCR . Amplicons were detected by reverse line blot hybridisation and visualised with chemiluminescence . Pathogen-specific detection of 16S rRNA gene sequences was successful in all 6 synovial fluid samples . No bacterial DNA was detected in the aspirates from the negative control horses using touchdown PCR followed by 25 additional cycles of amplification . The identity of the pathogens was confirmed by DNA sequencing of the amplicons . It can be concluded that broad range 16S rRNA gene PCR followed by reverse line blot hybridisation is a promising technique for detection of bacterial DNA in synovial fluid samples . Further research should aim at the detection of bacterial DNA in synovial fluid samples suspected of infection but having negative culture results . When the 16S PCR proves to be reliable and more sensitive than standard culturing techniques, it may become a powerful tool in the diagnosis of synovial infection. Trends Microbiol, 2004 Aug, 12(8), 366 - 72 Bacterial linguistic communication and social intelligence; Ben Jacob E et al.; Bacteria have developed intricate communication capabilities (e.g . quorum-sensing, chemotactic signaling and plasmid exchange) to cooperatively self-organize into highly structured colonies with elevated environmental adaptability . We propose that bacteria use their intracellular flexibility, involving signal transduction networks and genomic plasticity, to collectively maintain linguistic communication: self and shared interpretations of chemical cues, exchange of chemical messages (semantic) and dialogues (pragmatic) . Meaning-based communication permits colonial identity, intentional behavior (e.g . pheromone-based courtship for mating), purposeful alteration of colony structure (e.g . formation of fruiting bodies), decision-making (e.g . to sporulate) and the recognition and identification of other colonies - features we might begin to associate with a bacterial social intelligence . Such a social intelligence, should it exist, would require going beyond communication to encompass unknown additional intracellular processes to generate inheritable colonial memory and commonly shared genomic context. Biomaterials, 2005 Feb, 26(4), 419 - 31 Bacterial cellulose as a potential scaffold for tissue engineering of cartilage; Svensson A et al.; Tissue constructs for cartilage with native mechanical properties have not been described to date . To address this need the bacterial cellulose (BC) secreted by Gluconacetobacter xylinus (= Acetobacter xylinum) was explored as a novel scaffold material due to its unusual material properties and degradability . Native and chemically modified BC materials were evaluated using bovine chondrocytes . The results indicate that unmodified BC supports chondrocyte proliferation at levels of approximately 50% of the collagen type II substrate while providing significant advantages in terms of mechanical properties . Compared to tissue culture plastic and calcium alginate, unmodified BC showed significantly higher levels of chondrocyte growth . Chemical sulfation and phosphorylation of the BC, performed to mimic the glucosaminoglycans of native cartilage, did not enhance chondrocyte growth while the porosity of the material did affect chondrocyte viability . The BC did not induce significant activation of proinflammatory cytokine production during in vitro macrophage screening . Hence, unmodified BC was further explored using human chondrocytes . TEM analysis and RNA expression of the collagen II from human chondrocytes indicated that unmodified BC supports proliferation of chondrocytes . In addition, ingrowth of chondrocytes into the scaffold was verified by TEM . The results suggest the potential for this biomaterial as a scaffold for tissue engineering of cartilage. Cell Microbiol, 2004 Sep, 6(9), 849 - 65 Simultaneous analysis of host and pathogen interactions during an in vivo infection reveals local induction of host acute phase response proteins, a novel bacterial stress response, and evidence of a host-imposed metal ion limited environment; Motley ST et al.; A fundamental goal in the study of infections is to understand the dynamic interplay between host and pathogen; however, direct in vivo interrogation of this disease process via transcriptional profiling has been lacking . Here we describe the development and application of novel bacterial RNA amplification technology to simultaneously identify key elements of both host and pathogen responses in a murine infection model . On the bacterial side, we found induction of an unusual pattern of stress response genes, a response to host-induced metal ion limitation, and a failure to achieve stationary phase in vivo . On the mammalian side, we observed the surprising induction of several genes encoding acute phase response proteins including hepcidin, haptoglobin, complement C3 and metallothionein 1 at the site of infection, as well as other mediators of innate immunity . Thus, our results reveal host-pathogen cross-talk not predicted by previous in vitro analyses and provide the framework to eavesdrop on a broad array of host-pathogen interactions in vivo . As described here, the comprehensive examination of host-pathogen interactions during an infection is critical to the discovery of novel approaches for intervention not predicted by current models. Acta Crystallogr D Biol Crystallogr, 2004 Aug, 60(Pt 8), 1429 - 31 Epub 2004 Jul 21. Expression, crystallization and crystallographic analysis of DegS, a stress sensor of the bacterial periplasm; Grininger M et al.; Regulated proteolysis is a key event in transmembrane signalling between intercellular compartments . In Escherichia coli, a protein DegS has been identified as being a periplasmic stress sensor for unfolded or misfolded outer membrane proteins (OMPs) . Activation of DegS initiates a proteolytic cascade which results in the transcription of periplasmic genes under sigmaE control, most importantly chaperones and proteases . DegS has been cloned and expressed as full-length protein and in an N-terminally truncated form . Both proteins were tested for crystallization and two forms of well diffracting crystals of the truncated form were obtained . Crystals of form I diffract to 3.5 A and belong to space group P2(1)3, while crystals of form II diffract to 2.2 A and belong to space group I23 . Crystals of form II were soaked with a consensus peptide representing the C-termini of outer membrane proteins and data to 2.4 A resolution were collected . Molecular-replacement trials using a homologous protease domain indicate the presence of two molecules in the asymmetric unit of crystal form I . The correctness of the molecular-replacement solution was verified by identifying radiation-damage-induced structural changes. Biotechnol Lett, 2004 Jun, 26(12), 1007 - 11 Toxicity and degradation of metal-complexed cyanide by a bacterial consortium under sulfate-reducing conditions; Quan ZX et al.; Free cyanide at 1 mm decreased the initial sulfate reduction rate of a batch culture of granular sludge from 0.3 to 0.14 mmol d(-1) g(-1) SS (suspended solid), whereas 0.5 mm cyanide had a minimal effect (0.25 mmol d(-1) g(-1) SS) . The order of toxicity of metal-complexed cyanides to the sludge was as follows: zinc-complexed cyanide (most toxic) > free cyanide = nickel-complexed cyanide > copper-complexed cyanide (least toxic), which also corresponds well with the order of the stability (dissociation) constants of the metal-cyanide complexes . A consortium degrading cyanide was enriched using nickel cyanide as the sole nitrogen source . This consortium completely removed 0.5 mm of nickel-complexed cyanide under sulfate-reducing conditions in 11 d . Analysis of clone library of 16S rRNA genes shows that the consortium was composed of three major phylotypes including Desulfovibrio. J Biol Chem, 2004 Sep 24, 279(39), 40470 - 4 Epub 2004 Jul 20. Demethylation of 3-methylthymine in DNA by bacterial and human DNA dioxygenases; Koivisto P et al.; Rare DNA lesions that are chemically stable and refractory to repair may add disproportionately to the accumulation of mutations in long lived cells . 3-Methylthymine is a minor lesion that is induced by DNA-methylating agents and for which no repair process has been described previously . Here we demonstrate that this lesion can be directly demethylated in vitro by bacterial and human DNA dioxygenases . The Escherichia coli AlkB and human ABH3 proteins repaired 3-methylthymine in both single-stranded and double-stranded polydeoxynucleotides, whereas the human ABH2 protein preferred a duplex substrate . Thus, the known substrates of these enzymes now include 3-methylthymine in DNA, as well as 1-methyladenine and 3-methylcytosine, which all have structurally similar sites of alkylation . Repair of 3-methylthymine by AlkB and ABH3 was optimal at pH 6, but inefficient . At physiological pH, 3-methylthymine, which is a minor methylated lesion, was more slowly repaired than the major lesion generated in single-stranded DNA, 3-methylcytosine . Our data suggest that 3-methylthymine residues in DNA will be repaired inefficiently in vivo and therefore may occur at a low steady-state level, but the residues should not gradually accumulate to high levels in long lived cells . Nucl Med Commun, 2004 Aug, 25(8), 819 - 23 Dual time point 2-{18F}fluoro-2'-deoxyglucose positron emission tomography in chronic bacterial osteomyelitis; Sahlmann CO et al.; OBJECTIVE: Quantitative dual time point imaging with {F}fluorodeoxyglucose positron emission tomography (F-FDG PET) has recently been found to be more accurate than single time point scanning in the discrimination between benign lesions and malignancy in various conditions . In our study we investigated glucose metabolism in chronic bacterial osteomyelitis (COM) by using F-FDG PET and a dual time protocol . METHODS: Seventeen non-diabetic patients with histopathologically proven COM and four non-diabetic patients with malignant bone disease were prospectively investigated with dual time F-FDG PET . All lesions were detected by their increased F-FDG uptake 30 and 90 min after injection of 370 MBq of F-FDG . The maximum and mean lesional standardized uptake values (SUV(max) and SUV(mean) after 30 and 90 min were determined . RESULTS: The median SUV(max) and SUV(mean) values of all osteomyelitic lesions at 30 min were 1.85 (range, 0.45-3.45) and 1.1 (range, 0.21-1.99), respectively . The median SUV(max) and SUV(mean) values of all malignant lesions at 30 min were 3.19 (range, 2.31-4.7) and 2.82 (range, 2.4-3.71), respectively . At 90 min the median SUV(max) and SUV(mean) of all osteomyelitic lesions were 1.78 (range, 0.4-2.93) and 1.1 (range, 0.18-1.72), respectively . At the same time point the median SUV(max) and SUV(mean) of all malignant lesions were 4.1 (range, 3.52-5.32) and 3.34 (range, 2.81-4.12), respectively . In osteomyelitis the SUV(max) and SUV(mean) between 30 and 90 min post-injection remained stable or decreased in 16/17 patients . In these patients a median decrease of 6% for SUV(max) (range, 1-31%) and a median decrease of 8.5% for SUV(mean) (range, 0-24%) was observed . Changes of SUV(max) and SUV(mean) between 30 and 90 min were highly significant (P<0.05) . In one patient SUV(max) and SUV(mean) increased over the time . The histology of this patient revealed multiple foreign body granulomas in addition to a mononuclear infiltrate . In malignant lesions the SUV(max) and SUV(mean) between 30 and 90 min post-injection increased . CONCLUSION: Our preliminary results indicate that dynamic dual time point F-FDG PET provides a characteristic pattern in chronic osteomyelitis similar to inflammatory processes in other locations . This pattern may be of value in the differentiation between COM and malignant bone lesions. J Biol Chem, 2004 Oct 22, 279(43), 45162 - 74 Epub 2004 Jul 20. Crystal structure of a bacterial type III polyketide synthase and enzymatic control of reactive polyketide intermediates; Austin MB et al.; In bacteria, a structurally simple type III polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthlene synthase (THNS) catalyzes the iterative condensation of five CoA-linked malonyl units to form a pentaketide intermediate . THNS subsequently catalyzes dual intramolecular Claisen and aldol condensations of this linear intermediate to produce the fused ring tetrahydroxynaphthalene (THN) skeleton . The type III PKS-catalyzed polyketide extension mechanism, utilizing a conserved Cys-His-Asn catalytic triad in an internal active site cavity, is fairly well understood . However, the mechanistic basis for the unusual production of THN and dual cyclization of its malonyl-primed pentaketide is obscure . Here we present the first bacterial type III PKS crystal structure, that of Streptomyces coelicolor THNS, and identify by mutagenesis, structural modeling, and chemical analysis the unexpected catalytic participation of an additional THNS-conserved cysteine residue in facilitating malonyl-primed polyketide extension beyond the triketide stage . The resulting new mechanistic model, involving the use of additional cysteines to alter and steer polyketide reactivity, may generally apply to other PKS reaction mechanisms, including those catalyzed by iterative type I and II PKS enzymes . Our crystal structure also reveals an unanticipated novel cavity extending into the "floor" of the traditional active site cavity, providing the first plausible structural and mechanistic explanation for yet another unusual THNS catalytic activity: its previously inexplicable extra polyketide extension step when primed with a long acyl starter . This tunnel allows for selective expansion of available active site cavity volume by sequestration of aliphatic starter-derived polyketide tails, and further suggests another distinct protection mechanism involving maintenance of a linear polyketide conformation. Transfusion, 2004 Aug, 44(8), 1166 - 73 Detection of bacterial contamination of platelet components: six years' experience with the BacT/ALERT system; Munksgaard L et al.; BACKGROUND: Hemovigilance has shown that bacteria cause more fatalities than other infections together . Surveillance for detection of bacteria in platelets (PLTs) was initiated . Concomitantly, the storage period for PLTs was extended from 5 to 7 days to reduce cost . STUDY DESIGN AND METHODS: Analysis was performed of all cases of a positive signal in a screening procedure for contaminated PLTs taking into account results of confirmative cultures and results of culture from blood components including bacteria strains . Records were assessed from patients transfused with blood components issued before the screening culture became positive . RESULTS: Samples were collected from 22,057 PLT units . An initial reaction was seen in 84 (0.38%) . Growth was confirmed in 70 of these . Of the associated PLT units, 26 had been issued or outdated at the time when the culture was found to be reactive, in 27 bacteria were found, and in 17 cultures were negative . The bacteria found were mainly from normal skin flora . Sixty-six patients received 75 blood components issued before the screening system alarmed . None of these patients had a transfusion reaction reported . The outdating fell to less than 5 percent . CONCLUSION: A screening system for detection of bacterial contamination was implemented without increase in cost owing to extension of storage time to 7 days . Transfusion of several contaminated blood components was prevented. Prostate, 2004 Sep 1, 60(4), 282 - 8 Application of serum PSA to identify acute bacterial prostatitis in patients with fever of unknown origin or symptoms of acute pyelonephritis; Hara N et al.; BACKGROUND: Exclusion of prostatitis in screening for prostate cancer (Cap) is a matter of concern in the prostate-specific antigen (PSA) era . Yet, the identification of acute bacterial prostatitis (ABP), intentionally utilizing PSA in patients with pyrexia has been scarcely reported . METHODS: In total, 39 men, who presented at our department with a fever higher than 38.3 degrees C, were randomly selected . We investigated the fraction of patients who had serum PSA levels higher than 4.0 ng/ml and categorized them according to an initial diagnosis of pyelonephritis, ABP, other urogenital infections, and fever of unknown origin (FUO) . RESULTS: Six of nine cases initially diagnosed as pyelonephritis, presented with elevated PSA levels between 9.5 and 75.1 ng/ml . All six cases of clinically diagnosed prostatitis had PSA elevated between 4.1 and 13.6 ng/ml . In 8 of 18 FUO cases, PSA was elevated between 5.1 and 77.0 ng/ml . PSA levels significantly correlated with age (P < 0.005) . All 20 patients with elevated PSA received antibiotics, and serum PSA was significantly reduced in all cases (P < 0.001) together with the alleviation of fever and normalization of CRP . CONCLUSIONS: PSA is a prompt and steady diagnostic tool for identifying ABP that might be missed or misdiagnosed . We recommend the measurement of PSA in cases not only with urologic infection but also puzzling pyrexia. Turk J Gastroenterol, 2004 Mar, 15(1), 34 - 8 Comparison of cefotaxime and ofloxacin in treatment of spontaneous bacterial peritonitis; Taskiran B et al.; BACKGROUND/AIMS: Gold-standard treatment of spontaneous bacterial peritonitis currently involves 3rd generation cephalosporins . To evaluate the efficacy of ofloxacin in this infection, we compared a combined therapy with intravenous and oral ofloxacin to intravenous cefotaxime . METHODS: Thirty cirrhotic patients with spontaneous bacterial peritonitis were assigned to receive either intravenous (1 g/12 h) cefotaxime for 7 days (n=17) or intravenous (200 mg/12 h) ofloxacin for 2 days followed by oral (200 mg/12 h) ofloxacin for 5 days (n=13) . All cases had community-acquired spontaneous bacterial peritonitis . RESULTS: The infection resolution rate on the 7th day of therapy was 82.4% in the cefotaxime group and 92.3% in the ofloxacin group . Hospital survival rates were 82.4% and 100%, respectively . CONCLUSIONS: Oral ofloxacin after a short course of intravenous ofloxacin is effective in the treatment of uncomplicated spontaneous bacterial peritonitis . This regimen may allow physicians to treat these patients as outpatients as soon as their intravenous therapy is completed. Int Rev Cytol, 2004, 236, 181 - 249 Bacterial endocytobionts of ciliophora and their interactions with the host cell; Fokin SI; Ciliates may be hosts for numerous bacteria, which can occupy almost all cellular compartments of the protists . About 200 ciliate species are recorded as hosts of different intracellular bacteria, being a small part of the diversity for such types of endocytobiosis in nature . In the Paramecium genus alone close to 60 types of bacteria adapted for intracellular life are known . In this review extensive material concerning the variety of endocytobionts, their categories, and their interaction with host cells is presented . Special attention is paid to endocytobiosis in Paramecium with highly infectious bacteria Holospora, bacteria of the Caedibacter and Polynucleobacter genera, methanogenic bacteria, and "xenosomes" as well as to life cycles and strategies of bacterial endonucleobionts . The above model bacteria and their interactions with hosts have not been exhaustively studied . A number of unsolved problems concerning their interactions within an endocytobiotic system and their ecological implications remain to be studied. Ecotoxicol Environ Saf, 2004 Sep, 59(1), 127 - 32 Effect of fungicide iprodione on soil bacterial community; Wang YS et al.; The effect of the fungicide iprodione on soil bacterial communities was studied by treating two kinds of soils with different concentrations of iprodione . Degradation rates of iprodione in sterile and unsterile soils were also investigated . Residues of iprodione were measured by using high-performance liquid chromatography (HPLC) and a change of bacterial communities was performed with denaturing gradient gel electrophoresis (DGGE) by counting the 16S rDNA band on DGGE patterns . The degradation rate of iprodione was slower in sterile soil than in unsterile soil in both Da-Hu sandy loam and Kuan-Shi loam . After treatment with fungicide, soil bacterial communities were changed and recovered rapidly to the original status when incubated at a lower temperature (15 degrees C) and a lower iprodione concentration (5 microg/g) . At the same temperature but with more iprodione (50 microg/g) added, the soil bacterial community increases slowly and regains the original status slowly . However, when incubated at the higher temperature (30 degrees C), the soil bacterial community is more complex than that at the lower temperature . The response of the soil bacterial community to the iprodione is faster at the higher than at the lower temperature . At 30 degrees C and with 50 microg/g iprodione, the amounts of soil bacterial communities increased quickly but cannot be reduced to the original status after incubation for 23 days . Traffic, 2004 Aug, 5(8), 561 - 70 Chlamydia--host cell interactions: recent advances on bacterial entry and intracellular development; Dautry-Varsat A et al.; Bacteria of the Chlamydiales order are very successful intracellular organisms that grow in human and animal cells, and even in amoebae . They fulfill several essential functions to enter their host cells, establish an intracellular environment favorable for their multiplication and exit the host cell . They multiply in a unique organelle called the inclusion, which is isolated from the endocytic but not the exocytic pathway . A combination of host cell factors and of proteins secreted by the bacteria, from within the inclusion, contribute to the establishment and development of this inclusion . Here we review recent data on the entry mechanisms and maturation of the inclusion . Indian J Exp Biol, 2004 Jun, 42(6), 558 - 61 Immune haemolymph proteins in response to bacterial infection and identification of a putative bacteria binding protein in malaria vector Anopheles stephensi; Dixit RK et al.; Induction of haemolymph proteins in mosquito A . stephensi due to wounding or bacterial infection (E . coli) was analyzed using SDS-PAGE . Wounding response of pupa revealed subsequent induction of two polypeptides (21 and 74 kDa) . Two other polypeptides (44 and 57 kDa) were induced commonly in both pupa and adult female haemolymph upon bacterial infection . In vitro binding assay revealed identification of 44 kDa, a putative bacterial binding protein, a more relevant protein for further elucidation of molecular mechanism involved in host parasite interactions. World J Gastroenterol, 2004 Aug 1, 10(15), 2228 - 31 Early diagnosis of bacterial and fungal infection in chronic cholestatic hepatitis B; Wu XZ et al.; AIM: To investigate the early diagnostic methods of bacterial and fungal infection in patients with chronic cholestatic hepatitis B . METHODS: One hundred and one adult in-patients with chronic hepatitis B were studied and divided into 3 groups: direct bilirubin (DBil)/total bilirubin (TBil) > or = 0.5, without bacterial and fungal infection (group A, n=38); DBil/TBil <0.5, without bacterial and fungal infection (group B, n=23); DBil/TBil> or = 0.5, with bacterial or fungal infection (group C, n=40) . The serum biochemical index and pulse rate were analyzed . RESULTS: Level of TBil, DBil, alkaline phosphatase (ALP) and DBil/ALP in group A increased compared with that in group B . The level of ALP in group C decreased compared with that in group A, whereas the level of TBil, DBil and DBil/ALP increased (ALP: 156+/-43, 199+/-68, respectively, P<0.05; TBil: 370+/-227, 220+/-206, respectively, P<0.01; DBil: 214+/-143, 146+/-136, respectively, P<0.01; DBil/ALP: 1.65+/-1.05, 0.78+/-0.70, respectively, P<0.001) . The level of DBil and infection affected DBil/ALP . Independent of the effect of DBil, infection caused DBil/ALP to rise (P<0.05) . The pulse rate in group A decreased compared with that in group B (63.7+/-6.4, 77.7+/-11.4, respectively, P<0.001), and the pulse rate in group C increased compared with that in group A (81.2+/-12.2, 63.7+/-6.4, respectively, P<0.001) . The equation (infection=0.218 pusle rate +1.064 DBil/ALP -16.361), with total accuracy of 85.5%, was obtained from stepwise logistic regression . Pulse rate (> or =80/min) and DBil/ALP (> or =1.0) were used to screen infection . The sensitivity was 62.5% and 64.7% respectively, and the specificity was 100% and 82.8% respectively . CONCLUSION: Bacterial and fungal infection deteriorate jaundice and increase pulse rate, decrease serum ALP and increase DBil/ALP . Pulse rate, DBil/ALP and the equation (infection=0.218 pusle rate+1.064 DBil/ALP-16.361) are helpful to early diagnosis of bacterial and fungal infection in patients with chronic cholestatic hepatitis B. Shock, 2004 Aug, 22(2), 180 - 5 N(omega)-nitro-L-arginine methyl ester effects on neutrophil function and bacterial clearance; Stehr SN et al.; Nitric oxide synthase (NOS) inhibitors are considered promising as a therapeutic option in severe septic shock . The aim of this study was to investigate the effects of N-nitro-L-arginine methyl ester (L-NAME) application on neutrophil (PMN) respiratory burst, phagocytosis, and elimination of Escherichia coli from blood and tissue in rabbits . Twenty-eight female chinchilla rabbits were randomized to a treatment and control group . To quantify the bacterial clearance process, 10 colony forming units (CFU) of E . coli were injected intravenously into anesthetized rabbits . Animals in the L-NAME group had a significantly higher mortality compared with controls . NOS inhibition resulted in a significant delay of bacterial clearance (P < 0.001) . These findings correlated with a significant augmentation of all organ E . coli findings (P = 0.002-0.035) . PMN phagocytosis activity was notably reduced by L-NAME treatment during the experimental observation . Neutrophil burst, on the other hand, was amplified by NOS inhibition (P = 0.008) . Our findings point to an interference with the PMN-dependent immune mechanisms after L-NAME treatment . The augmented PMN burst reaction could be a compensatory mechanism, potentially leading to tissue damage . Therefore, in this model, we find sufficient evidence pointing to a possible cause for the deleterious effect of early nonselective NOS inhibition in critically ill patients. J Proteome Res, 2004 May-Jun, 3(3), 463 - 8 Sequential Peptide Affinity (SPA) system for the identification of mammalian and bacterial protein complexes; Zeghouf M et al.; A vector system is described that combines reliable, very low level, regulated protein expression in human cells with two affinity purification tags (Sequential Peptide Affinity, or SPA, system) . By avoiding overproduction of the target protein, this system allows for the efficient purification of natural protein complexes and their identification by mass spectrometry . We also present an adaptation of the SPA system for the efficient purification and identification of protein complexes in E . coli and, potentially, other bacteria. FEBS Lett, 2004 Jul 16, 570(1-3), 171 - 4 Temperature and cryoprotectant influence secondary quinone binding position in bacterial reaction centers; Pokkuluri PR et al.; We have determined the first de novo position of the secondary quinone QB in the Rhodobacter sphaeroides reaction center (RC) using phases derived by the single wavelength anomalous dispersion method from crystals with selenomethionine substitution . We found that in frozen RC crystals, QB occupies primarily the proximal binding site . In contrast, our room temperature structure showed that QB is largely in the distal position . Both data sets were collected in dark-adapted conditions . We estimate that the occupancy of the QB site is 80% with a proximal: distal ratio of 4:1 in frozen RC crystals . We could not separate the effect of freezing from the effect of the cryoprotectants ethylene glycol or glycerol . These results could have far-reaching implications in structure/function studies of electron transfer in the acceptor quinone complex because the above are the most commonly used cryoprotectants in spectroscopic experiments. Spine, 2004 Jul 15, 29(14), E298 - 9 A case with cauda equina syndrome due to bacterial meningitis of anterior sacral meningocele; Bal S et al.; STUDY DESIGN: A case of a patient with anterior sacral meningocele that was misdiagnosed as perianal abscess is presented . After the transrectal aspiration, the patient developed meningitis and cauda equina syndrome . OBJECTIVE: To report a rare case of anterior sacral meningocele in which primary presentation was mimicking perianal abscess . SUMMARY AND BACKGROUND DATA: Anterior sacral meningocele is a rare example of spinal dysraphism . It is created by a herniation of a dural sac through a defect in the sacral wall . It is usually asymptomatic until later decades . Most of the presenting symptoms are related to the pelvic organs . In the management of anterior sacral meningocele, surgical treatment is necessary . METHODS: The reported case is that of a 35-year-old female with cauda equina syndrome due to bacterial meningitis of the anterior sacral meningocele . At the beginning, she presented signs and symptoms resembling perianal abscess . She was misdiagnosed as anorectal abscess according to the computed tomography findings . The patient then underwent transrectal aspiration . Following the aspiration, meningitis and cauda equina syndrome were developed . Meningitis was treated with the appropriate antibiotics, and the patient underwent rehabilitation for paraplegia and bladder and bowel incontinence . RESULTS: Following the rehabilitation program, the patient has recovered completely within 8 months . CONCLUSION: This case represents a rare example of anterior sacral meningocele in which the patient was misdiagnosed as perianal abscess . Meningitis either iatrogenic or spontaneous may occur during the course of anterior sacral meningocele . Once it has occurred, it may result in severe morbidity and mortality . However, our case had been treated effectively, and she had regained her health with rehabilitation program. RNA, 2004 Aug, 10(8), 1225 - 35 Epub 2004 Jul 09. A reassessment of the response of the bacterial ribosome to the frameshift stimulatory signal of the human immunodeficiency virus type 1; Leger M et al.; HIV-1 uses a programmed -1 ribosomal frameshift to produce the precursor of its enzymes . This frameshift occurs at a specific slippery sequence followed by a stimulatory signal, which was recently shown to be a two-stem helix, for which a three-purine bulge separates the upper and lower stems . In the present study, we investigated the response of the bacterial ribosome to this signal, using a translation system specialized for the expression of a firefly luciferase reporter . The HIV-1 frameshift region was inserted at the beginning of the coding sequence of the luciferase gene, such that its expression requires a -1 frameshift . Mutations that disrupt the upper or the lower stem of the frameshift stimulatory signal or replace the purine bulge with pyrimidines decreased the frameshift efficiency, whereas compensatory mutations that re-form both stems restored the frame-shift efficiency to near wild-type level . These mutations had the same effect in a eukaryotic translation system, which shows that the bacterial ribosome responds like the eukaryote ribosome to the HIV-1 frameshift stimulatory signal . Also, we observed, in contrast to a previous report, that a stop codon immediately 3' to the slippery sequence does not decrease the frameshift efficiency, ruling out a proposal that the frameshift involves the deacylated-tRNA and the peptidyl-tRNA in the E and P sites of the ribosome, rather than the peptidyl-tRNA and the aminoacyl-tRNA in the P and A sites, as commonly assumed . Finally, mutations in 16S ribosomal RNA that facilitate the accommodation of the incoming aminoacyl-tRNA in the A site decreased the frameshift efficiency, which supports a previous suggestion that the frameshift occurs when the aminoacyl-tRNA occupies the A/T entry site. Nucleic Acids Res, 2004 Jun 24, 32(11), 3418 - 26 Print 2004. Extending the classification of bacterial transcription factors beyond the helix-turn-helix motif as an alternative approach to discover new cis/trans relationships; Rigali S et al.; Transcription factors (TFs) of bacterial helix-turn-helix superfamilies exhibit different effector-binding domains (EBDs) fused to a DNA-binding domain with a common feature . In a previous study of the GntR superfamily, we demonstrated that classifying members into subfamilies according to the EBD heterogeneity highlighted unsuspected and accurate TF-binding site signatures . In this work, we present how such in silico analysis can provide prediction tools to discover new cis/trans relationships . The TF-binding site consensus of the HutC/GntR subfamily was used to (i) predict target sites within the Streptomyces coelicolor genome, (ii) discover a new HutC/GntR regulon and (iii) discover its specific TF . By scanning the S.coelicolor genome we identified a presumed new HutC regulon that comprises genes of the phosphotransferase system (PTS) specific for the uptake of N-acetylglucosamine (PTS(Nag)) . A weight matrix was derived from the compilation of the predicted cis-acting elements upstream of each gene of the presumed regulon . Under the assumption that TFs are often subject to autoregulation, we used this matrix to scan the upstream region of the 24 HutC-like members of S.coelicolor . orf SCO5231 (dasR) was selected as the best candidate according to the high score of a 16 bp sequence identified in its upstream region . Our prediction that DasR regulates the PTS(Nag) regulon was confirmed by in vivo and in vitro experiments . In conclusion, our in silico approach permitted to highlight the specific TF of a regulon out of the 673 orfs annotated as 'regulatory proteins' within the genome of S.coelicolor. J Biotechnol, 2004 Aug 5, 111(3), 291 - 5 Development of an efficient enzymatic production of gamma-D-glutamyl-L-tryptophan (SCV-07), a prospective medicine for tuberculosis, with bacterial gamma-glutamyltranspeptidase; Suzuki H et al.; Gamma-D-Glutamyl-L-tryptophan (SCV-07) is a prospective medicine for the treatment of tuberculosis, according to the phase two clinical trial . Because gamma-D-glutamyl-L-tryptophan has several reactive groups in its molecule, consists of D- and L-amino acids, and is connected by gamma-glutamyl linkage, its chemical synthesis is complicated . An efficient enzymatic method to synthesize gamma-D-glutamyl-L-tryptophan from D-glutamine and L-tryptophan employing bacterial gamma-glutamyltranspeptidase was developed . The optimum reaction conditions were 50 mM D-glutamine, 50 mM L-tryptophan, and 0.2 U ml(-1) gamma-glutamyltranspeptidase, pH 9-9.5, and incubation at 37 degrees C for 5 h . After a 5 h incubation, 33 mM gamma-D-glutamyl-L-tryptophan was obtained, the conversion rate being 66% . The product was purified by Dowex 1 x 8 column and was considered to be gamma-D-glutamyl-L-tryptophan. Vaccine, 2004 Jul 29, 22(21-22), 2761 - 8 Maturation of dendritic cells by bacterial immunomodulators; Spisek R et al.; Dendritic cells (DC) become fully functional upon maturation by various stimuli . We tested whether an immunostimulatory effect of clinically used immunomodulators (Luivac, Biostim, Ribomunyl, Imudon, Bronchovaxom) is caused by direct DC activation . We found that Luivac, Biostim and Ribomunyl have a very high DC stimulatory potential in vitro . The level of DC activation was comparable or higher than DC maturation induced by standard maturation stimuli, Poly (I:C) or lipopolysaccharide . Treated DC had activated phenotype, reduced phagocytic activity and they induced the proliferation of allogeneic T lymphocytes . These results are important for understanding the physiology of action of these widely prescribed agents . Administration of bacterial immunomodulators should be considered with care to avoid the potential risk of inducing an autoimmune disease . They could also be used as well-defined maturating agents in the protocols used for the ex vivo production of DC-based vaccines for clinical trials. Eur J Radiol, 2004 Aug, 51(2), 102 - 13 Radiology of bacterial pneumonia; Vilar J et al.; Bacterial pneumonia is commonly encountered in clinical practice . Radiology plays a prominent role in the evaluation of pneumonia . Chest radiography is the most commonly used imaging tool in pneumonias due to its availability and excellent cost benefit ratio . CT should be used in unresolved cases or when complications of pneumonia are suspected . The main applications of radiology in pneumonia are oriented to detection, characterisation and follow-up, especially regarding complications . The classical classification of pneumonias into lobar and bronchial pneumonia has been abandoned for a more clinical classification . Thus, bacterial pneumonias are typified into three main groups: Community acquired pneumonia (CAD), Aspiration pneumonia and Nosocomial pneumonia (NP).The usual pattern of CAD is that of the previously called lobar pneumonia; an air-space consolidation limited to one lobe or segment . Nevertheless, the radiographic patterns of CAD may be variable and are often related to the causative agent . Aspiration pneumonia generally involves the lower lobes with bilateral multicentric opacities . Nosocomial Pneumonia (NP) occurs in hospitalised patients . The importance of NP is related to its high mortality and, thus, the need to obtain a prompt diagnosis . The role of imaging in NP is limited but decisive . The most valuable information is when the chest radiographs are negative and rule out pneumonia . The radiographic patterns of NP are very variable, most commonly showing diffuse multifocal involvement and pleural effusion . Imaging plays also an important role in the detection and evaluation of complications of bacterial pneumonias . In many of these cases, especially in hospitalised patients, chest CT must be obtained in order to better depict these associate findings. Bioorg Med Chem, 2004 Aug 1, 12(15), 4067 - 74 A bacterial selection for the directed evolution of pyruvate aldolases; Griffiths JS et al.; A novel bacterial in vivo selection for pyruvate aldolase activity is described . Pyruvate kinase deficient cells, which lack the ability to biosynthetically generate pyruvate, require supplementation of exogenous pyruvate when grown on ribose . Supplementation with pyruvate concentrations as low as 50 microM rescues cell growth . A known substrate of the KDPG aldolases, 2-keto-4-hydroxy-4-(2'-pyridyl)butyrate (KHPB), also rescues cell growth, consistent with retroaldol cleavage by KDPG aldolase and rescue through pyruvate release . An initial round of selection against 2-keto-4-hydroxyoctonate (KHO), a nonsubstrate for wild-type aldolase, produced three mutants with in