Schweiz Monatsschr Zahnmed, 2003, 113(9), 953 - 7 {Ribotyping Fusobacterium nucleatum isolates from healthy and diseased periodontium}; Berres F et al.; Fusobacterium nucleatum is one of the most frequently cultured bacteria from subgingival plaque . It can be isolated from active periodontal lesions as well as from healthy sites . Currently, F . nucleatum has been divided into five subspecies . An early hypothesis suggested an association between certain subspecies and periodontal disease or health . On the other hand, a broad heterogeneity between F . nucleatum isolates has been suggested, so that the subspeciation scheme and therefore the proposed association may not be valid . The purpose of the present study was to analyze if clonal differences exist between human F . nucleatum isolates from either periodontally healthy or diseased individuals . 23 F . nucleatum isolates from periodontally healthy and 17 isolates from periodontally diseased individuals were analysed by ribotyping . A broad genetic heterogeneity was present, although some of the isolates from periodontally healthy individuals were genotypically identical . However, no clonal differences between isolates from periodontally healthy or diseased individuals was detected. J Virol, 2003 Nov, 77(21), 11790 - 7 Norwalk virus nonstructural protein p48 forms a complex with the SNARE regulator VAP-A and prevents cell surface expression of vesicular stomatitis virus G protein; Ettayebi K et al.; Norwalk virus (NV), a reference strain of human calicivirus in the Norovirus genus of the family Caliciviridae, contains a positive-strand RNA genome with three open reading frames . ORF1 encodes a 1,789-amino-acid polyprotein that is processed into nonstructural proteins that include an NTPase, VPg, protease, and RNA-dependent RNA polymerase . The N-terminal protein p48 of ORF1 shows no significant sequence similarity to viral or cellular proteins, and its function in the human calicivirus replication cycle is not known . The lack of sequence similarity to any protein in the public databases suggested that p48 may have a unique function in the NV replication cycle or, alternatively, may perform a characterized function in replication by a unique mechanism . In this report, it is shown that p48 displays a vesicular localization pattern in transfected cells when fused to the fluorescent reporter EYFP . A predicted transmembrane domain at the C terminus of p48 was not necessary for the observed localization pattern, but this domain was sufficient to redirect localization of EYFP to a fluorescent pattern consistent with the Golgi apparatus . A yeast two-hybrid screen identified the SNARE regulator vesicle-associated membrane protein-associated protein A (VAP-A) as a binding partner of p48 . Biochemical assays confirmed that p48 and VAP-A interact and form a stable complex in mammalian cells . Furthermore, expression of the vesicular stomatitis virus G glcyoprotein on the cell surface was inhibited when cells coexpressed p48, suggesting that p48 disrupts intracellular protein trafficking. J Virol, 2003 Nov, 77(21), 11651 - 60 Murine leukemia virus particle assembly quantitated by fluorescence microscopy: role of Gag-Gag interactions and membrane association; Andrawiss M et al.; In order to track the assembly of murine leukemia virus (MLV), we used fluorescence microscopy to visualize particles containing Gag molecules fused to fluorescent proteins (FPs) . Gag-FP chimeras budded from cells to produce fluorescent spots, which passed through the same pore-size filters and sedimented at the same velocity as authentic MLV . N-terminal myristylation of Gag-FPs was necessary for particle formation unless wild-type Gag was coexpressed . By labeling nonmyristylated Gag with yellow FP and wild-type Gag with cyan FP, we could quantitate the coincorporation of two proteins into single particles . This experiment showed that nonmyristylated Gag was incorporated into mixed particles at approximately 50% the efficiency of wild-type Gag . Mutations that inhibit Gag-Gag interactions (K . Alin and S . P . Goff, Virology 216:418-424, 1996; K . Alin and S . P . Goff, Virology 222:339-351, 1996) were then introduced into the capsid (CA) region of Gag-FPs . The mutations P150L and R119C/P133L inhibited fluorescent particle formation by these Gag-FPs, but Gag-FPs containing these mutations could be efficiently incorporated into particles when coexpressed with wild-type Gag . When these mutations were introduced into nonmyristylated Gag-FPs, no incorporation into particles in the presence of wild-type Gag was detected . These data suggest that two independent mechanisms, CA interactions and membrane association following myristylation, cooperate in MLV Gag assembly and budding. Trends Microbiol, 2003 Oct, 11(10), 479 - 87 Multi-locus sequence typing: a tool for global epidemiology; Urwin R et al.; The characterization of pathogenic isolates plays a pivotal role in the epidemiology of infectious diseases, generating the information necessary for identifying, tracking, and intervening against disease outbreaks . In 1998 multi-locus sequence typing (MLST) was proposed as a nucleotide sequence-based approach that could be applied to many bacterial pathogens . It combined developments in high-throughput sequencing and bioinformatics with established population genetics techniques to provide a portable, reproducible, and scalable typing system that reflected the population and evolutionary biology of bacterial pathogens . MLST schemes have been developed for a variety of procaryotic and eucaryotic pathogens and the data generated have contributed to both epidemiological surveillance and fundamental studies of pathogen biology. Regul Pept, 2003 Oct 15, 115(3), 179 - 85 Expression and characterization of the preoptic regulatory factor-1 and -2 peptides; Nowak FV; The preoptic regulatory factors, PORF-1 and PORF-2, were originally detected as cDNAs from the rat preoptic area of the hypothalamus . Expression of the messenger RNAs is dependent on age, gender and hormonal status . PORF-1 is a putative transcription factor while PORF-2 affects cell growth . However, expression of peptides predicted from the PORF-1 and PORF-2 open reading frames has not been reported . The characterization of PORF-1 and PORF-2 peptides expressed in vitro and in vivo is described here . There are three potential PORF-1 peptides of 37, 59 and 86 amino acids, because of the presence of two TGA codons that can code either for termination or for selenocysteine incorporation in the PORF-1 open reading frame . All three peptides are detected after in vitro translation of full-length porf-1 brain mRNA . The two larger ones are found in normal male rat brain . The expression of a given peptide in bacteria depends upon the host strain . In contrast, a single 75 amino acid PORF-2 peptide is detected by in vitro translation, in transformed bacteria, and in rat brain . The PORF-1 and PORF-2 peptides have the charges and hydrophobicities predicted from their brain cDNA open reading frames . These small peptides may represent examples of multiple usage of single genetic loci in eukaryotes, giving rise to both a protein product and its regulatory peptide. Biochim Biophys Acta, 2003 Oct 17, 1607(1), 19 - 26 Identification of intramembrane hydrogen bonding between 13(1) keto group of bacteriochlorophyll and serine residue alpha27 in the LH2 light-harvesting complex; Braun P et al.; Intramembrane hydrogen bonding and its effect on the structural integrity of purple bacterial light-harvesting complex 2, LH2, have been assessed in the native membrane environment . A novel hydrogen bond has been identified by Raman resonance spectroscopy between a serine residue of the membrane-spanning region of LH2 alpha-subunit, and the C-13(1) keto carbonyl of bacteriochlorophyll (BChl) B850 bound to the beta-subunit . Replacement of the serine by alanine disrupts this strong hydrogen bond, but this neither alters the strongly red-shifted absorption nor the structural arrangement of the BChls, as judged from circular dichroism . It also decreases only slightly the thermal stability of the mutated LH2 in the native membrane environment . The possibility is discussed that weak H-bonding between the C-13(1) keto carbonyl and a methyl hydrogen of the alanine replacing serine(-4) or the imidazole group of the nearby histidine maintains structural integrity in this very stable bacterial light-harvesting complex . A more widespread occurrence of H-bonding to C-13(1) not only in BChl, but also in chlorophyll proteins, is indicated by a theoretical analysis of chlorophyll/polypeptide contacts at <3.5 A in the high-resolution structure of Photosystem I . Nearly half of the 96 chlorophylls have aa residues suitable as hydrogen bond donors to their keto groups. Biochim Biophys Acta, 2003 Oct 17, 1607(1), 1 - 4 Photosystem I trimers from Synechocystis PCC 6803 lacking the PsaF and PsaJ subunits bind an IsiA ring of 17 units; Kouril R et al.; We report a structural characterization by electron microscopy and image analysis of a supramolecular complex consisting of Photosystem I (PSI) and the chlorophyll-binding protein IsiA from a mutant of the cyanobacterium Synechocystis PCC 6803 lacking the PsaF and PsaJ subunits . The circular complex consists of a central PSI trimer surrounded by a ring of 17 IsiA units, one less than in the wild-type supercomplex . We conclude that PsaF and PsaJ are not obligatory for the binding of the IsiA ring, and that the size of the PSI complex determines the number of IsiA units in the ring . The resulting number of 17 copies implies that each PSI monomer has a different association to the IsiA ring. Vet Res, 2003 Sep-Oct, 34(5), 629 - 45 Polymorphonuclear proteolytic activity and milk composition change; Le Roux Y et al.; Relationships between Quarter Milk Cell Count (QMCC) and Tank Milk Cell Count (TMCC) with milk biochemical and technological parameters in milk and dairy products were investigated . All parameters measured were affected by the increase of TMCC and QMCC between 0 and 600 000 cells/mL . The variable effect of lactation stage which is different for different authors, is discussed . The three mechanisms, measured during the inflammation of the udder, implicated in the modification of milk quality are described (a decrease in synthesis, a decrease in the milk barrier permeability and an increase in proteolytic activities) . The direct effect of plasmin in caseinolysis is well known; the specific role of the increase of somatic cells (especially PMN) in the modification of milk quality is described . Several specific proteolytic activities of PMN are described and the impact of these activities on caseinolysis is evaluated . Two hypothetical mechanisms of caseinolysis by PMN are suggested and a synthetic scheme of the role of plasmin, bacteria and somatic cells in caseinolysis is discussed. Photochem Photobiol, 2003 Sep, 78(3), 225 - 7 UV damage and photoreactivation: timing and age are everything; Grad G et al.; Aquatic organisms, ranging from bacteria to fish, living in clear lakes are presently receiving damaging levels of UV radiation . Photoreactivation is a light-dependent mechanism by which some organisms deal with DNA damage caused by UV radiation . Yet, photoreactivation is a mechanism that confounds long-term predictive modeling of UV effects on the survival of these organisms . Here we show that a short-lived rotifer species, Asplanchna girodi, previously thought to have little to no photoreactivation, does indeed have a significant amount of it . The ability to undergo photoreactivation in A . girodi is dependent on age and becomes apparent only after several days of observation after UV exposure. Nat Neurosci, 2003 Nov, 6(11), 1194 - 200 Epub 2003 Oct 12. Activity-induced targeting of profilin and stabilization of dendritic spine morphology; Ackermann M et al.; Morphological changes in dendritic spines have been implicated in connective plasticity in brain circuitry, but the underlying pathway leading from synaptic transmission to structural change is unknown . Using primary neurons expressing GFP-tagged proteins, we found that profilin, a regulator of actin polymerization, is targeted to spine heads when postsynaptic NMDA receptors are activated and that actin-based changes in spine shape are concomitantly blocked . Profilin targeting was triggered by electrical stimulation patterns known to induce the long-term changes in synaptic responsiveness associated with memory formation . These results suggest that, in addition to electrophysiological changes, NMDA receptor activation initiates changes in the actin cytoskeleton of dendritic spines that stabilize synaptic structure. Appl Occup Environ Hyg, 2003 Nov, 18(11), 855 - 64 Summary of the findings from the exposure assessments for metalworking fluid mortality and morbidity studies; Woskie SR et al.; Since 1985, a number of North American researchers have evaluated the association between worker exposure to metalworking fluids (MWFs) and cancer mortality or respiratory morbidity . The studies have used different methods to measure the MWF aerosol concentration and to evaluate the exposures to the specific components of the MWF aerosol (bacteria, endotoxin, elements, metals, ethanolamines, polyaromatic hydrocarbons) . This diversity of approaches makes comparison of study results difficult and has impeded the development of an occupational exposure limit (OEL) for metalworking fluids . This article summarizes the exposures measured in these North American epidemiologic studies by estimating their thoracic and inhalable MWF particulate levels . In addition, issues that must be resolved before a universal sampling and analysis method for MWF can be recommended are reviewed, including: the use of gravimetric versus extractable analysis; whether the analytical limit of detection can support a lower occupational exposure limit; if the volatile components of mineral oils should also be collected; and whether there are components of specific concern in MWFs that should be regulated separately . Finally, recommendations for future directions in MWF exposure assessment and control are suggested. Biochem Pharmacol, 2003 Oct 15, 66(8), 1447 - 52 The prodigiosins, proapoptotic drugs with anticancer properties; Perez-Tomas R et al.; The family of natural red pigments, called prodigiosins (PGs), characterised by a common pyrrolylpyrromethene skeleton, are produced by various bacteria . Some members have immunosuppressive properties and apoptotic effects in vitro and they have also displayed antitumour activity in vivo . Understanding the mechanism of action of PGs is essential for drug development and will require the identification and characterisation of their still unidentified cell target . Four possible mechanisms of action have been suggested for these molecules: (i) PGs as pH modulators; (ii) PGs as cell cycle inhibitors; (iii) PGs as DNA cleavage agents; (iv) PGs as mitogen-activated protein kinase regulators . Here, we review the pharmacological activity of PG and related compounds, including novel synthetic PG derivatives with lower toxicity and discuss the mechanisms of action and the molecular targets of those molecules . The results reported in this review suggest that PGs are a new class of anticancer drugs, which hold out considerable promise for the Pharmacological Industry. J Dent, 2003 Nov, 31(8), 559 - 68 Direct detection of Actinomyces spp . from infected root canals in a Chinese population: a study using PCR-based, oligonucleotide-DNA hybridization technique; Tang G et al.; OBJECTIVES: The poor sensitivity of phenotypic identification techniques has hampered the taxonomic differentiation of Actinomyces . Hence we developed a sensitive and specific, PCR-based oligonucleotide-DNA hybridization technique to detect Actinomyces spp . and, used this method to detect these organisms in samples directly obtained from infected root canals . METHODS: A total of 32 samples from 28 Chinese patients, with primary root canal infections, aseptically exposed at the first patient visit, were studied . Whole bacterial genomic DNA was isolated directly from paper point samples . The variable regions of 16S ribosomal DNA of bacteria were amplified and labeled with digoxigenin for further hybridization and detection . A total of seven oligonucleotide probes specific for A . bovis, A . gerencseriae, A . israelii, A . meyeri, catalase-negative A . naeslundii (genospecies 1 and 2), catalase-positive A . naeslundii genospecies 2 and A . odontolyticus were used . RESULTS: 16 of the 32 teeth were infected with one or more Actinomyces species . The prevalence rates of the examined species were: A . odontolyticus 31.3%, A . meyeri 9.4%, A . naeslundii 9.4%, A . israelii 6.3% and A . gerencseriae 3.1%; no A . bovis was detected in any of the canals . Furthermore, A . odontolyticus was isolated more frequently from root canals with caries or a history of caries (Fisher's exact test: P=0.0496; Odds ratio=9.00, 95% confidence interval: 0.97-83.63), and A . naeslundii was significantly associated with traumatized teeth (Fisher's exact test: P=0.0121; Odds ratio=57.00, 95% confidence interval: 2.10-1546.90) . However, no significant correlation was found between Actinomyces spp . and clinical symptoms and signs, such as pain, swelling, percussion to tenderness, sinus and periapical radiolucency . CONCLUSION: Actinomyces spp . may be important pathogens of root canal infections . A . naeslundii in particular may be related with traumatized teeth . A . odontolyticus appears to be involved in infections related to caries, exposure of dentinal tubules during cavity preparation and/or leaking restoration, but further clarification with large samples is necessary. FEMS Microbiol Lett, 2003 Sep 26, 226(2), 397 - 403 Purification, characterization, and physiological response of a catalase-peroxidase in Mycobacterium sp . strain JC1 DSM 3803 grown on methanol; Ro YT et al.; A novel catalase-peroxidase (CP) from methanol-grown cells of Mycobacterium sp . strain JC1 was purified . The CP exhibited properties of both typical mycobacterial CPs (i.e . strict pH optimum, labile to heat treatment, capable of oxidizing NADH, and resistant to inhibition by 3-amino-1,2,4-triazole) and true catalases (i.e . stable against ethanol-chloroform treatment) . The enzyme oxidized methanol and shared common antigenic groups with other mycobacteria . Isoniazid had almost no effect on the growth and expression of CP but inhibited the enzyme activity to some extent . Sodium nitroprusside arrested the growth but strongly stimulated the expression of CP with a concomitant increase in activity after the mid-exponential growth phase. FEMS Microbiol Lett, 2003 Sep 26, 226(2), 267 - 71 Binding of Porphyromonas gingivalis fimbriae to Treponema denticola dentilisin; Hashimoto M et al.; Treponema denticola has been reported to coaggregate with Porphyromonas gingivalis and localize closely together in matured subgingival plaque . In this study of the interaction of T . denticola with P . gingivalis, the P . gingivalis fimbria-binding protein of T . denticola was identified by two-dimensional electrophoresis followed by a ligand overlay assay with P . gingivalis fimbriae, and was determined to be dentilisin, a chymotrypsin-like proteinase of T . denticola . The binding was further demonstrated with a ligand overlay assay using an isolated GST fusion dentilisin construct . Our results suggest that P . gingivalis fimbriae and T . denticola dentilisin are implicated in the coaggregation of these bacteria. Trends Immunol, 2003 Oct, 24(10), 534 - 9 The interferon in TLR signaling: more than just antiviral; Hertzog PJ et al.; The Toll-like receptor (TLR) system is responsible for the recognition of infectious agents leading to initiation of the primary innate, and later adaptive, immune response . Genetic technologies have enabled the discovery of new factors involved in these systems, their genetic manipulation and the global analyses of their effects on gene expression . Furthermore, this increased understanding has resulted in the need to reassess our preconceptions about the functions of well-known molecules . For example, type I interferons (IFNs), which were discovered as antiviral proteins, are now known to be produced in response to TLR activation by many pathogens, including bacteria . Should we be surprised? Has the inflammatory response unexpectedly highjacked the body's antiviral system? Or are we too easily blinkered by preconceptions from how a compound was discovered? PLoS Biol . 2003 Oct;1(1):E21 . Epub 2003 Oct 13. Developmental origin and evolution of bacteriocytes in the aphid-Buchnera symbiosis; Braendle C et al.; Symbiotic relationships between bacteria and insect hosts are common . Although the bacterial endosymbionts have been subjected to intense investigation, little is known of the host cells in which they reside, the bacteriocytes . We have studied the development and evolution of aphid bacteriocytes, the host cells that contain the endosymbiotic bacteria Buchnera aphidicola . We show that bacteriocytes of Acyrthosiphon pisum express several gene products (or their paralogues): Distal-less, Ultrabithorax/Abdominal-A, and Engrailed . Using these markers, we find that a subpopulation of the bacteriocytes is specified prior to the transmission of maternal bacteria to the embryo . In addition, we discovered that a second population of cells is recruited to the bacteriocyte fate later in development . We experimentally demonstrate that bacteriocyte induction and proliferation occur independently of B . aphidicola . Major features of bacteriocyte development, including the two-step recruitment of bacteriocytes, have been conserved in aphids for 80-150 million years . Furthermore, we have investigated two cases of evolutionary loss of bacterial symbionts: in one case, where novel extracellular, eukaryotic symbionts replaced the bacteria, the bacteriocyte is maintained; in another case, where symbionts are absent, the bacteriocytes are initiated but not maintained . The bacteriocyte represents an evolutionarily novel cell fate, which is developmentally determined independently of the bacteria . Three of five transcription factors we examined show novel expression patterns in bacteriocytes, suggesting that bacteriocytes may have evolved to express many additional transcription factors . The evolutionary transition to a symbiosis in which bacteria and an aphid cell form a functional unit, similar to the origin of plastids, has apparently involved extensive molecular adaptations on the part of the host cell. J Infect Dis, 2003 Oct 15, 188(8), 1098 - 104 Epub 2003 Oct 06. Selective up-regulation of matrix metalloproteinase-9 expression in human erythema migrans skin lesions of acute lyme disease; Zhao Z et al.; Despite the absence of enzymes that digest extracellular matrix, Borrelia burgdorferi spreads in the skin to form erythema migrans (EM) lesions and then disseminates to other organs . We studied the induction by bacteria of host matrix metalloproteinases (MMPs) in EM skin lesions of patients with acute Lyme disease . In blister fluid from the EM lesions, the expression of MMP-9 was selectively increased by 1900%+/-1037%, compared with blister fluid from the surrounding normal-appearing skin . The expression of all other MMP messenger RNAs was similar in the EM lesions and normal-appearing skin . Selective up-regulation of MMP-9 in the EM lesions was found . Fibroblasts and, to a lesser degree, mononuclear cells were the sources of local MMP-9 production . These results demonstrate specific up-regulation of MMP-9 in the EM skin lesions of patients with acute Lyme disease . Bacterial induction of host proteases may play a role in the dissemination of B . burgdorferi. J Infect Dis, 2003 Oct 15, 188(8), 1094 - 7 Epub 2003 Oct 01. Nuclear factor-kappaB activation in endothelium by Chlamydia pneumoniae without active infection; Baer JT et al.; Causative molecular mechanisms accounting for the potential link between Chlamydia pneumoniae and atherosclerosis are unknown . Formalin and heat-inactivated C . pneumoniae activated the transcription factor nuclear factor (NF)-kappaB in cultured porcine endothelium and up-regulated the expression of E-selectin messenger RNA and protein . This up-regulation was abolished by an IkappaB super-repressor, an NF-kappaB-specific inhibitor . Live bacteria are not necessary for the activation of endothelial NF-kappaB, and C . pneumoniae may contribute to atherogenesis without active infection. Circ Res, 2003 Oct 31, 93(9), 802 - 5 Epub 2003 Oct 09. Contrasting roles of NADPH oxidase isoforms in pressure-overload versus angiotensin II-induced cardiac hypertrophy; Byrne JA et al.; Increased production of reactive oxygen species (ROS) is implicated in the development of left ventricular hypertrophy (LVH) . Phagocyte-type NADPH oxidases are major cardiovascular sources of ROS, and recent data indicate a pivotal role of a gp91phox-containing NADPH oxidase in angiotensin II (Ang II)-induced LVH . We investigated the role of this oxidase in pressure-overload LVH . gp91phox-/- mice and matched controls underwent chronic Ang II infusion or aortic constriction . Ang II-induced increases in NADPH oxidase activity, atrial natriuretic factor (ANF) expression, and cardiac mass were inhibited in gp91phox-/- mice, whereas aortic constriction-induced increases in cardiac mass and ANF expression were not inhibited . However, aortic constriction increased cardiac NADPH oxidase activity in both gp91phox-/- and wild-type mice . Myocardial expression of an alternative gp91phox isoform, Nox4, was upregulated after aortic constriction in gp91phox-/- mice . The antioxidant, N-acetyl-cysteine, inhibited pressure-overload-induced LVH in both gp91phox-/- and wild-type mice . These data suggest a differential response of the cardiac Nox isoforms, gp91phox and Nox4, to Ang II versus pressure overload. Arch Pediatr, 2003 Oct, 10(10), 928 - 31 {Whooping cough: biological diagnosis}; Guiso N; Whooping cough is a respiratory infection particularly severe for infants . The agents of the disease are the bacteria Bordetella pertussis and B . parapertussis . Before vaccination, the disease affected majority of the children . Due to the high circulation of the bacteria, adolescents and adults were regularly in contact with the bacteria and had natural boosters . Forty years after generalized vaccination in some countries, a change in the transmission of the disease is observed . Children between 2 and 5-10 years of age are not infected (depending of the vaccine used and the vaccine calendar) because they were vaccinated in their infancy . However, adolescents and adults are becoming susceptible because they do not receive any vaccinal or natural boosters . They can be infected and contaminated infants are too young to be vaccinated . Clinical symptoms in adolescents and adults, previously vaccinated or infected, are very variable and for this reason biological diagnosis are now necessary to confirm the infection . These diagnostics are culture, PCR and serology. Autoimmun Rev, 2003 Oct, 2(6), 305 - 12 Primary sclerosing cholangitis; Cullen S et al.; Primary sclerosing cholangitis (PSC) is a fibrosing disease of the intra- and extra-hepatic bile ducts, and is closely associated with inflammatory bowel disease . It is immune mediated, rather than being a classical autoimmune disease . A range of immune abnormalities have been demonstrated in PSC, in particular the findings of a range of autoantibodies, a portal tract infiltrate of functional T cells, a restricted T-cell receptor repertoire, and aberrant expression of HLA molecules on biliary epithelial cells . The immunogenetics of PSC is currently under study and to date 4 key HLA haplotypes associated with PSC have been developed . The trigger factor for the initiation of the immune response may be the ingress of bacteria or other toxic metabolites into the portal circulation through a diseased and permeable bowel wall. Protein Expr Purif, 2003 Oct, 31(2), 265 - 70 Purification and characterization of hexahistidine-tagged elongation factor SelB; Thanbichler M et al.; The cotranslational incorporation of selenocysteine into proteins is mediated by a specialized elongation factor, named SelB . Its amino-terminal three domains show homology to elongation factor EF-Tu and accordingly bind GTP and selenocysteyl-tRNASec . In addition, SelB exhibits a long carboxy-terminal extension that interacts with a secondary structure of selenoprotein mRNAs (SECIS element) positioned immediately downstream of the in-frame UGA codons specifying the sites of selenocysteine insertion . In this report, a fast and efficient method for the purification of large amounts of hexahistidine-tagged SelB is presented . After two chromatographic steps, 10 mg pure protein was isolated from 12 g wet cell pellet . Biochemical analysis of the purified protein showed that the tag does not influence the interaction of SelB with guanine nucleotides, SECIS elements, and selenocysteyl-tRNASec . In addition, the fusion protein is fully functional in mediating UGA read-through in vivo . It therefore represents an excellent model for studying the function of SelB and the mechanisms of selenocysteine incorporation. FEBS Lett, 2003 Oct 9, 553(1-2), 205 - 8 Mitochondria from ejaculated human spermatozoa do not synthesize proteins; Diez-Sanchez C et al.; Sperm motility is dependent on mitochondrial ATP production that relies on the coordinated expression of the mitochondrial and nuclear genomes . It is generally accepted that mammalian ejaculated spermatozoa retain the ability to synthesize mtDNA-encoded proteins but not most of the nuclear ones . This implies an asynchronous regulation of the oxidative phosphorylation-related genes encoded by each genome . Trying to investigate this issue, we unexpectedly found that ejaculated human spermatozoa do not synthesize mtDNA-encoded proteins . Moreover, we estimated that the discrepancy between our observations and those published elsewhere was due to a chloramphenicol-sensitive protein synthesis attributed to mitochondria that instead corresponds to contaminating bacteria. FEBS Lett, 2003 Oct 9, 553(1-2), 179 - 82 pkn22 (alr2502) encoding a putative Ser/Thr kinase in the cyanobacterium Anabaena sp . PCC 7120 is induced by both iron starvation and oxidative stress and regulates the expression of isiA; Xu WL et al.; In cyanobacteria, the isiA gene is required for cell adaptation to oxidative damage caused by the absence of iron . We show here that a putative Ser/Thr kinase gene, pkn22 (alr2052), is activated by iron deficiency and oxidative damage in Anabaena sp . PCC 7120 . A pkn22 insertion mutant is unable to grow when iron is limiting . pkn22 regulates the expression of isiA (encoding CP43'), but not of isiB (encoding flavodoxin) and psbC (CP43) . Fluorescence measurement at 77 K reveals the absence of the typical signature of CP43' associated with photosystem I in the mutant under iron-limiting conditions . We propose that Pkn22 is required for the function of isiA/CP43' and constitutes a regulatory element necessary for stress response. FEBS Lett, 2003 Oct 9, 553(1-2), 68 - 72 Effects of glycerol and high temperatures on structure and function of phycobilisomes in Synechocystis sp . PCC 6803; Mao HB et al.; The effects of glycerol and high temperatures on structure and function of phycobilisomes (PBSs) in vivo were investigated in a chlL deletion mutant of the cyanobacterium Synechocystis sp . PCC 6803 . When the mutant was grown under light-activated heterotrophic growth conditions, it contained intact and functional PBSs, but essentially no chlorophyll and photosystems . So the structural and functional changes of the mutant PBSs in vivo can be handily detected by measurement of low temperature (77 K) fluorescence emission spectra . High concentration glycerol induced an obvious disassembly of PBSs and the dissociation of phycocyanins in the rod substructures into their oligomers and monomers . PBSs also disassembled at high temperatures and allophycocyanins were more sensitive to heat stress than phycocyanins . Our results demonstrate that the chlL(-) mutant strain is an advantageous model for studying the mechanisms of assembly and disassembly of protein complexes in vivo. Biotechnol Adv, 1990, 8(3), 515 - 37 Plasmid promiscuity and chastity and its uses; Iyer VN; Bacterial plasmids are obligate and intracellular genetic elements that replicate and are maintained autonomously from the chromosome . They are ubiquitous . Some of them are relatively more promiscuous than others . Plasmid genetic systems that contribute to relative promiscuity or chastity in naturally occurring plasmids are described and discussed . Both the promiscuity and the chastity of plasmid-based genetic systems have applications in bacterial molecular genetics, in the production of recombinant DNA products and in the breeding and use of desirable bacteria . The role of these systems in such applications is considered. Biotechnol Adv, 1994, 12(3), 489 - 523 Veterinary vaccines; Babiuk LA et al.; Vaccination of animals for the prevention of infectious diseases has been practised for a number of years with little change in product composition . Recent advances in molecular biology, pathogenesis and immunology have laid the groundwork for the development of a new generation of veterinary vaccines based on pure subunits as well as live vectored bacteria and viruses . Along with novel methods of antigen preparation, the use of new adjuvants and delivery systems will permit targeting of the appropriate immune response as well as offering flexibility in terms of vaccination protocols . These new technologies are also being applied to the development of vaccines to enhance animal productivity and to control reproduction. Biotechnol Adv, 1994, 12(4), 619 - 24 Vaccine design: future possibilities and potentials; Martin SJ; Recent developments in the understanding of the structure and replications of a wide range of pathogens, including viruses, bacteria and parasites have opened up ways of designing novel vaccines which should both improve the quality and extend the range and value of vaccines as major prophylactic and therapeutic tools of the future . Two main strategies have emerged, one involving the development of synthetic vaccines which are essentially composed of selected epitopes of the pathogenic agent that will elicit neutralising antibodies . The other strategy attempts to make use of chimeric agents that will allow live virus or bacteria to be used as vectors for carrying appropriate epitopes of the target pathogen . Current knowledge about the immunology and improvements in the presentation of antigen to the immune system will also play an important role in the rational design of vaccines . This review summarises present methods of producing vaccines and considers the development of more rational methods of vaccine design that will greatly influence the production of vaccines in the future. Biotechnol Adv, 1999 Sep, 17(2-3), 205 - 17 Bovine chymosin: production by rDNA technology and application in cheese manufacture; Mohanty AK et al.; Bovine chymosin, an aspartyl protease extracted from abomasum of suckling calves, is synthesized in vivo as preprochymosin and secreted as prochymosin which is autocatalytically activated to chymosin . Chymosin is bilobular, with Asp 32 and Asp 215 acting as the catalytic residues . Chymosin A and chymosin B have pH optima of 4.2 and 3.8, respectively, and act to initiate milk clotting by cleaving kappa-casein between Phe 105 and Met 106 . The gene encoding chymosin has been cloned and expressed in suitable bacteria and yeast hosts under the control of lac, trp, trp-beta, gly A genes, and serine hydroxymethyl-transferase promoters . Protein engineering of chymosin has also been attempted . A number of companies are now producing recombinant chymosin for commercial use in cheese manufacture. Biotechnol Adv, 1995, 13(3), 375 - 402 Antifreeze proteins and their potential use in frozen foods; Griffith M et al.; Antifreeze proteins (AFPs) are proteins that have the ability to modify the growth of ice, resulting in the stabilization of ice crystals over a defined temperature range and in the inhibition of the recrystallization of ice . AFPs are found in a wide range of organisms, including bacteria, fungi, plants, invertebrates and fish . Moreover, multiple forms of AFPs are synthesized within each organism . As a result, it should be possible to select an AFP with appropriate characteristics and a suitable level of activity for a particular food product . Antifreeze proteins may improve the quality of foods that are eaten while frozen by inhibiting recrystallization and maintaining a smooth texture . In foods that are frozen only for preservation, AFPs may inhibit recrystallization during freezing, storage, transport and thawing, thus preserving food texture by reducing cellular damage and also minimizing the loss of nutrients by reducing drip . Antifreeze proteins are naturally present in many foods consumed as part of the human diet . However, AFPs may be introduced into other food products either by physical processes, such as mixing and soaking, or by gene transfer. Andrologia, 2003 Oct, 35(5), 325 - 30 Chronic prostatitis and male accessory gland infection--is there an impact on male infertility (diagnosis and therapy)? Everaert K, Mahmoud A, Depuydt C, Maeyaert M, Comhaire F. The aim of this article was to discuss by means of a review of the literature and own study material the multifactorial aetiology of male infertility, extrapolate this hypothesis to male accessory gland infection (MAGI) and relate it to chronic prostatitis and its treatment . Infertility is a multifactorial disease and diagnosis and therapy must be oriented as such . Although the relationship between prostatitis and infertility remains unclear, bacteria, viruses, leucocytes, reactive oxygen species, cytokines, obstruction and immunological abnormalities must be seen as cofactors in the development of infertility in patients with MAGI and prostatitis . Infection, trauma, allergy, neurogenic damage, chemical or mechanical factors can lead to a long-lasting inflammation of the prostate or pelvic organs even after eradication of the aetiological agent, and is potentially related to infertility through cytokines . In relation to treatment of infertility, antibiotics play a role in bacterial prostatitis whereas in abacterial prostatitis other treatments like antioxidants, sacral nerve stimulation and anti-inflammatory treatment are worth to be studied in the future. J Forensic Sci, 2003 Sep, 48(5), 1014 - 34 AmpFlSTR profiler Plus short tandem repeat DNA analysis of casework samples, mixture samples, and nonhuman DNA samples amplified under reduced PCR volume conditions (25 microL); Fregeau CJ et al.; As part of the validation of the AmpFlSTR Profiler Plus short tandem repeat (STR) system, under reduced polymerase chain reaction (PCR) volume conditions (i.e., 25 microL), a total of 275 casework samples were processed . Examples of profiles are presented along with amplification conditions to improve the odds of obtaining balanced and complete profiles for samples showing partial results or profiles with a descending slope . Data collected and used to develop our interpretation guidelines are included . From the mixture studies, full profiles were obtained for minor contributors, using 2 ng of DNA, with ratios of 10:1 or 1:20 and using 1 ng of DNA, with ratios of 10:1 and 1:8 . The specificity of the Profiler Plus amplification reaction performed in 25 microL was examined and confirmed using a large spectrum of nonhuman DNAs . This report supports the use of the AmpFlSTR Profiler Plus STR system for casework DNA typing under reduced PCR volume conditions. J Forensic Sci, 2003 Sep, 48(5), 936 - 44 Development of an Alu-based, real-time PCR method for quantitation of human DNA in forensic samples; Nicklas JA et al.; Determining the amount of human DNA extracted from a crime scene sample is an important step in DNA profiling . The forensic community relies almost entirely upon a technique (slot blot) to quantitate human DNA that is imprecise, time consuming, and labor intensive . We have previously described a method for quantitation of human DNA based on PCR amplification of a repetitive Alu sequence that uses a fluorescence plate reader . This manuscript describes and validates a variation of this assay using real-time PCR and SYBR Green I for quantitation . The advantages of the real-time assay over the plate reader assay are: reduced hands-on time, lower assay cost, and a greater dynamic range . The main disadvantage is the cost of the real-time instrument . However, for those forensic laboratories with access to a real-time instrument, this Alu-based assay has a dynamic range of 16 ng to 1 pg, is sensitive, specific, fast, quantitative, and uses only 2 microL of sample. Microsc Res Tech, 2003 Nov 1, 62(4), 356 - 67 Molecular biology of demosponge axial filaments and their roles in biosilicification; Weaver JC et al.; For hundreds of years, the skeletal elements of marine and freshwater sponges have intrigued investigators with a diverse array of remarkably complex morphologies . Early studies of demosponge monaxonal megascleres revealed the presence of a central organic axial filament running their entire length . Until recently, however, the precise function of these axial filaments was largely unknown . The spicules from the temperate Eastern Pacific demosponge, Tethya aurantia, comprise approximately 75% of the dry weight of this species, facilitating the large-scale isolation and purification of the biosilica-associated proteins . Silicateins, the most abundant proteins comprising the axial filaments of these spicules, prove to be members of a well-known superfamily of proteolytic and hydrolytic enzymes and can be easily collected after silica demineralization with hydrofluoric acid . Consistent with these findings, the intact filaments are more than simple, passive templates; in vitro, they actively catalyze and spatially direct the hydrolysis and polycondensation of silicon alkoxides to yield silica at neutral pH and low temperature . Catalytic activity also is exhibited by the monomeric subunits obtained by disaggregation of the protein filaments and those produced from recombinant DNA templates cloned in bacteria . These proteins also can be used to direct the polymerization of organosilicon polymers (silicones) from the corresponding organically functionalized silicon alkoxides . Based on these observations, the silicateins are currently being used as models for the design of biomimetic agents with unique catalytic and structure-directing properties . The presence of axial filaments in a diversity of spicule types and the evolutionary implications of these findings are also discussed . J Biol Chem, 2003 Dec 19, 278(51), 51307 - 15 Epub 2003 Oct 08. Characterization of HdnoR, the transcriptional repressor of the 6-hydroxy-D-nicotine oxidase gene of Arthrobacter nicotinovorans pAO1, and its DNA-binding activity in response to L- and D-nicotine Derivatives; Sandu C et al.; Utilization of L-nicotine as growth substrate by Arthrobacter nicotinovorans pAO1 starts with hydroxylation of the pyridine ring at C6 . Next, the pyrrolidine ring is oxidized by 6-hydroxy-L-nicotine oxidase, which acts strictly stereo-specific on the L-enantiomer . Surprisingly, L-nicotine also induces the synthesis of a 6-hydroxy-d-nicotine-specific oxidase in the bacteria . Genes of nicotine-degrading enzymes are located on the catabolic plasmid pAO1 . The pAO1 sequence revealed that the 6-hydroxy-D-nicotine oxidase gene is flanked by two open reading frames with a similarity to amino acid permeases and a divergently transcribed open reading frame with a similarity to proteins of the tetracycline repressor TetR family . Reverse transcription PCR and primer extension analysis of RNA transcripts isolated from A . nicotinovorans pAO1 indicated that the 6-hydroxy-D-nicotine oxidase gene represents a transcriptional unit . DNA electromobility shift assays established that the purified TetR-similar protein represents the 6-hydroxy-D-nicotine oxidase gene repressor HdnoR and binds to the 6-hydroxy-D-nicotine oxidase gene operator with a Kd of 21 nM . The enantiomers 6-hydroxy-D- and 6-hydroxy-L-nicotine acted in vitro as inducers . In vivo analysis of 6-hydroxy-D-nicotine oxidase gene transcripts from bacteria grown with L- and D-nicotine confirmed this conclusion . The poor discrimination by HdnoR between the 6-hydroxy-L- and 6-hydroxy-D-nicotine enantiomers explains the presence of the 6-hydroxy-D-nicotine-specific enzyme in bacteria grown on L-nicotine. J Biol Chem, 2003 Dec 19, 278(51), 51291 - 300 Epub 2003 Oct 08. Surface-exposed glycopeptidolipids of Mycobacterium smegmatis specifically inhibit the phagocytosis of mycobacteria by human macrophages . Identification of a novel family of glycopeptidolipids; Villeneuve C et al.; Phagocytosis by macrophages represents the early step of the mycobacterial infection . It is governed both by the nature of the host receptors used and the ligands exposed on the bacteria . The outermost molecules of the nonpathogenic Mycobacterium smegmatis were extracted by a mechanical treatment and found to specifically and dose dependently inhibit the phagocytosis of both M . smegmatis and the opportunistic pathogen M . kansasii by human macrophages derived from monocytes . The inhibitory activity was attributed to surface lipids because it is extracted by chloroform and reduced by alkaline hydrolysis but not by protease treatment . Fractionation of surface lipids by adsorption chromatography indicated that the major inhibitory compounds consisted of phospholipids and glycopeptidolipids (GPLs) . Mass spectrometry and nuclear magnetic resonance spectroscopy analyses, combined with chemical degradation methods, demonstrated the existence of a novel family of GPLs that consists of a core composed of the long-chain tripeptidyl amino-alcohol with a di-O-acetyl-6-deoxytalosyl unit substituting the allo-threoninyl residue and a 2-succinyl-3,4-di-O-CH3-rhamnosyl unit linked to the alaninol end of the molecules . These compounds, as well as diglycosylated GPLs at the alaninol end and de-O-acylated GPLs, but not the non-serovar-specific di-O-acetylated GPLs, inhibited the phagocytosis of M . smegmatis and M . avium by human macrophages at a few nanomolar concentration without affecting the rate of zymosan internalization . At micromolar concentrations, the native GPLs also inhibit the uptake of both M . tuberculosis and M . kansasii . De-O-acylation experiments established the critical roles of both the succinyl and acetyl substituents . Collectively, these data provide evidence that surface-exposed mycobacterial glycoconjugates are efficient competitors of the interaction between macrophages and mycobacteria and, as such, could represent pharmacological tools for the control of mycobacterial infections. Braz J Infect Dis, 2003 Aug, 7(4), 268 - 72 A case study of disseminated histoplasmosis linked to common variable immunodeficiency; Rachid A et al.; Histoplasma capsulatum, a ground fungus, can infect humans, normally in endemic areas; the resulting disease can be asymptomatic or it can have a benign development, but in rare cases it can develop into a serious clinical condition and can even be fatal . Its most characteristic initial location is in the lungs, resembling tuberculosis, often accompanied by mediastinitis and an exuberant fibrotic response . The spread of this infection can be caused by the concomitance of another illness that alters the immunological balance . Sometimes such an association is not clear . Therefore, disseminated histoplasmosis is defined as a clinical condition where the fungus is present in more than one location . Common variable immunodeficiency is characterized by a generalized failure in the synthesis of antibodies, leading the affected individuals to present recurrent infections, especially those caused by encapsulated bacteria, most often involving the respiratory tract . We studied a serious case of disseminated histoplasmosis, accompanied by common variable immunodeficiency, observed at the Infirmary of the department of Medical Practice of the Federal University at Parana Hospital das Clinicas. J Environ Sci Health A Tox Hazard Subst Environ Eng, 2003, 38(11), 2549 - 63 Estimation of methane emission from rice cultivation in Korea; Kwun SK et al.; This study focused on estimating a methane (CH4) inventory and developing mitigation options in South Korea, and was performed jointly in an integrated national research program on CH4 from rice fields conducted by three National Agricultural Research Institutes, under the Rural Development Administration during 1993-1997 . Methane emissions were measured by a closed chamber method, in rice plots at three locations (Suwon, Iksan, and Milyang) with the single rice cropping system . All experimental data from 5 years of study were summarized and used for calculating nation-wide CH4 emissions . Temperature, soil type, cultural practices, water management, organic matter management, and cultivar selection significantly affected the fluctuations of CH4 emissions . The two most promising mitigation options for reducing emissions were altered water management, in particular mid-season aeration by short-term drainage, and improved organic matter management, by promotion of aerobic degradation through composing or soil incorporation . Annual total CH4 emission in Korea changed from 410Gg in 1990 to 339Gg in 2000, due mainly to a decrease in the cultivated area of paddy rice . If we convert annual CH4 emission to Global Warming Potential as CO2 equivalent, it amounts to 7.1 M CO2 t yr(-1) of greenhouse gas emitted to the atmosphere in 2000 from the rice fields in Korea, which is just 5.3% of the annual CO2 emission from the industry and energy sector . More importantly, the balance between CO2 uptake by photosynthesis and CO2 emission is positive (a net sink), so that rice culture actually has net benefits for the global atmospheric carbon issue . Further reductions in emission amounts, by following recommendations in this article, could make these benefits even greater. Adv Exp Med Biol, 2001, 491, 505 - 14 Protective immunity and gene expression related to pneumococcal glycoconjugate; Lee CJ et al.; The immunogenicity of most polysaccharides (PSs) contained in the pneumococcal vaccine is low in children less than 2 years of age . Enhancement of immune response in early life can be induced by immunization of pneumococcal glycoconjugate as well as plasma DNA coding for cell-surface protein antigen . Pneumococcal type 19F PS conjugated with inactivated pneumolysin (Ply) induced in mice remarkable antibody responses to both type 19F PS and the protein carrier . In addition, the conjugate was administered to pregnant mice during gestation and/or lactation, and to their offspring during early infancy . When the young mice were challenged with type 19F pneumococci, the bacteria were cleared more rapidly from the blood of immunized mice than from that of the control group . The mortality rate of young mice from immunized mothers was also significantly lower than the control group . These results indicate that the effective protective immunity against pneumococcal infection can be induced in young mice by the maternal immunization with the glycoconjugate during gestation and at early infancy . Studies have been conducted to express type 19A pneumolysin gene (ply) in mammalian cells . Ply DNA was inserted into the cloning site of a vector containing CMV promoter . The recombinant plasmid DNA containing ply was transfected in human rhabdomyosarcoma cells and the gene expression was confirmed by immunoblot . Injection of mice three times 50-100 ug per dose ply DNA produced high serum levels of Ply IgG and IgM antibodies and showed rapid bacterial clearance from the blood. Antonie Van Leeuwenhoek, 2003, 84(2), 89 - 98 Nitrile hydrolysing activities of deep-sea and terrestrial mycolate actinomycetes; Brandao PF et al.; Nitrile metabolising actinomycetes previously recovered from deep-sea sediments and terrestrial soils were investigated for their nitrile transforming properties . Metabolic profiling and activity assays confirmed that all strains catalysed the hydrolysis of nitriles by a nitrile hydratase/amidase system . Acetonitrile and benzonitrile, when used as growth substrates for enzyme induction experiments, had a significant influence on the biotransformation activities towards various nitriles and amides . The specific activities of selected deep-sea and terrestrial acetonitrile-grown bacteria against a suite of nitriles and amides were higher than those of the only other reported marine nitrile-hydrolysing R . erythropolis, isolated from a shallow sediment . The increase of nitrile chain length appeared to have negative influence on the nitrile hydratase activity of acetonitrile-grown bacteria, but the same was not true for benzonitrile-grown bacteria . The nitrile hydratases and amidases were constitutive in 10 of the 16 deep-sea and terrestrial actinomycetes studied, and one strain showed an inducible hydratase and a constitutive amidase . Most of the deep-sea strains had constitutive activities and showed some of the highest activities and broadest substrate specificities of organisms included in this study. Rev Gastroenterol Peru, 2003 Jul-Sep, 23(3), 177 - 83 {Omeprazole, amoxicillin and clarithromycin in the treatment of helicobacterpylori, in 7 and 10-day regimens}; Rodriguez W et al.; AIM: The most accepted treatment for infection by Helicobacter pylori is the proton pump inhibitor based therapy with two antibiotics . However, there is no consensus regarding the duration . The purpose here was to compare eradication percentages in the omeprazole+amoxicillin+clarithromycin regimen administered during 7 days versus 10 days and confront the results with a previous 14-day* experience in Peru . METHOD: Patients from the Central Military Hospital and Peruvian-Japanese Hospital evidencing chronic upper gastrointestinal tract symptoms were recruited . We excluded patients with peptic ulcer . Biopsies were taken for diagnosis, for urease and PCR tests, culture and coloring with silver . Omeprazole+clarithromycin+amoxicillin was used during 7 days versus 10 days . Control endoscopy was performed one month after treatment had been completed and molecular biology techniques were used to differentiate recurrences from new infections . Susceptibility to clarithromycin was assessed . RESULTS: 36 patients were included in each group . Eradication was the same in both groups: 86.1% (31/36) . In several patients in whom the bacteria persisted, the same initial nucleus was found . In a previous study* using this same regimen during 14 days, a 93% eradication was obtained . 91.18% of our samples were susceptible to clarithromycin . CONCLUSIONS: In Peru, the omeprazole+clarithromycin+amoxicillin combination gives results higher than 80% in the eradication of infection by Helicobacter pylori . The 7 and 10 days regimens eradicated the bacteria in 86% of our patients. J Clin Microbiol, 2003 Oct, 41(10), 4573 - 7 Fast and accurate quantitative detection of Helicobacter pylori and identification of clarithromycin resistance mutations in H . pylori isolates from gastric biopsy specimens by real-time PCR; Lascols C et al.; Rapid identification of patients infected with clarithromycin-resistant Helicobacter pylori without the need for culture can help to avoid useless prescriptions of clarithromycin . We developed and tested a routine real-time quantitative PCR assay dedicated to that purpose . One hundred ninety-six consecutive gastric biopsy specimens were examined by culture, histology performed by a trained physician, and rapid PCR with the LightCycler apparatus . Infection was defined as (i) positivity of culture, (ii) positivity of histology, or (iii) positivity of PCR if confirmed by positivity of a concomitant indirect test (serology or urea breath test) . Susceptibility to clarithromycin was tested by E-test and PCR . The prevalence of infection was 33.7% (66 of 196 samples) . The sensitivities of culture, histology, and PCR were 90.9% (60 of 66 samples), 87.9% (58 of 66 samples), and 97.0% (64 of 66 samples), respectively . The specificity of PCR was 94.6% (123 of 130 samples) . The linearity of the PCR results was achieved over a 6-log range of input DNA, and we were able to accurately quantify as few as 300 bacteria and to qualitatively detect as few as 30 bacteria per DNA sample . For clarithromycin susceptibility testing, there was 98.2% (55 of 56 samples) concordance between E-test and PCR . Forty-eight strains were clarithromycin susceptible, and 9 strains were clarithromycin resistant . The single discrepancy concerned a culture which was a mixture of mutant and wild type, with a susceptible-to-resistant ratio of 11.5: the resistant population was detected by E-test but not by PCR . Our PCR assay is accurate for fast detection of H . pylori as well as of clarithromycin resistance and is also able to objectively determine bacterial density. Appl Environ Microbiol, 2003 Oct, 69(10), 6264 - 7 Detection of the free-living forms of sulfide-oxidizing gill endosymbionts in the lucinid habitat (Thalassia testudinum environment); Gros O et al.; Target DNA from the uncultivable Codakia orbicularis endosymbiont was PCR amplified from sea-grass sediment . To confirm that such amplifications originated from intact bacterial cells rather than free DNA, whole-cell hybridization (fluorescence in situ hybridization technique) with the specific probe Symco2 was performed along with experimental infection of aposymbiotic juveniles placed in contact with the same sediment . Taken together, the data demonstrate that the sulfide-oxidizing gill endosymbiont of Codakia orbicularis is present in the environment as a free-living uncultivable form. Appl Environ Microbiol, 2003 Oct, 69(10), 6152 - 64 Patterns of community change among ammonia oxidizers in meadow soils upon long-term incubation at different temperatures; Avrahami S et al.; The effect of temperature on the community structure of ammonia-oxidizing bacteria was investigated in three different meadow soils . Two of the soils (OMS and GMS) were acidic (pH 5.0 to 5.8) and from sites in Germany with low annual mean temperature (about 10 degrees C), while KMS soil was slightly alkaline (pH 7.9) and from a site in Israel with a high annual mean temperature (about 22 degrees C) . The soils were fertilized and incubated for up to 20 weeks in a moist state and as a buffered (pH 7) slurry amended with urea at different incubation temperatures (4 to 37 degrees C) . OMS soil was also incubated with less fertilizer than the other soils . The community structure of ammonia oxidizers was analyzed before and after incubation by denaturing gradient gel electrophoresis (DGGE) of the amoA gene, which codes for the alpha subunit of ammonia monooxygenase . All amoA gene sequences found belonged to the genus Nitrosospira . The analysis showed community change due to temperature both in moist soil and in the soil slurry . Two patterns of community change were observed . One pattern was a change between the different Nitrosospira clusters, which was observed in moist soil and slurry incubations of GMS and OMS . Nitrosospira AmoA cluster 1 was mainly detected below 30 degrees C, while Nitrosospira cluster 4 was predominant at 25 degrees C . Nitrosospira clusters 3a, 3b, and 9 dominated at 30 degrees C . The second pattern, observed in KMS, showed a community shift predominantly within a single Nitrosospira cluster . The sequences of the individual DGGE bands that exhibited different trends with temperature belonged almost exclusively to Nitrosospira cluster 3a . We conclude that ammonia oxidizer populations are influenced by temperature . In addition, we confirmed previous observations that N fertilizer also influences the community structure of ammonia oxidizers . Thus, Nitrosospira cluster 1 was absent in OMS soil treated with less fertilizer, while Nitrosospira cluster 9 was only found in the sample given less fertilizer. Appl Environ Microbiol, 2003 Oct, 69(10), 6082 - 90 Diversity of Wolbachia endosymbionts in heteropteran bugs; Kikuchi Y et al.; An extensive survey of Wolbachia endosymbionts in Japanese terrestrial heteropteran bugs was performed by PCR detection with universal primers for wsp and ftsZ genes of Wolbachia, cloning of the PCR products, restriction fragment length polymorphism analysis of infecting Wolbachia types, and molecular phylogenetic characterization of all the detected Wolbachia strains . Of 134 heteropteran species from 19 families examined, Wolbachia infection was detected in 47 species from 13 families . From the 47 species, 59 Wolbachia strains were identified . Of the 59 strains, 16 and 43 were assigned to A group and B group in the Wolbachia phylogeny, respectively . The 47 species of Wolbachia-infected bugs were classified into 8 species with A infection, 28 species with B infection, 2 species with AA infection, 3 species with AB infection, 5 species with BB infection, and 1 species with ABB infection . Molecular phylogenetic analysis showed little congruence between Wolbachia phylogeny and host systematics, suggesting frequent horizontal transfers of Wolbachia in the evolutionary course of the Heteroptera . The phylogenetic analysis also revealed several novel lineages of Wolbachia . Based on statistical analyses of the multiple infections, we propose a hypothetical view that, in the heteropteran bugs, interactions between coinfecting Wolbachia strains are generally not intense and that Wolbachia coinfections have been established through a stochastic process probably depending on occasional horizontal transfers. Scand J Gastroenterol, 2003 Sep, 38(9), 931 - 7 Duodenogastric reflux sustains Helicobacter pylori infection in the gastric stump; Nakagawara H et al.; BACKGROUND: Duodenogastric reflux (DGR) and Helicobacter pylori infection have been suspected of being contributing agents to the genesis of gastritis and subsequent cancer, but compelling, conclusive data about the exact relationship have been lacking . METHODS: We investigated the effect of DGR on H . pylori infection in 95 gastrectomized subjects divided into four groups according to type of reconstruction: the jejunal pouch interposition group (JPI, n = 36); the Roux-en-Y group (RY, n = 17); the Billroth I group (B-I, n = 20); and the Billroth II group (B-II, n = 22) . The following items were examined for each group: the duration of DGR; the prevalence of H . pylori infection; other bacterial identification and quantity; and the severity of gastritis . RESULTS: The percent of total time of DGR was lower in the JPI (7%) and RY groups (28%) than in the B-I (59%) and B-II groups (88%) (P < 0.02) . The prevalence of H . pylori infection was lower in the JPI (28%) and RY groups (29%) than in the B-I (60%) and B-II groups (73%) (P < 0.02) . Inversely, the JPI and the RY groups had a higher quantity of other bacteria than the B-I group (P = 0.02) . For all four groups, the stomachs infected with H . pylori were fewer than those tested negative for the organism (P < 0.0001) . Inflammation scores were lower in both the JPI and RY groups than in the B-I and B-II groups (P < 0.05, respectively) . CONCLUSIONS: Duodenogastric reflux facilitates the survival of H . pylori in the gastric stump after a distal gastrectomy. Water Sci Technol, 2003, 48(4), 229 - 33 Continuous biogas production from fodder beet silage as sole substrate; Scherer PA et al.; Since April 2000 a two-step anaerobic plant with two subsequent 500 m3 reactors has been producing biogas from fodder beet silage (pH 3.4-4.1) as the sole substrate . The plant is located at Kirchlengern near Bielefeld, Germany . Initially the reactors were inoculated with swine manure at 37 degrees C . After a start-up phase the process was sustained at pH 7.5-8.0 by feeding the silage as sole substrate with an HRT of about 55 d twice a day . Parallel to the biogas plant at Kirchlengern four one-step laboratory reactors were continuously driven at temperatures of 37 degrees C, 45 degrees C, 60 degrees C and 65 degrees C . They were fed with the same silage, but only once per day (one impulse) . The organic loading rate (OLR) was adjusted to 3.9 g volatile solids (VS)/(l*d) with a concomitant hydraulic retention time (HRT) of 27 d . There was no problem with starting the reactors, but after 86 days the volumetric gas production of the 65 degrees C reactor ceased and a high amount of approximately 65 mM propionate could be determined . By decreasing the temperature down to 60 degrees C a stable reactor performance was recovered for a period of at least 600 further days . Interestingly microscopic analyses revealed that the morphology of methanogenic bacteria in the 60 degrees C was quite different from the 37 and 45 degrees C reactor exhibiting only rodlike methanogens whereas at 37 degrees C coccoid morphotypes besides rodlike methanogens were dominant . In a 55 degrees C reactor (separate experiment) a mixture of coccoid and rodlike methanogens established . During impulse feeding with 3.9 g (VS)/(l*d) it was observed that the quickest recovery of gas production, the pH, CH4 and CO2 content as well as the redox value could be observed at 37 degrees C or at 45 degrees C . Recovery of 75% gas volume (related to the value before or after impulse feeding) was obtained after 5.5 and 7.5 h of feeding time point whereas the 60 degrees C reactor needed 16 h . Slight significant differences were seen in the spectrum of volatile fatty acids (VFA) reaching at 37 degrees or 45 degrees C its maximum with 10-30 mM total VFA at 2-3 h after feeding . After this the VFA level declined to nearly zero (except for the 60 degrees C reactor) . Therefore the 37 degrees C reactor was favoured . A double experiment with a second 37 degrees C reactor was started by a somewhat different inoculation procedure from the remaining 3 reactors, but revealed similar results . An impulse feeding experiment with a very high OLR of 16.5 g (VS)/(l*d) lasting 1 week offered a stable reactor performance with a peak GPR of up to 24 l/(l reactor *d) and an HRT of 5.45d . Therefore a long term operation with an HRT of only 7.5 days and an OLR of 12 g (VS)/(l*d) should be possible . By increasing the temperature no significantly different specific gas production rates and methane yields could be observed, e.g . it gave 600-7001 biogas from 1 kg VS . The corresponding methane content ranged between 62-64% . With a methane content of 63 +/- 1% a yield of 40.1 +/- 2 m3 methane/ton fresh fodder beet silage was obtained. Water Sci Technol, 2003, 48(4), 189 - 94 Concentration of pig slurry by evaporation: anaerobic digestion as the key process; Bonmati A et al.; Nutrient redistribution between areas with a structural pig slurry surplus and those with a shortage, is limited by the high cost of transportation and spreading, due to the high water content in slurry (more than 90%) and its relative low nutrient concentration . Water can be removed from slurry by evaporation, through the application of waste heat from a power plant or from other processes . Apart from obtaining a concentrate with an obviously higher nutrient concentration than the original slurry, another objective is to obtain clean water as condensate . The objective of this work was to study the batch vacuum evaporation of pig slurry liquid fraction, to evaluate the economic feasibility and to evaluate condensate quality as a function of both pH (4, 5 and 6) and pig slurry type (fresh slurry and anaerobically digested slurry) . Results showed that condensate characteristics (ammonia nitrogen, VFA, COD) were strongly dependent on these variables . Previous anaerobic digestion presented clear advantages: it provided a fraction of the required energy and it removed organic matter, preventing its volatilisation in the evaporation process and providing higher quality condensates . These advantages make the combined treatment strategy economically more feasible than the evaporation process alone. Water Sci Technol, 2003, 48(4), 147 - 54 A distributed model of solid waste anaerobic digestion: sensitivity analysis; Vavilin VA et al.; A distributed model of anaerobic digestion of solid waste was developed to describe the balance between the rates of polymer hydrolysis and methanogenesis during the anaerobic conversion of rich and lean wastes in batch and continuous-flow reactors . Waste, volatile fatty acids (VFAs), methanogenic biomass and sodium concentrations are the model variables . Diffusion and advection of VFAs inhibiting both polymer hydrolysis and methanogenesis were considered . A sensitivity analysis by changing the key model parameter values was carried out . The model simulations showed that the effective distance between the areas of hydrolysis/acidogenesis and methanogenesis is very important . An initial spatial separation of rich waste and inoculum enhances the methane production and waste degradation at high waste loading if relatively low VFA diffusion into the methanogenic area is taking place . When both hydrolysis and methanogenesis are strongly inhibited by high levels of VFA, fluctuations in biomass concentration are thought to be responsible for initiating the expansion of methanogenic area over the reactor space. Water Sci Technol, 2003, 48(4), 85 - 96 Optimal process configuration for anaerobic digestion with ozonation; Goel R et al.; Economical source minimization of excess sludge production is an attractive option to deal with the problem of sludge disposal under strict disposal standards . In this paper long-term operational results for two different process configurations that combine oxidative ozone treatment with anaerobic sludge digestion are described . In the first configuration ozone pretreatment was combined with chemostat anaerobic digestion while in the second configuration ozone pre/post-treatments were combined with an anaerobic digester operated without solid removal . From the results of chemostat experiments, the ozone pretreatment solubilized around 19% and 37% of the solids at 0.015 and 0.05 gO3/gTS ozone dose respectively . The ozone pretreatment resulted in improved TVS reduction efficiencies and the degradation efficiencies were observed to depend on the applied ozone dose and system SRT . The TVS degradation efficiency for pre-ozonated sludge at an ozone dose of 0.05 gO3/gTS was 59% as compared to 31% for the control reactor fed with un-ozonated sludge . Test results with the second configuration indicated that overall TVS removal efficiencies for a process scheme with post-ozonation could be improved up to 85% with a minimum ozone dose of 0.045 gO3/gTVS-fed . However, since no solids (except that for sampling) were withdrawn in this configuration, the accumulated total solids in the reactors increased to 28 g/l to 30 g/l at pseudosteady state . The average specific methane recoveries were observed to be 0.36 lCH4/gTVS fed which were slightly lower than theoretically expected . Based on the experimental results, important points in the choice of process configuration are discussed. Water Sci Technol, 2003, 48(4), 69 - 75 High-rate anaerobic hydrolysis and acidogenesis of sewage sludge in a modified upflow reactor; Yu HQ et al.; Continuous experiments were conducted to study the hydrolysis and acidogenesis of sewage sludge in an upflow reactor with an agitator and a gas-liquid-solid separator . Results of this study showed that 34-78% of volatile suspended solids (VSS) in sewage sludge was hydrolyzed at pH in the range 4.0-6.5, 35 degrees C and 4-24 hours of hydraulic retention time (HRT) . About 31-65% of carbohydrate in sewage sludge, 20-45% of protein and 14-24% of lipid were acidified in this reactor . Hydrogen production was favored in lower pH and HRT, whereas methane production was encouraged at higher pH and HRT . Acetate, propionate, butyrate, and i-butyrate were the main aqueous acidogenic products . The distribution of these compounds in the effluent was more sensitive to pH, but was less sensitive to HRT . The maximu specific COD solubilization rate and specific volatile fatty acids production rate were 126 mg-COD/g-VSS x d and 102 mg-VFAIg-VSS x d, respectively . Compared with a CSTR, this modified upflow reactor was shown to be a more promising biosystem for the hydrolysis and acidogenesis of sewage sludge. Water Sci Technol, 2003, 48(4), 9 - 13 On-line NIR monitoring during anaerobic treatment of municipal solid waste; Hansson M et al.; An anaerobic digester (81) was fed with the organic fraction of municipal solid waste and monitored intermittently for two years with on-line near-infrared (NIR) spectroscopy and traditional chemical parameters analysed off-line . The dynamics that occurred due to changes in substrate composition (changed C:N ratio) and changes in operating conditions (overloading) could be followed using principal component analysis of the obtained NIR-spectra . In addition, process disturbances such as failed stirring and increased foaming were readily detected by the NIR-spectra . Using PLS regression the propionate concentration could be predicted in the range 0.1-3.6 g/l, RMSEP 0.53 g/l with slope 0.74 and correlation coefficient 0.85 . The response on changes in the digester fluid was reproducible and could be detected within 2.5 minutes, which can be considered as real-time monitoring. Nucleic Acids Res, 2003 Oct 15, 31(20), 5907 - 16 The complete nucleotide sequence and RNA editing content of the mitochondrial genome of rapeseed (Brassica napus L.): comparative analysis of the mitochondrial genomes of rapeseed and Arabidopsis thaliana; Handa H; The entire mitochondrial genome of rapeseed (Brassica napus L.) was sequenced and compared with that of Arabidopsis thaliana . The 221 853 bp genome contains 34 protein-coding genes, three rRNA genes and 17 tRNA genes . This gene content is almost identical to that of ARABIDOPSIS: However the rps14 gene, which is a pseudo-gene in Arabidopsis, is intact in rapeseed . On the other hand, five tRNA genes are missing in rapeseed compared to Arabidopsis, although the set of mitochondrially encoded tRNA species is identical in the two Cruciferae . RNA editing events were systematically investigated on the basis of the sequence of the rapeseed mitochondrial genome . A total of 427 C to U conversions were identified in ORFs, which is nearly identical to the number in Arabidopsis (441 sites) . The gene sequences and intron structures are mostly conserved (more than 99% similarity for protein-coding regions); however, only 358 editing sites (83% of total editings) are shared by rapeseed and ARABIDOPSIS: Non-coding regions are mostly divergent between the two plants . One-third (about 78.7 kb) and two-thirds (about 223.8 kb) of the rapeseed and Arabidopsis mitochondrial genomes, respectively, cannot be aligned with each other and most of these regions do not show any homology to sequences registered in the DNA databases . The results of the comparative analysis between the rapeseed and Arabidopsis mitochondrial genomes suggest that higher plant mitochondria are extremely conservative with respect to coding sequences and somewhat conservative with respect to RNA editing, but that non-coding parts of plant mitochondrial DNA are extraordinarily dynamic with respect to structural changes, sequence acquisition and/or sequence loss. J Mol Biol, 2003 Oct 17, 333(2), 355 - 65 Binders based on dimerised immunoglobulin VH domains; Sepulveda J et al.; Antibody binding to antigen is mediated by the surface formed by the association of the two variable (V) regions of the L (VL) and H (VH) chains . The capacity of VL to dimerise and the high structural similarity of VL and VH domains suggested the possibility that VH could also associate . We show here that spontaneous formation of VH dimers (VHD) is in many cases permissive, producing stable molecules with antigen binding specificity . VHD were displayed on filamentous phages for the selection of antigen-specific binders . VHD were expressed and secreted efficiently from both bacteria and mammalian cells in different formats, including single-chain (VH(1)-linker-VH(2)), double chain ((VH(2)) and IgG analogues having the VL replaced by VH . The affinity (Kd,app) achieved with a VH dimer expressed in the IgG format, specific for a glutenin subunit was around 30 nM measured by two different methods, which was about 20 times higher than that corresponding to the VL/VH counterpart. Curr Top Med Chem, 2003, 3(13), 1512 - 35 Natural products with anti-HIV activity from marine organisms; Tziveleka LA et al.; In order to combat the human immunodeficiency virus (HIV), diverse strategies have been developed to research on compounds which can be developed as therapeutic agents . Screening of natural products derived from numerous species has afforded metabolites with significant antiviral activity against the HIV . The marine environment representing approximately half of the global biodiversity offers an enormous resource for novel compounds . Currently more than 150 natural products with promising levels of anti-HIV activity have been isolated following bioassay guided protocols from aqueous or organic extracts of marine organisms . Some of the most characteristic marinemetabolites that have exhibited significant anti-HIV activity on different biochemical assays designed for chemotherapeutic strategies are: Cyanovirin-N, a protein from a blue green alga; various sulfated polysaccharides extracted from seaweeds (i.e . Nothogenia fastigiata, Aghardhiella tenera); the peptides tachyplesin and polyphemusin, which are highly abundant in hemocyte debris of the horseshoe crabs Tachypleus tridentatus and Limulus polyphemus; sponge metabolites such as avarol, avarone, ilimaquinone and several phloroglucinols; and a number of metabolites from marine fungi such as equisetin, phomasetin and integric acid . Considering that number of unique metabolites that have been isolated from a small extent of the ocean's biological and chemical diversity, the oceans represent a virtually untapped resource for the discovery of novel bioactive compounds. Comb Chem High Throughput Screen, 2003 Sep, 6(6), 489 - 500 Strategies for combinatorial biosynthesis with modular polyketide synthases; Donadio S et al.; Polyketides are assembled by the polyketide synthases (PKS) through a common mechanism, the condensation of small carboxylic acids . However, a large structural variety exists within these molecules, paralleled by their different bioactivities . Structural differences in polyketides mostly stem from variations in the number of elongation cycles, in the extender unit incorporated and the extent of processing occurring during each cycle . A significant fraction of polyketides is made in bacteria by modular PKSs, which direct polyketide synthesis on a protein template, where each module is responsible for selecting, incorporating and processing the appropriate carboxylate unit . Since their discovery in the early nineties, the architecture of modular PKSs and their modus operandi have attracted efforts by several laboratories to reprogram PKSs to produce tailor-made polyketides . The availability of a growing number of modular PKSs of defined sequence, and of well-developed model systems for the in vitro and in vivo analysis of these enzymes, has led to the successful production of many novel polyketides after genetic manipulation of the appropriate PKS . We discuss the different strategies that are followed for the construction of functional "hybrid" systems, with particular emphasis on what can be done in terms of generating chemical diversity, highlighting also the limitations of our current understanding . The prospects of generating novel useful polyketides by genetic engineering are also discussed. Biochemistry, 2003 Oct 14, 42(40), 11716 - 25 Receptor-regulated dynamic interaction between endothelial nitric oxide synthase and calmodulin revealed by fluorescence resonance energy transfer in living cells; Jobin CM et al.; The endothelial isoform of nitric oxide synthase (eNOS), a key regulator of vascular tone, is activated in endothelial cells by diverse Ca(2+)-mobilizing agonists, including vascular endothelial growth factor (VEGF) . Although the activation state of eNOS and the subcellular localization of the enzyme are both highly regulated, the relationship between enzyme activity and subcellular targeting remains obscure . We aim here to elucidate this relationship by direct dynamic imaging analysis of Ca(2+)/CaM-dependent eNOS activation in living endothelial cells, using high-resolution confocal microscopy and donor dequenching fluorescence resonance energy transfer (FRET) techniques . Confocal images show a complex pattern of eNOS subcellular distribution; the enzyme is concentrated in both the plasma membrane and internal membranes, with robust expression in the perinuclear region . We construct a fusion protein between eNOS and the FRET-based calcium sensor cameleon, and analyze the temporal and spatial pattern of VEGF-mediated calcium mobilization using donor dequenching FRET methods . We find that VEGF promotes rapid mobilization of intracellular calcium throughout the regions of the cell in which eNOS is distributed . We further create a series of fusion proteins and use FRET imaging methods to study the interactions between eNOS and its obligate allosteric activator protein calmodulin . We clone the FRET acceptor EYFP (enhanced yellow fluorescent protein) at the C-terminus of calmodulin, and the FRET donor ECFP (enhanced cyan fluorescent protein) into eNOS at a site adjacent to its calmodulin-binding domain . FRET imaging analysis of individual endothelial cells cotransfected with eNOS-ECFP and calmodulin-EYFP shows that VEGF induces interactions between eNOS and calmodulin wherever both are present in the cell . Our studies provide evidence that the pool of rapidly responsive receptor-activated eNOS is distributed throughout endothelial cells in both plasma membrane and internal membrane structures, and that this distribution parallels the localization of agonist-induced intracellular Ca(2+) changes in the vicinity of eNOS. Biochemistry, 2003 Oct 14, 42(40), 11707 - 15 Crystal structure of sulerythrin, a rubrerythrin-like protein from a strictly aerobic archaeon, Sulfolobus tokodaii strain 7, shows unexpected domain swapping; Fushinobu S et al.; Sulerythrin is the first rubrerythrin-like protein to be isolated from an aerobic organism, Sulfolobus tokodaii strain 7, and it lacks a C-terminal rubredoxin-like FeS(4) domain . The protein purified from Sulfolobus cells was crystallized, and the crystal structure was determined at 1.7 A resolution . The dimer of sulerythrin exhibited "domain-swapping" at the loop connecting alphaB and alphaC, hybrid four-helix bundles consisting of alphaA/B and alphaC/D being formed . The structure and atomic identity of the binuclear metal center were determined by means of anomalous scattering analysis . The site contained 1.0 mol of hexacoordinate Fe, 0.80-0.87 mol of tetracoordinate Zn, and 0.73-0.88 mol of putative O(2) per monomer . The metal ions were found at exchanged positions compared to those in the Fe/Zn-containing rubrerythrin from Desulfovibrio vulgaris . The results demonstrate that the binuclear metal center of rubrerythrin-like proteins is plastic in its ability to bind metal ions. Syst Appl Microbiol, 2003 Sep, 26(3), 438 - 44 Identification and differentiation of species and strains of Arthrobacter and Microbacterium barkeri isolated from smear cheeses with Amplified Ribosmal DNA Restriction Analysis (ARDRA) and pulsed field gel electrophoresis (PFGE); Hoppe-Seyler TS et al.; ARDRA (Amplified Ribosomal-DNA Restriction Analysis) was used to differentiate among species and genera of Arthrobacter and Microbacteria . Species-specific restriction patterns of PCR-products were obtained with NciI for Arthrobacter citreus (DSM 20133T), A . sulfureus (DSM 20167T), A . globiformis (DSM 20124T) and A . nicotianae strains (DSM 20123T, MGE 10D, CA13, CA14, isolate 95293, 95294, and 95299), A . rhombi CCUG 38813T, and CCUG 38812, and Microbacterium barkeri strains (DSM 30123T, MGE 10D, CA12 and CA15, isolate 95292, and isolate 95207) . All yellow pigmented coryneforme bacteria isolated from the smear of surface ripened cheeses were identified as either A . nicotianae or M . barkeri strains . Using pulsed field gel electrophoresis (PFGE) strain specific restriction pattern for all Arthrobacter species and Microbacteria tested were obtained with restriction enzymes AscI and SpeI. Nat Neurosci, 2003 Nov, 6(11), 1201 - 7 Epub 2003 Oct 05. Dendritic stability in the adult olfactory bulb; Mizrahi A et al.; In many regions of the adult mammalian brain, pronounced changes in synaptic input caused by lesions or severe sensory deprivation induce marked sprouting or retraction of neuronal dendrites . In the adult olfactory bulb, adult neurogenesis produces less pronounced, but continuously ongoing synapse turnover . To test the structural stability of adult dendrites in this context, we used two-photon microscopy to image dendrites of mitral and tufted (M/T) cells over prolonged periods in adult mice . Although pharmacologically increased activity could elicit morphological changes, under natural conditions such as ongoing neurogenesis, an odor-enriched environment or olfactory-based learning, M/T cell dendrites remained highly stable . Thus, in a context of ongoing adult synaptogenesis, dendritic stability could serve as a structural scaffold to maintain the organization of local circuits. Gene, 2003 Sep 18, 314, 149 - 55 Distribution of the MCS4 RNA genes in mycoplasmas belonging to the Mycoplasma mycoides cluster; Ushida C et al.; MCS4 RNA is one of the small stable RNAs found in Mycoplasma capricolum subsp . capricolum type strain California kid . This RNA has a sequence similarity to that of eukaryotic U6 snRNA . There are two genes encoding MCS4 RNA, designated mcs4a and mcs4b, in the genome . Homologous sequences of these genes were not found in databases of other bacterial sequences . We searched for MCS4 RNA and its genes in other bacteria by PCR and hybridization techniques . The results strongly suggested that this RNA exists only in a limited species of mycoplasmas belonging to the Mycoplasma mycoides cluster. FEBS Lett, 2003 Sep 25, 552(2-3), 135 - 40 Distal side tryptophan, tyrosine and methionine in catalase-peroxidases are covalently linked in solution; Jakopitsch C et al.; Distal side tryptophan and tyrosine have been shown to be essential in the catalase but not the peroxidase activity of bifunctional catalase-peroxidases (KatGs) . Recently published crystal structures suggest that both residues could be part of a novel adduct including in addition a conserved methionine . A mass spectrometric analysis of the tryptic peptides from recombinant wild-type Synechocystis KatG and the variants Trp122Phe, Tyr249Phe and Met275Ile confirms that this novel adduct really exists in solution and thus may be common to all KatGs . Exchange of either Trp122 or Tyr249 prevents cross-linking, whereas exchange of Met275 still allowed bond formation between Trp122 and Tyr249 . It is proposed that the covalent bond between Trp and Tyr may form before that between Tyr and Met . The findings are discussed with respect to the mechanism of cross-linking and its role in KatG catalysis. Int J Parasitol, 2003 Oct, 33(12), 1409 - 18 The protozoan parasite, Theileria annulata, induces a distinct acute phase protein response in cattle that is associated with pathology; Glass EJ et al.; Acute phase proteins (APP) are synthesised in the liver in response to the systemic presence of high levels of pro-inflammatory cytokines . Bacteria are considered to be strong inducers of APP whereas viruses are weak or non-inducers of APP . Very few reports have been published on APP induction by parasites . Here, we report that the tick-borne protozoan parasite of cattle, Theileria annulata, induced an atypical acute phase response in cattle . Following experimental infection, serum amyloid A (SAA) appeared first, followed by a rise in alpha(1) acid glycoprotein (alpha(1)AGP) in all animals, whereas haptoglobin, which is a major APP in cattle, only appeared in some of the animals, and generally at a low level . All three APP only became elevated around or after the appearance of schizonts in draining lymph nodes and after the first observed temperature rise . Increased alpha(1)AGP levels coincided with the appearance of piroplasms . The production of SAA and alpha(1)AGP correlated strongly with each other, and also with some clinical measures of disease severity including the time to fever, development of leucopaenia, parasitaemia and mortality . These results are consistent with the hypothesis that T . annulata causes severe pathology in susceptible cattle by inducing high levels of pro-inflammatory cytokines. Int J Parasitol, 2003 Oct, 33(12), 1363 - 71 Infection of human and bovine epithelial cells with Cryptosporidium andersoni induces apoptosis and disrupts tight junctional ZO-1: effects of epidermal growth factor; Buret AG et al.; The effects of Cryptosporidium andersoni on human or bovine epithelia are poorly defined . Epidermal growth factor inhibits colonisation of the gastrointestinal epithelium with bacteria and the enteric protozoan parasite Giardia lamblia . This study characterised whether C . andersoni infects human or bovine epithelial cells in vitro, assessed its impact on apoptosis and tight junctional Zonula-Occludens-1, and determined whether these effects may be altered by epidermal growth factor . Monolayers of human colonic CaCo(2) cells, SCBN (non-malignant small intestinal epithelial cells), and Madin Darby bovine kidney epithelial cell lines (MDBK and NBL-1) were grown to confluency in Dulbecco's Modified Eagle Medium . Monolayers were assigned to one of three experimental groups-(1) control: exposed to culture medium alone; (2) untreated: exposed to 10(3) live C . andersoni oocysts or (3) epidermal growth factor-treated: apically pre-treated with recombinant human epidermal growth factor and then exposed to Cryptosporidium . Oocyst viability, infection with Cryptosporidium, apoptosis, and integrity of tight junctional Zonula-Occludens-1 were assessed . In addition, live Cryptosporidium oocysts were incubated with epidermal growth factor to assess whether epidermal growth factor had cryptosporicidial activity . Cryptosporidium andersoni oocysts infected all human and bovine monolayers, increased nuclear fragmentation, and disrupted Zonula-Occludens-1 . Apical epidermal growth factor significantly reduced infection with C . andersoni in all cell lines and inhibited the Cryptosporidium-induced apoptosis and disruption of Zonula-Occludens-1 . Epidermal growth factor did not affect oocyst viability. Annu Rev Biochem, 2003, 72, 209 - 47 The many faces of vitamin B12: catalysis by cobalamin-dependent enzymes; Banerjee R et al.; Vitamin B12 is a complex organometallic cofactor associated with three subfamilies of enzymes: the adenosylcobalamin-dependent isomerases, the methylcobalamin-dependent methyltransferases, and the dehalogenases . Different chemical aspects of the cofactor are exploited during catalysis by the isomerases and the methyltransferases . Thus, the cobalt-carbon bond ruptures homolytically in the isomerases, whereas it is cleaved heterolytically in the methyltransferases . The reaction mechanism of the dehalogenases, the most recently discovered class of B12 enzymes, is poorly understood . Over the past decade our understanding of the reaction mechanisms of B12 enzymes has been greatly enhanced by the availability of large amounts of enzyme that have afforded detailed structure-function studies, and these recent advances are the subject of this review. Annu Rev Biochem, 2003, 72, 77 - 109 Function and structure of complex II of the respiratory chain; Cecchini G; Complex II is the only membrane-bound component of the Krebs cycle and in addition functions as a member of the electron transport chain in mitochondria and in many bacteria . A recent X-ray structural solution of members of the complex II family of proteins has provided important insights into their function . One feature of the complex II structures is a linear electron transport chain that extends from the flavin and iron-sulfur redox cofactors in the membrane extrinsic domain to the quinone and b heme cofactors in the membrane domain . Exciting recent developments in relation to disease in humans and the formation of reactive oxygen species by complex II point to its overall importance in cellular physiology. Annu Rev Microbiol, 2003, 57, 467 - 85 Natural selection and the emergence of a mutation phenotype: an update of the evolutionary synthesis considering mechanisms that affect genome variation; Caporale LH; Most descriptions of evolution assume that all mutations are completely random with respect to their potential effects on survival . However, much like other phenotypic variations that affect the survival of the descendants, intrinsic variations in the probability, type, and location of genetic change can feel the pressure of natural selection . From site-specific recombination to changes in polymerase fidelity and repair of DNA damage, an organism's gene products affect what genetic changes occur in its genome . Through the action of natural selection on these gene products, potentially favorable mutations can become more probable than random . With examples from variation in bacterial surface proteins to the vertebrate immune response, it is clear that a great deal of genetic change is better than "random" with respect to its potential effect on survival . Indeed, some potentially useful mutations are so probable that they can be viewed as being encoded implicitly in the genome . An updated evolutionary theory includes emergence, under selective pressure, of genomic information that affects the probability of different classes of mutation, with consequences for genome survival. Genome Res, 2003 Oct, 13(10), 2353 - 62 Comprehensive analysis of orthologous protein domains using the HOPS database; Storm CE et al.; One of the most reliable methods for protein function annotation is to transfer experimentally known functions from orthologous proteins in other organisms . Most methods for identifying orthologs operate on a subset of organisms with a completely sequenced genome, and treat proteins as single-domain units . However, it is well known that proteins are often made up of several independent domains, and there is a wealth of protein sequences from genomes that are not completely sequenced . A comprehensive set of protein domain families is found in the Pfam database . We wanted to apply orthology detection to Pfam families, but first some issues needed to be addressed . First, orthology detection becomes impractical and unreliable when too many species are included . Second, shorter domains contain less information . It is therefore important to assess the quality of the orthology assignment and avoid very short domains altogether . We present a database of orthologous protein domains in Pfam called HOPS: Hierarchical grouping of Orthologous and Paralogous Sequences . Orthology is inferred in a hierarchic system of phylogenetic subgroups using ortholog bootstrapping . To avoid the frequent errors stemming from horizontally transferred genes in bacteria, the analysis is presently limited to eukaryotic genes . The results are accessible in the graphical browser NIFAS, a Java tool originally developed for analyzing phylogenetic relations within Pfam families . The method was tested on a set of curated orthologs with experimentally verified function . In comparison to tree reconciliation with a complete species tree, our approach finds significantly more orthologs in the test set . Examples for investigating gene fusions and domain recombination using HOPS are given. Phys Rev E Stat Nonlin Soft Matter Phys . 2003 Aug;68(2 Pt 1):021913 . Epub 2003 Aug 22. Multifractal and correlation analyses of protein sequences from complete genomes; Yu ZG et al.; A measure representation of protein sequences similar to the measure representation of DNA sequences proposed in our previous paper {Yu et al., Phys . Rev . E 64, 031903 (2001)} and another induced measure are introduced . Multifractal analysis is then performed on these two kinds of measures of a large number of protein sequences derived from corresponding complete genomes . From the values of the D(q) (generalized dimensions) spectra and related C(q) (analogous specific heat) curves, it is concluded that these protein sequences are not completely random sequences . For substrings with length K=5, the D(q) spectra of all organisms studied are multifractal-like and sufficiently smooth for the C(q) curves to be meaningful . The C(q) curves of all bacteria resemble a classical phase transition at a critical point . But the "analogous" phase transitions of higher organisms studied exhibit the shape of double-peaked specific heat function . But for the classification problem, the multifractal property is not sufficient . When the measure representations of protein sequences from complete genomes are considered as time series, a method based on correlation analysis after removing some memory from the time series is proposed to construct a phylogenetic tree . This construction is shown to be reasonably satisfactory. Phys Rev E Stat Nonlin Soft Matter Phys . 2003 Sep;68(3 Pt 2):036108 . Epub 2003 Sep 12. Generalized thermodynamics and Fokker-Planck equations: applications to stellar dynamics and two-dimensional turbulence; Chavanis PH; We introduce a class of generalized Fokker-Planck equations that conserve energy and mass and increase a generalized entropy functional until a maximum entropy state is reached . Nonlinear Fokker-Planck equations associated with Tsallis entropies are a special case of these equations . Applications of these results to stellar dynamics and vortex dynamics are proposed . Our prime result is a relaxation equation that should offer an easily implementable parametrization of two-dimensional turbulence . Usual parametrizations (including a single turbulent viscosity) correspond to the infinite temperature limit of our model . They forget a fundamental systematic drift that acts against diffusion as in Brownian theory . Our generalized Fokker-Planck equations can have applications in other fields of physics such as chemotaxis for bacterial populations . We propose the idea of a classification of generalized entropies in "classes of equivalence" and provide an aesthetic connection between topics (vortices, stars, bacteria, em leader ) which were previously disconnected. Biotechnol Prog, 2003 Sep-Oct, 19(5), 1460 - 7 Chimeric gene library construction by a simple and highly versatile method using recombination-dependent exponential amplification; Ikeuchi A et al.; A simple and efficient method for the construction of chimeric gene libraries termed RDA-PCR (recombination-dependent exponential amplification polymerase chain reaction) was developed by modifying polymerase chain reaction . A chimeric gene library is generated from homologous parental genes with additional primer-annealing sequences at their "heads" and "tails" . Two primers ("skew primers") are designed to exclusively anneal to either the heads of maternal genes or the tails of paternal genes . During the RDA-PCR, short annealing/extension periods facilitate homologous recombination . The chimeric sequences can be exponentially amplified to form the chimeric gene library, whereas parental sequences without crossovers are not amplified . As a model, we constructed a chimeric gene library of yellow and green fluorescent protein (yfp and gfp, respectively) . The crossover point profile of RDA-PCR clones was compared with those obtained by (modified) family shuffling . PCR restriction fragment polymorphism (PCR-RFLP) analysis of the RDA-PCR clones showed a high content of chimeric genes in the library, whereas family shuffling required the modification using skew primers for selective enrichment of chimeric sequences . PCR-RFLP analysis also indicated that the crossover points of RDA-PCR chimeras were distributed over the entire protein-coding region . Moreover, as few as 2 bp of the continual identity of nucleotides were found at the crossover points at high frequency (30% of the tested clones), suggesting that RDA-PCR resulted in a higher diversity in crossover points than family shuffling. J Environ Sci Health A Tox Hazard Subst Environ Eng, 2003, 38(10), 2043 - 55 Anaerobic biodegradability and treatment of Egyptian domestic sewage; Elmitwalli T et al.; The anaerobic biodegradability of domestic sewage for four Egyptian villages and four Egyptian cities was determined in batch experiments . The results showed that the biodegradability of the Egyptian-villages sewage (73%) was higher than that of the cities (66%) . The higher biodegradability of the soluble COD of village sewage (69%) as compared to that of the cities (46%) was the reason for the higher biodegradability of the total COD of the villages sewage . The biodegradability of suspended COD was similar for the sewage of both villages and cities (73-74%) . The results of a mathematical-model . developed based on anaerobic digestion model number 1, indicate that at applying a UASB reactor for the treatment of Egyptian villages and cities sewage, an optimum HRT of, respectively . 16 and 8 h is required . At these HRTs, a total COD removal and a conversion to methane of, respectively, 62-70% and 59-64% can be achieved for the sewage of cities and, respectively, 71-77% and 67-69% for the villages sewage . The model results also show that in the treatment of villages sewage in a two-step (anaerobic filter + UASB reactor) system a higher total COD removal can be obtained (77-81%) at a short HRT of 10 h (4 + 6 h) . However, the excess sludge from the first-step of the two-step system will be less stabilized. Microbiol Immunol, 2003, 47(8), 553 - 6 The role of non-chromosomal histones in the host defense system; Kanai Y; Histone H1 is located at the inter-nucleosome and more correctly at both ends of the double-stranded DNA that protrude from the nucleosome unit . It has long been recognized to be localized only inside the nuclei as a constituent for packaging nucleosome into chromatin . Thus, it could be hardly believed that detatched or solubilized histone H1 plays the role of a host defense molecule . Given the old reports on histone-like basic proteins that show bacteriostatic functions, I herein chose some recent related articles and tried review them . Recent advances in research on the cell death mechanism makes it possible to understand that programmed cell death, (i.e . apoptosis) could serve as a good source of soluble histones . Some forms of them are highly probable to be bacteriostatic. J Am Mosq Control Assoc, 2003 Sep, 19(3), 206 - 10 Molecular and electron microscopic identification of Wolbachia in Culex pipiens complex populations from the Upper Rhine Valley, Germany, and Cebu City, Philippines; Mahilum MM et al.; We detected Wolbachia infections in Culex pipiens complex mosquitoes from the Upper Rhine Valley, Germany, and Cebu City, Philippines, with the use of polymerase chain reaction (PCR) amplification of the 16S rDNA of the bacteria and further confirmation by electron microscopy . Species of 5 different autogenous and anautogenous populations of Culex pipiens from Germany and 1 population of Culex quinquefasciatus from the Philippines were used in this study . Larvae were sampled from different localities and reared to the adult stage for further processing . Whole adult males and the heads of adult females were processed by PCR, whereas the abdomens of females were used for electron microscopic study when an infection was detected by PCR . All Culex populations assayed by PCR showed infection of Wolbachia at rates between 10 and 100% . Females from different populations exhibited higher infection rates than did the males, and, likewise, Cx . pipiens populations that were autogenous showed higher infection rates of Wolbachia than did anautogenous individuals . In Cx . quinquefasciatus populations, males were infected at 33.33% and females, only 10% . We studied the ultrastructure of Wolbachia in the ovaries of species belonging to Cx . pipiens complex by means of transmission electron microscopy . The bacteria exhibited typical morphology for Wolbachia with 3 enveloping membranes. In Silico Biol, 2003, 3(1-2), 197 - 204 The channel in transporters is formed by residues that are rare in transmembrane helices; Kalinina OV et al.; Transmembrane transport is an essential component of the cell life . Many genes encoding known or putative transport proteins are found in bacterial genomes . In most cases their substrate specificity is not experimentally determined and only approximately predicted by comparative genomic analysis . Even less is known about the 3D structure of transporters . Nevertheless, the published experimental data demonstrate that channel-forming residues determine the substrate specificity of secondary transporters and analysis of these residues would provide better understanding of the transport mechanism . We developed a simple computational method for identification of channel-forming residues in transporter sequences . It is based on the analysis of amino acids frequencies in bacterial secondary transporters . We applied this method to a variety of transmembrane proteins with resolved 3D structure . The predictions are in sufficiently good agreement with the real protein structure. Pol Merkuriusz Lek, 2003 Jun, 14(84), 566 - 8 {Bronchial asthma infectious exacerbations}; Halasa J et al.; Bronchial asthma and asthma-like form of COPD often undergo exacerbations with symptoms of infection . Currently, there is a general agreement, that most of these infections that exacerbate asthma especially in children are caused by viruses . Several "common cold" viruses are known to cause these exacerbations (RSV, parainfluenza virus, rhinoviruses) . To date, there is no certainty, if viruses exacerbate asthma alone or in combination with allergen . It is also unknown, whether they can induce primary asthma symptoms . There is a growing support for the opinion, that in infectious exacerbations of asthma also bacteria can play an important role . These include both typical as well as atypical bacterial strains (Chlamydia pneumoniae) . The effectiveness of antibiotics and bacterial immunotherapy with vaccines seems to support the role of bacteria in asthma exacerbations . Another very important problem is related to the role of infectious agents in prevention of atopy . Many facts have been gathered supporting the so called "hygienic theory". Pol Merkuriusz Lek, 2003 Jun, 14(84), 514 - 7 {Importance of atypical infection in asthma}; Carewicz R et al.; The category of atypical respiratory infection involves viruses, bacteria and rickettsia . These micro-organisms can play a marked role in the exacerbation an asthma course but also participate in its development . Authors discuses both aspects, reporting own observations . The knowledge of real importance of atypical infections in asthma remains to be established. Br Med Bull, 2003, 66, 141 - 59 Immune system and peripheral nerves in propagation of prions to CNS; Aguzzi A et al.; Prions are not only unique in the way they replicate . Also the sequence of events triggered by peripheral prion infection, generically termed 'peripheral pathogenesis', sets prions aside from all other known pathogens . Whereas most bacteria, parasites, and viruses trigger innate and adaptive immune responses, the mammalian immune system appears to be remarkably oblivious to prions . Transmissible spongiform encephalopathies (TSEs) do not go along with inflammatory infiltrates, and antibodies to the prion protein are not typically raised during the course of the disease . On the other hand, there is conspicuous involvement of lymphoid organs, which accumulate sizeable concentrations of the infectious agent early during disease . Moreover, various states of immune deficiency can abolish peripheral pathogenesis and prevent 'take' of infection when prions are administered to peripheral sites . Here, we critically re-visit the current evidence for an involvement of the immune system in prion diseases, and will attempt to trace the elaborate mechanisms by which prions, upon entry into the body from peripheral sites, reach the brain. FEMS Immunol Med Microbiol, 2003 Oct 15, 38(3), 193 - 8 Acute inflammatory response induced by Helicobacter pylori in the rat air pouch; Gambero A et al.; Infection by Helicobacter pylori elicits persistent neutrophil infiltration in the gastric mucosa and stimulates the release of substances that may contribute to the establishment of gastritis . In this study, we used the rat air pouch model to evaluate the acute inflammatory response to H . pylori, in vivo . A pronounced neutrophil infiltration was observed 6 h and 12 h after the injection of H . pylori into the air pouch . Strains with different genotypes were able to induce cellular influx . This response was dependent upon the amount of bacteria injected and still occurred when heat-killed bacteria were employed . An increase in prostaglandin E(2) levels was observed, indicating that H . pylori induced cyclooxygenase 2 in this model . The production of interleukin-1 beta and tumor necrosis factor-alpha by leukocytes was also enhanced, suggesting that this model may be useful for studying the direct activation of neutrophils by H . pylori in vivo. Biochim Biophys Acta, 2003 Oct 1, 1629(1-3), 53 - 9 Identification and characterization of mouse MTO1 gene related to mitochondrial tRNA modification; Li R et al.; The nucleotide modification in tRNA plays a pivotal role in the fidelity of translational process . The defects in nucleotide modification have often been observed in the mutated mitochondrial tRNAs associated with human diseases . Recently, MTO1-like protein in bacteria and yeast has been implicated to be a component of tRNA modification pathway . Here we report the identification and characterization of mouse MTO1 homolog . The mouse MTO1 gene containing 12 exons encodes a 669-residue protein with a strong homology to the MTO1-like proteins of bacteria and yeast, related to tRNA modification . Functional conservation of this protein is supported by the observation that the isolated mouse MTO1 cDNA can complement the respiratory-deficient phenotype of yeast mto1 cells carrying P(R)(454) mutation . MTO1 is ubiquitously expressed in various tissues, but with markedly elevated expression in tissues of high metabolic rates . Furthermore, we showed that mouse Mto1 localizes in mitochondrion . These observations suggest that the mouse MTO1 is a structural and functional homolog of yeast MTO1, thereby playing a role in the mitochondrial tRNA modification and protein synthesis. Microb Pathog, 2003 Nov, 35(5), 229 - 34 Roles of conserved nucleotide-binding domains in accessory proteins, HypB and UreG, in the maturation of nickel-enzymes required for efficient Helicobacter pylori colonization; Mehta N et al.; Helicobacter pylori synthesizes two nickel-containing enzymes (urease and hydrogenase), both of which are important pathogenesis factors . Among the many accessory proteins needed for maturation of these Ni-enzymes, are two proteins, HypB and UreG, each of which contain a conserved nucleotide-binding domain (GSGKT) . To address the role of this domain in the maturation process, site-directed mutations were introduced in both hypB and ureG . The hypB site-directed mutant strain (Lys59 to Ala59) lacked hydrogenase activity and had less than 1% of the parental urease activity . Hydrogenase activity was partially, and urease activity was fully restored in the hypB mutant strain when grown on nickel supplemented media . The hydrogenase activity of the ureG site-directed mutant strain (Lys14 to Ala14) was comparable to that of the parental strain . However, the ureG mutant strain lacked urease activity, and this deficiency could not be suppressed even when the strain was grown on nickel supplemented media . The expression of immunologically detectable HypB and UreG in the mutants was similar to the parental strain . Expression of the UreA and UreB subunits of urease in both the mutants was also normal . Purified UreG parental and mutant (Lys14 to Ala14) proteins had molecular masses of 27 kDa, but possessed negligible GTP hydrolyzing activity. Emerg Infect Dis, 2003 Sep,