Bacteria

Mikrobiologiia, 2000 Jul-Aug, 69(4), 553 - 8
{Potential activity of methane and ammonia oxidation by methanotropic communities from soda lakes of the southern Transbaikal}; Khmelenina VN et al.; Radioisotopic measurements of the methane consumption by mud samples taken from nine Southern Transbaikal soda lakes (pH 9.5-10.6) showed an intense oxidation of methane in the muds of lakes Khuzhirta, Bumalai Nur, Gorbunka, and Suduntuiskii Torom, with the maximum oxidation rate in the mud of lakes Khuzhirta (33.2 nmol/(ml day)) . The incorporation rate of the radioactive label from 14CH4 into 14CO2 was higher than into acid-stable metabolites . Optimum pH values for methane oxidation in water samples were 7-8, whereas mud samples exhibited two peaks of methane oxidation activity (at pH 8.15-9.4 and 5.8-7.0) . The majority of samples could oxidize ammonium to nitrites; the oxidation was inhibited by methane . The PCR amplification analysis of samples revealed the presence of genes encoding soluble and particulate methane monooxygenase and methanol dehydrogenase . Three alkaliphilic methanotrophic bacteria of morphotype I were isolated from mud samples in pure cultures, one of which, B5, was able to oxidize ammonium to nitrites at pH 7-11 . The data obtained suggest that methanotrophs are widely spread in the soda lakes of Southern Transbaikal, where they actively oxidize methane and ammonium.

Microbes Infect, 2000 Aug, 2(10), 1285 - 92
The divergent role of tumor necrosis factor receptors in infectious diseases; Schluter D et al.; Tumor necrosis factor (TNF) receptor types 1 and 2 are broadly expressed by most cell types and are activated by binding of either TNF or lymphotoxin-beta . TNF receptor-mediated immune reactions are critically important in the pathogenesis and control of a variety of infections caused by bacteria, viruses, protozoa, and fungi . This review summarizes recent findings on the role of TNF receptors in infectious diseases and discusses the divergent functions of these receptors in immune responses.

Microbes Infect, 2000 Aug, 2(10), 1207 - 14
Helicobacter pylori motility; O'Toole PW et al.; Motility is essential for Helicobacter pylori colonization . This review discusses the biochemistry, genetics and genomics of the H . pylori flagellum, and compares these features with well-characterized bacteria.

Avian Dis, 2000 Jul-Sep, 44(3), 549 - 55
Ornithobacterium rhinotracheale infection in turkeys: immunoprophylaxis studies; Sprenger SJ et al.; Ornithobacterium rhinotracheale has been shown to cause serious clinical illness and is a significant concern to the turkey industry because of its potential economic impact . In this study, 6-wk-old turkeys were vaccinated intranasally with a live or subcutaneously with a killed O . rhinotracheale vaccine . At 14 or 21 wk of age, the birds were challenged intratracheally with live O . rhinotracheale . Airsacculitis and pneumonia occurred less frequently in vaccinated birds than in unvaccinated birds after challenge with O . rhinotracheale . Ornithobacterium rhinotracheale was recovered from unvaccinated, challenged birds but not from vaccinated, challenged or from unchallenged birds . Thus, turkeys inoculated with live or killed O . rhinotracheale vaccine were protected from pathologic changes.

Proc R Soc Lond B Biol Sci, 2000 Aug 7, 267(1452), 1487 - 95
Evolutionary novelties in islands: Drosophila santomea, a new melanogaster sister species from São Tomé; Lachaise D et al.; The finding of new melanogaster sister species may help us in understanding more about how the emergence of genetic novelties, particularly in insular habitats, can result in speciation . Here we report on the discovery of Drosophila santomea, which is the first melanogaster sibling found off West-equatorial Africa, on Sao Tome, one of the Gulf of Guinea islands . Although the eight other melanogaster sister species are remarkably conservative in their morphology except for their terminalia, the new find has a morphological trait distinguishing it from all of these: a pure yellow body coloration of both sexes without the normal black abdominal banding . Evidence from the terminalia, polytene and mitotic chromosomes, period gene and allozymes are provided indicating that it is nonetheless the nearest relative of Drosophila yakuba with which it coexists on the island . The new find is a clear-cut taxon as shown by the production of sterile male hybrids, eventually with developmental defects, in both directions of cross with yakuba and by the existence of an altitudinal divide accompanied by a hybrid zone at mid-elevation on the island . Molecular and karyotypic data further support this conclusion . In contrast to the significant divergence of their nuclear DNAs, an intriguing similarity in their cytochrome b sequences was observed indicating a recent coalescence common to santomea, yakuba and also teissieri cytoplasms . These were shown to harbour the same Wolbachia endosymbiotic bacteria which could possibly be responsible for mitochondrial DNA hitchhiking across the species barrier.

Proc Natl Acad Sci U S A, 2000 Oct 10, 97(21), 11391 - 6
Proliferation is necessary for both repair and mutation in transgenic mouse cells; Bielas JH et al.; Proliferating cells are often presumed to be more mutable than quiescent cells because they have less time to repair DNA damage before DNA replication . Direct tests of this hypothesis have been confounded by the need for cell division before a mutation can be detected . We have avoided this problem by showing that the Big Blue mouse cell line permits the dynamic quantification of both lesions and mutations in the complete absence of cell division . These cells carry the bacterial lacI gene in a lambda shuttle vector . Mutant plaques recovered by in vitro packaging of the mouse DNA can arise from mutations sustained either in mouse cells or in the bacteria . The proportion of mutant phage contained within a mutant plaque can distinguish these two types of mutation . Mutations formed in mouse cells yield >90% mutant phage because both DNA strands are mutant . On the other hand, mutations formed in the bacteria from adducted DNA yield </=50% mutant phage, because one of the DNA strands is wild type . Immediately after exposure to a test mutagen, ethylnitrosourea, all induced mutations were formed in the bacteria, but after approximately one cell division, the reverse was true and all mutations arose in the mouse cells . Only one-fifth as many mutations were recovered from quiescent cells and all arose in the bacteria, showing that the mouse cells made no mutations in the absence of proliferation . Surprisingly, the mouse cells did not repair any of the premutagenic damage during 4 days of quiescence . When these quiescent cells were induced to proliferate, however, both repair and mutation fixation ensued.

J Periodontal Res, 2000 Oct, 35(5), 247 - 58
A novel closed-tube quantitative--PCR method for enumerating Porphyromonas gingivitis, Prevotella intermedia and Actinobacillus actinomycetemcomitans; Doung-udomdacha S et al.; Enumeration of specific periodontopathogens in subgingival plaque samples has been problematic because of either lack of sensitivity, specificity or the time taken to identify the organisms . These problems can be overcome using PCR, but quantification by this technique is more difficult . We report a simple quantitative PCR method developed for enumerating Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans in clinical samples . The method relies upon inclusion of ethidium bromide in a closed-tube PCR reaction and measurement of resultant amplicons by quantifying the fluorescence generated when UV-light is passed through the walls of the amplification tube . Hence, both PCR and detection are performed in the same tube . The technique was compared with a quantitative competitive PCR and a commercial colorimetric quantitative PCR and proved to be at least as sensitive, specific and reliable for enumeration of the target bacteria . However, its speed and convenience make it particularly useful for large-scale analyses in both clinical laboratories and epidemiological studies.

Verh K Acad Geneeskd Belg, 2000, 62(4), 245 - 67
{The immunological basis of the administration of DTP-polio vaccine}; Cohen H; The main subject of this essay is the analysis of the immunizing properties and possible side effects of DTP (whole cell)-polio vaccine, which since 1964 is the backbone of the immunization programme in the Netherlands . The concept, that the basis of the immunity conferred by diphtheria and tetanus toxoid and inactivated poliovirus is humoral, is now generally accepted . Assessment of the potency of these three components is based on their binding properties to specific immunoglobulines in standard-antisera . An example is the testing of neutralising antibodies against the three types of poliovirus in sera obtained from different groups of infants injected with increasing doses of this vaccine . Side-effects after injecting diphtheria- and tetanus toxoids and three types inactivated poliovaccine are negligible . For the pertussis component of the vaccine, this is not the case . The standardization of the potency of cellular pertussis vaccine is still based on the analysis of the protective capacity of a number of plain vaccine lots in the intercerebral mouse-protection test relative to the protection they confer in children . An international standard vaccine is available for this purpose, and the potency of individual lots can be expressed in International Units . At present acellular pertussis vaccines are also available for immunization against this disease . The components of such vaccines are at least for a number of components, arbitrarily chosen . Recently in a trial in Sweden, a five-component pertussis vaccine has given a similar protection of infants as a cellular vaccine of British origin . In addition this acellular vaccine induced less febrile reactions . However, acellular vaccines do not protect mice against intracerebral infection . because producers have decided to omit the "Outer Membrane Complex (OMC) as a component of the vaccine . In addition it has recently been shown that the presence of the cellular pertussis component in DPT vaccine will almost completely suppress tetanus antitoxins in the IgE fraction of the serum . This observation could indicate that this suppression is the result of a cellular immunity reaction by components in the bacteria.

Biochim Biophys Acta, 2000 Jul 14, 1480(1-2), 342 - 52
The role of tryptophan residues in substrate binding to catalytic domains A and B of xylanase C from Fibrobacter succinogenes S85; McAllister KA et al.; Oxidation of the isolated catalytic domain B of xylanase C (XynC-B) from Fibrobacter succinogenes with N-bromosuccinimide (NBS) resulted in the modification of five of the seven Trp residues present in the enzyme . Hydrolytic activity of the enzyme was rapidly lost upon initiation of oxidation as a molar ratio of about two NBS molecules per molar equivalent of protein was sufficient to cause 50% inhibition of enzyme activity, and the addition of five molar equivalents of NBS resulted in less than 10% activity . Pre-incubation of XynC-B with the competitive inhibitor D-xylose resulted in the apparent protection of two Trp residues from oxidation . Xylose protection of the enzyme also resulted in a maintenance of activity, with 60% activity still evident after addition of 8-9 molar equivalents of NBS . This protection from inactivation was enhanced by the inclusion of xylohexaose in reaction mixtures . Under these conditions, however, a further Trp residue was protected from NBS oxidation . The three protected Trp residues were identified as Trp135, Trp161 and Trp202 by differential labelling and peptide mapping of NBS-oxidized preparations of the xylanase employing a combination of electrospray mass spectroscopic analysis and N-terminal sequencing . By analogy to the known structures of the family 11 xylanases, the fully conserved Trp202 residue is located on the only alpha-helix present in the enzymes, at the interface between it and the back of the beta-sheet which forms the active site cleft . Trp135 represents a highly conserved aromatic residue in family 11, but it is replaced with Thr in domain A of F . succinogenes xylanase C . To investigate the role of Trp135 in conferring the different activity profile of domain B relative to domain A, the Trp135Thr and Trp135Ala derivatives of domain B were prepared by site-directed mutagenesis . However, the kinetic parameters of the two domain B derivatives were not significantly different compared to the wild-type enzyme as reflected by K(M) and k(cat) values and product distribution profiles . Similar results were obtained with the Trp161Ala derivative of domain B, indicating that these two residues do not directly participate in the binding of substrate but likely form the foundation for binding subsite 2.

Biochim Biophys Acta, 2000 Jul 24, 1492(2-3), 295 - 310
Differential developmental expression and cell type specificity of Dictyostelium catalases and their response to oxidative stress and UV-light; Garcia MX et al.; Cells of Dictyostelium discoideum are highly resistant to DNA damaging agents such as UV-light, gamma-radiation and chemicals . The genes encoding nucleotide excision repair (NER) and base excision repair (BER) enzymes are rapidly upregulated in response to UV-irradiation and DNA-damaging chemicals, suggesting that this is at least partially responsible for the resistance of this organism to these agents . Although Dictyostelium is also unusually resistant to high concentrations of H(2)O(2), little is known about the response of this organism to oxidative stress . To determine if transcriptional upregulation is a common mechanism for responding to DNA-damaging agents, we have studied the Dictyostelium catalase and Cu/Zn superoxide dismutase antioxidant enzymes . We show that there are two catalase genes and that each is differentially regulated both temporally and spatially during multicellular development . The catA gene is expressed throughout growth and development and its corresponding enzyme is maintained at a steady level . In contrast, the catB gene encodes a larger protein and is only expressed during the final stages of morphogenesis . Cell type fractionation showed that the CatB enzyme is exclusively localized to the prespore cells and the CatA enzyme is found exclusively in the prestalk cells . Each enzyme has a different subcellular localization . The unique developmental timing and cell type distribution suggest that the role for catB in cell differentiation is to protect the dormant spores from oxidative damage . We found that exposure to H(2)O(2) does not result in the induction of the catalase, superoxide dismutase, NER or BER mRNAs . A mutant with greatly reduced levels of catA mRNA and enzyme has greatly increased sensitivity to H(2)O(2) but normal sensitivity to UV . These results indicate that the natural resistance to oxidative stress is not due to an ability to rapidly raise the level of antioxidant or DNA repair enzymes and that the response to UV-light is independent from the response to reactive oxygen compounds.

Biochim Biophys Acta, 2000 Aug 15, 1459(2-3), 390 - 6
Effects of nitric oxide and peroxynitrite on the cytochrome oxidase K(m) for oxygen: implications for mitochondrial pathology; Cooper CE et al.; This review summarises current knowledge about the effect of oxygen on cytochrome oxidase activity in vitro and in vivo . Cytochrome oxidase normally operates above its K(m) for oxygen in vivo . However, decreases in the intracellular oxygen concentration (hypoxia) under physiological extremes, or during pathophysiology, can cause mitochondrial respiration to become oxygen limited . Inhibitors that raise the enzyme's K(m) will induce oxygen limitation under apparently normoxic conditions . It is known that the concentrations of nitric oxide and peroxynitrite are raised in a number of pathophysiological conditions . These compounds are capable of reversibly and irreversibly raising the cytochrome oxidase K(m) for oxygen . Therefore, measurements of cell and mitochondrial respiration in vitro that fail to systematically vary oxygen through the range of physiological concentrations are likely to underestimate the effects of nitric oxide and peroxynitrite in vivo.

Biochim Biophys Acta, 2000 Aug 15, 1459(2-3), 310 - 5
Catalysis in fumarate reductase; Reid GA et al.; In the absence of oxygen many bacteria are able to utilise fumarate as a terminal oxidant for respiration . In most known organisms the fumarate reductases are membrane-bound iron-sulfur flavoproteins but Shewanella species produce a soluble, periplasmic flavocytochrome c(3) that catalyses this reaction . The active sites of all fumarate reductases are clearly conserved at the structural level, indicating a common mechanism . The structures of fumarate reductases from two Shewanella species have been determined . Fumarate, succinate and a partially hydrated fumarate ligand are found in equivalent locations in different crystals, tightly bound in the active site and close to N5 of the FAD cofactor, allowing identification of amino acid residues that are involved in substrate binding and catalysis . Conversion of fumarate to succinate requires hydride transfer from FAD and protonation by an active site acid . The identity of the proton donor has been open to question but we have used structural considerations to suggest that this function is provided by an arginine side chain . We have confirmed this experimentally by analysing the effects of site-directed mutations on enzyme activity . Substitutions of Arg402 lead to a dramatic loss of activity whereas neither of the two active site histidine residues is required for catalysis.

Biochim Biophys Acta, 2000 Aug 15, 1459(2-3), 274 - 83
Diversity and origin of alternative NADH:ubiquinone oxidoreductases; Kerscher SJ; Mitochondria from various organisms, especially plants, fungi and many bacteria contain so-called alternative NADH:ubiquinone oxidoreductases that catalyse the same redox reaction as respiratory chain complex I, but do not contribute to the generation of transmembrane proton gradients . In eucaryotes, these enzymes are associated with the mitochondrial inner membrane, with their NADH reaction site facing either the mitochondrial matrix (internal alternative NADH:ubiquinone oxidoreductases) or the cytoplasm (external alternative NADH:ubiquinone oxidoreductases) . Some of these enzymes also accept NADPH as substrate, some require calcium for activity . In the past few years, the characterisation of several alternative NADH:ubiquinone oxidoreductases on the DNA and on the protein level, of substrate specificities, mitochondrial import and targeting to the mitochondrial inner membrane has greatly improved our understanding of these enzymes . The present review will, with an emphasis on yeast model systems, illuminate various aspects of the biochemistry of alternative NADH:ubiquinone oxidoreductases, address recent developments and discuss some of the questions still open in the field.

Biochim Biophys Acta, 2000 Aug 15, 1459(2-3), 248 - 57
Solution structure of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase from Rhodospirillum rubrum; Jeeves M et al.; Transhydrogenase is a proton pump found in the membranes of bacteria and animal mitochondria . The solution structure of the expressed, 21.5 kDa, NADP(H)-binding component (dIII) of transhydrogenase from Rhodospirillum rubrum has been solved by NMR methods . This is the first description of the structure of dIII from a bacterial source . The protein adopts a Rossmann fold: an open, twisted, parallel beta-sheet, flanked by helices . However, the binding of NADP(+) to dIII is profoundly different to that seen in other Rossmann structures, in that its orientation is reversed: the adenosine moiety interacts with the first betaalphabetaalphabeta motif, and the nicotinamide with the second . Features in the structure that might be responsible for changes in nucleotide-binding affinity during catalysis, and for interaction with other components of the enzyme, are identified . The results are compared with the recently determined, high-resolution crystal structures of human and bovine dIII which also show the reversed nucleotide orientation.

An Esp Pediatr, 2000 Mar, 52(3), 245 - 50
{Experience with ventriculitis at a neonatology department}; Nieto Del Rincon N et al.; OBJECTIVE: To study the characteristics, treatment and follow-up of patients with ventriculitis in our neonatal unit . PATIENTS AND METHODS: Retrospective study of patients diagnosed with ventriculitis from January 1990 to December 1997 . Diagnostic criteria were the identification of any bacteria in the ventricular fluid and pleocytosis (> or = 100 leukocytes per microl) . Personal history, clinical and analytical findings and evolution after diagnosis were studied . RESULTS: We recorded ten cases of ventriculitis in nine patients . Six of them occurred as a complication of previous meningitis and four occurred after neurosurgical treatment . The mean age at diagnosis was of 38.8 days (range 8-130), and mean gestational age was 29.4 weeks (range 25-38) . Clinical and ventricular fluid anomalies were seen in six cases and in four the diagnosis was made at autopsy . Treatment was systemic antibiotics . In two cases intraventricular antibiotics were added . Six patients died, ventriculitis being the direct cause of death in five . Of the three survivors, one had mental retardation and cerebral palsy and the other two had minor disabilities . CONCLUSIONS: During the neonatal period, a high degree of clinical suspicion and techniques for an early diagnosis and treatment are needed for ventriculitis.

Mol Cell Biol, 2000 Oct, 20(20), 7784 - 97
Mmf1p, a novel yeast mitochondrial protein conserved throughout evolution and involved in maintenance of the mitochondrial genome; Oxelmark E et al.; A novel protein family (p14.5, or YERO57c/YJGFc) highly conserved throughout evolution has recently been identified . The biological role of these proteins is not yet well characterized . Two members of the p14.5 family are present in the yeast Saccharomyces cerevisiae . In this study, we have characterized some of the biological functions of the two yeast proteins . Mmf1p is a mitochondrial matrix factor, and homologous Mmf1p factor (Hmf1p) copurifies with the soluble cytoplasmic fraction . Deltammf1 cells lose mitochondrial DNA (mtDNA) and have a decreased growth rate, while Deltahmf1 cells do not display any visible phenotype . Furthermore, we demonstrate by genetic analysis that Mmf1p does not play a direct role in replication and segregation of the mtDNA . rho(+) Deltammf1 haploid cells can be obtained when tetrads are directly dissected on medium containing a nonfermentable carbon source . Our data also indicate that Mmf1p and Hmf1p have similar biological functions in different subcellular compartments . Hmf1p, when fused with the Mmf1p leader peptide, is transported into mitochondria and is able to functionally replace Mmf1p . Moreover, we show that homologous mammalian proteins are functionally related to Mmf1p . Human p14.5 localizes in yeast mitochondria and rescues the Deltammf1-associated phenotypes . In addition, fractionation of rat liver mitochondria showed that rat p14.5, like Mmf1p, is a soluble protein of the matrix . Our study identifies a biological function for Mmf1p and furthermore indicates that this function is conserved between members of the p14.5 family.

J Dairy Sci, 2000 Sep, 83(9), 2139 - 48
Whole-herd optimization with the Cornell Net Carbohydrate and Protein System . I . Predicting feed biological values for diet optimization with linear programming; Tedeschi LO et al.; We developed a diet optimizer for least-cost diet formulation with the Cornell Net Carbohydrate and Protein System (CNCPS) using linear programming . The CNCPS model is intrinsically nonlinear, and feed biological values vary with animal and feed characteristics . To allow linear diet optimization, we first used the CNCPS model to generate biological values to characterize the energy and protein content of each feed for the specific group for which the diet was being formulated . The biological values used were metabolizable energy (Mcal/kg), metabolizable protein {(% dry matter (DM)}, passage rate (%/h), bacteria yield efficiencies (g/g), and degradation rate of the carbohydrate B2 fraction (%/h) . In addition, the ruminal balances for nitrogen and peptides were included in the optimizer to optimize ruminal degradation of fiber . The objective function was to minimize diet cost subject to animal requirement and feed availability constraints . The animal constraints were set by requirements for DM intake (kg/d), metabolizable energy (Mcal/kg), metabolizable protein (%DM), and effective neutral detergent fiber (%DM) for a given level of production . Data from a dairy farm were used to evaluate this linear diet optimizer . Across all classes of dairy cattle, the CNCPS 4.0 model typically obtained a solution in less than six iterations that met the requirements with nearly 100% accuracy . We conclude this linear optimizer can be used to accurately formulate least-cost diets with the CNCPS model.

Parasitol Res, 2000 Sep, 86(9), 724 - 8
Direct amplification of Entamoeba histolytica DNA from amoebic liver abscess pus using polymerase chain reaction; Zaman S et al.; An important and serious complication of intestinal infection with Entamoeba histolytica is the involvement of the liver (hepatic amoebiasis) . Hepatic amoebiasis is usually diagnosed by the clinical picture (pain in the right upper quadrant and fever), ultrasound examination and positive serology . However, none of these tests are definitive and the picture overlaps with pyogenic liver abscess caused by bacteria . It is for this reason that the feasibility of using polymerase chain reaction (PCR) for the detection of E . histolytica DNA in liver abscess pus was investigated . A comparative study was done to verify the sensitivity of ten pairs of primers specific for detecting E . histolytica in stools . Samples of liver abscess pus from 22 serology-positive patients were collected under ultrasound guidance; and these were used directly in PCR assays without any prior pre-treatment of the samples . Of the ten pairs of previously published primers tested, two pairs of primers (PI + P2 and P11 + P12) were found to give 100% sensitivity . Based on these results, we recommend that PCR assay can be successfully used to confirm the diagnosis of amoebic liver abscess with the primers identified.

J Med Virol, 2000 Oct, 62(2), 140 - 3
Cytomegalovirus infection is not necessarily a poor prognostic factor in adult T-cell leukemia/lymphoma; Fujiwara H et al.; The relationship between cytomegalovirus (CMV) antigenemia and the clinical course was examined in 57 patients with adult T-cell leukemia/lymphoma (ATLL) . All patients included had the acute/lymphoma type of ATL according to the criteria of the Japan Lymphoma Study Group (LSG) . CMV antigenemia was assessed on admission and at the time when the patients had fever higher than 37 . 5 degrees C, which did not respond to antibiotics for longer than 3 days . The incidence of CMV antigenemia was 44% . Approximately 90% of patients with CMV antigenemia died of infections with viruses, bacteria, and/or fungi, while approximately 40% of patients without CMV antigenemia died of deterioration of ATLL (P<0.0001) . In this study, the patients with CMV antigenemia tended to survive longer than those negative for it (321.4 days vs . 266.2 days), although there was no statistical significance (P=0.09) . Kaplan-Meier analysis revealed that CMV antigenemia was not a poor prognostic factor . When the disease status of ATLL was evaluated by thymidine kinase (TK) and soluble interleukin 2 receptor (sIL-2R), both had lower titers during CMV antigenemia (TK: P=0.01, sIL-2R: P=0.03, respectively) . Therefore, CMV infections in ATLL patients seemed to have bimodal meanings; CMV infection at the end of clinical course were life-threatening, but infection during the first half of clinical course seemed to suppress ATLL activity and to contribute to the longer survival of the patients .

Biotechnol Appl Biochem, 2000 Oct, 32 ( Pt 2), 95 - 107
Design and production of recombinant subunit vaccines; Hansson M et al.; The development of subunit vaccines is presently the main strategy being evaluated for prevention of infectious diseases . The use of recombinant-DNA techniques has facilitated the development of new principles for design and production of subunit vaccines . First of all, the properties of a target protein immunogen can be improved by the use of gene-fusion technology or by the creation of specific changes, to generate 'second-generation protein vaccines' . Properties that can be modified include protein solubility, protein stability, in vivo half-lives, etc . In addition, for subunit protein vaccine candidates, the immunogenic properties can be significantly augmented by the addition of immunopotentiating tags or by means of targeting to immunoreactive sites . The recombinant subunit vaccine can furthermore be adapted by gene-fusion technology, to be efficiently incorporated into immunopotentiating adjuvant systems . Also in passive vaccination strategies, i.e . the use of antibodies or antibody fragments for prevention of infectious diseases, the recombinant strategies have become increasingly important . Humanized antibodies and antibody fusion proteins represent common present anti-infectious-disease agents . The selected examples will indicate that recombinant strategies will indeed have an impact on the design, selection and production of recombinant proteins to be used in the prevention of infectious diseases.

J Nucl Med Technol, 2000 Sep, 28(3), 162 - 4
The impact of brushing teeth on carbon-14 urea breath test results; Higazy E et al.; OBJECTIVE: The 14C urea breath test noninvasively detects the presence of the urease-producing bacteria Helicobacter pylori in the stomach . Several sources of errors have been identified to cause false or indeterminate results on the test . The objective of this study was to identify whether brushing teeth affects the test results . METHODS: We performed the 14C urea breath test on 168 patients, with breath samples counted at 10 and 20 min after oral administration of 2 microCi (74 kBq) 14C urea . Ninety-four patients brushed their teeth before the test while 74 did not . RESULTS: Thirty-six of the 74 patients (49%) who did not brush their teeth had positive results at 10 min, which became negative at 20 min . None of the 94 patients who brushed their teeth before testing showed this pattern with agreement of results at 10 and 20 min . CONCLUSION: We recommend brushing teeth before the 14C urea breath test since it significantly decreased the ambiguous results of the test in our laboratory.

J Biotechnol, 2000 Sep 29, 83(1-2), 77 - 84
How CD4(+) T cells may eliminate extracellular gastric Helicobacter?
Aebischer T, Lucas B, Koesling J, Bumann D, Meyer TF.
Helicobacter pylori is recognised as a causal agent in the pathogenesis of gastritis, gastric and duodenal ulcer disease as well as gastric cancers . Eradication of the bacteria with antibiotics is currently used to treat symptomatic, infected individuals . Theoretically the infection could also be controlled by vaccination . Several immunisation protocols were developed in small animal models and primates in order to validate this approach . Recently making use of mice with defined genetic defects, H . pylori-specific CD4(+) T cells were found to be crucial for protective vaccination . This was unexpected and poses the question of how activation of CD4(+) T cells leads to the elimination of bacteria that reside primarily in the mucin layer behind a barrier of epithelial cells . CD4(+) T cells fulfil their effector function by secreting lymphokines and by engaging specific surface ligands on interacting cells . Here we propose that phagocytes and epithelial cells stimulated either by direct interaction with CD4(+) T cells or by soluble mediators such as cytokines or neuropeptides are the ultimate effector populations in protective immunity induced by vaccination.

J Biotechnol, 2000 Sep 29, 83(1-2), 51 - 5
The porcine gastrointestinal lamina propria: an appropriate target for mucosal immunisation?
Stokes CR, Bailey M.
During the course of a lifetime, it has been calculated that we may consume between 100 and 700 tons of food . For the average British citizen, this is likely to include some 550 poultry, 36 pigs, 36 sheep, eight oxen, 10000 eggs and dairy products (milk, butter cheese, etc.) equivalent to 18 tonnes of milk . As if that were not sufficient enough a challenge, the homeostasis within the intestine is further complicated by the presence of 10(5)-10(11) bacteria (pathogenic and non-pathogenic) per gram of mucus and the constant turnover of gut epithelial cells . Given such a magnitude of challenge, which, at least in health is heavily biased in favour of harmless antigens, it can be reasonably hypothesised that the default response of the intestinal mucosal immune system would appear to be set heavily in favour of non-responsiveness and oral tolerance . The purpose of this review is to briefly describe recent progress from studies of the pig that support this hypothesis and to discuss the implications for future mucosal vaccine design.

Ann Genet, 2000 Apr-Jun, 43(2), 69 - 74
Human TIP49b/RUVBL2 gene: genomic structure, expression pattern, physical link to the human CGB/LHB gene cluster on chromosome 19q13.3; Parfait B et al.; Bacterial DNA helicase RuvB protein is an essential component in homologous recombination and DNA double-strand break repair . Here, we report the gene structure of TIP49b/RUVBL2, a second putative human homologue of the bacterial RuvB gene . This gene contains 15 exons and 14 introns . The TIP49b/RUVBL2 open reading frame encodes a protein of 463 amino acids, showing 43% identity with the RUVBL1 protein . The TIP49b/RUVBL2 gene is physically linked to the human CGB/LHB gene cluster on chromosome 19q13.3 . Genomic sequence analysis revealed that the TIP49b/RUVBL2 gene is very close (55 nucleotides in length) to the LHB gene, in the opposite orientation . The very close co-location of the mouse homologues of the human TIP49b/RUVBL2 and LHB genes was also conserved on mouse chromosome 7 . Co-ordinated transcriptional regulation between the TIP49b/RUVBL2 and LHB genes was not observed . TIP49b/RUVBL2, like RUVBL1, was expressed ubiquitously in all human tissues examined and more strongly in testis . As TIP49b/RUVBL2 is expected to be involved in recombination repair and is located in a chromosome region frequently amplified in breast cancer, we quantified TIP49b/RUVBL2 gene expression by using real-time quantitative RT-PCR in a series of breast tumour samples . None of the tumour samples showed an altered TIP49b/RUVBL2 transcription level relative to normal breast tissue.

Emerg Infect Dis, 2000 Sep-Oct, 6(5), 449 - 57
Antigenic variation in vector-borne pathogens; Barbour AG et al.; Several pathogens of humans and domestic animals depend on hematophagous arthropods to transmit them from one vertebrate reservoir host to another and maintain them in an environment . These pathogens use antigenic variation to prolong their circulation in the blood and thus increase the likelihood of transmission . By convergent evolution, bacterial and protozoal vector-borne pathogens have acquired similar genetic mechanisms for successful antigenic variation . Borrelia spp . and Anaplasma marginale (among bacteria) and African trypanosomes, Plasmodium falciparum, and Babesia bovis (among parasites) are examples of pathogens using these mechanisms . Antigenic variation poses a challenge in the development of vaccines against vector-borne pathogens.

Biochem J, 2000 Oct 1, 351(Pt 1), 281 - 8
Cloning and functional expression of a gene encoding a vacuolar-type proton-translocating pyrophosphatase from Trypanosoma cruzi; Hill JE et al.; Acidocalcisomes are acidic Ca(2+)-storage organelles found in trypanosomatids that are similar to organelles known historically as volutin granules . Acidification of these organelles is driven in part by a vacuolar H(+)-pyrophosphatase (V-H(+)-PPase), an enzyme that is also present in plant vacuoles and in some bacteria . Here, we report the cloning and sequencing of a gene encoding the acidocalcisomal V-H(+)-PPase of Trypanosoma cruzi . The protein (T . cruzi pyrophosphatase, TcPPase) predicted from the nucleotide sequence of the gene has 816 amino acids and a molecular mass of 85 kDa . Several sequence motifs found in plant V-H(+)-PPases were present in TcPPase, explaining its sensitivity to N-ethylmaleimide and N,N'-dicyclohexylcarbodi-imide . Heterologous expression of the cDNA encoding TcPPase in the yeast Saccharomyces cerevisiae produced a functional enzyme . Phylogenetic analysis of the available V-H(+)-PPase sequences indicates that TcPPase is nearer to the vascular plant cluster and the branch containing Chara, a precursor to land plants, than to any of the other pyrophosphatase sequences included in the analysis . The apparent lack of such a V-H(+)-PPase in mammalian cells may provide a target for the development of new drugs.

Anal Biochem, 2000 Oct 1, 285(1), 113 - 20
Selective extraction and purification of a mycobacterial outer membrane protein; Heinz C et al.; MspA forms water-filled channels in the mycolic acid layer of Mycobacterium smegmatis thereby allowing the diffusion of hydrophilic solutes through this permeability barrier into the periplasm . MspA is the first member of a new family of porins and is extremely stable against chemical and thermal denaturation . We developed a purification procedure based on selective extraction of MspA with detergents from whole cells of M . smegmatis at high temperatures . Anion-exchange and size-exclusion chromatography yielded about 230 microg apparently pure and highly active MspA per liter of culture . This was a 20-fold increased yield compared to previous purification protocols . Similar amounts of pure MspA were obtained with the detergents isotridecylpolyethyleneglycolether, lauryldimethylamine oxide, and octylpolyethylene oxide indicating that this purification procedure is not restricted to a specific detergent . This study will promote the structural and functional analysis of MspA and might be valuable for the isolation of porins from other mycolic acid-containing bacteria .

Mol Microbiol, 2000 Sep, 37(6), 1470 - 9
Interdependence of environmental factors influencing reciprocal patterns of gene expression in virulent Borrelia burgdorferi; Yang X et al.; The paradigm for differential antigen expression in Borrelia burgdorferi, the agent of Lyme disease, is the reciprocal expression of its outer surface (lipo)proteins (Osp) A and C; as B . burgdorferi transitions from its arthropod vector into mammalian tissue, ospC is upregulated, and ospA is downregulated . In the current study, using B . burgdorferi cultivated under varying conditions in BSK-H medium, we found that a decrease in pH, in conjunction with increases in temperature (e.g . 34 degrees C or 37 degrees C) and cell density, acted interdependently for the reciprocal expression of ospC and ospA . The lower pH (6.8), which induced the reciprocal expression of ospC and ospA in BSK-H medium, correlated with a drop in pH from 7.4 to 6.8 of tick midgut contents during tick feeding . In addition to ospC and ospA, other genes were found to be regulated in reciprocal fashion . Such genes were either ospC-like (e.g . ospF, mlp-8 and rpoS) (group I) or ospA-like (lp6.6 and p22) (group II); changes in expression occurred at the mRNA level . That the expression of rpoS, encoding a putative stress-related alternative sigma factor (sigma(s)), was ospC-like suggested that the expression of some of the group I genes may be controlled through sigma(s) . The combined results prompt a model that allows for predicting the regulation of other B . burgdorferi genes that may be involved in spirochaete transmission, virulence or mammalian host immune responses.

Structure Fold Des, 2000 Aug 15, 8(8), 897 - 904
Crystal structure of archaeal RNase HII: a homologue of human major RNase H; Lai L et al.; BACKGROUND: RNases H are present in all organisms and cleave RNAs in RNA/DNA hybrids . There are two major types of RNases H that have little similarity in sequence, size and specificity . The structure of RNase HI, the smaller enzyme and most abundant in bacteria, has been extensively studied . However, no structural information is available for the larger RNase H, which is most abundant in eukaryotes and archaea . Mammalian RNase H participates in DNA replication, removal of the Okazaki fragments and possibly DNA repair . RESULTS: The crystal structure of RNase HII from the hypothermophile Methanococcus jannaschii, which is homologous to mammalian RNase H, was solved using a multiwavelength anomalous dispersion (MAD) phasing method at 2 A resolution . The structure contains two compact domains . Despite the absence of sequence similarity, the large N-terminal domain shares a similar fold with the RNase HI of bacteria . The active site of RNase HII contains three aspartates: Asp7, Asp112 and Asp149 . The nucleotide-binding site is located in the cleft between the N-terminal and C-terminal domains . CONCLUSIONS: Despite a lack of any detectable similarity in primary structure, RNase HII shares a similar structural domain with RNase HI, suggesting that the two classes of RNases H have a common catalytic mechanism and possibly a common evolutionary origin . The involvement of the unique C-terminal domain in substrate recognition explains the different reaction specificity observed between the two classes of RNase H.

EXS, 2000, 89, 227 - 37
Delivery systems and adjuvants for vaccination against HIV; Velin D et al.; Epidemiological studies have revealed that HIV-1 infections occur through contact with contaminated blood or during unprotected vaginal or anal intercourse . Hence, to protect against HIV infection, vaccines should ideally induce both mucosal and systemic immune responses . We present a brief review of the different delivery systems and adjuvants which can be used to elicit mucosal immune responses . Oral or nasal administration of recombinant attenuated bacteria or viruses can induce both mucosal and systemic immune responses against the carried antigen . The oral delivery of mucosal adjuvants (such as cholera toxin) in association with antigens has been shown to enhance mucosal and systemic immune responses against them . Recently developed vaccination strategies using naked DNA or other antigen delivery systems are also discussed.

Biotechniques, 2000 Sep, 29(3), 602 - 4, 606-8, 609
Assessing the binding and endocytosis activity of cellular receptors using GFP-ligand fusions; Medina-Kauwe LK et al.; We have developed a simple scheme for characterizing ligand-receptor binding and post-binding activity on living cells . Our approach makes use of green fluorescent protein (GFP) as an auto-fluorescent tag to label protein ligands . We have constructed GFP-tagged ligands that can be expressed in bacteria as soluble fusion proteins . A cell-binding assay using fluorescence-activated cell sorting (FACS) demonstrates that GFP-tagged proteins retain their wild-type receptor-binding specificity . Using this assay, we measure ligand binding on unfixed cells and demonstrate receptor specificity using specific competitors . To determine the ability of receptor targets to internalize, we developed a second FACS-based assay to detect the rate and percentage of internalized ligand in living cells . Noninternalizing control ligands and fluorescence microscopy of treated cells confirm that our assay is reliable for determining receptor internalization activity.

Med Clin (Barc), 2000 Jul 1, 115(5), 176 - 7
{Analysis using ELISA test of antibody response to Fusobacterium nucleatum and Eikenella corrodens in subjects with periodontal disease}; Gutierrez J et al.; BACKGROUND: To know the synthesis of IgG, IgA, and IgM to Fusobacterium nucleatum and Eikenella corrodens in serum, crevicular liquid and saliva in subjects with periodontal disease, using ELISA test . PATIENTS AND METHODS: We studied 26 patients with high bias to the disease aged less than 35 years and 30 individuals with comparable age and with scarce bias to the disease . RESULTS: No differences were found for IgG and IgM titers between the groups, except for IgM to E . corrodens . For IgA, differences were found when the saliva and crevicular liquid were studied for both bacteria, as occurred in serum IgA compared to E . corrodens . In all the cases antibody levels were lower in the patients . CONCLUSIONS: The lower synthesis of IgM and IgA, fundamentally the latter, to F . nucleatum and E . corrodens in patients with periodontal disease would contribute to the pathogenesis of this illness.

Comp Biochem Physiol A Mol Integr Physiol, 2000 Sep, 127(1), 1 - 20
Silk produced by hornets: thermophotovoltaic properties-a review; Kirshboim S et al.; This article deals with the silk weave produced by pupating larvae of the Oriental hornet and its electric properties . Larvae of this hornet commence pupation at approximately 2 weeks of age . Creation of the cocoonal silk weave requires a number of hours and the encased pupa remains in the cocoon for approximately 2 more weeks before ecloding as an adult . The silk weave is initially of a creamish white color, but gradually becomes brown-gray owing to the activity of certain bacteria secreted in the silk . The silk weave is composed of fibers arranged in multiple layers with interposed surfaces occupying a considerable part of the area and containing pockets of bacteria . The spun silk contains both metallic and non-metallic elements, mostly K and Cl but also Mg, P, S, Ca, Ti and V . Shaped as a dome, the silk projects considerably beyond the cell proper, contributing importantly to its total volume and providing a shield for the contained pupa against predators, parasites, or extreme changes in temperature, as well as affording a 'sterile and clean room' in which the pupa can form its new cuticle without the interference of contaminating dust particles or the turbulence of air currents . The silk is endowed with electric properties . Inter alia, a thermoelectric phenomenon was observed in the dark, namely, upon increase in temperature the current rose to several hundred nano Amperes (nA); in light, a photovoltaic effect was observed involving voltages of several dozen millivolts (mV), with a sharp transition between the current and voltage during transition from darkness to light . Also recorded was a very high electric capacitance, amounting to scores of milli farads (mF) . In all, the pupal silk behaves like an organic semiconductor, in that its electric properties are temperature-dependent, and it also displays ferroelectric properties . Additionally, a luminescence phenomenon was recorded on the silk, wherein excitation at wavelengths within the UV(i.e . 249, 290 and 312 nm) range yielded an emission spectrum at a wavelength of 450 and of 530 nm . The silk caps are anisotropic in that the emission from the outside is lower than that from the inside . By way of recap, the various mentioned properties of the pupal silk are discussed from their biological and physical aspects.

Immunopharmacology, 2000 Sep, 49(3), 419 - 23
Interleukin 2 maintains biologic stability and sterility over prolonged time; Safar M et al.; The FDA approved interleukin 2 (IL2) for clinical use in 1992 in a high-dose bolus intravenous infusion schedule . IL2 administered by continuous low- and intermediate-dose infusion can result in a variety of immunologic effects including the expansion of the Natural Killer (NK) cell pool and immune reconstitution in immune-deficient hosts . These immune modifications are essential for augmentation of both currently available and evolving immunotherapies . The manufacturer's data indicate stability of the IL2 for a period of 6 days . This time frame is not practical for prolonged infusional schemes necessitating frequent changes of drug depots . We tested the biologic stability and sterility of the commercially available recombinant IL2 preparation (aldesleukin; Proleukin, Chiron) under clinical conditions for up to 30 days . Our results confirm that IL2 retains its biologic activity and sterility under these conditions for prolonged periods . This information will simplify IL2 outpatient regimens, allowing for convenient intervals for drug depot renewal, leading to improved patient compliance and conserved health care expenditures.

Genomics, 2000 Sep 15, 68(3), 343 - 7
Tissue-specific splicing of Omi stress-regulated endoprotease leads to an inactive protease with a modified PDZ motif; Faccio L et al.; Omi is a human serine protease whose catalytic domain is homologous to a bacterial heat shock endoprotease (HtrA), a protein indispensable to the survival of bacteria at elevated temperatures . Omi is expressed ubiquitously, and its protein product is predominantly localized in the endoplasmic reticulum of mammalian cells . Here we present the genomic structure of Omi, consisting of eight exons located on human chromosome 2p12-p13 . Furthermore, we describe an alternatively splice form of Omi (D-Omi) that is expressed predominantly in the kidney, colon, and thyroid . D-Omi lacks peptide sequence encoded by two exons (exons III and VII) . The absence of exon VII leads to a protein with a modified PDZ domain unable to interact with a known partner, the Mxi2 protein . The absence of exon III affects the catalytic domain and leads to a protein with no detectable protease activity . Our studies suggest that D-Omi may have a unique role in the normal function of kidney, colon, and thyroid .

Proc Natl Acad Sci U S A, 2000 Sep 26, 97(20), 10899 - 904
Borrelia burgdorferi periplasmic flagella have both skeletal and motility functions; Motaleb MA et al.; Bacterial shape usually is dictated by the peptidoglycan layer of the cell wall . In this paper, we show that the morphology of the Lyme disease spirochete Borrelia burgdorferi is the result of a complex interaction between the cell cylinder and the internal periplasmic flagella . B . burgdorferi has a bundle of 7-11 helically shaped periplasmic flagella attached at each end of the cell cylinder and has a flat-wave cell morphology . Backward moving, propagating waves enable these bacteria to swim in both low viscosity media and highly viscous gel-like media . Using targeted mutagenesis, we inactivated the gene encoding the major periplasmic flagellar filament protein FlaB . The resulting flaB mutants not only were nonmotile, but were rod-shaped . Western blot analysis indicated that FlaB was no longer synthesized, and electron microscopy revealed that the mutants were completely deficient in periplasmic flagella . Wild-type cells poisoned with the protonophore carbonyl cyanide-m-chlorophenylhydrazone retained their flat-wave morphology, indicating that the periplasmic flagella do not need to be energized for the cell to maintain this shape . Our results indicate that the periplasmic flagella of B . burgdorferi have a skeletal function . These organelles dynamically interact with the rod-shaped cell cylinder to enable the cell to swim, and to confer in part its flat-wave morphology.

Am J Reprod Immunol, 2000 Aug, 44(2), 73 - 9
Infection of polarized primary epithelial cells from rat uterus with Chlamydia trachomatis: cell-cell interaction and cytokine secretion; Kaushic C et al.; PROBLEM: The objective of this study was to examine the susceptibility of rat uterine epithelial cells (UEC) to infection with Chlamydia trachomatis and to study the epithelial-stromal interactions following infection . METHOD OF STUDY: UEC were isolated from adult rats and grown in culture . Polarized, confluent monolayers of UEC were infected with 10(6) IFU/well C . trachomatis (MoPn) . In order to confirm infection, MoPn was labeled with a fluorescent tracking dye, PKH-26, and then used in epithelial cell infections . Transepithelial resistances were measured prior to and following infection to test the effect of Chlamydia on the integrity of the epithelial monolayers . In other experiments, polarized epithelial cultures were infected in the presence and absence of stromal cells . Media was collected from the apical and basolateral compartments of the cultures before and after infection and analyzed for cytokines IL-1alpha and TNF-alpha . RESULTS: Epithelial cell cultures infected with PKH-26 labeled MoPn were examined 4-5 days later . Bacterial inclusions were detected inside epithelial cells indicating infection had occurred . Co-localization of PKH-26 labeled bacteria with FITC-labelled anti-Chlamydia antibody on the epithelial cells confirmed infection . No changes were found in resistance across the monolayers of epithelial cells in the presence or absence of infection . ELISA results indicate that UEC secrete IL-1alpha constitutively in citro . Stromal cells secrete very little IL-1alpha . When stromal cells were co-incubated with epithelial cells there was a decrease in the amount of IL-1alpha secreted by epithelial cells 48 hr post-infection . On the other hand, maximum TNF-alpha was found in stromal cells . both with and without infection . Epithelial cells, in these studies made very little TNF-alpha . CONCLUSIONS: These results show that primary rat epithelial cells can be infected with Chlamydia in vitro . Epithelial and stromal cells from uteri of adult rats make IL-1alpha and TNF-alpha in vitro both prior to and following infection with Chlamydia . This system can be used to analyze the role played by epithelial-stromal interactions in providing protection on this mucosal surface.

Prikl Biokhim Mikrobiol, 2000 Jul-Aug, 36(4), 484 - 7
{Granulated preparations of azotobacter in a clay-mineral base}; Kirdish IK et al.; Interaction of Azotobacter chroococcum 20 cells with clay minerals increased their viability at supraoptimal temperatures . Therefore, clay minerals were used to develop granular bacterial preparations with high viable cell counts and stable compositions during long-term storage . The titers of viable bacteria in the preparations remained 60-70% of the initial level after 12-month storage.

J Pharm Pharm Sci, 2000 May-Aug, 3(2), 228 - 33
Pharmaceutical interference with the {14C} carbon urea breath test for the detection of Helicobacter pylori infection; Abrams DN et al.; Helicobacter pylori bacteria reside in the mucosal lining of the stomach where, due to a variety of factors, the infection predisposes patients to peptic ulcer disease . Detection of H . pylori is important in the treatment and follow-up of patients with peptic ulcer disease and the urea breath test is the method of choice . This article will briefly review the methods for diagnosing H . pylori, emphasizing the {(14)C}urea breath test . The agents which can interfere with the results of the breath test will be reviewed and the role of the consulting pharmacist will be discussed.

J Mol Biol, 2000 Sep 29, 302(4), 761 - 75
Analysis of a cell-cycle promoter bound by a response regulator; Ouimet MC et al.; Caulobacter crescentus CtrA protein, an OmpR-type response regulator, receives cell-cycle signals and binds the proposed consensus TTAA-N7-TTAA present inside the chromosome replication origin and cell-cycle transcription promoters . We synthesized a 42 bp cell-cycle promoter based on this consensus and elements of the fliL promoter . Over 100 promoter mutations were assayed for transcription directed by CtrA . Although both CtrA binding half-sites cooperate and the N7 spacing is critical for transcription, the upstream half-site is relatively flexible . The downstream CtrA half-site is less flexible and more important for cell-cycle regulation . A CtrA binding site and a -10 promoter element are sufficient for cell-cycle transcription, and both sequences cooperate and compensate for respective defects . Mutations in the CtrA binding site, but not in the -10 promoter sequence, perturb cell-cycle transcription . A single base-pair change switches a cell-cycle promoter into a strong conventional promoter . We propose rules for CtrA binding and promoter interactions implying how CtrA evolved into a versatile regulator of cell-cycle functions including flagellar biogenesis, cell division, DNA methylation, and chromosome replication .

Biosci Biotechnol Biochem, 2000 Aug, 64(8), 1771 - 3
Preliminary communication . MRNA expression of MT1-MMP, MMP-9, cathepsin K, and TRAP in highly enriched osteoclasts cultured on several matrix proteins and ivory surfaces; Uemura T et al.; We demonstrated that the transcriptional expression of MT1-MMP, MMP-9, cathepsin K, and TRAP in highly enriched osteoclasts were regulated by different matrix proteins that bind to integrin on osteoclast, such as collagen type I (CoI), fibronectin (FN), vitronectin (VN), osteopontin (OPN), and ivory . Results suggested that the OPN-integrin alphavbeta3 binding plays a more important role than CoI-alpha2beta1 binding in the regulation of osteoclast activity.

Nature, 2000 Sep 7, 407(6800), 78 - 80
Uptake of dissolved organic carbon and trace elements by zebra mussels; Roditi HA et al.; Zebra mussels (Dreissena polymorpha) are widespread and abundant in major freshwater ecosystems in North America, even though the phytoplankton food resources in some of these systems seem to be too low to sustain them . Because phytoplankton biomass is greatly depleted in ecosystems with large D . polymorpha populations and bacteria do not seem to be an important food source for this species, exploitation of alternative carbon sources may explain the unexpected success of D . polymorpha in such environments . Here we examine the possibility that absorption of dissolved organic carbon (DOC) from water could provide a nutritional supplement to zebra mussels . We find that mussels absorb 14C-labelled DOC produced by cultured diatoms with an efficiency of 0.23%; this indicates that DOC in natural waters could contribute up to 50% of the carbon demand of zebra mussels . We also find that zebra mussels absorb some dissolved metals that have been complexed by the DOM; although absorption of dissolved selenium was unaffected by DOC, absorption of dissolved cadmium, silver and mercury by the mussels increased 32-, 8.7- and 3.6-fold, respectively, in the presence of high-molecular-weight DOC.

Rev Med Liege, 2000 Jun, 55(6), 527 - 30
{Bites by terrestrial vertebrates}; Henry F et al.; Bites by terrestrial vertebrates, reptiles or mammals, represent a special risk in tropical regions . Envenomation is possible by a few lizards and many snakes . For mammals, tissular destructions due to the bite can be severe . Whatever is the offending animal, bites can further become infected by transmitted viruses or bacteria.

Gac Med Mex, 2000 Jul-Aug, 136(4), 329 - 34
{Characteristics of peritoneal fluid in children with severe diarrhea}; Gomez-Alcala AV; BACKGROUND: A significant number of acute diarrheal diseases produce severe damage to the bowel wall that can lead the patient to death . Peritoneal fluid (PF) analysis has been proposed as a tool to establish an early diagnosis of these injuries . MATERIAL AND METHOD: Thirty patients with a diagnosis of acute diarrhea and suspected enteric perforation or gangrene were submitted to paracentesis . LP was classified according to its appearance immediately after obtention by the surgeon, and determinations of leukocytes, proteins, glucose, and chloride were done . Clinical diagnosis was established during laparotomy or necropsy, or after an uneventual recovery . Data were analyzed by means of diagnostic test statistics . RESULTS: Clear and yellow PF was considered a sign of the absence of enteric perforation or gangrene, with a sensitivity (S) of 87%, specificity (SP) and positive predictive value (PPV) of 100%, and a negative predictive value (NPV) of 91.6%, with a predictive accuracy (PA) of 95% . A bloody PF was considered a sign of enteric gangrene, with an S of 54%, SP and PPV of 100%, and NPV of 72% (PA 80%) . More than 1000 leukocytes per mm3 of PF were considered an sign of some kind of macroscopic injury of the bowel wall, with an S of 68%, a SP of 88%, a PPV of 91% and a NPV of 61% (PA 76%) . PF glucose level less than 40 mg/dl was considered a sign of enteric gangrene (S 42%, SP 78%, PPV 71%, NPV 50%, with a PA of 57%), and also of bacteria viability in PF cultures (S and NPV 100%, SP 80%, PPV 67% and PA of 86%) . Chloride levels greater than 90 meq/L were considered a sign of enteric perforation or gangrene, with an S and NPV of 100%, a SP of 60%, and a PPV de 82% (PA 86%) . CONCLUSION: PF analysis is of great value in the evaluation of the child with acute severe diarrhea in which enteric perforation or gangrene is suspected . A clear yellow fluid with less than 1000 leukocytes per mm3, more than 40 mg/dL of glucose, and less than 90 meq/L of chloride suggest that enteritis has not caused irreversible injury to the bowel wall.

Infect Immun, 2000 Oct, 68(10), 6069 - 72
Differential effects of virulent versus avirulent Legionella pneumophila on chemokine gene expression in murine alveolar macrophages determined by cDNA expression array technique; Nakachi N et al.; The cDNA expression array technique is a powerful tool to determine, at one time from many genes, specific gene messages modulated by infection . In the present study, we identified genes modulated in response to virulent versus avirulent Legionella pneumophila infection of the alveolar macrophage cell line MH-S by the cDNA expression array technique . Many macrophage genes were found to be modulated after 5 h of in vitro infection with L . pneumophila . In particular, it was found that the monocyte chemotactic protein 3 (MCP-3) gene expression was significantly induced by infection with virulent L . pneumophila but not with avirulent L . pneumophila . In contrast, other chemokine genes, such as macrophage inflammatory protein (MIP) 1alpha, were induced by both virulent and avirulent L . pneumophila . Reverse transcription (RT)-PCR assay of total RNA isolated from macrophages infected with the bacteria for 5 or 24 h confirmed the differential induction of the chemokine genes by virulent versus avirulent L . pneumophila . Thus, the cDNA expression array technique readily revealed differential induction by L . pneumophila infection of select chemokine genes of macrophages from more than 1,100 genes . These results also indicate that certain chemokine genes may be selectively induced by virulent bacteria.

Infect Immun, 2000 Oct, 68(10), 5960 - 9
Infection of human endothelial cells with Bartonella bacilliformis is dependent on Rho and results in activation of Rho; Verma A et al.; Bartonella bacilliformis was continuously internalized into human endothelial cells beginning shortly after addition of the bacteria and continuing for at least 24 h after infection in vitro, with a major increase in uptake occurring between 16 and 24 h . Preincubation of endothelial cells with C3 exoenzyme, which inactivated intracellular Rho-GTPase, blocked internalization of the bacteria . Addition of C3 exoenzyme at any time after addition of the bacteria blocked further internalization of bacteria, including the major uptake of bacteria internalized at 16 to 24 h . Rho, a key signaling protein in pathways involving actin organization, was directly shown to be activated in endothelial cells undergoing infection with B . bacilliformis, with maximal activation and translocation to the plasma membrane at 12 to 16 h . At late times of infection, most of the bacteria were found in a perinuclear location . Staining of the Golgi complex with specific markers, anti-human Golgin-97, anti-KDEL receptor, and BODIPY-TR ceramide, showed colocalization of bacteria in the Golgi complex region . Disruption of the Golgi complex with brefeldin A scattered the bacteria from this perinuclear location and resulted in inhibition of internalization of the bacteria in endothelial cells.

Infect Immun, 2000 Oct, 68(10), 5575 - 80
Construction and characterization of a Mycobacterium tuberculosis mutant lacking the alternate sigma factor gene, sigF; Chen P et al.; The alternate RNA polymerase sigma factor gene, sigF, which is expressed in stationary phase and under stress conditions in vitro, has been deleted in the virulent CDC1551 strain of Mycobacterium tuberculosis . The growth rate of the DeltasigF mutant was identical to that of the isogenic wild-type strain in exponential phase, although in stationary phase the mutant achieved a higher density than the wild type . The mutant showed increased susceptibility to rifampin and rifapentine . Additionally, the DeltasigF mutant displayed diminished uptake of chenodeoxycholate, and this effect was reversed by complementation with a wild-type sigF gene . No differences in short-term intracellular growth between mutant and wild-type organisms within human monocytes were observed . Similarly, the organisms did not differ in their susceptibilities to lymphocyte-mediated inhibition of intracellular growth . However, mice infected with the DeltasigF mutant showed a median time to death of 246 days compared with 161 days for wild-type strain-infected animals (P < 0.001) . These data indicate that M . tuberculosis sigF is a nonessential alternate sigma factor both in axenic culture and for survival in macrophages in vitro . While the DeltasigF mutant produces a lethal infection of mice, it is less virulent than its wild-type counterpart by time-to-death analysis.

J Biol Chem, 2000 Dec 8, 275(49), 38254 - 60
H2O2-sensitive fur-like repressor CatR regulating the major catalase gene in Streptomyces coelicolor; Hahn JS et al.; Streptomyces coelicolor produces three distinct catalases to cope with oxidative and osmotic stresses and allow proper growth and differentiation . The major vegetative catalase A (CatA) is induced by H(2)O(2) and is required for efficient aerobic growth . In order to investigate the H(2)O(2)-dependent regulatory mechanism, an H(2)O(2)-resistant mutant (HR40) overproducing CatA was isolated from S . coelicolor A3(2) . Based on the genetic map location of the mutated locus in HR40, the wild type catR gene was isolated from the ordered cosmid library of S . coelicolor by screening for its ability to suppress the HR40 phenotype . catR encodes a protein of 138 amino acids (15319 Da), with sequence homology to ferric uptake regulator (Fur)-like proteins . Disruption of catR caused CatA overproduction as observed in the HR40 mutant, confirming the role of CatR as a negative regulator of catA expression . The levels of catA and catR transcripts were higher in HR40 than in the wild type, implying that CatR represses transcription of these genes . Transcripts from the catA and catR genes were induced within 10 min of H(2)O(2) treatment, suggesting that the repressor activity of CatR may be directly modulated by H(2)O(2) . A putative CatR-binding site containing an inverted repeat of 23 base pairs was localized upstream of the catA and catR gene, on the basis of sequence comparison and deletion analysis . CatR protein purified in the presence of dithiothreitol bound to this region, whereas oxidized CatR, treated with H(2)O(2) or diamide, did not . The redox shift of CatR involved thiol-disulfide exchange as judged by modification of free thiols with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonate . From these results we propose that CatR regulates its downstream target genes as a repressor whose DNA binding ability is directly modulated by redox changes in the cell.

Bull Mem Acad R Med Belg, 1999, 154(6 Pt 2), 371 - 5
{Effect of ammonia on the pig respiratory system . Study of the dose-response relation}; Gustin P; The effects of ammonia on the respiratory system and somatic growth have been measured in piglets taking into account the interactions with endotoxins from gram-bacteria and dust . Dose-response curves were constructed for the inflammatory reactions induced in nasal cavities, the smooth muscle hyperresponsiveness in the trachea, the permeability of alveolo-capillary barrier, the pulmonary vasomotricity, the cough reflex and the systemic effects . An acceptable level of pollution was proposed.

Phys Rev Lett, 2000 Jun 5, 84(23), 5435 - 8
Long-lived amide I vibrational modes in myoglobin; Xie A et al.; Pump-probe experiments in the infrared measure vibrational relaxation rates . Myoglobin, which is almost entirely alpha helix in secondary structure, has an unusually long, nonexponential excited state relaxation generated by optically pumping at the blue side ( 5 . 85 microm) of the amide I band . The amino acid alanine and the predominantly beta sheet protein photoactive yellow protein do not have such a long-lived state, suggesting that the alpha helix in proteins can support nonlinear states of 15 ps characteristic times.

Carbohydr Res, 2000 Aug 7, 327(4), 447 - 53
An improved model for calculating the optical rotation of simple saccharides; Stroyan EP et al.; A calculational model for the optical rotation (OR) at the sodium D-line of simple saccharides has been developed that eliminates deficiencies of a previous model . Conformational conclusions based on the earlier model are not affected, as established by a recalculation of the OR phi,psi-map of methyl 3-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside . The model relocates the strong long-wavelength sigma-sigma* circular dichroism (CD) component, which is mainly responsible for the NaD OR from 160 to below 130 nm, where it is now known to occur . That correction allows future modeling of CD bands of different origins that appear in the 150-190 nm region . In order to demonstrate the utility of the revised model, it was applied to calculating the OR of methyl 3-O-(alpha-L-rhamnopyranosyl)-alpha-L-rhamnopyranoside . The results provide experimental support for conformational conclusions derived from a molecular mechanics study of that molecule.

Transfusion, 2000 Sep, 40(9), 1041 - 7
Survival of Ehrlichia chaffeensis in refrigerated, ADSOL-treated RBCs; McKechnie DB et al.; BACKGROUND: The purpose of this study was to investigate the persistence of viable Ehrlichia chaffeensis in ADSOL-treated RBCs stored at 4 to 6 degrees C . STUDY DESIGN AND METHODS: The continuous monocytic cell lines THP-1 and DH82 were infected with E . chaffeensis (St . Vincent isolate) . Packed RBC units were inoculated in separate experiments with E . chaffeensis-infected cells as final concentrations of 8.02 x 10(4) (DH82) and 1.43 x 10(4) (THP-1) infected cells per mL . Aliquots were stored at 4 to 6 degrees C for 1 to 42 days . At selected intervals, nucleated cells from the RBC aliquots were obtained by using a ficoll-isopaque separation procedure . Uninfected DH82 cell cultures were inoculated with the harvested nucleated cells or supernatant . The cell cultures were evaluated for infection by weekly examination of Wright's (Diff-Quik) stained cytocentrifuged slides . PCR amplification was also used to test the harvested nucleated cells or supernatant for the presence of E . chaffeensis DNA . RESULTS: In both types of infected cell lines, E . chaffeensis was reisolated in DH82 cells for as long as 11 days from the cellular fraction and for up to 5 days from the supernatant fraction . PCR results were positive throughout the 42-day testing period . CONCLUSION: Cell-associated E . chaffeensis remains viable in ADSOL-treated RBCs stored at 4 to 6 degrees C for at least 11 days . These data suggest that transfusion-acquired infection is possible . Successful reisolation was achieved from the supernatant fraction, which suggests that RBC products treated with a WBC-reduction procedure may still present a risk for transfusion transmission . No correlation between PCR positivity and viability of bacteria was noted.

MMWR Morb Mortal Wkly Rep, 2000 Sep 1, 49(34), 777 - 8
Legionnaires' Disease associated with potting soil--California, Oregon, and Washington, May-June 2000.
{How does one treat the osteitis and osteoarthritis of the extremities in older leprosy patients using granulated table sugar?}
Grauwin MY, Cartel JL, Lepers JP.

Institut de Leprologie Appliquee, Dakar, SenegalA common problem of osteitis and septic arthritis is the recurrent bone infection after surgical debridement, a problem frequently encountered in patients with sequela leprosy . In these cases the authors propose the use of an ancient method of post surgical wound care based on the treatment with ordinary granulated sugar . The hyperosmolar climate created this way in the wounds inhibits the bacterial growth, enhances bacterial death and therefore permits the growth of granulation tissue in order to recover the debrided nude bones . At ILAD (Leprosy Institute of Dakar), 36 osteitis and septic arthritis were treated and healed during the last 2 years from March 1995 to March 1997 using this technic . All the wounds healed in the mean-time of 44 days . Only two of them needed a second debridement and healed afterwards . Up to now the method using ordinary sugar was applied in the treatment of infected wounds, eschars and postsurgical infections . Our experience shows that it also can be indicated to treat bone infections . This method is easy to apply also under often difficult field conditions and is very cheap.

Pediatr Int, 2000 Aug, 42(4), 428 - 39
Rotavirus infection in children in Japan; Zhou Y et al.; Currently, a high morbidity of rotavirus diarrhea has been seen in children in developed and developing countries . Improvement of the vaccines is necessary in order to reduce the burden of diarrhea caused by rotavirus . A survey of rotavirus infection from diarrheal stool specimens in children of seven regions in Japan was conducted from 1984 to 1999 . The present study discusses the survey results and reviews the national and international data of more than 23 papers and congress proceedings about rotavirus infection in Japan . We analyze the prevalence of rotavirus infection in acute diarrheal in- and outpatients, the distribution of rotavirus G-serotypes and surveillance data for seasonality and age groups in Japan . The data indicated that rotavirus is the most important cause of diarrhea in Japan among young children, with the prevalence ranging from approximately 9.7 to 88% . The most common rotavirus strains belonged to serotype G1, specifically since 1993 . Serotypes G2, G3 and G4 had also been documented to be predominantly based in the area and year before 1992 . However, untypeable rotavirus strains had been found each year, with a prevalence up to 56.7% which suggests that rare serotypes (except G1-4) or new serotypes might exist . Unexpectedly, in Tokyo and Sapporo from 1998 to 1999, G9 was found to be the first most prevailing serotype with a high prevalence of 52.9 and 71.4%, respectively . Despite these data from different geographic areas, the year under investigation was relatively clear in respect to seasonality, with a peak of rotavirus activity in late winter (February) through early spring (March) . Age distribution had also characterized that the infection was predominant among children aged 1-2 years of age, although it was also common in children of 2-3 years . In addition, mixed infection with bacteria was documented.

Mutat Res, 2000 Oct 10, 470(1), 85 - 92
Determination of the vaporization of solutions of mutagenic antineoplastic agents at 23 and 37 degrees C using a desiccator technique; Connor TH et al.; This study evaluated the ability of mutagenic antineoplastic agents to vaporize at room temperature (23 degrees C) and 37 degrees C . A bacterial mutagenicity assay was used to determine the mutagenicity of these agents in the vapor phase . Open plates of bacteria were exposed to varying amounts of drug solutions in sealed glass containers for 24h . The drug solutions were prepared as they would be for patient treatment and were tested at 0.25, 0.5 and 1.0 ml of each drug solution per 10 l of air . Following exposure, the plates exposed at 23 degrees C were incubated an additional 48 h at 37 degrees C to allow for expression of mutations . Those exposed at 37 degrees C were incubated for an additional 24h at 37 degrees C . Carmustine, cyclophosphamide, ifosfamide, thiotepa, and mustargen demonstrated vaporization at 37 degrees C . Carmustine and mustargen also demonstrated significant vaporization at 23 degrees C, while cyclophosphamide demonstrated a 50% increase in revertants at this temperature . In addition, sodium azide, a known mutagen used as a control was also mutagenic as a vapor at both temperatures . Doxorubicin, cisplatin, etoposide, 5-fluorouracil and mitomycin were not detected as vaporizing in this assay . The study found that vaporization of standard solutions of some antineoplastic agents is possible at room temperature and increases as the temperature increases . Therefore, vaporization of spilled antineoplastic agents may present an additional route of exposure to healthcare workers through inhalation.

J Bacteriol, 2000 Oct, 182(19), 5615 - 9
The Synechococcus strain PCC 7942 glnN product (glutamine synthetase III) helps recovery from prolonged nitrogen chlorosis; Sauer J et al.; We report the cloning and sequencing of the glnN gene encoding a class III glutamine synthetase from the cyanobacterium Synechococcus strain PCC 7942 . Mapping of the transcriptional start site revealed a DNA sequence in the promoter region that resembles an imperfect NtcA binding motif . Expression of glnN is impaired in NtcA- and P(II)-deficient mutants . The only parameter which was negatively affected in the glnN mutant compared to the wild type was the recovery rate of prolonged nitrogen-starved cells with low concentrations of combined nitrogen.

J Bacteriol, 2000 Oct, 182(19), 5433 - 9
DNA polymerase I is essential for growth of Methylobacterium dichloromethanicum DM4 with dichloromethane; Kayser MF et al.; Methylobacterium dichloromethanicum DM4 grows with dichloromethane as the unique carbon and energy source by virtue of a single enzyme, dichloromethane dehalogenase-glutathione S-transferase . A mutant of the dichloromethane-degrading strain M . dichloromethanicum DM4, strain DM4-1445, was obtained by mini-Tn5 transposon mutagenesis that was no longer able to grow with dichloromethane . Dichloromethane dehalogenase activity in this mutant was comparable to that of the wild-type strain . The site of mini-Tn5 insertion in this mutant was located in the polA gene encoding DNA polymerase I, an enzyme with a well-known role in DNA repair . DNA polymerase activity was not detected in cell extracts of the polA mutant . Conjugation of a plasmid containing the intact DNA polymerase I gene into the polA mutant restored growth with dichloromethane, indicating that the polA gene defect was responsible for the observed lack of growth of this mutant with dichloromethane . Viability of the DM4-1445 mutant was strongly reduced upon exposure to both UV light and dichloromethane . The polA'-lacZ transcriptional fusion resulting from mini-Tn5 insertion was constitutively expressed at high levels and induced about twofold after addition of 10 mM dichloromethane . Taken together, these data indicate that DNA polymerase I is essential for growth of M . dichloromethanicum DM4 with dichloromethane and further suggest an important role of the DNA repair machinery in the degradation of halogenated, DNA-alkylating compounds by bacteria.

Med Hypotheses, 2000 Sep, 55(3), 185 - 6
Monoselenolipoic acid may be an outstanding pharmaceutical antioxidant with direct thioredoxin-like activity; McCarty MF; Owing to the low pKa of its selenohydryl group, reduced monoselenolipoic acid (MSL) can be expected to be a very versatile antioxidant with direct thioredoxin-like activity . Since MSL supports the growth of lipoate-dependent bacteria, it can be anticipated that MSL will be susceptible to reversible reduction by one or more of the enzymes that reduces lipoic acid - thus greatly potentiating MSL's utility as an antioxidant . If it is not metabolized to release toxic free selenide, MSL may have interesting pharmaceutical potential .

Arch Microbiol, 2000 Jul-Aug, 174(1-2), 35 - 41
Transformation of ortho-substituted biphenyls by Methylosinus trichosporium OB3b: substituent effects on oxidation kinetics and product formation; Lindner AS et al.; The ability of Methylosinus trichosporium OB3b, expressing soluble methane monooxygenase, to oxidize a range of ortho-substituted biphenyls was examined to better understand how substituents affect both the rate and products of oxidation in comparison to biphenyl . Inhibition of oxidation was observed over the tested substrate range for both biphenyl and ortho-halogenated biphenyls (2-chloro-, 2-bromo-, and 2-iodobiphenyl) . No inhibition was observed during the oxidation of 2-hydroxybiphenyl and 2-methylbiphenyl . Analysis of the products of oxidation showed that, depending on the substituent, ring hydroxylation, substituent oxidation, and elimination pathways could occur . The type and abundance of products formed along with the relatively high kinetic isotope effect observed for deuterated vs . nondeuterated biphenyl (k(h)/k(d) = 3.4+/-0.02) are consistent with mechanisms that include both hydrogen abstraction and NIH-shift pathways . Knowledge of these substituent-dependent reaction rates and mechanisms enhances our understanding of the methanotrophic aryl transformation potential and allows for better prediction of the formation of oxidized intermediates by methanotrophic bacteria.

Int J Tuberc Lung Dis, 2000 Sep, 4(9), 807 - 26
Case management of childhood pneumonia in developing countries: recent relevant research and current initiatives; Rasmussen Z et al.; Acute respiratory infections (ARI), mostly pneumonia, are one of the leading causes of death in young children in developing countries, accounting for 28% of childhood mortality . This paper provides a summary of the research and technical development efforts made in the last 15 years which contributed to improving the effectiveness of the case management strategy to reduce mortality from pneumonia in children in developing countries . Community intervention studies provided strong evidence that the strategy was feasible and effective in producing a substantial impact on pneumonia mortality . Clinical studies provided the rationale for improving the sensitivity and specificity of key signs of pneumonia, and for enhancing the therapeutic efficacy of standard home treatment . Research also provided data to deal with the problem of the clinical overlap of pneumonia and malaria in children . Technological initiatives succeeded in making appropriate diagnostic and therapeutic devices available . An important body of socio-cultural knowledge about family practices regarding pneumonia and ARI in children was built up and provided orientation on effective communication between health workers and families about home care of children with ARI . Health systems research focused on methods for surveillance of bacterial drug resistance and methodologies for evaluating the control programmes . Despite advances in the development of vaccines against respiratory bacteria and in the prevention of risk factors for pneumonia in children, case management will continue to be a central strategy for preventing mortality . Current international research initiatives are looking into measures that can improve the referral of severe pneumonia and effective management of severe pneumonia at first level hospitals.

Bioessays, 2000 Oct, 22(10), 947 - 53
Redox control and the evolution of multicellularity; Blackstone NW; Redox chemistry, involving the transfer of electrons and hydrogen atoms, is central to energy conversion in respiration; in addition, control of gene expression by redox state commonly occurs in bacteria, allowing a rapid response to environmental changes, such as altered food supply . Colonial metazoans often encrust surfaces over which the food supply varies in time or space; hence, in these organisms redox control of the development of feeding structures and gastrovascular connections could be similarly adaptive, allowing colonies to adjust the timing of development and spacing of structures in response to a variable food supply and other environmental factors . Experimental perturbations of redox state in colonial hydroids support this notion of adaptive redox control, and redox signaling in metazoans may have evolved in this ecological context . At the same time, redox signaling has important consequences for the evolutionary transition from unicellular to multicellular organisms . Unlike protein or peptide signaling, redox signaling acting in concert with programmed cell death may automatically inflict a cost on those cells that "defect," that is, selfishly favor their own replication rate over that of the multicellular group . In this way, redox signaling may have allowed multicellular individuality to evolve and more easily be maintained.

Proc Natl Acad Sci U S A, 2000 Sep 26, 97(20), 10855 - 60
Postsymbiotic plasmid acquisition and evolution of the repA1-replicon in Buchnera aphidicola; Van Ham RC et al.; Buchnera aphidicola is an obligate, strictly vertically transmitted, bacterial symbiont of aphids . It supplies its host with essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap . Several lineages of Buchnera show adaptation to their nutritional role in the form of plasmid-mediated amplification of key-genes involved in the biosynthesis of tryptophan (trpEG) and leucine (leuABCD) . Phylogenetic analyses of these plasmid-encoded functions have thus far suggested the absence of horizontal plasmid exchange among lineages of Buchnera . Here, we describe three new Buchnera plasmids, obtained from species of the aphid host families Lachnidae and Pemphigidae . All three plasmids belong to the repA1 family of Buchnera plasmids, which is characterized by the presence of a repA1-replicon responsible for replication initiation . A comprehensive analysis of this family of plasmids unexpectedly revealed significantly incongruent phylogenies for different plasmid and chromosomally encoded loci . We infer from these incongruencies a case of horizontal plasmid transfer in Buchnera . This process may have been mediated by secondary endosymbionts, which occasionally undergo horizontal transmission in aphids.

J Allergy Clin Immunol, 2000 Sep, 106(3), 409 - 18
Recombinant allergens for diagnosis and therapy of allergic disease; Chapman MD et al.; Many of the problems associated with using natural allergenic products for allergy diagnosis and treatment can be overcome with use of genetically engineered recombinant allergens . Over the past 10 years, the most important allergens from mites, pollens, animal dander, insects, and foods have been cloned, sequenced, and expressed . In many cases the three-dimensional allergen structure has been determined and B-cell and T-cell epitopes have been mapped . These studies show that allergens have diverse biologic functions (they may be enzymes, enzyme inhibitors, lipocalins, or structural proteins) and that as a rule the allergen function is unrelated to its ability to cause IgE antibody responses . High-level expression systems have been developed to produce recombinant allergens in bacteria, yeast, or insect cells . Recombinant allergens show comparable IgE antibody binding to their natural counterparts (where available) and show excellent reactivity on skin testing and in in vitro diagnostic tests . Cocktails of recombinant allergens can be formulated with predetermined and uniform allergen levels, which could replace natural allergens and result in the development of innovative, patient-based tests for allergy diagnosis . Recombinant allergens also offer the exciting possibility of developing new forms of allergen immunotherapy, including the use of hypoallergens, allergens coupled to IgE suppressive adjuvants, and peptide-based therapies . The production of recombinant allergens as defined molecular entities makes it feasible to consider the possibility of developing prophylactic allergen vaccines . The introduction of recombinant allergens in research and in clinical trials should lead to significant improvements in allergy diagnosis and treatment.

Nature, 2000 Aug 31, 406(6799), 998 - 1001
A role for the C3a anaphylatoxin receptor in the effector phase of asthma; Humbles AA et al.; Asthma is a chronic inflammatory disease of the airways and lung mucosa with a strong correlation to atopy and acquired (IgE) immunity . However, many features of bronchial asthma, such as smooth muscle contraction, mucus secretion and recruitment of inflammatory cells, are consistent with the actions of complement anaphylatoxins, in particular C3a and C5a . Complement activation forms a central core of innate immune defence against mucosal bacteria, viruses, fungi, helminths and other pathogens . As a system of 'pattern-recognition molecules', foreign surface antigens and immune complexes lead to a proteolytic cascade culminating in a lytic membrane attack . The anaphylatoxins C3a and C5a are liberated as activation byproducts and are potent pro-inflammatory mediators that bind to specific cell surface receptors and cause leukocyte activation, smooth muscle contraction and vascular permeability . Here we show that in a murine model of allergic airway disease, genetic deletion of the C3a receptor protects against the changes in lung physiology seen after allergen challenge . Furthermore, human asthmatics develop significant levels of ligand C3a following intra-pulmonary deposition of allergen, but not saline . We propose that, in addition to acquired immune responses, the innate immune system and complement (C3a in particular) are involved in the pathogenesis of asthma.

Proc R Soc Lond B Biol Sci, 2000 Jul 22, 267(1451), 1469 - 73
Male-killing Wolbachia in a flour beetle; Fialho RF et al.; The bacteria in the genus Wolbachia are cytoplasmically inherited symbionts of arthropods . Infection often causes profound changes in host reproduction, enhancing bacterial transmission and spread in a population . The reproductive alterations known to result from Wolbachia infection include cytoplasmic incompatibility (CI), parthenogenesis, feminization of genetic males, fecundity enhancement, male killing and, perhaps, lethality Here, we report male killing in a third insect, the black flour beetle Tribolium madens, based on highly female-biased sex ratios of progeny from females infected with Wolbachia . The bias is cytoplasmic in nature as shown by repeated backcrossing of infected females with males of a naturally uninfected strain . Infection also lowers the egg hatch rates significantly to approximately half of those observed for uninfected females . Treatment of the host with antibiotics eliminated infection, reverted the sex ratio to unbiased levels and increased the percentage hatch . Typically Wolbachia infection is transmitted from mother to progeny, regardless of the sex of the progeny; however, infected T . madens males are never found . Virgin females are sterile, suggesting that the sex-ratio distortion in T . madens results from embryonic male killing rather than parthenogenesis . Based on DNA sequence data, the male-killing strain of Wolbachia in T . madens was indistinguishable from the CI-inducing Wolbachia in Tribolium confusum, a closely related beetle . Our findings suggest that host symbiont interaction effects may play an important role in the induction of Wolbachia reproductive phenotypes.

Nurs Times, 1999 Nov 3-9, 95(44), 62, 65 - 6
Infection control . Unhappy nappy-changing; Chudleigh J et al.; This study was conducted in a special care baby unit (SCBU) and concentrated on hand decontamination and glove-wearing during nappy-changing . The aims were to determine whether or not nurses were adhering to existing infection control policies and guidelines as well as determining the most appropriate product to use for hand decontamination . The study demonstrated that not all nurses were adhering to existing infection control policies and guidelines . On the majority of occasions, alcohol was significantly better than soap at removing bacteria from the hands, but gloves provided the best protection overall for both babies and nurses by preventing the acquisition of bacteria.

J Zoo Wildl Med, 2000 Jun, 31(2), 255 - 8
Zinc-responsive dermatosis in a red wolf (canis rufus); Kearns K et al.; An 18-mo-old male red wolf (Canis rufus) presented with footpad hyperkeratosis, suppurative paronychia, distal limb pyoderma, and peripheral lymphadenopathy . Diet for the previous 11 mo consisted of a mixture of two commercially prepared dog foods with a mineral supplement containing primarily calcium . Culture of the draining tracts on the distal limbs yielded a mixed population of opportunistic bacteria . Histopathologic findings were consistent with a diagnosis of zinc deficiency . Medical therapy consisted of 15 mg/kg amoxicillin p.o . b.i.d . and 10 mg/kg zinc sulfate p.o . s.i.d . Calcium supplementation was discontinued . Clinical signs resolved by 10 wk after the initiation of treatment.

FEMS Microbiol Lett, 2000 Sep 1, 190(1), 103 - 8
Evaluation of strain-specific primer sequences from an abortifacient strain of ovine Chlamydophila abortus (Chalmydia psittaci) for the detection of EAE by PCR; Creelan JL et al.; Strain-specific primer sequences derived from the helicase gene of an ovine abortifacient strain (S26/3) of Chlamydophila abortus (Chlamydia psittaci) were evaluated for the diagnosis of enzootic abortion in ewes (EAE) by polymerase chain reaction (PCR) . C abortus DNA was amplified from tissues submitted from ovine abortion cases using genus-specific and strain-specific primers in a standard thermal cycler . Amplification was followed by Southern blotting and hybridisation with a strain-specific probe . Real-time PCR was also evaluated using strain-specific primers in a microvolume fluorimeter-based thermal cycler (LightCycler) . Detection using both PCR methods was compared with other diagnostic methods against the standard of McCoy cell culture isolation . In this paper we report the application of strain-specific PCR as a fast, sensitive, specific method for the detection of EAE.

Curr Opin Struct Biol, 2000 Aug, 10(4), 443 - 7
beta-Barrel membrane proteins; Schulz GE; beta-Barrel proteins are found in the outer membranes of bacteria, mitochondria and chloroplasts . The presently known sizes range from small eight-stranded to large twenty-two-stranded beta barrels existing as monomers and oligomers . Their functions are as diverse as active ion transport, passive nutrient intake, membrane anchors, membrane-bound enzymes and defense against attack proteins . Of further interest are the folding process, the crystallization, the observed limited structural diversity and the manifold channel engineering options of these beta-barrel proteins.

Int J Cardiol, 2000 Aug 31, 75 Suppl 1, S37 - 45; discussion S47-52
Animal models of infection-mediated vasculitis: implications for human disease; Dal Canto AJ et al.; The human vasculitides including Takayasu's arteritis are idiopathic syndromes for which both autoimmune and infectious etiologies have been proposed . Although proof of a correlation between infection and human vasculitis would aid in patient management, it is difficult to confirm causality . To study infection-mediated vascular disease, different animal models have been developed . Infections with the bacteria C . pneumoniae, an RNA virus, and herpesviruses all cause vascular pathology and will be reviewed here . Many aspects of the human diseases are recapitulated in these models, so further animal studies may help elucidate mechanisms of infection-mediated vasculitis . Such results may improve management, and potentially, prevention of these important human diseases . Importantly, the animal models provide an opportunity to define how the immune and inflammatory processes function in the great vessels and the molecular basis for the selectivity of certain viral infections for the great elastic arteries.

Int J Parasitol, 2000 Aug, 30(9), 995 - 1000
Anhydrobiotic potential and long-term storage of entomopathogenic nematodes (Rhabditida: Steinernematidae); Grewal PS; Anhydrobiosis is considered to be an important means of achieving storage stability of entomopathogenic nematodes that are used in biological control . This study explored the effects of anhydrobiosis on longevity and infectivity of infective juveniles (IJs) of three species of entomopathogenic nematodes Steinernema carpocapsae, Steinernema feltiae, and Steinernema riobrave at 5 and 25 degrees C . Anhydrobiosis was induced in water-dispersible granules (WG) at 0.966-0.971 water activity and 25 degrees C following a 7-day preconditioning of IJs at 5 degrees C in tap water . Survival and infectivity of the desiccated (anhydrobiotic) IJs was compared with non-desiccated IJs stored in water for different periods . Anhydrobiosis increased longevity of S . carpocapsae IJs by 3 months and of S . riobrave by 1 month in WG at 25 degrees C as compared with IJs stored in water . However, desiccation decreased S . feltiae longevity at 25 degrees C and of all three species at 5 degrees C . These results demonstrate a shelf-life of 5 months for S . carpocapsae at 25 degrees C and 9 months at 5 degrees C in WG with over 90% IJ survival . For S . feltiae, over 90% survival occurred only for 2 months at 25 degrees C and 5 months at 5 degrees C in WG . Steinernema riobrave had over 90% survival only for 1 month at 25 degrees C and the survival dropped below 85% within 1 month at 5 degrees C . Induction of anhydrobiosis in WG resulted in 85, 79 and 76% reduction in oxygen consumption by S . carpocapsae, S . feltiae, and S . riobrave IJs, respectively . Differences in IJ longevity among three species in water at 25 degrees C were related both to the initial lipid content and the rate of lipid utilisation, but not at 5 degrees C . The one-on-one infection bioassays indicated that desiccation had no negative effect on the infectivity of any of the nematode species suggesting no harmful effect on the IJs and/or their symbiotic bacteria . The species differences in IJ longevity and desiccation survival at different temperatures are discussed in relation to their foraging strategy and temperature adaptation.

J Am Soc Echocardiogr, 2000 Sep, 13(9), 876 - 81
Echocardiography in nonbacterial thrombotic endocarditis: from autopsy to clinical entity; Reisner SA et al.; Bacteria-free verrucae, frequently termed "non-bacterial thrombotic endocarditis," have been recognized in autoimmune disorders as well as in neo-plastic diseases . The antemortem diagnosis of non-bacterial thrombotic endocarditis is rare, and most existing data result from postmortem examinations . In 3 prospective echocardiographic studies we found typical cardiac valvular lesions in patients with primary antiphospholipid syndrome, myelo-proliferative disorders, and solid malignant tumors . Cardiac lesions associated with these 3 different entities had common echocardiographic appearance and correlated positively with thromboembolic events . The possibility of common pathogenesis is suggested, and clinical significance is discussed.

J Infect Dis, 2000 Oct, 182(4), 1264 - 7 Epub 2000 Sep 05.
Reduced ex vivo chemokine production by polymorphonuclear cells after in vivo exposure of normal humans to endotoxin; Schultz MJ et al.; Monocytes from patients with sepsis have a reduced capacity to produce cytokines, a state referred to as immunoparalysis . To determine whether polymorphonuclear leukocytes (PMNL) can be rendered hyporesponsive, PMNL from 6 healthy volunteers intravenously challenged with lipopolysaccharide (LPS; 4 ng/kg) were stimulated ex vivo with heat-killed bacteria or LPS, and the release of the CXC chemokines interleukin-8, epithelial-derived neutrophil attractant-78, and growth-related oncogen-alpha was measured . At 1 and 2 h after LPS administration in vivo, PMNL produced fewer CXC chemokines after stimulation with bacteria or LPS (all P<.05) . Serum obtained 2 h after in vivo administration of LPS did not influence chemokine production by PMNL from 6 healthy volunteers not previously exposed to LPS . Thus, intravenous injection of LPS induces a refractory state of PMNL that is not caused by soluble factors produced in response to in vivo exposure to LPS.

Biocell, 2000 Aug, 24(2), 123 - 32
The laticifer system of Chamaesyce thymifolia: a closed host environment for plant trypanosomatids; da Cunha M et al.; Specimens of Chamaesyce thymifolia (Euphorbiaceae) infected and uninfected by Phytomonas sp., a parasite of the Trypanosomatidae family, were anatomically and ultrastructurally analyzed with special emphasis on the laticifer system . C . thymifolia presents branched non-articulated laticifers and was heavily infected by Phytomonas sp . in all collection sites . Infection was often observed in the initial stages inside the vacuole, when the latex particles could be seen . In intermediary stages of laticifer differentiation, Phytomonas sp . were found free in the cytoplasm, inside small vacuoles or in the central vacuole . In differentiated laticifers that had only the plasma membrane, Phytomonas sp . were free in the latex and close to the cell membrane . Infected and uninfected plants showed identical anatomy and ultrastructure and the starch grain numbers in the latex were not reduced in the presence of this flagellate . Biochemical analysis of the latex of infected and uninfected plants presented similar levels of protein, carbohydrate and beta-1,3-glucanase, suggesting that this species is not pathogenic for the host . Besides, all infected plants complete its life cycle . Plants infected with Phytomonas presented occasionally virus like particles and bacteria inside the laticifer tubes.

Isr Med Assoc J, 2000 Aug, 2(8), 580 - 2
Association of Ureaplasma urealyticum colonization in male urethra and Condyloma acuminatum; Zvulunov A et al.; BACKGROUND: The frequent coexistence of two or more sexually transmitted diseases in one patient has been reported in non-dermatological literature, mostly in languages other than English . Identification of Ureaplasma urealyticum, Chlamydia trachomatis and Mycoplasma hominis in men with other STDs is important, since these bacteria have been implicated in a variety of diseases such as non-gonococcal urethritis, premature rupture of fetal membranes, and infertility in female sexual partners of these patients . OBJECTIVE: To assess the frequency of concomitant STD, particularly urethral colonization of U . urealyticum, C . trachomatis and M . hominis, in men consulting for suspected STD-related symptoms . METHODS: All patients attending our dermatology clinic for STD-related symptoms during a 12 month period in 1996-97 underwent systematic clinical and laboratory screening for syphilis, gonorrhea, NGU, prostatitis, genital herpes simplex infection, Condyloma acuminatum, urethral carriage of U . urealyticum, C . trachomatis and M . hominis, as well as serological screening for HIV, and hepatitis B and C infections . RESULTS: A total of 169 men with STD-related symptoms were enrolled in the study . The following clinical diagnoses were established: NGU in 109 men, C . acuminatum in 40, genital herpes simplex in 10, prostatitis in 7, latent syphilis in 6, primary syphilis in 1, and Behcet's disease in 1 . No clinical evidence of STD was found in 13 patients . Of the 169 patients, 39 (23%) had two or more concomitant STDs, of whom 27 (69%) had C . acuminatum associated with one or more of the urethral pathogens . A positive U . urealyticum culture was found in 67.5% (27/40) of the men with C . acuminatum as compared to 42% (40/96) among the patients with NGU who did not have C . acuminatum (P = 0.004, chi 2 test) . Conversely, the prevalence of C . acuminatum among patients positive for U . urealyticum was significantly higher than the prevalence among those who were negative--27/75 (36%) vs . 13/94 (14%), P < 0.0009, chi 2 test . About half of the U . urealyticum-positive patients with C . acuminatum had no clinical signs or symptoms of urethritis . CONCLUSION: Our findings suggest that patients with C . acuminatum should be assessed for U . urealyticum carriage and, when identified, their sexual contacts should be actively sought and treated.

Nihon Kokyuki Gakkai Zasshi, 2000 Jun, 38(6), 490 - 3
{Pulmonary dirofilariasis with cavity formation and pleural effusion}; Sano A et al.; A 61-year-old man visited a community hospital because of hemosputum . A solitary nodule in the left lower lung field was pointed out on a chest roentgenogram . The patient was treated with antibiotics, but the solitary nodule increased in size . He was referred to our hospital because of high fever and observations of cavity formation and pleural effusion on a chest roentgenogram . The pleural effusion showed no cytologic evidence of malignancy, and cultures were also negative for bacteria . An increased percentage of lymphocytes was detected in the pleural effusion, but slight eosinophilia was found in blood samples . Dot enzyme-linked immunosorbent assay and Ouchterlony's double-diffusion test yielded a diagnosis of pulmonary dirofilariasis . After drainage of the pleural effusion by thoracentesis, spontaneous regression was observed . Cavity formation, pleural effusion, and spontaneous regression are in general rare in patients with pulmonary dirofilariasis.

Biochemistry, 2000 Sep 5, 39(35), 10684 - 94
Molecular architecture of the mutagenic active site of human immunodeficiency virus type 1 reverse transcriptase: roles of the beta 8-alpha E loop in fidelity, processivity, and substrate interactions; Weiss KK et al.; Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a putative source of the genomic hypermutation that promotes rapid evolution of HIV-1 . To understand the molecular strategies that create a highly mutagenic DNA polymerase active site in HIV-1 RT, we investigated the roles of four residues in the beta 8-alpha E loop . Gln151, which interacts with the sugar of the incoming dNTP, and Lys154, which interacts with the template, yielded site-directed mutants with increased fidelity, suggesting that these residues are directly involved in the mutagenic architecture of the active site . Mutations at Gln151 and Lys154 also reduced processivity . Q151N RT showed enhanced ability to discriminate between TTP and AZT triphosphate, consistent with the observation that the Q151M mutation confers AZT resistance in vivo . Mutations at Gly152 greatly decreased RT activity; molecular modeling suggests that Gly152 is critical for the proper geometric alignment that permits base-pairing of the incoming dNTP with the template . Mutations at Trp153 reduced the expression level, and presumably the stability, of RT proteins in bacteria . These observations support the conclusion that interactions of active site residues in the beta 8-alpha E loop with incoming dNTPs and the template are determinants of the accuracy, processivity, and substrate selectivity of HIV-1 RT.

Cell Mol Biol (Noisy-le-grand), 2000 Jul, 46(5), 895 - 913
New techniques in fast time-resolved structure determination; Perman B et al.; New techniques in fast time-resolved X-ray crystallography provide a different approach to understanding the structural basis of protein function . Two biological systems have been studied as part of the refinement of these techniques, and have actually spurred new ideas in time-resolved structural studies . The dissociation of carbon monoxide from carbon-monoxy myoglobin has earlier been investigated over a time range spanning 18 orders of magnitude (femtoseconds to hours) using spectroscopic methods . Rapid time-resolved determination of the entire myoglobin structure made it possible to determine both the position of the CO after photodissociation and the entire globin structure, over a time range from nanoseconds to milliseconds, during which the heme and globin relax and the carbon monoxide rebinds . Photoactive yellow protein, a relative newcomer to biophysical research, has a fully-reversible photocycle containing several spectrally distinct intermediates . Identifying and solving the structures of each intermediate is the initial goal in time-resolved studies on this protein and will contribute to a greater understanding of the biological process of light driven signal transduction.

Am J Vet Res, 2000 Sep, 61(9), 1145 - 9
Detection of cell detachment activity induced by Moraxella bovis; Marrion RM et al.; OBJECTIVE: To characterize the effect that filtrate obtained from cultures of Moraxella bovis has on cultured corneal epithelial cells and other types of cultured mammalian cells . SAMPLE POPULATION: Cultured hamster corneal epithelial cells, bovine epithelial cells, and several transformed cell lines exposed to culture filtrate from a pathogenic isolate of M bovis . PROCEDURE: Moraxella bovis was cultured, and bacteria were removed by filtration . The resulting bacterial culture filtrate was incubated with various types of cultured cells, and effects of the filtrate on detachment of various mammalian cell types was quantified by the use of neutral red dye . Additionally, bacterial culture filtrate was treated with protease inhibitors as well as trypsin and heat prior to incubation with cultured mammalian cells . RESULTS: Bacterial culture filtrate of M bovis caused all types of cultured cells to detach from each other and from the substrate, with the maximal effect evident 2 hours after initiating incubation . Detached cells were alive, and detachment was reversible . Serine protease inhibitors (phenylmethylsulfonylfluoride and alpha2-macroglobulin) inhibited cell detachment attributable to bacterial culture filtrate . Heating and treatment with trypsin also inhibited cell detachment . CONCLUSIONS AND CLINICAL RELEVANCE: Moraxella bovis produces a soluble factor that causes reversible detachment of cultured cells . This activity may play a role in the pathogenesis of infectious bovine keratoconjunctivitis.

J Biol Chem, 2000 Dec 8, 275(49), 38302 - 10
Redox switch of hsp33 has a novel zinc-binding motif; Jakob U et al.; The chaperone activity of the heat shock protein Hsp33 is regulated by reversible disulfide bond formation . Oxidized Hsp33 is active, and reduced Hsp33 is inactive . We show that zinc binding is essential for the function of this redox switch . Our results reveal that Hps33 contains a new, high affinity (K(a) > 10(17) m(-)(1)), zinc-binding motif in the form Cys-X-Cys-X(27-32)-Cys-X-X-Cys . All four conserved cysteines within this motif act to coordinate a single zinc atom . Experiments where reduced wild type Hsp33 is reconstituted with cobalt or cadmium demonstrate that the metal-coordinating cysteines are present as highly reactive thiolate anions . This ionization may allow for the fast and successful activation of the chaperone function of Hsp33 upon incubation in oxidizing agents.

Protein Sci, 2000 Aug, 9(8), 1503 - 18
Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping--a novel approach to assess intermolecular protein contacts; Bennett KL et al.; The intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical cross-linking with differential MALDI-MS analyses . ParR dimers were modified in vitro with the thiol-cleavable cross-linker 3,3'-dithio-bis(succinimidylproprionate) (DTSSP), proteolytically digested with trypsin and analyzed by MALDI-MS peptide mapping . Comparison of the peptide maps obtained from digested cross-linked ParR dimers in the presence and absence of a thiol reagent strongly supported a "head-to-tail" arrangement of the monomers in the dimeric complex . Glycoprotein fusion constructs CD28-IgG and CD80-Fab were cross-linked in vitro by DTSSP, characterized by nonreducing SDS-PAGE, digested in situ with trypsin and analyzed by MALDI-MS peptide mapping (+/- thiol reagent) . The data revealed the presence of an intermolecular cross-link between the receptor regions of the glycoprotein constructs, as well as a number of unexpected but nonetheless specific interactions between the fusion domains of CD28-IgG and the receptor domain of CD80-Fab . The strategy of chemical cross-linking combined with differential MALDI-MS peptide mapping (+ thiol reagent) enabled localization of the interface region(s) of the complexes studied and clearly demonstrates the utility of such an approach to obtain structural information on interacting noncovalent complexes.

Protein Sci, 2000 Aug, 9(8), 1439 - 54
Blue copper proteins: a comparative analysis of their molecular interaction properties; De Rienzo F et al.; Blue copper proteins are type-I copper-containing redox proteins whose role is to shuttle electrons from an electron donor to an electron acceptor in bacteria and plants . A large amount of experimental data is available on blue copper proteins; however, their functional characterization is hindered by the complexity of redox processes in biological systems . We describe here the application of a semiquantitative method based on a comparative analysis of molecular interaction fields to gain insights into the recognition properties of blue copper proteins . Molecular electrostatic and hydrophobic potentials were computed and compared for a set of 33 experimentally-determined structures of proteins from seven blue copper subfamilies, and the results were quantified by means of similarity indices . The analysis provides a classification of the blue copper proteins and shows that (I) comparison of the molecular electrostatic potentials provides useful information complementary to that highlighted by sequence analysis; (2) similarities in recognition properties can be detected for proteins belonging to different subfamilies, such as amicyanins and pseudoazurins, that may be isofunctional proteins; (3) dissimilarities in interaction properties, consistent with experimentally different binding specificities, may be observed between proteins belonging to the same subfamily, such as cyanobacterial and eukaryotic plastocyanins; (4) proteins with low sequence identity, such as azurins and pseudoazurins, can have sufficient similarity to bind to similar electron donors and acceptors while having different binding specificity profiles.

Comb Chem High Throughput Screen, 2000 Aug, 3(4), 315 - 27
Nucleic acid sequence based amplification (NASBA) of Chlamydia pneumoniae major outer membrane protein (ompA) mRNA with bioluminescent detection; Coombes BK et al.; Chlamydia pneumoniae has been associated with chronic conditions such as atherosclerosis and coronary heart disease but the precise role of this intracellular bacteria in the pathogenesis of these diseases is not well defined . Several techniques have been developed for detection of C . pneumoniae in atheromatous lesions, however it remains unclear whether persistent forms of the organism and/or actively replicating bacteria contribute to associated pathology . The aim of this study was to utilize nucleic acid sequence based amplification (NASBA) technology together with a highly sensitive aequorin bioluminescent hybridization assay for the detection of C . pneumoniae ompA mRNA transcripts . A NASBA targeting the ompA gene of C . pneumoniae was developed, and the sensitivity was evaluated using both C . pneumoniae ompA RNA generated in vitro, and purified C . pneumoniae inclusion forming units (IFU) . C . pneumoniae NASBA was capable of detecting between 100 and 1000 ompA RNA molecules and could detect 0.2 IFU of C . pneumoniae using the aequorin bioluminescent assay . The sensitivity of the bioluminescent assay was at least 10-fold higher than Northern blot detection . The linearity of NASBA amplification was assessed in time-course amplification experiments with different input numbers of RNA molecules . When NASBA products were analyzed during the linear phase of amplification, the dynamic range of bioluminescent detection extended over 8-log units of input RNA copy number . NASBA amplification coupled with bioluminescent detection may prove to be a useful molecular tool for the detection, quantitation and analysis of differentially expressed chlamydial genes during various stages of infection and disease pathology or for other mRNAs of interest in different disease processes.

Microbiology, 2000 Sep, 146 ( Pt 9), 2229 - 36
Fructose operon mutants of Spiroplasma citri; Gaurivaud P et al.; Fructose-negative mutants of Spiroplasma citri wild-type strain GII-3 were selected by two methods . The first method is based on the selection of spontaneous xylitol-resistant mutants, xylitol being a toxic fructose analogue . Five such mutants were obtained, but only one, xyl3, was unable to use fructose and had no phosphoenolpuryvate:fructose phosphotransferase system (fructose-PTS) activity . Amplification and sequencing of the fructose permease gene of mutant xyl3 revealed the presence of an adenylic insertion leading to a truncated permease . The second method is based on inactivation of fruA and/or fruK by homologous recombination involving one crossing-over between the chromosomal genes and inactivated genes carried by replicative plasmids . Fructose-negative mutants were obtained at a frequency of about 10% . Fructose-PTS activity and 1-phosphofructokinase activity were not detected in four representative mutants that were characterized (H31, H45, E38 and E53) . In strain H31, Southern blot analysis and PCR showed that the result of homologous recombination was, as expected, the presence in the chromosome of two mutated fruA-fruK copies with the plasmid sequence in between . Only the mutated copy, under control of the fructose operon promoter, was transcribed . This work describes for the first time the use of two methods to obtain fructose-auxotrophic mutants of S . citri . The method involving homologous recombination is a general procedure for gene disruption in S . citri.

Microbiology, 2000 Sep, 146 ( Pt 9), 2199 - 207
Inhibition of chromosome replication in Mycobacterium smegmatis: effect of the rpmH-dnaA promoter region; Salazar L; In a previous study a functional mycobacterial origin of replication, oriC, was isolated on a plasmid . However, it was found that origin function was inhibited by the presence of the adjacent dnaA gene or its regulatory region, so that plasmids containing both of these regions next to the origin did not yield transformants . This inhibition could be due either to overexpression of dnaA on a plasmid being toxic, the transcription of dnaA into the downstream origin topologically inhibiting its function, or to the DnaA boxes upstream of dnaA somehow interacting with the DnaA boxes in the origin to prevent its function . To distinguish between these possibilities, plasmids were constructed lacking different parts of the dnaA gene: the promoter, the DnaA boxes, or both . Additionally, the putative dnaA promoter region was replaced by mycobacterial sequences that exhibit weaker or null promoter activity . The results indicate that the rpmH-dnaA promoter region, but not the DnaA boxes, is the principal cause of the incompatibility observed and suggest that this region could be playing a role in the inhibition of chromosome replication.

Nat Biotechnol, 2000 Sep, 18(9), 989 - 94
Antibody arrays for high-throughput screening of antibody-antigen interactions; de Wildt RM et al.; We have developed a novel technique for high-throughput screening of recombinant antibodies, based on the creation of antibody arrays . Our method uses robotic picking and high-density gridding of bacteria containing antibody genes followed by filter-based enzyme-linked immunosorbent assay (ELISA) screening to identify clones that express binding antibody fragments . By eliminating the need for liquid handling, we can thereby screen up to 18,342 different antibody clones at a time and, because the clones are arrayed from master stocks, the same antibodies can be double spotted and screened simultaneously against 15 different antigens . We have used our technique in several different applications, including isolating antibodies against impure proteins and complex antigens, where several rounds of phage display often fail . Our results indicate that antibody arrays can be used to identify differentially expressed proteins.

Scand J Immunol, 2000 Sep, 52(3), 240 - 8
Role of J chain in secretory immunoglobulin formation; Johansen FE et al.; The joining (J) chain is a small polypeptide, expressed by mucosal and glandular plasma cells, which regulates polymer formation of immunoglobulin (Ig)A and IgM . J-chain incorporation into polymeric IgA (pIgA, mainly dimers) and pentameric IgM endows these antibodies with several salient features . First, a high valency of antigen-binding sites, which makes them suitable for agglutinating bacteria and viruses; little or no complement-activating potential, which allows them to operate in a noninflammatory fashion; and, most importantly, only J-chain-containing polymers show high affinity for the polymeric Ig receptor (pIgR), also known as transmembrane secretory component (SC) . This epithelial glycoprotein mediates active external transfer of pIgA and pentameric IgM to exocrine secretions . Thus, secretory IgA (SIgA) and SIgM, as well as free SC, are generated by endoproteolytic cleavage of the pIgR extracellular domain . The secretory antibodies form the 'first line' of defence against pathogens and noxious substances that favour the mucosae as their portal of entry . The J chain is involved in creating the binding site for pIgR/SC in the Ig polymers, not only by determining the polymeric quaternary structure but apparently also by interacting directly with the receptor protein . Therefore, both the J chain and the pIgR/SC are key proteins in secretory immunity.

Proc R Soc Lond B Biol Sci, 2000 Jul 7, 267(1450), 1277 - 85
Wolbachia infection frequencies in insects: evidence of a global equilibrium?
Werren JH, Windsor DM.
Wolbachia are a group of cytoplasmically inherited bacteria that cause reproduction alterations in arthropods, including parthenogenesis, reproductive incompatibility, feminization of genetic males and male killing . Previous general surveys of insects in Panama and Britain found Wolbachia to be common, occurring in 16-22% of species . Here, using similar polymerase chain reaction methods, we report that 19.3% of a sample of temperate North American insects are infected with Wolbachia, a frequency strikingly similar to frequencies found in two other studies in widely separated locales . The results may indicate a widespread equilibrium of Wolbachia infection frequencies in insects whose maintenance remains to be explained . Alternatively, Wolbachia may be increasing in global insect communities . Within each of the three geographic regions surveyed, Hymenoptera are more frequently infected with A group Wolbachia and Lepidoptera more frequently infected with B group Wolbachia.

Arch Latinoam Nutr, 1999 Sep, 49(3 Suppl 2), 40S - 46S
{Role of iron in immunity and its relation with infections}; Soyano A et al.; Experimental evidence in the last decades show that iron is a fundamental element for normal development of the immune system . Its deficiency affects the capacity to have an adequate immune response . The role of iron in immunity is necessary for immune cells proliferation and maturation, particularly lymphocytes, associated with the generation of a specific response to infection . The body has the capacity to reduce the iron availability to be consumed by infectious elements by proteins such as transferrin and lactoferrin . Also, iron is essential for the proliferation of bacteria, parasites, and neoplastic cells . Thus excess iron could potentially facilitate the development of infections and the invasion of tumoral cells . The immune system has bacteriostatic mechanisms that reduce the availability of the metal, interfering with bacterial growth . Additionally the system uses iron as the intermediary in the production of bacteriostatic cells.

Insect Mol Biol, 2000 Aug, 9(4), 393 - 405
Long PCR improves Wolbachia DNA amplification: wsp sequences found in 76% of sixty-three arthropod species; Jeyaprakash A et al.; Bacteria belonging to the genus Wolbachia are associated with a variety of reproductive anomalies in arthropods . Allele-specific polymerase chain reaction (= Standard PCR) routinely has been used to amplify Wolbachia DNA from arthropods . While testing the two-spotted spider mite Tetranychus urticae and other arthropods known to be infected with Wolbachia, Standard PCR frequently produced false negatives, perhaps because the DNA from the arthropod host interfered with amplification by Taq DNA polymerase . Long PCR, which uses two enzymes (Taq and Pwo), consistently amplified Wolbachia DNA and a sensitivity analysis indicated that Long PCR was approximately six orders of magnitude more sensitive than Standard PCR in amplifying plasmid DNA spiked into insect genomic DNA . A survey indicated that 76% of sixty-two arthropod species and two subspecies in thirteen orders tested positive for the Wolbachia wsp sequence by Long PCR, which is considerably higher than the rate of 16.9% obtained previously for the ftsZ sequence using Standard PCR (Werren, J.H., Windsor, D . and Gao, L . (1995a) Proc R Soc Lond B 262: 197-204) . A subsample of Long PCR products from fourteen arthropod species and two subspecies were sequenced, both directly and after cloning . Two A- and eleven B-Wolbachia strains were detected and their wsp sequences displayed a maximum of 23.7% sequence divergence at this locus . Two new groups (named Fus and Ten) were identified in addition to nineteen reported earlier (Zhou, W., Rousset, F . and O'Neill, S.L . (1998) Proc R Soc Lond B 265: 1-7; van Meer, M.M.M., Witteveldt, J . and Stouthamer, R . (1999) Insect Mol Biol 8: 399-408), because they displayed more than 2.5% sequence divergence from other Wolbachia wsp sequences . PCR products from seventeen of twenty-nine (59%) arthropod species analysed could not be sequenced directly due to apparent infection by multiple Wolbachia strains . The wsp sequences cloned from two such species (Plutella xylostella and Trichoplusia ni) indicated both A- and B-Wolbachia were present in a single individual . Hence, superinfection also may be more widespread than the 1.2% incidence previously estimated.

Eur J Biochem, 2000 Sep, 267(18), 5657 - 64
A tribute to sulfur; Beinert H; Recent progress in a number of areas of biochemistry and biology has drawn attention to the critical importance of sulfur in the biosynthesis of vital cofactors and active sites in proteins, and in the complex reaction mechanisms often involved . This brief review is intended as a broad overview of this currently rapidly moving field of sulfur biochemistry, for those who are interested or are involved in one or the other aspect of it, a synopsis by one who has stumbled into this field from several directions in the course of time . Only for iron are metal-sulfur relationships discussed in detail, as the iron-sulfur subfield is one of the most active areas.

EMBO J, 2000 Sep 1, 19(17), 4503 - 12
CtrA mediates a DNA replication checkpoint that prevents cell division in Caulobacter crescentus; Wortinger M et al.; Coordination of DNA replication and cell division is essential in order to ensure that progeny cells inherit a full copy of the genome . Caulobacter crescentus divides asymmetrically to produce a non-replicating swarmer cell and a replicating stalked cell . The global response regulator CtrA coordinates DNA replication and cell division by repressing replication initiation and transcription of the early cell division gene ftsZ in swarmer cells . We show that CtrA also mediates a DNA replication checkpoint of cell division by regulating the late cell division genes ftsQ and ftsA . CtrA activates transcription of the P(QA) promoter that co-transcribes ftsQA, thus regulating the ordered expression of early and late cell division proteins . Cells inhibited for DNA replication are unable to complete cell division . We show that CtrA is not synthesized in pre-divisional cells in which replication has been inhibited, preventing the transcription of P(QA) and cell division . Replication inhibition prevents the activation of the ctrA P2 promoter, which normally depends on CtrA phosphorylation . This suggests the possibility that CtrA phosphorylation may be affected by replication inhibition.

Tuber Lung Dis, 2000, 80(3), 131 - 40
Immunologic diagnosis of tuberculosis: a review; Chan ED et al.; The diagnosis of tuberculosis (TB) principally rests on the sputum examination and culture . However, the sensitivity of sputum smear for acid-fast bacteria is only approximately 50% and sputum culture has a relatively long turnaround time . As a result, a number of studies have been conducted in an attempt to find a rapid and accurate diagnostic test for TB . They include serological assays against various mycobacterial antigens . Here we review the merits and deficiencies of the serological tests for TB . In general, serological assays have a high negative predictive value, making them potentially useful as a screening test to rule out active TB although in HIV-positive individuals, low sensitivity and low negative predictive value compromises the accuracy of the seroassays in this group of individuals . In populations where the prevalence of latent TB infection is high, the relatively low positive predictive value of the tests reduces their specificity for active TB . Furthermore, the higher costs and greater training required in performing these tests makes it important that future studies also assess whether their use affects patient outcomes in management of TB .

Midwifery, 2000 Sep, 16(3), 237 - 45
Cord-care practice in Scotland; Ireland J et al.; OBJECTIVE: To identify the range of policies, practices and rationale for umbilical-cord stump care in the NHS in Scotland . DESIGN: A postal questionnaire survey completed in two stages . The first stage elicited the views of Heads of Midwifery/Senior Midwives, and the second stage the views of midwives, enrolled nurses and nursery nurses . SETTING: NHS units in Scotland providing intrapartum care . They were separated into large and small units with small units having < or = 1000 deliveries per year (n = 178), and large units > 1000 deliveries per year (n = 300) . PARTICIPANTS: The Heads of Midwifery/Senior Midwives from the 51 units were invited to participate in the study and 49 (96.1%) replied . In stage two 512 maternity unit employees were sent questionnaires and 390 (76.2%) replied . These were six enrolled nurses, 20 nursery nurses and 360 midwives and four respondents of unspecified occupation . MEASUREMENTS: The existence of cord-care policies and their rationale . FINDINGS: About half of the units that responded had a written policy . Large units were four times more likely than small units to have a written policy . Both managers and staff reported that the most common policy/agreed practice was no specific care (cord observed and only cleaned if soiled) . Where a written policy existed, less than one-half of the Heads of Midwifery/Senior Midwives and less than one third of the staff reported that the basis for this policy was research . KEY CONCLUSIONS: Units with a written policy are in the minority and small units are far less likely to have such a policy . Wide variation exists in policy, practice and rationale . Diversity within and between units creates anxiety and disillusionment for practitioners . It may also cause confusion for patients who are exposed to different cord-care practices either as these change over time or because they use different units . IMPLICATIONS FOR PRACTICE: The midwifery profession must examine this area of practice and determine how to address this lack of evidence . Further research is required to determine the most effective method of cord care and how best to put the findings into practice . Outstanding questions which beg further investigation are: How do cords heal and separate and what bacteria are naturally involved in this process? What constitutes an infected cord as opposed to a colonised cord?

Immunogenetics, 2000 Aug, 51(10), 856 - 62
Evolutionary history of the Rh blood group-related genes in vertebrates; Kitano T et al.; Rh and its homologous Rh50 gene products are considered to form heterotetramers on erythrocyte membranes . Rh protein has Rh blood group antigen sites, while Rh50 protein does not, and is more conserved than Rh protein . We previously determined both Rh and Rh50 gene cDNA coding regions from mouse and rat, and carried out phylogenetic analyses . In this study, we determined Rh50 gene cDNA coding regions from African clawed frog and Japanese medaka fish, and examined the long-term evolution of the Rh blood group and related genes . We constructed the phylogenetic tree from amino acid sequences . Rh50 genes of African clawed frog and Japanese medaka fish formed a cluster with mammalian Rh50 genes . The gene duplication time between Rh and Rh50 genes was estimated to be about 510 million years ago based on this tree . This period roughly corresponds to the Cambrian, before the divergence between jawless fish and jawed vertebrates . We also BLAST-searched an amino acid sequence database, and the Rh blood group and related genes were found to have homology with ammonium transporter genes of many organisms . Ammonium transporter genes can be classified into two major groups (amt alpha and amt beta) . Both groups contain genes from three domains (bacteria, archaea, and eukaryota) . The Rh blood group and related genes are separated from both amt alpha and beta groups.

Bioorg Med Chem Lett, 2000 Aug 21, 10(16), 1839 - 43
Synthesis of the nucleoside moiety of liposidomycins: elucidation of the pharmacophore of this family of MraY inhibitors; Dini C et al.; Tunicamycins (TCMs) and liposidomycins (LPMs) are naturally occurring inhibitors of the bacterial translocase (MraY) . Based on structure-activity relationship (SAR) studies, a molecular model has been proposed for their inhibitory mechanism . This study points out the importance of the nucleoside moiety of liposidomycins in the inhibition of MraY . A simplified molecule (I) based on the liposidomycin core structure has been synthesised and tested on MraY . The compound displayed a moderate inhibitory activity (IC50 = 50 microM) . The validation of the molecular model was then performed by synthesising higher homologues of I, containing an additional stereocentre in the 5' position (XIV and XV) . In agreement with the prediction, only the (S) isomer XV showed significant activity against MraY (IC50 = 5 microM).

Cancer Res, 2000 Aug 15, 60(16), 4311 - 4
Inhibition of tumor angiogenesis by a single-chain antibody directed against vascular endothelial growth factor; Vitaliti A et al.; Monoclonal antibody (Ab) directed against the vascular endothelial growth factor, one of the major inducers of angiogenesis, can inhibit tumor growth in mice . Treatment of cancer patients with monoclonal Ab requires large-scale production of the clean Ab and frequent application of the Ab . This might be improved by using single-chain Ab fragments (scFvs), which can be produced in large quantities in bacteria and are attractive for gene therapeutic approaches . Here we describe anti-vascular endothelial growth factor scFvs derived from a human phage-display library able to block the vascularization of the chorioallantoic membrane of chick embryos and reduce the growth of s.c . tumors in nude mice . This work opens the way to develop gene therapy-based strategies using a scFv to treat angiogenesis-dependent diseases.

Microb Pathog, 2000 Sep, 29(3), 191 - 200
Residual virulence of Brucella abortus in the absence of the cytochrome bc(1)complex in a murine model in vitro and in vivo; Ko J et al.; To maintain survival in macrophages, Brucella must overcome a hostile phagosomal environment defined as low pH, limited nutrition and low oxygen tension . The specific mechanisms utilized by Brucella to surmount such unfavorable environmental factors in phagosomes are not well understood . In general, to adapt to a change in environmental oxygen tension, bacteria use different terminal oxidases that have different oxygen affinity . To survive in phagosomes where low oxygen tension exists, Brucella, like other bacteria, may require high oxygen affinity terminal oxidases that can accept electrons through a cytochrome bc(1)complex dependent or independent pathway . Using a Brucella abortus cytochrome bc(1)complex deficient mutant, delta fbcF, the requirement for a high oxygen affinity terminal oxidase governed by the cytochrome bc(1)complex dependent pathway was tested . The number of cfu from RAW 264.7 macrophage cells and spleens of BALB/c mice infected with wild-type or the cytochrome bc(1)complex deficient mutant was similar during the course of infection . These results suggest that B . abortus contains no essential terminal oxidase utilized at low oxygen tension in phagosomes requiring the cytochrome bc(1)complex . Alternatively, other branched cytochrome bc(1)complex independent respiratory mechanisms that contain the high oxygen affinity terminal oxidases likely exist to facilitate Brucella survival in phagosomes . This is the first investigation regarding the Brucella respiratory system at the molecular level and the involvement of a respiratory system in Brucella pathogenesis .

J Autoimmun, 2000 Sep, 15(2), 227 - 30
New developments in viral peptides and APL induction; Gharavi AE et al.; The associations of antiphospholipid antibodies (aPL) with thrombosis and fetal death are well recognized, but the mechanism(s) that induce their production are not . We demonstrated induction of pathogenic aPL antibodies by immunization with foreign beta(2)-GPI, or synthetic peptides representing the PL-binding site of the beta(2)-GPI . These antibodies caused intrauterine fetal death and transverse myelopathy due to spinal cord infarction in mice, and activated endothelial cells in vitro . We also introduced aPL in mice by immunization with PL-binding viral peptides and observed their pathogenic effects . This study demonstrated that pathogenic effects of aPL antibodies induced by immunization with a human CMV-derived PL-binding synthetic peptide . We hypothesize that in APS patients aPL is induced by beta(2)-GPL-like PL-binding products of human common bacteria or viruses .

J Biol Inorg Chem, 2000 Aug, 5(4), 527 - 34
Novel structure and redox chemistry of the prosthetic groups of the iron-sulfur flavoprotein sulfide dehydrogenase from Pyrococcus furiosus; evidence for a {2Fe-2S} cluster with Asp(Cys)3 ligands; Hagen WR et al.; The consecutive structural genes for the iron-sulfur flavoenzyme sulfide dehydrogenase, sudB and sudA, have been identified in the genome of Pyrococcus furiosus . The translated sequences encode a heterodimeric protein with an alpha-subunit, SudA, of 52598 Da and a beta-subunit, SudB, of 30686 Da . The alpha-subunit carries a FAD, a putative nucleotide binding site for NADPH, and a {2Fe-2S}2+,+ prosthetic group . The latter exhibit EPR g-values, 2.035, 1.908, 1.786, and reduction potential, Em,8 = +80 mV, reminiscent of Rieske-type clusters; however, comparative sequence analysis indicates that this cluster is coordinated by a novel motif of one Asp and three Cys ligands . The motif is not only found in the genome of hyperthermophilic archaea and hyperthermophilic bacteria, but also in that of mesophilic Treponema pallidum . The beta-subunit of sulfide dehydrogenase contains another FAD, another putative binding site for NADPH, a {3Fe-4S}+,0 cluster, and a {4Fe-4S}2+,+ cluster . The 3Fe cluster has an unusually high reduction potential, Em,8 = +230 mV . The reduced 4Fe cluster exhibits a complex EPR signal, presumably resulting from magnetic interaction of its S = 1/2 spin with the S=2 spin of the reduced 3Fe cluster . The 4Fe cluster can be reduced with deazaflavin/EDTA/light but not with sodium dithionite; however, it is readily reduced with NADPH . SudA is highly homologous to KOD1-GO-GAT (or KOD1-GltA), a single-gene encoded protein in Pyrococcus kodakaraensis, which has been putatively identified as hyperthermophilic glutamate synthase . However, P . furiosus sulfide dehydrogenase does not have glutamate synthase activity . SudB is highly homologous to HydG, the gamma-subunit of P . furiosus NiFe hydrogenase . The latter enzyme also has sulfide dehydrogenase activity . The P . furiosus genome contains a second set of consecutive genes, sudY and sudX, with very high homology to the sudB and sudA genes, and possibly encoding a sulfide dehydrogenase isoenzyme . Each subunit of sulfide dehydrogenase is a primary structural paradigm for a different class of iron-sulfur flavoproteins.

J Biol Inorg Chem, 2000 Aug, 5(4), 469 - 74
Synthesis, spectroscopic, and structural characterization of the first aqueous cobalt(II)-citrate complex: toward a potentially bioavailable form of cobalt in biologically relevant fluids; Matzapetakis M et al.; Citric acid represents a class of carboxylic acids present in biological fluids and playing key roles in biochemical processes in bacteria and humans . Its ability to promote diverse coordination chemistries in aqueous media, in the presence of metal ions known to act as trace elements in human metabolism, earmarks its involvement in a number of physiological functions . Cobalt is known to be a central element of metabolically important biomolecules, such as B12, and therefore its biospeciation in biological fluids constitutes a theme worthy of chemical and biological perusal . In an effort to unravel the aqueous chemistry of cobalt in the presence of a physiologically relevant ligand, citrate, the first aqueous, soluble, mononuclear complex has been synthesized and isolated from reaction mixtures containing Co(II) and citrate in a 1:2 molar ratio at pH approximately 8 . The crystalline compound (NH4)4{Co(C6H5O7)2} (1) has been characterized spectroscopically (UV/vis, EPR) and crystallographically . Its X-ray structure consists of a distorted octahedral anion with two citrate ligands fulfilling the coordination requirements of the Co(II) ion . The magnetic susceptibility measurements of 1 in the range from 6 to 295 K are consistent with a high-spin complex containing Co(II) with a ground state S=3/2 . Corroborating this result is the EPR spectrum of 1, which shows a signal consistent with the presence of a Co(II) system . The spectroscopic and structural properties of the complex signify its potential biological relevance and participation in speciation patterns arising under conditions consistent with those employed for its synthesis and isolation.

Crit Rev Immunol, 2000, 20(3), 223 - 44
Interaction between the human NK receptors and their ligands; Vales-Gomez M et al.; NK cells are physiologically important in a number of contexts: mediating immunity against viruses, intracellular bacteria and parasites, and in anti-tumour immune responses . Moreover, in addition to these overtly immune protective functions, NK cells also mediate a variety of homeostatic functions, particularly in the regulation of haematopoesis and they may have an important role to play in the maintenance and development of placentation; certainly NK cells are a major component of the lymphocyte population of the decidua . The behaviour of the NK cell in these various situations is regulated by a large number of distinct receptors that transmit positive and negative signals . The balance of these signals determines whether the NK cell does nothing or is activated to proliferate, kill or secrete a wide range of cytokines and chemokines . In this review the structure and function of a number of molecules found on the NK cell surface are discussed, particular emphasis being placed on the molecular details of the recognition of target cell classical class I HLA molecules by Killer cell Immunoglobulin-like Receptors (KIR) and the binding of the non-classical class I molecule HLA-E to the heterodimer formed by the association of CD94 with various members of the NKG2 proteins.

Rozhl Chir, 2000 Jun, 79(6), 239 - 43
{The moist wound healing system in an experiment and clinical practice}; Brychta P et al.; We compared two types of moisture-retentive dressings (dressings that are capable of maintaining a moist environment) in wound healing . The conventional method of using an impregnated gauze in combination with a moist wound dressing was compared with TenderWet . First, we report results from an in vitro study and an animal experiment that included assessment of reepithelization and incidence of wound infection . Secondly, we discuss our experience using TenderWet in a clinical setting . Two cases of deep dermal burns are described and documented in detail . The results of our work suggest that a moist wound environment is more effective in facilitating wound healing than conventional methods.

FEBS Lett, 2000 Aug 25, 480(1), 32 - 6
Genome-wide protein interaction maps using two-hybrid systems; Legrain P et al.; Automated sequence technology has rendered functional biology amenable to genomic scale analysis . Among genome-wide exploratory approaches, the two-hybrid system in yeast (Y2H) has outranked other techniques because it is the system of choice to detect protein-protein interactions . Deciphering the cascade of binding events in a whole cell helps define signal transduction and metabolic pathways or enzymatic complexes . The function of proteins is eventually attributed through whole cell protein interaction maps where totally unknown proteins are partnered with fully annotated proteins belonging to the same functional category . Since its first description in the late 1980's, several versions of the Y2H have been developed in order to overcome the major limitations of the system, namely false positives and false negatives . Optimized versions have been recently applied at multi-molecular and genomic scale . These genome-wide surveys can be methodologically divided into two types of approaches: one either tests combinations of predefined polypeptides (the so-called matrix approach) using various short-cuts to speed up the process, or one screens with a given polypeptide (bait) for potential partners (preys) present in complex libraries of genomic or complementary DNA (library screening) . In the former strategy, one tests what one knows, for example pair-wise interactions between full-length open reading frames from recently sequenced and annotated genomes . Although based on a one-by-one scheme, this method is reported to be amenable to large-scale genomics thanks to multicloning strategies and to the use of small robotics workstations . In the latter, highly complex cDNA or genomic libraries of protein domains can be screened to saturation with high-throughput screening systems allowing the discovery of yet unidentified proteins . Both approaches have strengths and drawbacks that will be discussed here . None yields a full proteome-wide screening since certain proteins (e.g . some transcription factors) are not usable in Y2H . Novel two-hybrid assays have been recently described in bacteria . Applications of these time- and cost-effective assays to genomic screening will be discussed and compared to the Y2H technology.

Microbes Infect, 2000 Jul, 2(9), 1089 - 100
DNA polymorphism in the genus Brucella; Vizcaino N et al.; The genus Brucella has been described as consisting of six species, three of them including several biovars, which display a high degree of DNA homology by DNA-DNA hybridization . However, DNA polymorphism able to differentiate the six Brucella species and some of their biovars has been shown to exist . This work reviews the DNA variability in the genus Brucella and discusses the relationships between its members according to this genetic variability and a proposal for their evolution based on genetic diversity of the omp2 locus.

Dent Mater, 2000 Nov, 16(6), 432 - 40
Factors influencing pulpal response to cavity restorations; Camps J et al.; OBJECTIVES: The purposes of this retrospective work were: (1) to determine the relative importance of bacteria on cavity walls, remaining dentin thickness and post-operative time on pulpal inflammation after cavity restoration; (2) to compare the respective influences of bacterial microleakage and the restorative material itself on pulp reaction severity . METHODS: 317 class V cavities, in human bicuspids scheduled for extraction for orthodontic reasons were used for this study . Nine different materials were included . The severity of the pulpal reaction was ranked on hematoxylin/eosin stained sections according to FDI standards . The further parameters recorded were: (1) the presence or absence of bacteria on the cavity walls was noted on Brown and Brenn stained sections; (2) the remaining dentin thickness was measured and the teeth classified into three groups (< 500, 500-1000, > 1000 microns); and (3) the post-operative delay before extraction was recorded and classified as short time (< 5 weeks) or long time (> 5 weeks) . Three two-way analyses of variance (ANOVA) followed by Kruskall and Wallis tests evaluated the influence of the three parameters on pulpal reaction severity . The third ANOVA also compared pulpal reactions under the different materials when the teeth were pooled, on bacteria free teeth and on bacteria contaminated teeth . RESULTS: The first ANOVA ranked by decreasing order of importance: the presence of bacteria (p < 0.0001), the remaining dentin thickness (p = 0.02) and the post-operative delay (p = 0.04) . The second ANOVA showed no difference among the restorative materials when bacteria were present on the cavity walls . SIGNIFICANCE: The presence of bacteria on the cavity walls is the main factor influencing pulpal reaction under restorative materials, but does not account for 100% of the cases.

Front Biosci, 2000 Sep 01, 5, D796 - 812
Osmoadaptation and osmoregulation in archaea; Roberts MF; The response of archaea to changes in external NaCl is reviewed and compared to what is known about osmoadaptation and osmoregulation in bacteria and eukaryotes . Cells placed in altered external NaCl exhibit short term and long term responses . The earliest events are likely to be water movement through aquaporin-like channels (efflux if external NaCl has been increased, influx into the cell if the external NaCl has been decreased) and ion movement (e.g., K+ moving in the direction opposite to water flow) through channels sensitive to osmotic pressure . Accumulation of organic solutes, either by uptake from the medium or de novo synthesis, is triggered after these initial changes . Archaea have some unique organic solutes (osmolytes) that are not used by other organisms . These as well as other more common solutes have a role in stabilizing macromolecules from denaturation . Many osmolytes are distinguished by their stability in the cell and their lack of strong interactions with cellular components . A cell may respond by accumulating one or more temporary osmolytes, then over time readjust the intracellular solute distribution to what is optimal for cell growth under the new conditions . Coupled with the movement and accumulation of solutes is the induction of stress proteins (e.g., chaperonins) and, in some cases, transcriptional regulation of key enzymes . The response to NaCl stress of Methanococcus thermolithotrophicus is presented as an example of how one particular archaeon responds and adapts to altered osmotic pressure . Clearly, the detailed response of other archaea to osmotic stress will be needed in order to identify features (aside from some of the organic osmolytes) unique to the organisms in this kingdom.

Front Biosci, 2000 Sep 01, 5, D780 - 6
Stressors, stress and survival: overview; de Macario EC et al.; This overview introduces the contributions in this Special Issue with the aim of presenting an integrated picture of it . The contributions cover several important areas: protein stability and function under extreme conditions, osmotic stress and osmoadaptation, the structural features of the cell membrane and their possible significance with regard to heat stress, the molecular chaperone machine and multicellular structures as anti-stress mechanisms, peptidyl-prolyl cis-trans isomerases, proteases and the proteasome, and oxidative stress and the role of superoxide dismutase . These topics are briefly discussed to explain the basic concepts underpinning them, quoting for the most part introductory articles or reviews that might help the non-specialist to become familiar with the central themes of the Special Issue . As mentioned in the Preface every effort has been made to discuss the archaeal features within the context of other disciplines and biology in general, against the background of what is known for bacteria and eucarya . Hopefully, this approach will help the reader in understanding what is unique to the archaea, what is shared between them and the members of the other two phylogenetic domains, and how studies in archaea impact on other fields of science.

J Mol Biol, 2000 Sep 1, 301(5), 1163 - 78
Specificity in protein-protein interactions: the structural basis for dual recognition in endonuclease colicin-immunity protein complexes; Kuhlmann UC et al.; Bacteria producing endonuclease colicins are protected against their cytotoxic activity by virtue of a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease . DNase binding by the immunity protein occurs through a "dual recognition" mechanism in which conserved residues from helix III act as the binding-site anchor, while variable residues from helix II define specificity . We now report the 1.7 A crystal structure of the 24.5 kDa complex formed between the endonuclease domain of colicin E9 and its cognate immunity protein Im9, which provides a molecular rationale for this mechanism . Conserved residues of Im9 form a binding-energy hotspot through a combination of backbone hydrogen bonds to the endonuclease, many via buried solvent molecules, and hydrophobic interactions at the core of the interface, while the specificity-determining residues interact with corresponding specificity side-chains on the enzyme . Comparison between the present structure and that reported recently for the colicin E7 endonuclease domain in complex with Im7 highlights how specificity is achieved by very different interactions in the two complexes, predominantly hydrophobic in nature in the E9-Im9 complex but charged in the E7-Im7 complex . A key feature of both complexes is the contact between a conserved tyrosine residue from the immunity proteins (Im9 Tyr54) with a specificity residue on the endonuclease directing it toward the specificity sites of the immunity protein . Remarkably, this tyrosine residue and its neighbour (Im9 Tyr55) are the pivots of a 19 degrees rigid-body rotation that relates the positions of Im7 and Im9 in the two complexes . This rotation does not affect conserved immunity protein interactions with the endonuclease but results in different regions of the specificity helix being presented to the enzyme .

Eur J Intern Med, 2000 Aug, 11(4), 210 - 214
Musculoskeletal manifestations in inflammatory bowel disease; Salvarani C et al.; Musculoskeletal manifestations are the most common extra-intestinal complication of inflammatory bowel disease (IBD) . They are part of the clinical spectrum of spondylarthropathies and include different articular manifestations . In addition to axial symptoms, peripheral findings such as seronegative oligoarthritis, dactylitis, and enthesopathy commonly occur, sometimes representing the only manifestation . Wide ranges of prevalence have been reported, depending on the criteria used to define spondyloarthropathy and on the selection of patients . In an inceptional cohort of newly diagnosed IBD patients, we observed musculoskeletal manifestations in 30.7% of the patients . The clinician should, therefore, carefully evaluate any rheumatological findings in order to provide an accurate and early diagnosis, and to establish an adequate therapy . In this article, epidemiological, clinical, and diagnostic aspects are discussed . Furthermore, the contribution of intestinal bacteria and immunogenetic factors to the pathogenesis of arthritis is briefly reviewed . Finally, we summarize the available therapeutic options.

Annu Rev Biochem, 2000, 69, 617 - 50
Aminoacyl-tRNA synthesis; Ibba M et al.; Aminoacyl-tRNAs are substrates for translation and are pivotal in determining how the genetic code is interpreted as amino acids . The function of aminoacyl-tRNA synthesis is to precisely match amino acids with tRNAs containing the corresponding anticodon . This is primarily achieved by the direct attachment of an amino acid to the corresponding tRNA by an aminoacyl-tRNA synthetase, although intrinsic proofreading and extrinsic editing are also essential in several cases . Recent studies of aminoacyl-tRNA synthesis, mainly prompted by the advent of whole genome sequencing and the availability of a vast body of structural data, have led to an expanded and more detailed picture of how aminoacyl-tRNAs are synthesized . This article reviews current knowledge of the biochemical, structural, and evolutionary facets of aminoacyl-tRNA synthesis.

Annu Rev Biochem, 2000, 69, 277 - 302
Structural insights into microtubule function; Nogales E; Microtubules are polymers that are essential for, among other functions, cell transport and cell division in all eukaryotes . The regulation of the microtubule system includes transcription of different tubulin isotypes, folding of / inverted question mark-tubulin heterodimers, post-translation modification of tubulin, and nucleotide-based microtubule dynamics, as well as interaction with numerous microtubule-associated proteins that are themselves regulated . The result is the precise temporal and spatial pattern of microtubules that is observed throughout the cell cycle . The recent high-resolution analysis of the structure of tubulin and the microtubule has brought new insight to the study of microtubule function and regulation, as well as the mode of action of antimitotic drugs that disrupt normal microtubule behavior . The combination of structural, genetic, biochemical, and biophysical data should soon give us a fuller understanding of the exquisite details in the regulation of the microtubule cytoskeleton.

Appl Environ Microbiol, 2000 Sep, 66(9), 3924 - 30
Additional evidence that juvenile oyster disease is caused by a member of the Roseobacter group and colonization of nonaffected animals by Stappia stellulata-like strains; Boettcher KJ et al.; Juvenile oyster disease (JOD) causes significant annual mortalities of hatchery-produced Eastern oysters, Crassostrea virginica, cultured in the Northeast . We have reported that a novel species of the alpha-proteobacteria Roseobacter group (designated CVSP) was numerically dominant in JOD-affected animals sampled during the 1997 epizootic on the Damariscotta River, Maine . In this study we report the isolation of CVSP bacteria from JOD-affected oysters during three separate epizootics in 1998 . These bacteria were not detected in nonaffected oysters at the enzootic site, nor in animals raised at a JOD-free site . Animals raised at the JOD enzootic site that were unaffected by JOD were stably and persistently colonized by Stappia stellulata-like strains . These isolates (designated M1) inhibited the growth of CVSP bacteria in a disk-diffusion assay and thus may have prevented colonization of these animals by CVSP bacteria in situ . Laboratory-maintained C . virginica injected with CVSP bacteria experienced statistically significant elevated mortalities compared to controls, and CVSP bacteria were recovered from these animals during the mortality events . Together, these results provide additional evidence that CVSP bacteria are the etiological agent of JOD . Further, there are no other descriptions of specific marine alpha-proteobacteria that have been successfully cultivated from a defined animal host . Thus, this system presents an opportunity to investigate both bacterial and host factors involved in the establishment of such associations and the role of the invertebrate host in the ecology of these marine alpha-proteobacteria.

Appl Environ Microbiol, 2000 Sep, 66(9), 3823 - 7
Ferrous iron-dependent volatilization of mercury by the plasma membrane of Thiobacillus ferrooxidans; Iwahori K et al.; Of 100 strains of iron-oxidizing bacteria isolated, Thiobacillus ferrooxidans SUG 2-2 was the most resistant to mercury toxicity and could grow in an Fe(2+) medium (pH 2.5) supplemented with 6 microM Hg(2+) . In contrast, T . ferrooxidans AP19-3, a mercury-sensitive T . ferrooxidans strain, could not grow with 0.7 microM Hg(2+) . When incubated for 3 h in a salt solution (pH 2.5) with 0.7 microM Hg(2+), resting cells of resistant and sensitive strains volatilized approximately 20 and 1.7%, respectively, of the total mercury added . The amount of mercury volatilized by resistant cells, but not by sensitive cells, increased to 62% when Fe(2+) was added . The optimum pH and temperature for mercury volatilization activity were 2.3 and 30 degrees C, respectively . Sodium cyanide, sodium molybdate, sodium tungstate, and silver nitrate strongly inhibited the Fe(2+)-dependent mercury volatilization activity of T . ferrooxidans . When incubated in a salt solution (pH 3.8) with 0.7 microM Hg(2+) and 1 mM Fe(2+), plasma membranes prepared from resistant cells volatilized 48% of the total mercury added after 5 days of incubation . However, the membrane did not have mercury reductase activity with NADPH as an electron donor . Fe(2+)-dependent mercury volatilization activity was not observed with plasma membranes pretreated with 2 mM sodium cyanide . Rusticyanin from resistant cells activated iron oxidation activity of the plasma membrane and activated the Fe(2+)-dependent mercury volatilization activity of the plasma membrane.

Appl Environ Microbiol, 2000 Sep, 66(9), 3814 - 22
Molecular analysis of diazotroph diversity in the rhizosphere of the smooth cordgrass, Spartina alterniflora; Lovell CR et al.; N(2) fixation by diazotrophic bacteria associated with the roots of the smooth cordgrass, Spartina alterniflora, is an important source of new nitrogen in many salt marsh ecosystems . However, the diversity and phylogenetic affiliations of these rhizosphere diazotrophs are unknown . Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified nifH sequence segments was used in previous studies to examine the stability and dynamics of the Spartina rhizosphere diazotroph assemblages in the North Inlet salt marsh, near Georgetown, S.C . In this study, plugs were taken from gel bands from representative DGGE gels, the nifH amplimers were recovered and cloned, and their sequences were determined . A total of 59 sequences were recovered, and the amino acid sequences predicted from them were aligned with sequences from known and unknown diazotrophs in order to determine the types of organisms present in the Spartina rhizosphere . We recovered numerous sequences from diazotrophs in the gamma subdivision of the division Proteobacteria (gamma-Proteobacteria) and from various anaerobic diazotrophs . Diazotrophs in the alpha-Proteobacteria were poorly represented . None of the Spartina rhizosphere DGGE band sequences were identical to any known or previously recovered environmental nifH sequences . The Spartina rhizosphere diazotroph assemblage is very diverse and apparently consists mainly of unknown organisms.

Appl Environ Microbiol, 2000 Sep, 66(9), 3674 - 9
Response of atmospheric methane consumption by maine forest soils to exogenous aluminum salts; Nanba K et al.; Atmospheric methane consumption by Maine forest soils was inhibited by additions of environmentally relevant levels of aluminum . Aluminum chloride was more inhibitory than nitrate or sulfate salts, but its effect was comparable to that of a chelated form of aluminum . Inhibition could be explained in part by the lower soil pH values which resulted from aluminum addition . However, significantly greater inhibition by aluminum than by mineral acids at equivalent soil pH values indicated that inhibition also resulted from direct effects of aluminum per se . The extent of inhibition by exogenous aluminum increased with increasing methane concentration for soils incubated in vitro . At methane concentrations of >10 ppm, inhibition could be observed when aluminum chloride was added at concentrations as low as 10 nmol g (fresh weight) of soil(-1) . These results suggest that widespread acidification of soils and aluminum mobilization due to acid precipitation may exacerbate inhibition of atmospheric methane consumption due to changes in other parameters and increase the contribution of methane to global warming.

Cell, 2000 Aug 18, 102(4), 463 - 73
Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis; Ikura T et al.; It is well known that histone acetylases are important chromatin modifiers and that they play a central role in chromatin transcription . Here, we present evidence for novel roles of histone acetylases . The TIP60 histone acetylase purifies as a multimeric protein complex . Besides histone acetylase activity on chromatin, the TIP60 complex possesses ATPase, DNA helicase, and structural DNA binding activities . Ectopic expression of mutated TIP60 lacking histone acetylase activity results in cells with defective double-strand DNA break repair . Importantly, the resulting cells lose their apoptotic competence, suggesting a defect in the cells' ability to signal the existence of DNA damage to the apoptotic machinery . These results indicate that the histone acetylase TIP60-containing complex plays a role in DNA repair and apoptosis.

Med Pregl, 2000 Mar-Apr, 53(3-4), 164 - 8
{Clinical significance of analysis of immunoglobulin A levels in saliva}; Bokor-Bratic M; SALIVA COLLECTION: Whole saliva is a product of secretion of 3 major glands (parotid, submandibular, sublingual) and many minor glands (labial, buccal, palatal) . Unstimulated saliva is usually obtained as the patient spits out every 60 sec . or by forward bended head the patient allows saliva to drip off the lower lip into a cylinder . By collection of saliva in the tube the flow rate per unit time can be measured . When volume measurement is not required the saliva can be collected on cotton rolls, gauze or filter paper . For evaluating salivary gland function or when large volumes of saliva are required for analytic purposes, stimulated whole saliva is used . Method of collection is the same as for unstimulated saliva . The usual masticatory stimuli are paraffin wax or a washed rubber band . A standard gustatory stimulus is obtained by 2% citric acid applied directly to the tongue every 15 to 60 sec . Parotid saliva can be collected by aspiration from the duct opening with a micropipette . Parotid saliva is best collected with Lashley's vacuum chamber . Submandibular and sublingual saliva can be collected by cannulation of the duct with micropipette, but in practice this is both uncomfortable for the patients and technically difficult since the duct orifice is mobile and has a strong sphincter . Because of that, alginate and silicone impression material is used for retention of the collecting tube . As alternative and simple technique is to block off secretion from the parotid glands with absorbent swabs and collect mixed submandibular and sublingual saliva by pipette from the floor of the mouth . Saliva from labial and palatal glands can be collected by filter paper disc or disc of other synthetic materials . SALIVARY IMMUNOGLOBULIN A: The most significant characteristics of the salivary immunoglobulin system are quantitative domination of immunoglobulin A, local synthesis and specific structure . Immunofluorescence studies have shown that immunoglobulin A is produced by plasma cells locally in the salivary glands . There is still little convincing evidence for the origin of predominantly immunoglobulin A secreting plasma cells in salivary glands . DETECTION OF IMMUNOGLOBULIN A IN SALIVA: Radial immunodiffusion (RID) was the most applicable method for detecting salivary immunoglobulin A . However, there are more sensitive and automatic methods such as nephelometry and ELISA . A standard level of immunoglobulin in saliva is still in question since the concentration varies in relation to origin of saliva, method of collection and stimulation of secretion (Table 1) . PERIODONTAL DISEASE: Studies of the salivary immunoglobulin A in patients with periodontal disease and healthy persons showed that there are differences which can be used in detection of high-risk groups and individuals . If the bacterial adherence to the mucosa is a prerequisite for bacterial evolution in subgingival or any other region of the oral cavity respectively introduction in periodontitis development, than it is to be presumed that the basic function of salivary immunoglobulin A is inhibition of bacterial adherence rather than antigens destruction . Several bacterial species frequently isolated from the oral cavity of patients with periodontitis have been identified as producers of IgA protease . These enzymes cleave serum IgA and secretory IgA equally well . Additionally, most of the IgA proteases studied have cleaved the A1 and A2 subclass . Several studies have demonstrated that cleavage of human IgA occurs in vivo, resulting in generation of intact Fab alpha and (Fc alpha)2 fragment . Moreover, when bacteria are exposed to Fab alpha fragments released from IgA after cleavage by IgA protease, their surface antigens are likely to be occupied by Fab alpha fragments . These Fab alpha fragments left on the bacterial surface may mediate adhesion . Together, these results indicate that IgA proteases, by promoting adherence, contribute the pathogenic potential of bacteria in the oral c

Mol Med, 2000 Mar, 6(3), 155 - 64
Structural basis of Bloom syndrome (BS) causing mutations in the BLM helicase domain; Rong SB et al.; BACKGROUND: Bloom syndrome (BS) is characterized by mutations within the BLM gene . The Bloom syndrome protein (BLM) has similarity to the RecQ subfamily of DNA helicases, which contain seven conserved helicase domains and share significant sequence and structural similarity with the Rep and PcrA DNA helicases . We modeled the three-dimensional structure of the BLM helicase domain to analyze the structural basis of BS-causing mutations . MATERIALS AND METHODS: The sequence alignment was performed for RecQ DNA helicases and Rep and PcrA helicases . The crystal structure of PcrA helicase (PDB entry 3PJR) was used as the template for modeling the BLM helicase domain . The model was used to infer the function of BLM and to analyze the effect of the mutations . RESULTS: The structural model with good stereochemistry of the BLM helicase domain contains two subdomains, 1A and 2A . The electrostatic potential of the model is highly negative over most of the surface, except for the cleft between subdomains 1A and 2A which is similar to the template protein . The ATP-binding site is located inside the model between subdomains 1A and 2A; whereas, the DNA-binding region is situated at the surface cleft, with positive potential between 1A and 2A . CONCLUSIONS: The three-dimensional structure of the BLM helicase domain was modeled and applied to interpret BS-causing mutations . The mutation I841T is likely to weaken DNA binding, while the mutations C891R, C901Y, and Q672R presumably disturb the ATP binding . In addition, other critical positions are discussed.

Jpn J Cancer Res, 2000 Aug, 91(8), 769 - 73
Marked enhancement by fish meal of Helicobacter pylori-induced gastritis in Mongolian gerbils; Tanigawa T et al.; In a search for dietary factors influencing Helicobacter pylori-induced gastritis, the effects of fish meal in the diet were examined in Mongolian gerbils . When a conventional diet containing 10% fish meal was given to Mongolian gerbils for 4 weeks after inoculation of H . pylori, edematous thickening with severe neutrophil and mononuclear cell infiltration in both the mucosa and submucosa was observed in the glandular stomach of 19 out of the 20 animals, and hemorrhagic spots were evident in 11 cases . These gastric lesions were enhanced by a 20% fish meal supplement, and edema and hemorrhage in the gastric mucosa were observed in 19 and 17 out of 20 animals, respectively . Although almost the same levels of viable bacteria were detected independent of the diet, edema and hemorrhage were seen in only 2 and 1 of 20 gerbils fed a diet containing 10% casein, instead of 10% fish meal, respectively . Neither edema nor hemorrhage was observed in 10% beef diet animals . These results suggest that fish meal contains factors which greatly enhance H . pylori-induced gastritis in Mongolian gerbils . Since the incidences of gastritis and gastric cancer are very high throughout the world, it is very important to identify these gastritis-enhancing factors.

J Neurosci, 2000 Sep 1, 20(17), 6333 - 9
Cbln3, a novel member of the precerebellin family that binds specifically to Cbln1; Pang Z et al.; Precerebellin (Cbln1) is the precursor of the brain-specific hexadecapeptide cerebellin . Although cerebellin has properties of a conventional neuropeptide, its function is controversial because Cbln1 has structural features characteristic of circulating atypical collagens . Cbln1 is related to the three subunits of the complement C1q complex . Therefore, we hypothesized that Cbln1 participated in analogous heteromeric complexes with precerebellin-related proteins . Using LexA-Cbln1 as bait in a yeast two-hybrid screen, we isolated a cDNA encoding a novel Cbln1-related protein, designated Cbln3 . The gene encoding cbln3 had the same intron-exon structure as cbln1 but mapped to a different mouse chromosome (14) . The deduced amino acid sequence of Cbln3 was 55% identical to Cbln1 and also contained a C1q signature domain and signal sequence for secretion . In addition to binding avidly to Cbln3, Cbln1 also formed homomeric complexes . In contrast, Cbln3 homomeric association was weak . These interactions exhibited specificity because C1qB bound to neither Cbln1 nor Cbln3 . Like cbln1, cbln3 was expressed in the cerebellum and dorsal cochlear nucleus in which it was detected in granule neurons . Because Cbln1 and Cbln3 are coexpressed in the brain and interact avidly, they may function as a secreted heteromeric complex in vivo.

J Mol Biol, 2000 Sep 8, 302(1), 49 - 63
Characterization of a Tn5 pre-cleavage synaptic complex; Bhasin A et al.; Protein catalyzed DNA rearrangements typically require assembly of complex nucleoprotein structures . In transposition and integration reactions, these structures, termed synaptic complexes, are mandatory for catalysis . We characterize the Tn5 pre-cleavage synaptic complex, the simplest transposition complex described to date . We identified this complex by gel retardation assay using short, linear fragments and have shown that it contains a dimer of transposase, two DNA molecules, and is competent for DNA cleavage in the presence of Mg(2+) . We also used hydroxyl radical footprinting and interference techniques to delineate the protein-DNA contacts made in the Tn5 synaptic and monomer complexes . All positions (except position 1) of the end sequence are contacted by transposase in the synaptic complex . We have determined that positions 2-5 of the end sequence are specifically required for synaptic complex formation as they are not required for monomer complex formation . In addition, in the synaptic complex, there is a strong, local distortion centered around position 1 which likely facilitates cleavage .

Anal Biochem, 2000 Sep 10, 284(2), 201 - 16
Single-step gas chromatography-mass spectrometry analysis of glycolipid constituents as heptafluorobutyrate derivatives with a special reference to the lipid portion; Pons A et al.; In a previous work (Zanetta et al . Glycobiology 9, 255-266 (1999)), it was reported that all constituents of gangliosides could be obtained as heptafluorobutyrate derivatives after methanolysis in a single gas chromatography analysis . This report demonstrates that gas chromatography coupled with mass spectrometry in the electron impact mode allows identification and quantification of long-chain bases and fatty acids without interference from monosaccharides . On the basis of ions specific for families and for individual compounds, sphingosines, sphinganines, and phytosphingosines (including ramified, unsaturated, hydroxylated, and etherified compounds) can be identified . Fatty acid methyl esters, including linear, ramified, unsaturated, and hydroxylated species, are identified and quantified in the same way . Possible extensions of this method to the fatty moiety of other lipids (alkylacylglycerol and dimethyl acetal) are discussed .

Proc Natl Acad Sci U S A, 2000 Aug 29, 97(18), 10126 - 31
Virus neutralization by germ-line vs . hypermutated antibodies; Kalinke U et al.; Mice infected with vesicular stomatitis virus (VSV), a cytopathic virus closely related to rabies virus, mount a virus-neutralizing antibody response protecting against lethal disease . VSVneutralizing monoclonal IgGs isolated from primary immune responses were devoid of somatic mutations, whereas most secondary and all hyperimmune response IgGs tested were hypermutated . A comparative analysis of recombinant single-chain antibody fragments (scFv-Ckappa) revealed that even the germ-line precursor of one hypermutated antibody bound and neutralized VSV . Four somatic amino acid substitutions in V(H) increased by 300-fold the binding strength of monovalent scFv-Ckappa . The multivalent binding avidity of germ-line scFv-Ckappa was increased by more than 10-fold compared with the monovalent binding strength . In contrast, hypermutated scFv-Ckappa did not show such avidity effects . Thus the overall binding difference between the germ-line and the hypermutated VSV-neutralizing antibody was only 10- to 15-fold . This may explain why primary germ-line antibodies and secondary hypermutated antibodies directed against pathogens such as viruses and bacteria expressing repetitive antibody determinants show rather similar binding qualities, whereas monovalently binding hapten-specific antibodies can show "affinity maturation" effects of up to 1000-fold.

Nature, 2000 Aug 17, 406(6797), 782 - 7
Toll-like receptors in the induction of the innate immune response; Aderem A et al.; The innate immune response is the first line of defence against infectious disease . The principal challenge for the host is to detect the pathogen and mount a rapid defensive response . A group of proteins that comprise the Toll or Toll-like family of receptors perform this role in vertebrate and invertebrate organisms . This reflects a remarkable conservation of function and it is therefore not surprising that studies of the mechanism by which they act has revealed new and important insights into host defence.

Biochem Soc Trans, 2000, 28(4), 283 - 96
The chemical and biological versatility of riboflavin; Massey V; Since their discovery and chemical characterization in the 1930s, flavins have been recognized as being capable of both one- and two-electron transfer processes, and as playing a pivotal role in coupling the two-electron oxidation of most organic substrates to the one-electron transfers of the respiratory chain . In addition, they are now known as versatile compounds that can function as electrophiles and nucleophiles, with covalent intermediates of flavin and substrate frequently being involved in catalysis . Flavins are thought to contribute to oxidative stress through their ability to produce superoxide, but at the same time flavins are frequently involved in the reduction of hydroperoxides, products of oxygen-derived radical reactions . Flavoproteins play an important role in soil detoxification processes via the hydroxylation of many aromatic compounds, and a simple flavoprotein in liver microsomes catalyses many reactions similar to those carried out by cytochrome P450 enzymes . Flavins are involved in the production of light in bioluminescent bacteria, and are intimately connected with light-initiated reactions such as plant phototropism and nucleic acid repair processes . Recent reports also link them to programmed cell death . The chemical versatility of flavoproteins is clearly controlled by specific interactions with the proteins with which they are bound . One of the main thrusts of current research is to try to define the nature of these interactions, and to understand in chemical terms the various steps involved in catalysis by flavoprotein enzymes.

Blood, 2000 Sep 1, 96(5), 1836 - 43
Activation of macrophage cytostatic effector mechanisms during acute graft-versus-host disease: release of intracellular iron and nitric oxide-mediated cytostasis; Nestel FP et al.; During acute graft-versus-host disease (GVHD) the activation of macrophages (Mphi) is mediated by 2 signals, interferon (IFN)-gamma and bacteria-derived lipopolysaccharide (LPS) . A cascade of inflammatory responses that includes the release of mediators of tissue injury follows Mphi activation . Among the tissues characteristically targeted during acute GVHD are epithelial tissues of the skin and gastrointestinal tract that normally undergo continuous proliferation and are therefore sensitive to cytostatic processes . We have investigated whether Mphi can mediate cytostatic mechanisms capable of interrupting cell proliferation during acute GVHD . GVHD was induced in nonirradiated C57BL/6XAF(1) (B6AF(1)) mice by the injection of 60 x 10(6) (acute GVHD) or 30 x 10(6) (nonlethal GVHD) C57BL/6 (B6) lymphoid cells . Mphi from animals undergoing acute GVHD could be triggered by normally insignificant quantities of LPS to mediate a cytostatic effect on target cells, resulting in the complete shutdown of cellular proliferation . The same amounts of LPS had no effect on Mphi from normal or syngeneically transplanted animals . Mphi mediated the release of significant quantities of intracellular iron from target cells undergoing cytostasis . Reversal of cytostasis occurred following inhibition of nitric oxide (NO) production by N(G)-monomethyl-L-arginine (NMMA) . Production of NO by LPS-triggered Mphi reflected the severity of GVHD . NO release increased significantly during acute GVHD but was only transiently increased during nonlethal GVHD . The results provide evidence that, as a result of activation during acute GVHD, Mphi produce NO and induce the release of iron from target cells, resulting in a potent cytostatic effect that inhibits cellular proliferation . (Blood . 2000;96:1836-1843)

Dig Dis Sci, 2000 Jul, 45(7), 1330 - 6
Selection of lower cutoff point of {13C}urea breath test is helpful to monitor H . pylori eradication after proton pump inhibitor-based triple therapy; Sheu BS et al.; Our objectives were to test the efficacy of {13C} urea breath test (UBT) for H . pylori infection in patients before and after proton pump inhibitor (PPI) based triple therapy, and thus to trace the optimal cutoff value of UBT to monitor H . pylori eradication; and to analyze the histologic bacterial density and distribution of H . pylori in patients with equivocal UBT . A total of 441 dyspeptic cases patients enrolled and completed the study design, including 120 noninfected and 321 H . pylori-infected patients . All 441 cases had received the same protocol of UBT . in which the baseline and 15-min gas samples after ingestion of 100 mg 13C-labeled urea were analyzed for excess 13CO2/12CO2 ratio (ECR) . In addition, a first endoscopy was performed in each patient to evaluate the initial status of H . pylori by culture and histology . Of the 321 H . pylori-infected patients, 286 received a second endoscopy and a second UBT six weeks after completing any one of four regimens of the PPI-based triple therapy to document the success of H . pylori eradication . During both sessions of endoscopy, topographic gastric biopsies for histology were sampled to evaluate the distribution and density of H . pylori . Based on the diagnostic standard by culture and histology, the sensitivity and specificity of the first UBT achieved most optimally was 97.5% and 96.7%, respectively, by setting the cutoff point of ECR at 4.0 . In contrast, using the same cutoff point of 4.0, the sensitivity and specificity of the second UBT in patients after therapy achieved just 80% and 97.6%, respectively . By applying cutoff points downward of 4.0, 3.5, 3, and 2.5 for the second UBT, the sensitivity was elevated to 80%, 82.8%, 88.6%, and 94.3%, respectively, while the specificity was preserved at more than 95.2% . The overall eradication rate of H . pylori was 87.8% (251/286) . Seven of 35 patients with failure of therapy had equivocal ECR at the second UBT (range 2-5), and this accounted for the false negative result . All seven patients had low bacterial densities, and three patients had bacteria distributed only in high body near the cardia . In conclusion, selection of a lower cutoff value of ECR at 2.5 is helpful to maintain the diagnostic accuracy of UBT for monitoring the H . pylori eradication . The equivocal ECR of UBT after therapy would be related to the low bacterial load and its distribution.

Dig Dis Sci, 2000 Jul, 45(7), 1255 - 9
Carbohydrate malabsorption syndromes and early signs of mental depression in females; Ledochowski M et al.; Fructose and lactose malabsorption are characterized by impaired duodenal fructose transport or by the deficiency of mucosal lactase, respectively . As a consequence, the nonabsorbed saccharides reach the colon, where they are broken down by bacteria to short fatty acids, CO2, and H2 . Bloating, cramps, osmotic diarrhea, and other symptoms of irritable bowel syndrome are the consequence and can be seen in about 50% of carbohydrate malabsorbers . We have previously shown that fructose as well as lactose malabsorption were associated with signs of mental depression . It was therefore of interest to investigate possible interactions between fructose and lactose malabsorption and their influence on the development of signs of depression . In all, 111 otherwise healthy volunteers (81 females and 30 males) with gastrointestinal complaints were analyzed by measuring breath H2 concentrations after an oral dose of 50 g lactose and of 50 g fructose one week apart . They were classified as normals, isolated fructose malabsorbers, isolated lactose malabsorbers, and combined fructose/lactose malabsorbers . All patients filled out a Beck's depression inventory-questionnaire . Twenty-five individuals (22.5%) were neither fructose nor lactose malabsorbers (group 1), 69 (62.2%) were only fructose malabsorbers (group 2), 4 (3.6%) were only lactose malabsorbers (group 3), and 13 (11.7%) presented with fructose and lactose malabsorption together (group 4) . Isolated fructose malabsorption and combined fructose/lactose malabsorption was significantly associated with a higher Beck's depression score . Further analysis of the data show that this association was strong in females (P < 0.01), but there was no such association between carbohydrate malabsorption and early signs of depression in males . In conclusion, the data confirm that fructose malabsorption may play a role in the development of mental depression in females and additional lactose malabsorption seems to further increase the risk for development of mental depression.

Infection, 2000 Jul-Aug, 28(4), 219 - 22
Tropheryma whippelii DNA in saliva of patients without Whipple's disease; Dutly F et al.; Tropheryma whippelii is the causative agent of Whipple's disease, a difficult to diagnose systemic illness . Amplification of part of its 16S ribosomal RNA gene(s) has become a standard diagnostic method because of increased sensitivity as compared to classical histopathological analysis . Recently, we demonstrated the presence of T . whippelii DNA by PCR in duodenal biopsies and/or gastric juice of a considerable fraction of individuals without clinical signs of Whipple's disease . In this follow-up study, saliva and dental plaques of the same patients were screened for the presence of T . whippelii DNA . Six out of the 14 previously PCR-positive persons but none of the 17 controls had T . whippelii DNA in their saliva . Our results suggest that Whipple bacteria are ubiquitous environmental or commensal organisms causing Whipple's disease only in a particular subset of individuals, possibly those with an as yet uncharacterized immunological defect.

Eur J Cell Biol, 2000 Jul, 79(7), 513 - 9
In vitro production of superoxide and nitric oxide (as nitrite and nitrate) by Mytilus galloprovincialis haemocytes upon incubation with PMA or laminarin or during yeast phagocytosis; Arumugam M et al.; The phagocytic process is one of the most important elements of the self-defence system in mammals as well as in molluscs . In mammalian phagocytes, superoxide participates in the innate defence system by combining with nitric oxide to generate peroxynitrite, a strong oxidant that possesses highly cytotoxic properties against bacteria . To evidence a role of nitric oxide in the self-defence system of the marine bivalve Mytilus galloprovincialis similar to the role observed in the mammalian defence system, we measured the generation of superoxide and nitrite/nitrate (the stable end products of nitric oxide) upon in vitro stimulation of M . galloprovincialis haemocytes with PMA, laminarin, LPS and by phagocytosis of Saccharomyces cerevisiae (yeast cells) . We show that stimulation with PMA, laminarin and yeast cell phagocytosis promotes superoxide and nitrite/nitrate generation from M . galloprovincialis haemocytes . Inhibitors of NADPH oxidase and inhibitors of NO synthase decreased the nitrite/nitrate levels generated by M . galloprovincialis haemocytes showing that both NADPH oxidase and NO synthase pathways are involved in the self-defence system of M . galloprovincialis.

J Dent, 2000 Sep, 28(7), 515 - 28
An ultrastructural study of the application of dentine adhesives to acid-conditioned sclerotic dentine; Kwong SM et al.; OBJECTIVE: This in vitro study examined the ultrastructure of resin-infiltrated sclerotic dentine following the application of a self-etching primer, with or without the adjunctive use of phosphoric acid pre-conditioning . MATERIALS AND METHODS: Non-carious, natural cervical sclerotic lesions were hand-cleaned with a slurry of pumice and chlorhexidine and bonded without further cavity preparation . One group was bonded using Clearfil Liner Bond 2V (Kuraray Co . Ltd, Osaka, Japan) alone . Specimens from the other group were pre-conditioned with K-etchant (40% phosphoric acid gel, Kuraray) prior to the application of the same self-etching primer . Artificially prepared wedge-shaped lesions were also made in sound bicuspids, bonded using the two methods, and used as controls for the two groups . For SEM examination, each specimen was cryofractured into two halves through a pre-formed slit made on the lingual surface, after the respective conditioning treatment . Different locations within the lesions were examined after rinsing of the phosphoric acid/self-etching primer and specimen dehydration . For TEM investigation, specimens were bonded with the adhesive . Both demineralised and undemineralised ultrathin sections were prepared from the occlusal, gingival and deepest part of the wedge-shaped bonded lesions following specimen fixation, dehydration and resin embedding . RESULTS: A hypermineralised surface layer was present on the surface of etched sclerotic dentine . This layer was thicker in the deepest part of the natural lesions, where bacterial colonisation of the lesion surface was also apparent . Both treatment protocols were unable to effectively dissolve sclerotic casts that occluded the dentinal tubules . Depending upon the thickness of the surface layers at different locations in the natural lesion, self-etching primer treatment alone resulted in reduction of the thickness of the authentic hybrid layer (i.e . hybridised intertubular dentine) . This was also true of phosphoric acid pre-conditioning along the deepest part of the natural lesions . Within this region, intertubular dentine completely devoid of an authentic hybrid layer could be seen in both treatment groups . Resin tags were also sparsely observed in such regions . CONCLUSIONS: Adhesive strategies that rely mostly on micromechanical retention alone may be compromised by the sporadic absence of the hybrid layer and resin tags in sclerotic dentine . Based on the ultrastructural features presented, it is further speculated that adaptive strategies such as removal of the surface layers and extended etching time may not be completely effective in improving bonding efficacy in highly sclerotic dentine . Interdisciplinary research should be continued to develop alternative procedures for bonding resins equally well to sound and sclerotic dentine.

Nucleosides Nucleotides Nucleic Acids, 2000 Apr, 19(4), 705 - 22
Pyrazole related nucleosides 5 . Synthesis and biological activity of 2'-deoxy-2',3'-dideoxy- and acyclo-analogues of 4-iodo-1-beta-D-ribofuranosyl-3-carboxymethyl pyrazole (IPCAR); Manfredini S et al.; Continuing our studies on the structure-activity relationships (SAR) of 4-iodo-1-beta-D-ribofuranosyl-3-carboxymethyl pyrazole (IPCAR), the ribofuranosyl moiety has been substituted with acyclic chains, namely 1-{(2-hydroxyethoxy)methyl}- and 1-{(1,3-dihydroxy-2-propoxy)methyl}-pyrazole derivatives (4, 5 and 8, 9 respectively), with the 2'-deoxy-beta-D-ribofuranosyl group (12 and 13) and finally with the 2',3'-dideoxy-D-glycero-pentofuranosyl-moiety (16 and 17) . None of the new compounds display any interesting biological activity.

J Periodontol, 2000 Jul, 71(7), 1167 - 73
Induction of experimental periodontitis in mice with Porphyromonas gingivalis-adhered ligatures; Kimura S et al.; BACKGROUND: Little information is available on the colonization of periodontopathic bacteria and alveolar bone loss in a mouse system, because of the difficulty in establishing bacteria in the oral cavity . The aim of this study was to establish experimental periodontitis in mice by applying a Porphyromonas gingivalis-adhered ligature onto the molars . METHODS: Specific pathogen-free C3H/HeN mice were divided into 3 groups: 80 infected, 80 sham-infected, and 48 non-treated control mice . Sterile silk ligatures were preincubated with and without P . gingivalis 381 in vitro and then physically tied on the right maxillary first molar of infected and sham-infected mice, respectively . Ten mice from the infected and sham-infected groups and 6 from the control group were sacrificed at 2-week intervals for up to 15 weeks after infection . RESULTS: Plaque samples were collected at the time of sacrifice and alveolar bone loss was examined . The results indicated that P . gingivalis was recovered from the plaque samples in 95% of the infected mice after 1 week and then gradually dropped to 58% after 15 weeks of infection, whereas P . gingivalis was not isolated in either sham-infected or control mice throughout the experimental period . The infected mice showed significant P . gingivalis-induced bone loss at the sites where the ligature was tied weeks 13 to 15 . A linear regression analysis revealed a significant positive correlation between the number of P . gingivalis recovered and alveolar bone loss at 15 weeks after infection (P <0.01) . CONCLUSIONS: The use of a P . gingivalis-adhered ligature supported a long-lasting infection of P . gingivalis in mice, resulting in P . gingivalis-induced alveolar bone breakdown.

Scand J Infect Dis, 2000, 32(4), 407 - 10
Risk and case fatality rate of meningitis in patients with liver cirrhosis; Molle I et al.; Bacterial infections are major causes of morbidity and death in patients with liver cirrhosis . The risk of bacterial meningitis in these patients is unknown, however . In this study on a nation-wide cohort of 22,743 patients with liver cirrhosis in Denmark an incidence rate of bacterial meningitis of 54.4 per 100,000 was found {95% confidence interval (CI) 40.3-71.9} . The highest incidence rate was found in patients with alcoholic cirrhosis, 65.3 per 100,000 person-years (95% CI 46.2-89.6), compared with 34.6 per 100,000 person-years (95% CI 17.3-61.9) in patients with non-alcoholic cirrhosis . The 30-d case fatality rate was 53.1% (95% CI 38.3-67.5), and high age and alcoholic cirrhosis were associated with the highest case fatality rates . The main bacterial pathogens were pneumococci and unspecified bacteria . These findings suggest that patients with liver cirrhosis are at increased risk of bacterial meningitis with a poor prognosis.

Adv Exp Med Biol, 2000, 480, 259 - 77
Immune surveillance of mammary tissue by phagocytic cells; Paape MJ et al.; The leukocytes in milk consist of lymphocytes, neutrophil polymorphonuclear leukocytes (PMN) and macrophages . Lymphocytes together with antigen-presenting cells function in the generation of an effective immune response . Lymphocytes can be divided into two distinct subsets, T- and B-lymphocytes, that differ in function and protein products . The professional phagocytic cells of the bovine mammary gland are PMN and macrophages . In the normal mammary gland macrophages are the predominate cells which act as sentinels to invading mastitis causing pathogens . Once the invaders are detected, macrophages release chemical messengers called chemoattractants that cause the directed migration of PMN into the infection . Migration of neutrophils into mammary tissue provides the first immunological line of defense against bacteria that penetrate the physical barrier of the teat canal . However, their presence is like a double-edged sword . While the PMN are phagocytosing and destroying the invading pathogens, they inadvertently release chemicals which induces swelling of secretory epithelium cytoplasm, sloughing of secretory cells, and decreased secretory activity . Permanent scarring will result in a loss of milk production . Resident and newly migrated macrophages help reduce the damage to the epithelium by phagocytosing PMN that undergo programmed cell death through a process called apoptosis . Specific ligands on the neutrophil surface are required for directed migration and phagocytosis . In response to infection, freshly migrated leukocytes express greater numbers of cell surface receptors for immunoglobulins and complement and are more phagocytic than their counterparts in blood . However, phagocytic activity rapidly decreases with continued exposure to inhibitory factors such as milk fat globules and casein in mammary secretions . Compensatory hypertrophy in non-mastitic quarters partially compensates for lost milk production in diseased quarters . Advances in molecular biology are making available the tools, techniques, and products to study and modulate host-parasite interactions . For example the cloning and expression of proteins that bind endotoxin may provide ways of reducing damaging effects of endotoxin during acute coliform mastitis . The successful formation of bifunctional monoclonal antibodies for the targeted lysis of mastitis causing bacteria represents a new line of therapeutics for the control of mastitis in dairy cows.

Adv Exp Med Biol, 2000, 480, 247 - 58
Cells and cytokines in inflammatory secretions of bovine mammary gland; Riollet C et al.; In response to invading bacteria, the mammary gland is protected by a variety of defence mechanisms, which can be separated into two distinct categories: innate immunity and specific immunity . Milk somatic cells consist of several cell types, including neutrophils, macrophages, lymphocytes and a smaller percentage of epithelial cells . In the healthy lactating mammary gland, macrophages are the predominant cell type whereas neutrophils are the major cell population during early inflammation . Following a bacteria invasion, neutrophil recruitment is elicited by inflammatory mediators that are produced in the infected gland by cells, possibly macrophages, activated by bacteria phagocytosis or responding to bacterial toxins or metabolites . Several cytokines, including interleukin- (IL-) 1 beta, IL-6, IL-8, tumour necrosis factor- (TNF-) alpha and interferon- (IFN-) gamma are known to be important to elicit the acute phase response and allow the accumulation of leukocytes at the site of infection . In addition to their role in early non-specific defences, macrophages also play a key role in the specific immune system, as antigen processing and presenting cells for the T cells . Few lymphocytes are found in milk of healthy glands where the predominant phenotype is CD8+ T cells . During the inflammatory reaction, T cells are recruited in milk and CD4+ cells become the predominant phenotype . The understanding of the specific and nonspecific immune mechanisms involved in the mammary gland defence against invading bacteria may lead to the development of new vaccines and to the use of cytokines to design immunomodulatory strategies for the control of bovine mastitis.

J Microbiol Methods, 2000 Aug, 41(3), 227 - 34
Electrospray ionization/mass spectrometry compatible reversed-phase separation of phospholipids: piperidine as a post column modifier for negative ion detection; Lytle CA et al.; An electrospray ionization (ESI) compatible separation of phospholipids (PL), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC), was performed on a C18 column by reversed phase High Performance Liquid Chromatography (HPLC) with minimal ESI suppression . The mobile phase, used isocratically, consisted of methanol and water . ESI was used to efficiently transfer the ions present in solution to the gas phase for mass spectrometric (MS) detection . Formation of negative ions was reinforced by incorporating piperidine post column . Limits of detection (LOD) and limits of quantitation (LOQ) were experimentally determined to be 20 and 60 fmol/microl, respectively, when acquiring data in the selected ion monitoring (SIM) mode monitoring three ions with a single quadrupole MS . When acquiring data from m/z 110-900 in the scanning mode, the LOD and LOQ were experimentally determined to be 1 pmol/microl and 3 pmol/microl . When acquiring product ion spectra for m/z 747, the LOD and LOQ were experimentally determined to be 446 attomol/microl and 1.3 fmol/microl, respectively.

Mol Cell Biol, 2000 Sep, 20(18), 6646 - 58
Meiotic segregation, synapsis, and recombination checkpoint functions require physical interaction between the chromosomal proteins Red1p and Hop1p; Woltering D et al.; In yeast, HOP1 and RED1 are required during meiosis for proper chromosome segregation and the consequent formation of viable spores . Mutations in either HOP1 or RED1 create unique as well as overlapping phenotypes, indicating that the two proteins act alone as well as in concert with each other . To understand which meiotic processes specifically require Red1p-Hop1p hetero-oligomers, a novel genetic screen was used to identify a single-point mutation of RED1, red1-K348E, that separates Hop1p binding from Red1p homo-oligomerization . The Red1-K348E protein is stable, phosphorylated in a manner equivalent to Red1p, and undergoes efficient homo-oligomerization; however, its ability to interact with Hop1p both by two-hybrid and coimmunoprecipitation assays is greatly reduced . Overexpression of HOP1 specifically suppresses red1-K348E, supporting the idea that the only defect in the protein is a reduced affinity for Hop1p . red1-K348E mutants exhibit reduced levels of crossing over and spore viability and fail to undergo chromosome synapsis, thereby implicating a role for Red1p-Hop1p hetero-oligomers in these processes . Furthermore, red1-K348E suppresses the sae2/com1 defects in meiotic progression and sporulation, indicating a previously unknown role for HOP1 in the meiotic recombination checkpoint.

Genome Res, 2000 Aug, 10(8), 1172 - 84
Eukaryote-specific domains in translation initiation factors: implications for translation regulation and evolution of the translation system; Aravind L et al.; Computational analysis of sequences of proteins involved in translation initiation in eukaryotes reveals a number of specific domains that are not represented in bacteria or archaea . Most of these eukaryote-specific domains are known or predicted to possess an alpha-helical structure, which suggests that such domains are easier to invent in the course of evolution than are domains of other structural classes . A previously undetected, conserved region predicted to form an alpha-helical domain is delineated in the initiation factor eIF4G, in Nonsense-mediated mRNA decay 2 protein (NMD2/UPF2), in the nuclear cap-binding CBP80, and in other, poorly characterized proteins, which is named the NIC (NMD2, eIF4G, CBP80) domain . Biochemical and mutagenesis data on NIC-containing proteins indicate that this predicted domain is one of the central adapters in the regulation of mRNA processing, translation, and degradation . It is demonstrated that, in the course of eukaryotic evolution, initiation factor eIF4G, of which NIC is the core, conserved portion, has accreted several additional, distinct predicted domains such as MI (MA-3 and eIF4G ) and W2, which probably was accompanied by acquisition of new regulatory interactions.

Genome Res, 2000 Aug, 10(8), 1108 - 14
GT repeats are associated with recombination on human chromosome 22; Majewski J et al.; The rate of meiotic recombination is not a constant function of physical distance across chromosomes . This variation is manifested by recombination hot spots and cold spots, observed in all organisms ranging from bacteria to humans . It is generally believed that factors such as primary and secondary DNA sequence, as well as chromatin structure and associated proteins, influence the frequency of recombination within a specific region . Several such factors, for example repetitive sequences, gene promoters, or regions with the ability to adopt Z-DNA conformation, have been hypothesized to enhance recombination . However, apart from specific examples, no general trends of association between recombination rates and particular DNA sequence motifs have been reported . In this paper, we analyze the complete sequence data from human chromosome 22 and compare microsatellite repeat distributions with mitotic recombination patterns available from earlier genetic studies . We show significant correlation between long tandem GT repeats, which are known to form Z-DNA and interact with several components of the recombination machinery, and recombination hot spots on human chromosome 22.

J Math Biol, 2000 Jul, 41(1), 1 - 23
Life and death near a windy oasis; Dahman KA et al.; We propose a simple experiment to study delocalization and extinction in inhomogeneous biological systems . The nonlinear steady state for, say, a bacteria colony living on and near a patch of nutrient or favorable illumination ("oasis") in the presence of a drift term ("wind") is computed . The bacteria, described by a simple generalization of the Fisher equation, diffuse, divide A --> A + A, die A --> 0, and annihilate A + A --> 0 . At high wind velocities all bacteria are blown into an unfavorable region ("desert"), and the colony dies out . At low velocity a steady state concentration survives near the oasis . In between these two regimes there is a critical velocity at which bacteria first survive . If the "desert" supports a small nonzero population, this extinction transition is replaced by a delocalization transition with increasing velocity . Predictions for the behavior as a function of wind velocity are made for one and two dimensions.

J Agric Food Chem, 2000 Aug, 48(8), 3470 - 5
Shelf-life prediction of processed milk by solid-phase microextraction, mass spectrometry, and multivariate analysis; Marsili RT; A technique based on solid-phase microextraction, mass spectrometry, and multivariate analysis (SPME-MS-MVA) was used to predict the shelf life of pasteurized and homogenized reduced-fat milk and whole-fat chocolate milk sampled over a 7 month period . Using SPME-MS-MVA, which is essentially a mass spectrometry-based electronic-nose instrument, volatile bacterial metabolites were extracted from milk with SPME (Carboxen-PDMS) and injected into a GC capillary column at elevated temperature . Mass fragmentation profiles from the unresolved milk volatile components were normalized to the intensity of a chlorobenzene internal standard mass peak (m/z 112) and subjected to MVA . Prediction models based on partial least-squares regression of mass intensity lists were able to predict the shelf life of samples to approximately +/-1 day, with correlation coefficients greater than 0.98 for the two types of milk samples . Using principal component analysis techniques, the procedure was also useful for classifying samples that were rendered unpalatable by nonmicrobial sources (contamination by copper and sanitizer) as well as by bacteria.

Vet Rec, 2000 Jul 22, 147(4), 93 - 7
Pheasant ataxia: a new condition in pheasant poults; de B Welchman D et al.; Between 1995 and 1997 a neurological condition in pheasant poults from 24 sites in England and Scotland was investigated . Affected birds showed varying degrees of ataxia and incoordinated movements and, in severe cases, recumbency, but generally remained alert with their heads held upright . The condition characteristically affected poults from seven weeks of age and the incidence on any one site was low . No significant bacteria were isolated consistently from brain tissue . The condition was characterised histologically by a non-suppurative meningoencephalitis, in which lesions were found predominantly in the cerebellum in 61 of 81 samples examined (75.3 per cent) . A non-suppurative myelitis was recorded in 16 of 20 spinal cords examined . No lesions were recorded in peripheral neural tissue and lesions were rare in other tissues . The condition appeared not to have been recorded previously in pheasants . A viral aetiology was suspected but Newcastle disease virus was not involved.

Mod Pathol, 2000 Aug, 13(8), 909 - 13
Necrotizing sarcoid granulomatosis mimicking an intracranial neoplasm: clinicopathologic features and review of the literature; Strickland-Marmol LB et al.; We present a unique case of biopsy-proven necrotizing sarcoidosis involving the central nervous system (CNS) in a 52-year-old woman . The patient presented with a 3-month history of left-sided headache and sharp, shooting pains on the left side of her face . She also has a previous history of sarcoidosis, histopathologically confirmed on parotid gland biopsy 24 years before . Imaging studies of the present lesion revealed a 1.8 x 1.4-cm mass in the left temporal lobe with signal intensity suggestive of meningioma or low-grade glial neoplasm . Surgical resection was initiated, and intraoperative consultation with frozen sections revealed granulomata . The lesion was biopsied, and surgical intervention was terminated . Permanent sections failed to reveal bacteria, mycobacteria, fungi, or foreign bodies . A diagnosis of necrotizing neurosarcoidosis was rendered . The patient was administered steroid therapy and clinically responded favorably . At the most recent follow-up almost 2 years later, there was no evidence of recurrence or progression . Necrotizing sarcoidosis has been reported most commonly in the lungs and rarely in other organ systems . We report the first histologically proven case involving the CNS as well as a rare example of sarcoidosis and necrotizing sarcoid granulomatosis in the same patient . Sarcoidosis and its necrotizing variant should be considered in the differential diagnosis of a granulomatous mass lesion involving the CNS, particularly in the context of a history of systemic disease.

J Biol Chem, 2000 Nov 10, 275(45), 34873 - 80
FlaA1, a new bifunctional UDP-GlcNAc C6 Dehydratase/ C4 reductase from Helicobacter pylori; Creuzenet C et al.; FlaA1 is a small soluble protein of unknown function in Helicobacter pylori . It has homologues that are essential for the virulence of numerous medically relevant bacteria . FlaA1 was overexpressed as a histidine-tagged protein and purified to homogeneity by nickel chelation and cation exchange chromatography . Spectrophotometric assays, capillary electrophoresis, and mass spectrometry analyses showed that FlaA1 is a novel bifunctional C(6) dehydratase/C(4) reductase specific for UDP-GlcNAc . It converts UDP-GlcNAc into a UDP-4-keto-6-methyl-GlcNAc intermediate, which is stereospecifically reduced into UDP-QuiNAc . Substrate conversions as high as 80% were obtained at equilibrium . The K(m) and V(max) for UDP-GlcNAc were 159 microm and 65 pmol/min, respectively . No exogenous cofactor was required to obtain full activity of FlaA1 . Additional NADH was only used with poor efficiency for the reduction step . The biochemical characterization of FlaA1 is important for the elucidation of biosynthetic pathways that lead to the formation of 2,6-deoxysugars in medically relevant bacteria . It establishes unambiguously the first step of the pathway and provides the means of preparing the substrate UDP-QuiNAc, which is necessary for the study of downstream enzymes.

Nucleic Acids Res, 2000 Sep 1, 28(17), 3310 - 5
RNA-binding characteristics of the chloroplast S1-like ribosomal protein CS1; Shteiman-Kotler A et al.; The chloroplast ribosomal protein CS1, the homolog of the bacterial ribosomal protein S1, is believed to be involved in the process of ribosome binding to mRNA during translation . Since translation control is an important step in chloroplast gene expression, and in order to study initiation complex formation, we studied the RNA-binding properties of CS1 protein . We found that most of the CS1 protein in spinach chloroplast co-purified with the 30S ribosomal subunit . The relative binding affinity of RNA to CS1 was determined using the UV-crosslinking competition assay . CS1 protein binds the ribohomopolymer poly(U) with a relatively high binding affinity . Very low binding affinities were obtained for the other ribohomopolymers, poly(G), poly(A) and poly(C) . In addition, no specific binding of CS1, either in the 30S complex or as a recombinant purified protein, was obtained to the 5'-untranslated region of the mRNA in comparison to the other parts . RNA-binding experiments, in which the N- and C-termini of the protein were analyzed, revealed that the RNA-binding site is located in the C-terminus half of the protein . These results suggest that CS1 does not direct the 30S complex to the initiation codon of the translation site by specific binding to the 5'-untranslated region . In bacteria, specific binding is derived by base pairing between 16S rRNA and the Shine-Dalagarno sequences . In the chloroplast, nuclear encoded and gene-specific translation factors may be involved in the determination of specific binding of the 30S subunit to the initiator codon.

Baillieres Best Pract Res Clin Rheumatol, 1999 Mar, 13(1), 179 - 92
Septic and aseptic arthritis: a continuum?
Taylor-Robinson D, Keat A.
This chapter considers the likelihood that a wide spectrum of infection-provoked arthritis exists, ranging from overt sepsis to apparently aseptic chronic arthritis in which very small numbers of causal bacteria can be detected only by using highly sensitive techniques . It asks whether joints are, as conventionally held, normally devoid of micro-organisms and how to judge the significance of bacteria detected within apparently sterile joints . Through a consideration of known septic, probably infective and apparently aseptic forms of arthritis, a set of criteria for attributing causality to putative arthritogenic micro-organisms is proposed .

Br J Pharmacol, 2000 Aug, 130(8), 1949 - 59
Intestinal anti-inflammatory activity of UR-12746, a novel 5-ASA conjugate, on acute and chronic experimental colitis in the rat; Galvez J et al.; The present study was undertaken to investigate the intestinal anti-inflammatory effects of UR-12746 on the acute and chronic stages of a trinitrobenzene sulphonic acid (TNBS) experimental model of inflammatory bowel disease (IBD) in the rat . UR-12746 is a novel, locally-acting compound which combines, through an azo bond, 5-aminosalicylic (5-ASA) and UR-12715, a potent platelet activating factor (PAF)-antagonist . UR-12746 oral pretreatment of colitic rats (50 and 100 mg kg(-1)) reduced acute colonic damage when evaluated 2 days after colonic insult . Postreatment for 4 weeks with UR-12746 (50 and 100 mg kg(-1)) resulted in a faster recovery of the damaged colonic mucosa, which was macroscopically significant from the third week . The intestinal anti-inflammatory effect of UR-12746 was associated with a decrease in leukocyte infiltration in the colonic mucosa, which was evidenced both biochemically, by a reduction in myeloperoxidase activity, and histologically, by a lower leukocyte count after morphometric analysis . This effect was higher than that seen with sulphasalazine, when assayed at the same doses and in the same experimental conditions . Several mechanisms can be involved in the beneficial effects showed by UR-12746: inhibition of leukotriene B(4) synthesis in the inflamed colon, improvement of the altered colonic oxidative status, and reduction of colonic interleukin-1beta production . The results suggest that the intestinal anti-inflammatory activity of UR-12746 can be attributed to the additive effects exerted by 5-ASA and UR-12715, the PAF antagonist compound, that are released in the colonic lumen after reduction of the azo bond by the intestinal bacteria.

Anal Chem, 2000 Aug 1, 72(15), 3497 - 503
Synthesis and application of submicrometer fluorescence sensing particles for lysosomal pH measurements in murine macrophages; Ji J et al.; Phagocytosis of bioparticles such as bacteria and viruses by macrophages is a critical component of the immune response against infections . In this paper we describe the synthesis of submicrometer fluorescent particles with pH sensing capability . The particles are used to measure the pH and to monitor the effect of chloroquine, an antimalarial drug, on the pH in the lysosome, the cellular organelle involved in the phagocytosis process . The synthesis of the pH sensing particles is realized by the covalent attachment of amine reactive forms of Oregon Green (pH sensitive dye) and Texas Red (pH insensitive dye) to the surface of amino-modified submicrometer polystyrene particles . The particles are absorbed by J774 Murine Macrophages through phagocytosis and directed to lysosomes . Despite the high lysosomal levels of digestive enzymes and acidity, the absorbed particles remain stable for 12 h in the cells when they are stored in a PBS buffer solution at pH 7.4 . The pH dynamic range of the sensing particles is between pH 4.5 and 7 with a sensitivity of 0.1 pH units . Exposure of the cells to chloroquine increases the lysosomal pH from 4.8 to 6.5 . The effect is concentration-dependent.

Jpn Circ J, 2000 Aug, 64(8), 619 - 22
Unclassified connective tissue disease presenting as cardiac tamponade: a case report; Oishi Y et al.; This report describes a case of cardiac tamponade as the initial manifestation of unclassified connective tissue disease (UCTD) . A 68-year-old Japanese woman was admitted to hospital because of dyspnea and edema . She had undergone a radical left mastectomy for the treatment of breast cancer 18 years before . On admission, bilateral leg edema, hepatomegaly, and a paradoxical pulse were noted on physical examination . The erythrocyte sedimentation rate was elevated and the C-reactive protein was 2.8 mg/dl . Antinuclear antibodies and anti-SS-A/Ro antibodies were present . The scl-70 and anticentromere antibodies were elevated . Chest radiography showed cardiomegaly . Echocardiography revealed a large pericardial effusion, but the pericardial fluid did not contain malignant cells or bacteria . She did not meet the diagnostic criteria for any known connective tissue diseases, so was diagnosed with cardiac tamponade due to UCTD . Prednisolone (30 mg/day) was administered, which resulted in a gradual resolution of the pericardial effusion . Although connective tissue diseases are known to cause pericardial effusion, cardiac tamponade as the initial manifestation of the disease in the absence of other symptoms is quite rare.

Jpn J Pharmacol, 2000 Jul, 83(3), 167 - 74
DNA vaccines; Koide Y et al.; DNA vaccination or genetic immunization is a rapidly developing technology that offers new approaches for the prevention and therapy of disease . Regarding the inoculation method of DNA vaccine, we recommend the gene gun delivery system, which is a highly reliable method compared to intramuscular inoculation . DNA vaccines could have potential advantages over other types of vaccines in that these vaccines can induce strong cellular immune responses, cytotoxic T lymphocytes and type 1 helper T cells, without resorting to live organisms or complicated protein formulation . The cellular immune responses are especially required for the protection against infections with intracellular pathogens such as viruses and Mycobacterium tuberculosis and protection against cancers, suggesting that they seem to be suitable targets of DNA vaccines . We describe here that their application to bacterial infections requires optimization of codon usage in the DNA vaccines to the host animal to improve translational efficiencies of the bacteria genes . DNA vaccines for a variety of pathogens and cancers have now entered phase I/II human clinical trials.

Am J Vet Res, 2000 Aug, 61(8), 928 - 30
Prevalence of papillomatous digital dermatitis among culled adult cattle in the southeastern United States; Brown CC et al.; OBJECTIVE: To determine prevalence of papillomatous digital dermatitis (PDD) among culled adult dairy and beef cattle in the southeastern United States . ANIMALS: 815 cattle examined during 4 visits to a slaughterhouse . PROCEDURE: The left hind foot of each animal was examined for gross lesions of PDD . Breed and sex of the animals were recorded . Lesions were examined histologically for pathologic changes and bacteria, especially spirochetes . RESULTS: 22 of 76 (29%) dairy cattle and 29 of 739 (4%) beef cattle had gross lesions of PDD . Detection of lesions was not associated with sex of dairy cattle, but male beef cattle were more likely to have lesions of PDD than were female beef cattle . Histologically, acute and chronic lesions were seen; the most severe changes were localized to the stratum corneum . Spirochetes were seen in lesions from 31 of 51 (61%) cattle . CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that PDD is common among culled adult cattle . Prevalence was higher in culled adult dairy cattle than in culled adult beef cattle.

Genesis, 2000 Jul, 27(3), 95 - 8
FACS for the isolation of individual cells from transgenic mice harboring a fluorescent protein reporter; Hadjantonakis AK et al.; Flow cytometry is extensively used for the isolation of discreet populations of cells from complex pools . The advent of autofluorescent (AFP) reporters such as wild type Green Fluorescent Protein (wtGFP) (Chalfie et al., 1994) and its variants, including enhanced green fluorescent protein (EGFP) and enhanced yellow fluorescent protein (EYFP) (Cormack et al., 1996), as vital reporters opens up the possibility of sorting live reporter-expressing cells . Moreover the use of these reporters in transgenics (Okabe et al., 1997) or mice carrying homologously targeted loci (Godwin et al., 1998) should enable the direct isolation of reporter-expressing cells from any desired lineage . Here we have assessed this approach in transgenic mice . ES cell-mediated transgenesis was used for generating a line of mice that express an autofluorescent EYFP reporter in the heart and part of the neural tube at midgestation . Pools of fluorescent cells harboring and expressing the EYFP reporter were isolated from defined regions of embryos and their origin confirmed by assaying the expression of domain-defined marker genes . Such a tool should prove useful for gaining access to any given lineage that can be fluorescent protein reporter tagged.

J Invest Dermatol, 2000 Aug, 115(2), 183 - 92
Topical ALA-photodynamic therapy for the treatment of acne vulgaris; Hongcharu W et al.; Topical aminolevulinic acid is converted into a potent photosensitizer, protoporphyrin, in human hair follicles and sebaceous glands . Photodynamic therapy with topical aminolevulinic acid was tested for the treatment of acne vulgaris, in an open-label prospective human study . Each of 22 subjects with acne on the back was treated in four sites with aminolevulinic acid plus red light, aminolevulinic acid alone, light alone, and untreated control . Half of the subjects were treated once; half were treated four times . Twenty percent topical aminolevulinic acid was applied with 3 h occlusion, and 150 J per cm2 broad-band light (550-700 nm) was given . Sebum excretion rate and auto-fluorescence from follicular bacteria were measured before, and 2, 3, 10, and 20 wk after, treatment . Histologic changes and protoporphyrin synthesis in pilosebaceous units were observed from skin biopsies . Aminolevulinic acid plus red light caused a transient acne-like folliculitis . Sebum excretion was eliminated for several weeks, and decreased for 20 wk after photodynamic therapy; multiple treatments caused greater suppression of sebum . Bacterial porphyrin fluorescence was also suppressed by photodynamic therapy . On histology, sebaceous glands showed acute damage and were smaller 20 wk after photodynamic therapy . There was clinical and statistically significant clearance of inflammatory acne by aminolevulinic acid plus red light, for at least 20 wk after multiple treatments and 10 wk after a single treatment . Transient hyperpigmentation, superficial exfoliation, and crusting were observed, which cleared without scarring . Topical aminolevulinic acid plus red light is an effective treatment of acne vulgaris, associated with significant side-effects . Aminolevulinic acid plus red light causes phototoxicity to sebaceous follicles, prolonged suppression of sebaceous gland function, and apparent decrease in follicular bacteria after photodynamic therapy . Potentially, aminolevulinic acid plus red light may be useful for some patients with acne.

Eur J Biochem, 2000 Sep, 267(17), 5561 - 70
Biosynthesis of the cyanobacterial reserve polymer multi-L-arginyl-poly-L-aspartic acid (cyanophycin): mechanism of the cyanophycin synthetase reaction studied with synthetic primers; Berg H et al.; Biosynthesis of the cyanobacterial nitrogen reserve cyanophycin (multi-L-arginyl-poly-L-aspartic acid) is catalysed by cyanophycin synthetase, an enzyme that consists of a single kind of polypeptide . Efficient synthesis of the polymer requires ATP, the constituent amino acids aspartic acid and arginine, and a primer like cyanophycin . Using synthetic peptide primers, the course of the biosynthetic reaction was studied . The following results were obtained: (a) sequence analysis suggests that cyanophycin synthetase has two ATP-binding sites and hence probably two active sites; (b) the enzyme catalyses the formation of cyanophycin-like polymers of 25-30 kDa apparent molecular mass in vitro; (c) primers are elongated at their C-terminus; (d) the constituent amino acids are incorporated stepwise, in the order aspartic acid followed by arginine, into the growing polymer . A mechanism for the cyanophycin synthetase reaction is proposed; (e) the specificity of the enzyme for its amino-acid substrates was also studied . Glutamic acid cannot replace aspartic acid as the acidic amino acid, whereas lysine can replace arginine but is incorporated into cyanophycin at a much lower rate.

J Infect Dis, 2000 Sep, 182(3), 895 - 901 Epub 2000 Aug 15.
Evaluation of human antimycobacterial immunity using recombinant reporter mycobacteria; Kampmann B et al.; A novel in vitro whole blood model was developed to study human antimycobacterial immunity . Recombinant reporter mycobacteria were used to enumerate the bacteria, and interactions between host immune cells and mycobacteria were studied using whole blood rather than cell fractions . The ability of healthy tuberculin-positive and tuberculin-negative individuals to restrict mycobacterial growth was compared . Growth of luminescent mycobacteria was significantly lower in blood samples of tuberculin-positive individuals than in blood samples of tuberculin-negative individuals (P=.005) . Restricted mycobacterial growth was associated with significantly higher production of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma (P=.01 and.004, respectively) . Inhibition of the TNF-alpha and IFN-gamma response pathways by neutralizing monoclonal antibodies increased mycobacterial growth in whole blood . This model is the first functional assay in which individual variations in cell-mediated immunity are shown to correlate with differences in ability to control mycobacterial growth . It provides a new tool for studying human mycobactericidal mechanisms and, potentially, for the evaluation of improved vaccines.

Amino Acids, 2000, 18(4), 339 - 52
Determination of L- and D-amino acids in foodstuffs by coupling of high-performance liquid chromatography with enzyme reactors; Voss K et al.; A technique is described for the enantiomeric determination of L- and D-amino acids . It works on the principle that the separation efficiency of high-performance liquid chromatography is coupled with the specificity of enzymes and the sensitivity of electrochemical detection . After separation on a lithium cation-exchange column the amino acids are converted into keto acids and hydrogen peroxide under catalyzation of L- or D-amino acid oxidase . Hydrogen peroxide is detected amperometrically . The method has been tested by the analysis of beer, port, sherry, wine and fruit juice . A main emphasis was put onto the determination of D-alanine which can serve as an indicator for bacterial contamination . It is shown that a coupling of HPLC with enzyme reactors is a suitable technique for the rapid detection of this marker.

Cell Mol Life Sci, 2000 Jan 20, 57(1), 106 - 27
Radical catalysis of B12 enzymes: structure, mechanism, inactivation, and reactivation of diol and glycerol dehydratases; Toraya T; Enzymatic radical catalysis is defined as a mechanism of catalysis by which enzymes catalyze chemically difficult reactions by utilizing the high reactivity of free radicals . Adenosylcobalamin (coenzyme B12) serves as a cofactor for enzymatic radical reactions . The recent structural analysis of adenosylcobalamin-dependent diol dehydratase revealed that the substrate 1,2-propanediol and an essential potassium ion are located inside a (beta/alpha)8 barrel . Two hydroxyl groups of the substrate coordinate directly to the potassium ion which binds to the negatively charged inner part of the cavity . Cobalamin bound in the base-on mode covers the cavity to isolate the active site from solvent . Based on the three-dimensional structure and theoretical calculations, a new mechanism for diol dehydratase is proposed in which the potassium ion plays a direct role in the catalysis . The mechanisms for generation of a catalytic radical by homolysis of the coenzyme Co-C bond and for protection of radical intermediates from undesired side reactions during catalysis are discussed based on the structure . The reactivating factors for diol and glycerol dehydratases have been identified . These factors are a new type of molecular chaperone which participate in reactivation of the inactivated holoenzymes by mediating ATP-dependent exchange of the modified coenzyme for free intact coenzyme.

J Biotechnol, 2000 Jul 14, 80(3), 203 - 15
Analysis and description of the evolution of alginate immobilised cells systems; Laca A et al.; Different immobilised cells models, including very simple ones, can be useful in the fitting of experimental results . However, goodness or the ability to extrapolate results needs to be in accordance with basic observations and these will also suggest models to be proposed . In this paper, observations of calcium alginate/bacteria systems are used to show the ability of basic models to fit classic observations, as well as how new observations, in this case from electronic microscopy, oblige us to think about more complex mechanisms and mathematical treatments . Nevertheless it is not only important to discuss the model type, but also the type of kinetics assumed in the interior of the beads, as well as the internal structure, the boundary conditions related to bead shredding and cell escape and finally, geometrical effects.

Paediatr Perinat Epidemiol, 2000 Jul, 14(3), 248 - 56
Childhood brain tumours and exposure to animals and farm life: a review; Yeni-Komshian H et al.; Brain tumours are the most common solid tumour in children . However, little is known about their aetiology, and only a small percentage of cases can be attributed to established risk factors . Exposure to farm animals and pets have been considered as possible risk factors for childhood brain tumour (CBT) development for several reasons . Numerous factors associated with farm life, including bacteria, pesticides, solvents and some animal oncogenic viruses, have been found to induce brain tumour formation in animals . Some studies have found viral gene sequences in human brain tumours . Epidemiological studies of brain tumours in adults have reported an increased risk among veterinarians and farmers . In this review, data are examined from seven case-control studies published between 1979 and 1998 that considered a possible relationship between fetal or childhood exposure to farm animals or pets and CBT . Five of the seven studies examined childhood farm residence or exposure of mother or child to farm animals and, of these five, four reported elevated risk for CBT with odds ratios (OR) ranging from 0.9 to 2.5 for maternal exposures and from 0.6 to 6.7 for children's exposures . Later studies that were larger subsequently examined histological type and reported excess risk for primitive neuroectodermal tumours (PNETs) with farm residence prenatally (OR = 3.7, CI = 0.8, 24) or in childhood (OR = 5.0, CI = 1.1, 4.7) . Increased risk of PNET was also associated with maternal exposure to pigs (OR = 12, CI = .1, 47) or poultry (OR = 4.0, CI = 1.2, 13) . The results of these studies showed few other consistent relationships between farm life or farm animals and CBT.

Bull Entomol Res, 2000 Feb, 90(1), 9 - 21
Prospects for the biological control of subterranean termites (Isoptera: rhinotermitidae), with special reference to Coptotermes formosanus; Culliney TW et al.; Costs associated with subterranean termite damage and control are estimated to approach $2 billion annually in the United States alone . The Formosan subterranean termite, Coptotermes formosanus Shiraki, is one of the more economically important subterranean species . In recent years, the shortcomings associated with conventional chemical control methods have prompted policymakers and scientists to evaluate the potential for biological control of subterranean termites (C . formosanus in particular), that is, to determine the potential for natural enemies - predators, parasitoids and pathogens - to suppress termite populations . Ants are the greatest predators of termites, and may have a considerable local impact on termite populations in some areas of the world . A few parasitoids of termites are known, but their potential for regulating termite populations seems negligible . Characteristics of the colony, such as a protected, underground location (and, for the C . formosanus nest, its modular and dispersed nature), are likely to limit the impact predators and parasitoids have on subterranean termites . Thus, there seems little potential for use of these agents for subterranean termite control . For various reasons, pathogenic organisms, such as viruses, bacteria, Protozoa, nematodes and most fungi, have shown little promise for use in biological termite control . However, research suggests that strains of two well-studied, endoparasitic fungi, Beauveria bassiana and Metarhizium anisopliae, when employed in baiting schemes, may offer the potential for at least some measure of subterranean termite control, although their successful use is compromised by a number of inherent biological limitations and logistical problems that have yet to be solved . Although not strictly in the realm of classical biological control, recent studies suggest that natural products, such as ant semiochemicals and fungal metabolites (siderophores), or their synthetic analogues, eventually might find a use in termite control programmes as repellents or insecticides in wood treatments or soil applications if stable formulations can be developed.

Plant Cell, 2000 Aug, 12(8), 1441 - 53
The Chlamydomonas reinhardtii Nar1 gene encodes a chloroplast membrane protein involved in nitrite transport; Rexach J et al.; A key step for nitrate assimilation in photosynthetic eukaryotes occurs within chloroplasts, where nitrite is reduced to ammonium, which is incorporated into carbon skeletons . The Nar1 gene from Chlamydomonas reinhardtii is clustered with five other genes for nitrate assimilation, all of them regulated by nitrate . Sequence analysis of genomic DNA and cDNA of Nar1 and comparative studies of strains having or lacking Nar1 have been performed . The deduced amino acid sequence indicates that Nar1 encodes a chloroplast membrane protein with substantial identity to putative formate and nitrite transporters in bacteria . Use of antibodies against NAR1 has corroborated its location in the plastidic membrane . Characterization of strains having or lacking this gene suggests that NAR1 is involved in nitrite transport in plastids, which is critical for cell survival under limiting nitrate conditions, and controls the amount of nitrate incorporated by the cells under limiting CO(2) conditions.

J Biol Chem, 2000 Nov 17, 275(46), 36350 - 7
DIK, a novel protein kinase that interacts with protein kinase Cdelta . Cloning, characterization, and gene analysis; Bhr C et al.; A novel serine/threonine kinase, termed DIK, was cloned using the yeast two-hybrid system to screen a cDNA library from the human keratinocyte cell line HaCaT with the catalytic domain of rat protein kinase Cdelta (PKCdelta(cat)) cDNA as bait . The predicted 784-amino acid polypeptide with a calculated molecular mass of 86 kDa contains a catalytic kinase domain and a putative regulatory domain with ankyrin-like repeats and a nuclear localization signal . Expression of DIK at the mRNA and protein level could be demonstrated in several cell lines . The dik gene is located on chromosome 21q22.3 and possesses 8 exons and 7 introns . DIK was synthesized in an in vitro transcription/translation system and expressed as recombinant protein in bacteria, HEK, COS-7, and baculovirus-infected insect cells . In the in vitro system and in cells, but not in bacteria, various post-translationally modified forms of DIK were produced . DIK was shown to exhibit protein kinase activity toward autophosphorylation and substrate phosphorylation . The interaction of PKCdelta(cat) and PKCdelta with DIK was confirmed by coimmunoprecipitation of the proteins from HEK cells transiently transfected with PKCdelta(cat) or PKCdelta and DIK expression constructs.

Infect Immun, 2000 Sep, 68(9), 5321 - 8
Chronic Helicobacter pylori infection induces an apoptosis-resistant phenotype associated with decreased expression of p27(kip1); Shirin H et al.; Helicobacter pylori infection is associated with the development of gastric cancer . In short-term coculture with AGS gastric cells, H . pylori inhibits cell cycle progression and induces dose-dependent apoptosis . Based on the concept that an imbalance between proliferation and apoptosis may contribute to the emergence of gastric cancer, we chronically exposed AGS cells to H . pylori as a model of chronic exposure in humans . The AGS derivatives selected by this process were stably resistant not only to H . pylori-induced apoptosis but also to apoptosis induced by other enteric bacteria and by several toxic agents including radiation and cancer chemotherapy . Like the parental AGS cells, the derivatives underwent G(1)/S-phase cell cycle inhibition in response to H . pylori . The AGS derivatives displayed a marked decrease in cellular levels of the cell cycle control protein p27(kip1) . We found a similar decrease in epithelial cell p27(kip1) expression in gastric biopsy specimens from H . pylori-infected patients . These findings are consistent with observations that link decreases in the p27(kip1) level to increased susceptibility to cancer in mice with p27(kip1) deleted and to a poor prognosis of gastric cancer in humans . This is the first demonstration that bacterial infection can lead to apoptosis resistance and to cross-resistance to other inducers of apoptosis such as bacteria, chemotherapeutic agents, and radiation . The development of apoptosis resistance and downmodulation of p27(kip1) may contribute to the increased risk for gastric cancer observed in humans chronically exposed to H . pylori.

Infect Immun, 2000 Sep, 68(9), 5314 - 20
Impaired control of Brucella melitensis infection in Rag1-deficient mice; Izadjoo MJ et al.; After intranasal inoculation, Brucella melitensis chronically infects the mononuclear phagocyte system in BALB/c mice, but it causes no apparent illness . Adaptive immunity, which can be transferred by either T cells or antibody from immune to naive animals, confers resistance to challenge infection . The role of innate, non-B-, non-T-cell-mediated immunity in control of murine brucellosis, however, is unknown . In the present study, we documented that BALB/c and C57BL/6 mice had a similar course of infection after intranasal administration of 16M, validating the usefulness of the model in the latter mouse strain . We then compared the course of infection in Rag1 knockout mice (C57BL/6 background) (referred to here as RAG-1 mice) which have no B or T cells as a consequence of deletion of Rag1 (recombination-activating gene 1), with infection in normal C57BL/6 animals after intranasal administration of B . melitensis 16M . C57BL/6 mice cleared brucellae from their lungs by 8 to 12 weeks and controlled infection in the liver and spleen at a low level . In contrast, RAG-1 mice failed to reduce the number of bacteria in any of these organs . From 1 to 4 weeks after inoculation, the number of splenic bacteria increased from 2 to 4.5 logs and remained at that level . In contrast to the consistently high numbers of brucellae observed in the spleens, the number of bacteria rose in the livers sampled for up to 20 weeks . Immunohistologic examination at 8 weeks after infection disclosed foci of persistent pneumonia and large amounts of Brucella antigen in macrophages in lung, liver, and spleen in RAG-1, but not C57BL/6, mice . These studies indicate that T- and B-cell-independent immunity can control Brucella infection at a high level in the murine spleen, but not in the liver . Immunity mediated by T and/or B cells is required for clearance of bacteria from spleen and lung and for control of bacterial replication in the liver.

Infect Immun, 2000 Sep, 68(9), 5183 - 9
Shiga toxin-induced tumor necrosis factor alpha expression: requirement for toxin enzymatic activity and monocyte protein kinase C and protein tyrosine kinases; Foster GH et al.; Infections with Shiga toxin (Stx)-producing bacteria cause bloody diarrhea which may progress to life-threatening complications, including acute renal failure and neurological abnormalities . The precise mechanism of disease progression is unclear, although evidence suggests that the localized production of the host proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-1 may exacerbate toxin-mediated vascular damage . Purified Stxs have been demonstrated to elicit proinflammatory cytokine synthesis from human peripheral blood mononuclear cells and monocytic cell lines in vitro . To understand toxin-monocyte interactions required for cytokine synthesis, we have treated differentiated THP-1 cells with purified wild-type toxins, enzymatic mutants, or B subunits and measured TNF-alpha production . Our data suggest that A subunit enzymatic activity is essential for cytokine production . THP-1 cells were treated with a series of protein kinase C (PKC), PKA, and protein tyrosine kinase inhibitors to examine the role of intracellular signaling molecules in Stx-mediated cytokine production . Treatment of cells with PKC and tyrosine kinase inhibitors blocked TNF-alpha secretion by Stx-stimulated THP-1 cells . Stx treatment directly activated PKC, which occurred at a point upstream of transcriptional activation of the gene encoding TNF-alpha.

Infect Immun, 2000 Sep, 68(9), 5132 - 8
A novel factor isolated from Actinobacillus actinomycetemcomitans stimulates mouse B cells and human peripheral blood mononuclear cells; Jeong SJ et al.; A novel immunostimulating factor (ISTF) of Actinobacillus actinomycetemcomitans ATCC 29522 was isolated and characterized as inducing proliferation of mouse B cells and human peripheral blood mononuclear cells . This factor was isolated from the bacterial culture medium and purified by size exclusion chromatography, dye-ligand affinity chromatography, immunoaffinity chromatography using monoclonal antibodies, and preparative electrophoresis . Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified ISTF migrated as a single band corresponding to a molecular mass of 13 kDa . ISTF was a proteinaceous material distinct from lipopolysaccharide; it directly induced the proliferation of B lymphocytes but had no effect on the proliferation of T lymphocytes, even in the presence of antigen-presenting cells . A B-lymphocyte-mitogenic activity of ISTF was also shown by flow cytometric analysis of responding cell subpopulations . Immunoblot analysis revealed that ISTF was a component of the outer membranes of bacteria, could exist as a soluble form, and was released by growing and/or lysed bacteria . These results suggest that ISTF produced by A . actinomycetemcomitans may play an important role in immunopathologic changes associated with A . actinomycetemcomitans infections.

Cell Mol Life Sci, 2000 Jun, 57(6), 914 - 31
DNA repeat expansions and human disease; Usdin K et al.; The repeat expansion diseases are genetic disorders caused by intergenerational expansions of a specific tandem DNA repeat . These disorders range from mildly to severely debilitating or fatal, and all have limited treatment options . How expansion occurs and causes disease is only now beginning to be understood . Efforts to model expansion in mice have so far met with only limited success, perhaps due to a requirement for specific cis- or trans-acting factors . In vitro studies and data from bacteria and yeast suggest that in addition to secondary structures formed by the repeats, components of the DNA replication and recombination machinery are important determinants of instability . The consequences of expansion differ depending on where in the gene the repeat tract is located, and range from reduction of transcription initiation to protein toxicity . Recent advances are beginning to make rational approaches to the development of therapies possible.

Mutat Res, 2000 Aug 21, 469(1), 23 - 34
ENU induces mutations in the heart of lacZ transgenic mice; Cruz-Munoz W et al.; The use of transgenic mouse models as somatic mutation assays allows determination of mutation in all tissues of the mouse, including non-dividing tissues . In this regard, these models can be used to study the possibility that mutations can be induced in mitotically quiescent organs such as the heart . Mutations are generally thought to be associated with mitotic processes of DNA replication . Mutations, however, are also postulated to occur in the absence of mitosis as the result of DNA repair . In order to determine whether or not mutations could be induced in the heart, we analyzed the mutant frequency in the hearts of F(1) (Muta Mouse X SWR) mice that had been treated acutely with 250 mg/kg ENU and sampled at days 10, 35, and 70 post-treatment . A significant increase in mutant frequency at day 70 shows that mutations can be induced in the heart . Since the heart contains small numbers of non-muscle cells, additional mechanisms that could explain these results were also considered . The effect of ENU-induced cell proliferation or a sub-population of rapidly dividing cells is ruled out by C(14)-thymidine uptake studies which showed minimal proliferation . By the same token, the influence of ex vivo mutations (i.e., DNA adducts fixed as mutations during replication in the bacteria) is ruled out by the observed time course of mutations, as well as experimental evidence showing that such mutations are not detected in the lacZ assay.

Free Radic Biol Med, 2000 Jun 15, 28(12), 1771 - 9
Immunological responses to oxidized LDL; Horkko S et al.; Considerable evidence now points to an important role for the immune system in experimental models of atherosclerosis . We have reviewed the growing body of evidence that oxidation of LDL generates a wide variety of neoself determinants that lead to cellular and humoral immune responses . In particular, we have demonstrated that at least some of the oxidation-specific epitopes generated on the oxidized LDL particle, such as oxidized phospholipid epitopes, are also generated on apoptotic cells and are also present on the surface of some bacteria . Many of these same epitopes serve as important ligands mediating the binding and clearance of oxidatively damaged lipoprotein particles and apoptotic cells, and the innate immune response to these epitopes can be seen as a concerted response to effect their removal . In addition, other epitopes of OxLDL also undoubtedly play a role in the immune activation that characterizes the progressive atherosclerotic plaque . It will be of great importance to define the importance of the role of these responses and to understand which are beneficial and which deleterious . Such information could lead one day to novel therapeutic approaches to inhibit atherogenesis that take advantage of the ability to manipulate the immune response.

Biochimie, 2000 Jun-Jul, 82(6-7), 603 - 14
Assembly of chloroplast cytochromes b and c; Nakamoto SS et al.; The synthesis of holocytochromes in plastids is a catalyzed process . Several proteins, including plastid CcsA, Ccs1, possibly CcdA and a thioredoxin, plus at least two additional Ccs factors, are required in sub-stoichiometric amounts for the conversion of apocytochromes f and c(6) to their respective holoforms . CcsA, proposed to be a heme delivery factor, and Ccs1, an apoprotein chaperone, are speculated to interact physically in vivo . The formation of holocytochrome b(6) is a multi-step pathway in which at least four, as yet unidentified, Ccb factors are required for association of the b(H) heme . The specific requirement of reduced heme for in vitro synthesis of a cytochrome b(559)-derived holo-beta(2) suggests that cytochrome b synthesis in PSII might also be catalyzed in vivo.

Biochimie, 2000 Jun-Jul, 82(6-7), 583 - 601
Translation in chloroplasts; Zerges W; The discovery that chloroplasts have semi-autonomous genetic systems has led to many insights into the biogenesis of these organelles and their evolution from free-living photosynthetic bacteria . Recent developments of our understanding of the molecular mechanisms of translation in chloroplasts suggest selective pressures that have maintained the 100-200 genes of the ancestral endosymbiont in chloroplast genomes . The ability to introduce modified genes into chloroplast genomes by homologous recombination and the recent development of an in vitro chloroplast translation system have been exploited for analyses of the cis-acting requirements for chloroplast translation . Trans-acting translational factors have been identified by genetic and biochemical approaches . Several studies have suggested that chloroplast mRNAs are translated in association with membranes.

J Surg Res, 2000 Sep, 93(1), 182 - 96
Regulation of intestinal blood flow; Matheson PJ et al.; The gastrointestinal system anatomically is positioned to perform two distinct functions: to digest and absorb ingested nutrients and to sustain barrier function to prevent transepithelial migration of bacteria and antigens . Alterations in these basic functions contribute to a variety of clinical scenarios . These primary functions intrinsically require splanchnic blood flow at both the macrovascular and microvascular levels of perfusion . Therefore, a greater understanding of the mechanisms that regulate intestinal vascular perfusion in the normal state and during pathophysiological conditions would be beneficial . The purpose of this review is to summarize the current understanding regarding the regulatory mechanisms of intestinal blood flow in fasted and fed conditions and during pathological stress .

Genomics, 2000 Jul 1, 67(1), 69 - 77
Chromosomal location and genomic structure of the human translin-associated factor X gene (TRAX; TSNAX) revealed by intergenic splicing to DISC1, a gene disrupted by a translocation segregating with schizophrenia; Millar JK et al.; Two candidate genes, DISC1 and DISC2 on chromosome 1, are disrupted by a translocation that segregates with major psychiatric illness . Several DISC1 transcripts contain TRAX (HGMW-approved symbol TSNAX) sequence at the 5' end . These transcripts initiate at the 5' end of TRAX and terminate at the final exon of DISC1 . Five species of transcript resulting from intergenic splicing have been identified; one encodes a novel TRAX/DISC1 fusion protein . The remaining four transcripts are bicistronic and encode a series of novel truncated isoforms of TRAX and DISC1 . Demonstration that the various TRAX/DISC1 transcripts are translated awaits further experimentation . As a consequence of the observation of intergenic splicing, the human TRAX gene has been mapped at least 35 kb proximal to DISC1 and within approximately 150-250 kb of the translocation breakpoint at 1q42.1 . The TRAX gene consists of six exons with a putative CpG island at the 5' end . Four major transcripts are produced from this gene, of which the smallest, at 2.7 kb, had previously been identified.

J Vet Med Sci, 2000 Jul, 62(7), 771 - 3
Immunohistochemistry and polymerase chain reaction for the detection of Lawsonia intracellularis in porcine intestinal tissues with proliferative enteropathy; Kim J et al.; Detection method of Lawsonia intracellularis was studied in formalin-fixed paraffin-embedded intestinal tissues from 5 naturally infected pigs by immunohistochemistry with a monoclonal antibody against outer membrane protein of L . intracellularis . Warthin-Starry silver stain revealed clusters of argyrophilic, slightly curved rod-shaped organisms in the apical cytoplasm of enterocytes . Immunohistochemical staining with a L . intracellularis-specific monoclonal antibody confirmed the presence of the organism in the apical cytoplasm of hyperplastic enterocytes . The presence of L . intracellularis in the ileum of pig with proliferative enteropathy was confirmed by polymerase chain reaction (PCR) further on the basis of amplification of 319 base pair products specific for porcine L . intracellularis chromosomal DNA . Immunohistochemistry and PCR may be a complementary method to confirm the diagnosis of L . intracellularis infection in pigs.

Anat Rec, 2000 Aug 15, 261(4), 141 - 52
Biological time and in vivo research: a field guide to pitfalls; Burns ER; Biological rhythmicity is a fundamental characteristic of all life forms, from primitive bacteria to man . The molecular biology, genetics, and the neurobiology of the biological clock(s) are being elucidated . Daily (circadian) statistically significant fluctuations occur in all of the normal biological variables studied in the experimental animal and the human . Many researchers, however, are not aware of the negative impact biological rhythmicity can have on experimental design and/or data interpretation . This article serves not as a review, but as a "field guide" to the pitfalls that can occur when research is performed in the absence of an understanding of biological rhythmicity . The major topics discussed are: 1) data transfer from the diurnally in-active/resting/sleeping lab animal to the diurnally active human, 2) frequency of sampling, 3) free-running vs . synchronization, 4) alternating periods of resistance and susceptibility, 5) phase shifting of a rhythm, 6) the assumption that one mean +/- S.E . from control animals can be "stretched" across an experimental time span, and 7) plotting data on an "hours after treatment" format vs . a "time of day" format . The hope is that by avoiding the pitfalls, biological time will become an ally in the endeavor to understand human biology.

Acta Crystallogr D Biol Crystallogr, 2000 Aug, 56 ( Pt 8), 973 - 85
Towards automated Laue data processing: application to the choice of optimal X-ray spectrum; Bourgeois D et al.; Laue data reduction has now reached a level of sophistication that allows nearly automated processing to be performed . The software described enables complete reduction of the data with essentially no user intervention, making Laue processing almost as straightforward as monochromatic data processing . Interactive work is limited to the indexing of only one Laue pattern . More importantly, it is shown that the data quality is substantially enhanced when soft-limited predictions are used . Further improvement obtained by taking advantage of the structure-factor amplitudes from a known closely related structure is described . To determine the most suitable type of insertion device to be used for time-resolved Laue crystallography, the technique described was applied to Laue data sets collected from photoactive yellow protein under identical conditions but with three different insertion devices: a wiggler, a broad-bandpass undulator and a single-line undulator . Although the optimal choice may ultimately be dictated by sample parameters (such as mosaic spread) and by the type of experiment (repeatable or non-repeatable reactions), the results here show that the use of single-line undulators will generally yield by far the best compromise between data quality, acquisition time and radiation damage.

Clin Otolaryngol, 2000 Jun, 25(3), 181 - 94
The aetiology of otitis media with effusion: a review; Kubba H et al.; Otitis media with effusion (OME) is the most common cause of deafness in children in the developed world . In this article we aim to present an overview of current research developments on the aetiology of OME and the resulting implications for treatment . In the model we describe, the primary event is inflammation of the middle ear mucosa, usually due to the presence of bacteria . This leads to the release of inflammatory mediators, which cause secretion of a mucin-rich effusion by up-regulating mucin genes . Prolonged stimulation of the inflammatory response and poor mucociliary clearance lead to persistence of the middle ear fluid, giving rise to the clinical presentation of OME . We describe OME in the following sequence: the initial production of the effusion, the composition of the effusion produced, and factors impairing clearance of the effusion.

J Photochem Photobiol B, 2000 Apr-May, 55(2-3), 109 - 12
UV-B-induced generation of free radicals in blood platelets; Bednarska K et al.; UV-B irradiation of blood-platelet concentrates is used in transfusion practice to prevent the development of post-transfusion alloimmunization and inactivate viruses and bacteria in the concentrates . UV-B radiation may affect the blood-platelet metabolism and function; therefore we have investigated the effect of UV-B irradiation on free radical production in blood platelets . Our results show that exposure of pig blood platelets to UV-B radiation (0.36 and 1.08 J/cm2) induces the generation of free radicals measured by the chemiluminescence method (respectively 28 and 148.6% above the control) . The superoxide radical level after UV-B irradiation measured by the cytochrome c reduction method shows only a slight increase (p > 0.05) . Free radical generation induced by UV-B radiation is dependent partly on blood-platelet activation and enzymatic pathways, since we have shown that wortmannin, an inhibitor of phosphatidylinositide 3-kinase, reduces the level of radicals formed in blood platelets after UV-B irradiation . This indicates that free radicals generated in blood platelets after stimulation by UV-B radiation are involved in platelet activation and metabolism of platelet polyphosphoinositides.

J Mol Microbiol Biotechnol, 1999 Aug, 1(1), 101 - 5
Adaptation of proteins from hyperthermophiles to high pressure and high temperature; Robb FT et al.; Further clarification of the adaptations permitting the persistence of life at temperatures above 100 degrees C depends in part on the analysis of adaptive mechanisms at the protein level . The hyperthermophiles include both Bacteria and Archaea, although the majority of isolates growing at or above 100 degrees C are Archaea . Newly described adaptive features of hyperthermophiles include proteins whose structural integrity persists at temperatures up to 200 degrees C, and under elevated hydrostatic pressure, which in some cases adds significant increments of stability.

Int J Biochem Cell Biol, 2000 Aug, 32(8), 855 - 63
Evidence for the existence of two soluble NAD(+) kinase isoenzymes in Euglena gracilis Z; Stephan C et al.; Two soluble NAD(+) kinase isoenzymes (isoenzymes 1 and 2) from Euglena gracilis were separated by preparative electrophoresis and characterized . They display several similar properties: both have an identical apparent molecular weight of 68 kDa and their activities are independent on calmodulin, insensitive to 2-mercaptoethanol but inhibited by p-chloromercurybenzoate, 5, 5'-dithiobis(2-nitrobenzoate) and, surprisingly, by low dithiothreitol concentrations, the inhibition by dithiothreitol being irreversible for isoenzyme 1 but reversible for isoenzyme 2 . Nevertheless, the two isoenzymes mainly differ by their specificities towards triphosphate nucleotides and their catalytic mechanisms . Isoenzyme 1 is as active in the presence of ATP as of GTP and acts by a ping-pong mechanism with a k(M) for NAD(+) of 0.26 mM and a k(M) for low MgATP(2-)concentrations of 0.03 mM . Isoenzyme 2 is three-fold more active in the presence of GTP than of ATP and operates by a sequential mechanism with k(M)s for NAD(+) and MgGTP(2-) of 1.03 and 0.20 mM, respectively . This study shows the evidence for the existence of two structurally similar but catalytically different NAD(+) kinase isoenzymes in E . gracilis . One resembles the enzyme previously described in bacteria . The other displays a catalytic mechanism identical to that of NAD(+) kinase from other organisms but remains unique among all the NAD(+) kinases studied to-date regarding its specificity towards GTP.

Gene, 2000 Aug 8, 253(2), 259 - 69
Molecular characterization of the gyrB region of the oral spirochete, Treponema denticola; Greene SR et al.; The nucleotide (nt) sequence of the Treponema denticola (Td) DNA gyrase beta-subunit gene (gyrB) has been determined . Southern blot analysis of Td chromosomal DNA indicated that gyrB is present as a single copy . Approximately 3.2kb of the nt sequence 5' and 0.7kb of nucleotide sequence 3' of gyrB were obtained . Analysis of the deduced amino acid (aa) sequence revealed two complete open reading frames (ORFs) (ORF1 and ORF3) and a truncated ORF (ORF4') . ORF1 has no homology to sequences in the databases, whereas ORF3 and ORF4' have significant homology to several bacterial DnaA (replication initiator) and DnaE (DNA polymerase III) proteins respectively . RT-PCR data showed that orf1-gyrB are co-transcribed, while dnaA-dnaE are co-transcribed but in the opposite direction . These data indicated that the gene organization of the Td gyrB region is unique compared with that of other bacteria . Eighteen putative DnaA boxes with several AT-rich regions were identified in the dnaA-dnaE intergenic region, and three putative DnaA boxes were identified in the gyrB-dnaA intergenic region . Spontaneous coumermycin A(1)-resistant Td mutants were isolated and characterized . The mutants have a >20-fold higher resistance to coumermycin A(1) than wild-type Td . A single point mutation in gyrB that changed GyrB Lys(136) to Glu or Thr appears to be responsible for the coumermycin A(1) resistance.

Neurosci Biobehav Rev, 2000 Aug, 24(6), 627 - 38
The effects of hormones on sex differences in infection: from genes to behavior; Klein SL; Males of many species are more susceptible than females to infections caused by parasites, fungi, bacteria, and viruses . One proximate cause of sex differences in infection is differences in endocrine-immune interactions . Specifically, males may be more susceptible to infection than females because sex steroids, specifically androgens in males and estrogens in females, modulate several aspects of host immunity . It is, however, becoming increasingly more apparent that in addition to affecting host immunity, sex steroid hormones alter genes and behaviors that influence susceptibility and resistance to infection . Thus, males may be more susceptible to infection than females not only because androgens reduce immunocompetence, but because sex steroid hormones affect disease resistance genes and behaviors that make males more susceptible to infection . Consideration of the cumulative effects of sex steroid hormones on susceptibility to infection may serve to clarify current discrepancies in the literature and offer alternative hypotheses to the view that sex steroid hormones only alter susceptibility to infection via changes in host immune function.

Int J Syst Evol Microbiol, 2000 Jul, 50 Pt 4, 1487 - 93
Nocardia abscessus sp . nov; Yassin AF et al.; Chemotaxonomic and 16S rRNA gene sequence analyses of four bacterial strains isolated from clinical material clearly demonstrated that these bacteria belong to the genus Nocardia . DNA-DNA hybridization data as well as the physiological characteristics of the isolates indicated that they are closely related and belong to a single species that differs from previously described members of the genus . The name Nocardia abscessus sp . nov . is proposed for these organisms represented by strain IMMIB D-1592T (= DSM 44432T) . Strain IMMIB D-1592T exhibits 56.8 and 60.0% DNA-DNA relatedness to Nocardia asteroides ATCC 19247T and Nocardia paucivorans DSM 44386T, respectively.

Int J Syst Evol Microbiol, 2000 Jul, 50 Pt 4, 1457 - 61
Polyphasic evidence for the reclassification of Rhodothermus obamensis Sako et al . 1996 as a member of the species Rhodothermus marinus Alfredsson et al . 1988; Silva Z et al.; DNA-DNA reassociation studies, 16S rRNA gene sequence comparisons and fatty acid analysis were used to reassess the taxonomic status of the type strain of Rhodothermus obamensis and several strains of the genus Rhodothermus isolated from widely distributed shallow marine hot springs . The results show that the type strain of R . obamensis, JCM 9785T, has a DNA-DNA reassociation value of 78% with the type strain of R . marinus, DSM 4252T . The other strains examined had DNA-DNA reassociation values that varied between about 68 and 94% with R . marinus . The 165 rRNA gene sequence was determined for the type strain of R . obamensis and found to share 99.5% similarity with the type strain of R . marinus . The fatty acid composition of R . obamensis was slightly different from that of the other strains examined, but indicated that this strain is very closely related to the other strains examined in this study . On the basis of DNA-DNA reassociation values, 16S rRNA gene sequence comparison and fatty acid profiles, it was concluded that R . obamensis and R . marinus represent the same species and that the name Rhodothermus obamensis should be regarded as a junior synonym of Rhodothermus marinus.

Int J Syst Evol Microbiol, 2000 Jul, 50 Pt 4, 1435 - 40
Actinoalloteichus cyanogriseus gen . nov., sp . nov; Tamura T et al.; Phylogenetic analysis of 'Actinoalloteichus cyanogriseus' based on its 16S rDNA sequence confirmed that the organism belongs to the family Pseudonocardiaceae . It contains glutamic acid, alanine and meso-diaminopimelic acid as cell wall amino acids, and menaquinone 9 (H4) . Its taxonomic characteristics differ from those of the previously described organisms of the family Pseudonocardiaceae . On the basis of these morphological, physiological, chemotaxonomic and phylogenetic analyses, a new genus, Actinoalloteichus, is proposed, along with a new species, namely Actinoalloteichus cyanogriseus sp . nov . The type strain is A . cyanogriseus IFO 14455T (= AS 4.1159T = JCM 6095T).

Vox Sang, 2000, 78 Suppl 2, 211 - 5
The use of riboflavin for the inactivation of pathogens in blood products; Goodrich RP; BACKGROUND AND OBJECTIVES: In recent years, the desire to develop methods to inactivate pathogens in blood components has continued to grow . Several of these proposed approaches have been introduced or are currently in clinical studies . The use of chemical inactivating agents must be considered in terms of the current safety of the blood supply and the potential risks that the introduction of new chemical entities into blood components may carry . The impact which these treatment procedures have on the in vitro and in vivo performance of these products must also be considered relative to the potential benefit of the pathogen inactivation potential they offer . This paper will discuss one possible approach for inactivating pathogens in blood using vitamin B2, Riboflavin, and light . MATERIALS AND METHODS: We have used Riboflavin for treating plasma and platelets and evaluated protein quality and platelet function in vitro . Initial toxicology tests to assess the impact of infusion of photoproducts generated in these processes have also been conducted in rodents . Cytotoxicity evaluations have been used to assess the possible impact of photoproduct toxicity in vivo . Virus and bacteria spiking studies using a variety of human and animal model pathogens have been conducted in order to asses the efficacy of this process . RESULTS: Initial toxicology assessment of the photoproducts of Riboflavin generated under the proposed treatment conditions have been favorable . Virus and bacteria clearance studies have demonstrated efficacy of the procedure against a wide range of human and animal pathogens, including intracellular HIV-1 . Studies with platelet and plasma function indicated reductions in vitro comparable to other proposed treatment approaches . CONCLUSION: The use of Riboflavin in a photochemical decontamination process for blood components shows promise.

Vox Sang, 2000, 78 Suppl 2, 183 - 6
Platelet substitutes; Blajchman MA; Many experimental approaches have been explored to produce hemostatically active novel human platelet products and substitutes capable of long-term storage . These include: platelet storage in the frozen state; storage in the cold (4 degrees C) in the liquid state; photochemical methods for the inactivation of viruses, bacteria and protozoa; infusible platelet membranes; and rehydrated lyophilized platelets . In addition to products using human platelets as their primary manufacturing source, other approaches have been used to make non-platelet-derived substitutes that might be capable of in vivo hemostasis . These include production of red blood cells, or liposomes, bearing hemostatically active agents on their surfaces; and fibrinogen-coated albumin microcapsules or microspheres . The development of platelet substitutes is an increasing scientific and technological endeavor and there are many reasons to believe that platelet substitutes will become more efficacious and safer to use over time . Ultimately, such work will result in safe and effective platelet substitutes being available for clinical use in thrombocytopenic or other patients with increased risk for bleeding.

Platelets, 2000 Feb, 11(1), 6 - 22
Heat-shock proteins and platelet function; Polanowska-Grabowska R et al.; Heat-shock proteins are found in organisms as diverse as slime moulds, bacteria, plants and higher eukarycotes . They play fundamental roles in cell function, ranging from protein folding to transmembrane protein movement, to serving as scaffolds or frameworks for the assembly of enzyme signalling complexes such as the steroid receptors . Intracellular concentrations may be high, in the range of structural proteins such as actin, with which they often interact . Therefore, it is not surprising that heat-shock proteins are present in blood platelets, and recent studies point to important roles in platelet function . The small heat-shock protein, hsp27, becomes phosphorylated following cell stimulation with thrombin and associates with the actin-rich cytoskeleton . Phosphorylation results from activation of a protein kinase cascade involving the p38 mitogen-activated protein kinase (MAPK), the MAPKAP-K2 kinase, as well as PRAK, or p38-regulated protein kinase . Intriguingly, platelet hsp27 can associate with platelet factor XIII, suggesting a role for regulation of transglutaminase activity in stabilizing fibrin-platelet clots . The higher molecular-weight heat-shock proteins hsc70 and hsp90 are also present in platelets, being found in a large phosphorylated complex that contains the catalytic and myosin-targeting subunits of protein phosphatase 1 (PP1) . Platelet adhesion to collagen via the alpha 2 beta 1 integrin causes the rapid dissociation of this complex and dephosphorylation of components . These results suggest that hsc70 and hsp90 can serve as signalling scaffolds, helping regulate function, including platelet adhesion and spreading via modulation of protein phosphatase activity . Hsp27, on the other hand, may be more involved in controlling actin polymerization during the platelet shape change and subsequent aggregation.

Bull Math Biol, 2000 Jul, 62(4), 759 - 74
Individual- and population-based diversity in restriction-modification systems; Pagie L et al.; Restriction-modification (RM) systems are cognate gene complexes that code for an endonuclease and a methylase . They are often thought to have developed in bacteria as protection against invading genetic material, e.g., phage DNA . The high diversity of RM systems, as observed in nature, is often ascribed to the coevolution of RM systems (which 'invent' novel types) and phages . However, the extent to which phages are insensitive to RM systems casts doubts on the effectiveness of RM systems as protection against infection and thereby on the reason for the diversity of RM systems . We present an eco-evolutionary model in order to study the evolution of the diversity of RM systems . The model predicts that in general diversity of RM systems is high . More importantly, the diversity of the RM systems is expressed either at the individual level or at the population level . In the first case all individuals carry RM systems of all sequence specificities, whereas in the second case they carry only one RM system or no RM systems at all . Nevertheless, in the second case the same number of sequence specificities are present in the population.

Enzyme Microb Technol, 2000 Sep 1, 27(6), 390 - 398
Influence of dissolved oxygen tension and agitation speed on alginate production and its molecular weight in cultures of Azotobacter vinelandii*
Pena C, Trujillo-Roldan MA, Galindo E.
The alginate production by Azotobacter vinelandii, as well as the molecular weight of the polymer, are strongly influenced by the dissolved oxygen tension (DOT) and stirring speed of the culture . Under high DOT (5% of air saturation), the bacteria produced more alginate (4.5 g/l) than that obtained at low (0.5%) oxygen tension (1.0 g/l) in cultures conducted at 300 rpm . On the other hand, under constant DOT (3%), the higher the stirring speed (from 300 to 700 rev./min), the higher the specific growth rate and the alginate production rate . However, low agitation speed (300 rev./min) lead the culture to produce a polymer of high molecular weight (680 000 g/g mol) whereas a low molecular weight (352 000 g/g mol) alginate was isolated from cultures conducted at high (700 rev./min) stirring speed . At 700 rev./min, the MMW increased to a plateau between 1 and 3% DOT and then decreased to a minimum of 0.11 x 10(6) g/g mol at 7% . Microscopic observations revealed the presence of cell aggregates (one order of magnitude larger than individual cells) when the culture was conducted at 300 rev./min . Oxygen gradients occurring within the aggregates could be responsible of this phenomenon . At high agitation rate, the MMW of the alginate dropped towards the end of the culture in all conditions evaluated . Alginase activity was detected, which would be responsible for this phenomenon.

Metab Eng, 1999 Jul, 1(3), 243 - 54
Metabolic modeling as a tool for evaluating polyhydroxyalkanoate copolymer production in plants; Daae EB et al.; The production of polyhydroxyalkanoates in plants is an interesting commercial prospect due to lower carbon feedstock costs and capital investments . The production of poly-(3-hydroxybutyrate) has already been successfully demonstrated in plant plastids, and the production of more complex polymers is under investigation . Using a mathematical simulation model this paper outlines the theoretical prospects of producing the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) {P(3HB-3HV)} in plant plastids . The model suggests that both the 3HV/3HB ratio and the copolymer production rate will vary considerably between dark and light conditions . Using metabolic control analysis we predict that the beta-ketothiolase predominately controls the copolymer production rate, but that the activity of all three enzymes influence the copolymer ratio . Dynamic simulations further suggest that controlled expression of the three enzymes at different levels may enable desirable changes in both the copolymer production rate and the 3HV/3HB ratio . Finally, we illustrate that natural variations in substrate and cofactor levels may have a considerable impact on both the production rate and the copolymer ratio, which must be taken into account when constructing a production system.

Biol Chem, 2000 May-Jun, 381(5-6), 377 - 87
GTPases mechanisms and functions of translation factors on the ribosome; Rodnina MV et al.; The elongation factors (EF) Tu and G and initiation factor 2 (IF2) from bacteria are multidomain GTPases with essential functions in the elongation and initiation phases of translation . They bind to the same site on the ribosome where their low intrinsic GTPase activities are strongly stimulated . The factors differ fundamentally from each other, and from the majority of GTPases, in the mechanisms of GTPase control, the timing of Pi release, and the functional role of GTP hydrolysis . EF-Tu x GTP forms a ternary complex with aminoacyl-tRNA, which binds to the ribosome . Only when a matching codon is recognized, the GTPase of EF-Tu is stimulated, rapid GTP hydrolysis and Pi release take place, EF-Tu rearranges to the GDP form, and aminoacyl-tRNA is released into the peptidyltransferase center . In contrast, EF-G hydrolyzes GTP immediately upon binding to the ribosome, stimulated by ribosomal protein L7/12 . Subsequent translocation is driven by the slow dissociation of Pi, suggesting a mechano-chemical function of EF-G . Accordingly, different conformations of EF-G on the ribosome are revealed by cryo-electron microscopy . GTP hydrolysis by IF2 is triggered upon formation of the 70S initiation complex, and the dissociation of Pi and/or IF2 follows a rearrangement of the ribosome into the elongation-competent state.

Evolution Int J Org Evolution, 2000 Jun, 54(3), 731 - 9
Stability and evolution of overlapping genes; Krakauer DC; When the same sequence of nucleotides codes for regions of more than one functional polypeptide, this sequence contains overlapping genes . Overlap is most common in rapidly evolving genomes with high mutation rates such as viruses, bacteria, and mitochondria . Overlap is thought to be important as: (1) a means of compressing a maximum amount of information into short sequences of structural genes; and (2) as a mechanism for regulating gene expression through translational coupling of functionally related polypeptides . The stability of overlapping codes is examined in relation to the information cost of overlap and the mutation rate of the genome . The degree of overlap in a given population will tend to become monomorphic . Evolution toward partial overlap of genes is shown to depend on a convex cost function of overlap . Overlap does not evolve when expression of overlapping genes is mutually exclusive and produced by rare mutations to the wild-type genome . Assuming overlap increases coupling between functionally related genes, the conditions favoring overlap are explored in relation to the kinetics of gene activation and decay . Coupling is most effective for genes in which the gene overlapping at its 5' end (leading gene) decays rapidly, while the gene overlapping at the 3' end (induced gene) decays slowly . If gene expression can feedback on itself (autocatalysis), then high rates of activation favor overlap.

Kidney Int Suppl, 2000 Aug, 76, S60 - 5
Adsorption in hemodialysis; Botella J et al.; The use of sorbents in different blood purification techniques is reviewed . The sorbents used in these therapies are divided into two groups: (1) Adsorption occurs fundamentally because of the hydrophobic properties of the sorbents . In this group, the sorbents used in different dialysis techniques are charcoal and nonionic macroporous resins . (2) Adsorption occurs by chemical affinity, such as ion exchange resins and chemisorbents . Sorbents were initially used in hemoperfusion, which caused many adverse events; later, with the use of coated charcoal, these undesired effects decreased or disappeared, but the adsorptive properties, water control, and acid-base balance still created problems . For these reasons, the use of sorbents in the treatment of chronic renal failure was almost totally discontinued . Little by little, interest in these substances has reappeared, and at present, they have been used in combination with other blood purification techniques such as hemodialysis, hemofiltration, peritoneal dialysis, and finally, hemodiafiltration . Within the various hemodiafiltration techniques, paired filtration dialysis-charcoal is being used to regenerate the ultrafiltrate, which is used as the replacement fluid . Charcoal regenerates the ultrafiltrate and transforms it into a physiological solution with a normal electrolyte composition, calcium, bicarbonate, and glucose, having eliminated the majority of both middle and large molecule uremic toxins . If regeneration is done properly, this replacement fluid is bacteria and endotoxin free . Studies currently are underway on the adsorption of different inflammatory substances in the ultrafiltrate, which could lead to improvement in the biocompatibility of the system.

Diabetes Res Clin Pract, 2000 Sep, 50(1), 27 - 34
Type 2 diabetes and oral health: a comparison between diabetic and non-diabetic subjects; Sandberg GE et al.; A controlled cross-sectional study with the aim of studying oral health in patients with type 2 diabetes was carried out in a health care district in Sweden . The study included 102 randomly sampled diabetic patients and 102 age- and gender-matched non-diabetic subjects from the same geographical area, treated at the same Public Dental Service clinics . Oral conditions were measured at clinical and X-ray examinations . Diabetes-related variables were extracted from medical records . Diabetic patients suffered from xerostomia (dry mouth) to a significantly higher degree than non-diabetic controls did (53.5 vs . 28.4%; P=0.0003) . Sites with advanced periodontitis were more frequent in the diabetic group (P=0.006) as were initial caries lesions (P=0.02) . Diabetic subjects showed a greater need of periodontal treatment (P=0.05), caries prevention (P=0.002) and prosthetic corrections (P=0.004) . Diabetes duration or metabolic control of the disease was not related to periodontal status . However, patients with longer duration of diabetes had more manifest caries lesions (P=0.05) as had those on insulin treatment when compared with patients on oral/diet or combined treatment (P=0.0001) . The conclusion is that individuals with type 2 diabetes in some oral conditions exhibited poorer health . Close collaboration between the patient, the primary health care and oral health professionals could be a way of improving the diabetic patient's general and oral health.

Genomics, 2000 Aug 1, 67(3), 351 - 5
Cloning and characterization of human DDX24 and mouse Ddx24, two novel putative DEAD-Box proteins, and mapping DDX24 to human chromosome 14q32; Zhao Y et al.; DEAD-box proteins are a large group of putative RNA helicases that exist ubiquitously in organisms ranging from bacteria to humans . They are likely to play important roles in many different RNA metabolic processes . In this paper, we report the cloning of human DDX24, a putative DEAD-box protein, and its ortholog, Ddx24 in mouse . The deduced proteins encoded by these two cDNAs share 78.7% identity at the amino acid level and possess all the well-conserved motifs of DEAD-box proteins . However, little homology can be found between them and other DEAD-box proteins, even in their core region (identity <40%) . Northern blot analysis showed that a 3.0-kb transcript of human DDX24 exists ubiquitously in the 16 human tissues examined and was most abundant in heart and brain, but with lowest levels in thymus and small intestine . The mouse Ddx24, whose transcript is 4.0 kb, was also expressed widely in 10 tissues tested with the highest level in heart and testis . By radiation hybrid mapping, the human DDX24 gene was localized to human chromosome 14q32 between the markers D14S81 and D14S265 . Moreover, the gene structure of DDX24 was determined by comparing its cDNA and genomic sequence from BAC R-1089B7, which showed that the gene spanned a 30-kb region and consisted of at least nine exons .

Chin J Biotechnol, 1999, 15(3), 145 - 51
Site-directed mutagenesis analysis of draTG genes and their downstream region from Azospirillum brasilense Yu62; Ma L et al.; draT and draG genes are involved in posttranslational regulation of nitrogenase activity of Azospirillum brasilense Yu62 . Both genes and their downstream region were mutagenized by Kmr cassette insertions . Analysis of mutations introduced into the draTG region on the A . brasilense Yu62 chromosome showed that mutants affected in draT were incapable of regulating nitrogenase activity in response to ammonium . In contrast, a mutant with an insertion in draG was still capable of ADP-ribosylating dinitrogenase reductase in response to ammonium but was no longer able to recover activity after ammonium depletion . Analysis of mutations introduced into the draTG downstream region (the mutagenized site is about 2 kb downstream from draG) showed that the mutant had higher nitrogenase activity than the wild strain while growing in nitrogen-free medium and medium with 2 mmol/L ammonium . These results reveal that there is no gene required for nitrogen fixation in this mutagenized region, but it is possible that there are genes which play a role in regulating nitrogen fixation . The results of monitoring the expression of transcriptional nifH-lacZ gene fusion in the mutant YZ4 showed that the transcriptional regulation of nif gene in the mutant YZ4 was the same as that in the wild type.

Rev Latinoam Microbiol, 1999 Oct-Dec, 41(4), 267 - 72
{Prevalence of Chlamydia trachomatis in newborn infants with respiratory problems}; Lopez-Hurtado M et al.; The prevalence of C . trachomatis in neonates with respiratory distress was studied after 24 h of birth, nine patients were positive for C . trachomatis culture (12.9%) . The chest radiographs showed six with hyaline membrane disease and two with pneumonia . One patient with treatment of ventilation mechanics developed bronchopulmonary dysplasia and was C . trachomatis positive in a second cell culture . Of the nine patients with C . trachomatis, eight were neonates preterm with low weight to the birth and with leukocytosis . Six patients were delivered by cesarean section . These results suggest that C . trachomatis can participate in an important way in the development of the distress respiratory in infants preterm.

Nat Biotechnol, 2000 Aug, 18(8), 888 - 92
Metabolic engineering of astaxanthin production in tobacco flowers; Mann V et al.; Using metabolic engineering, we have modified the carotenoid biosynthesis pathway in tobacco (Nicotiana tabacum) to produce astaxanthin, a red pigment of considerable economic value . To alter the carotenoid pathway in chromoplasts of higher plants, the cDNA of the gene CrtO from the alga Haematococcus pluvialis, encoding beta-carotene ketolase, was transferred to tobacco under the regulation of the tomato Pds (phytoene desaturase) promoter . The transit peptide of PDS from tomato was used to target the CRTO polypeptide to the plastids . Chromoplasts in the nectary tissue of transgenic plants accumulated (3S,3'S) astaxanthin and other ketocarotenoids, changing the color of the nectary from yellow to red . This accomplishment demonstrates that plants can be used as a source of novel carotenoid pigments such as astaxanthin . The procedures described in this work can serve as a platform technology for future genetic manipulations of pigmentation of fruits and flowers of horticultural and floricultural importance.

Clin Exp Immunol, 2000 Aug, 121(2), 289 - 94
Growth of Brucella abortus in macrophages from resistant and susceptible mouse strains; Sathiyaseelan J et al.; C57Bl/10 mice have a superior ability to control chronic infections with virulent strains of the intracellular bacteria Brucella abortus compared with BALB/c mice . While a number of differences in the cytokines produced by lymphocytes following infection of these two strains of mice have been shown, macrophages have not been evaluated for their role in conveying relative resistance . The importance of macrophages in control of brucella infections is demonstrated by the observations that intracellular survival of various strains of B . abortus directly correlates with their virulence in vivo, and the ability of macrophages to control brucellae in vitro has been shown to correlate with a brucella-resistant phenotype in ruminants . While both BALB/c and C57Bl are Nramp-susceptible mouse strains, additional differences in macrophage function outside of the Nramp1 gene effects could influence susceptibility to brucellosis . The studies conducted here comparing the ability of macrophages from C57Bl/10 and BALB/c mice indicate that the macrophages from resistant mice did not control intracellular growth of B . abortus strain 2308 more efficiently than those from the susceptible mice, either in the absence of, or following, interferon-gamma activation or iron supplementation . A number of different conditions for culturing macrophages were evaluated to rule out the influence of antibiotics on the conclusions drawn from the results.

Br J Haematol, 2000 Jul, 110(1), 116 - 24
Increased platelet activation and abnormal membrane glycoprotein content and redistribution in myeloproliferative disorders; Jensen MK et al.; Chronic myeloproliferative disorders (MPDs) are characterized by a high incidence of thrombohaemorrhagic complications, possibly caused by platelet dysfunction . In an attempt to define platelet functional abnormalities, we assessed the expression of activation-dependent membrane proteins in unstimulated and agonist {ADP and thrombin receptor-activating peptide (TRAP)}-stimulated platelets using quantitative whole blood flow cytometry in samples from 50 MPD patients and 30 controls . The receptor densities of activation markers and glycoproteins (GPs) were quantified using standardized fluorescent beads . Compared with controls, the mean percentage of P-selectin-positive (15.3% vs . 7.2%; P < 0.001) and thrombospondin (TSP)-positive (6.6% vs . 3.7%; P = 0.003) platelets was increased in unstimulated platelets from patients . Patients having experienced a thrombotic event had a higher mean percentage of TSP-positive non-stimulated platelets than patients without a history of thrombosis (9.0% vs . 4.6%; P = 0.02) and a higher GPIV molecules of equivalent fluorochrome (MEF) value (33113 vs . 24471 MEF; P = 0.02) . Mean MEF values of monoclonal antibodies (mAbs) against GPIb (34055 vs . 38945 MEF; P < 0.001) and GPIIb/IIIa (1416 vs . 1648 MEF; P < 0 . 001) were significantly reduced among patients, whereas surface expression of GPIV was increased in patients (28273 vs . 16258 MEF; P < 0.001) . In TRAP (10 micromol/l) stimulated whole blood, the MEF of P-selectin (9611 vs . 13293 MEF; P = 0.004) and CD63 (2385 vs . 5177 MEF; P < 0.001) and the ratio of PAC-1/GPIIb/IIIa MEF (0.98 vs . 2 . 00; P < 0.001) was reduced in patients, indicating either a reduced granule GP content or an intrinsic cellular defect in receptor-mediated granule secretion and activation of the GPIIb/IIIa complex . Expressed as the relative change of MEF compared with unstimulated platelets, TRAP induced decrease of GPIb (7.8% vs . 45%; P < 0.001) and increase of GPIIb/IIIa (49.1% vs . 95.7%; P < 0.001) and GPIV expression (17.8% vs . 55.2%; P < 0.001) was attenuated in patients.

FEMS Microbiol Lett, 2000 Aug 15, 189(2), 259 - 64
Extracellular polysaccharide composition of Azospirillum brasilense and its relation with cell aggregation; Burdman S et al.; The exopolysaccharide (EPS) and capsular polysaccharide (CPS) composition of four Azospirillum brasilense strains differing in their aggregation capacity was analyzed by high performance anion exchange chromatography . When growing the different strains in an aggregation inducing medium containing a high carbon:nitrogen (C:N) ratio, both EPS and CPS showed a positive correlation between aggregation and the relative amount of arabinose . Arabinose was not detected in polysaccharides from Sp72002, a pleiotrophic Tn5 mutant strain impaired in aggregation . Arabinose was also not detected in extracellular polysaccharides of bacteria grown in a low C:N ratio, non-inducing aggregation medium, with exception for a relatively small amount found in the CPS of FAJ0204, a super-aggregating mutant strain . The only monosaccharides able to significantly inhibit aggregation at low sugar concentration when tested in a bioassay were arabinose (at a higher extent) and galactose . The possibility that residues of arabinose present in the extracellular polysaccharides are involved in the aggregation of A . brasilense is discussed.

J Clin Invest, 2000 Aug, 106(3), 339 - 47
Local pH elevation mediated by the intrabacterial urease of Helicobacter pylori cocultured with gastric cells; Athmann C et al.; Helicobacter pylori resists gastric acidity by modulating the proton-gated urea channel UreI, allowing for pH(out)-dependent regulation of urea access to intrabacterial urease . We employed pH- and Ca(2+)-sensitive fluorescent dyes and confocal microscopy to determine the location, rate, and magnitude of pH changes in an H . pylori-AGS cell coculture model, comparing wild-type bacteria with nonpolar ureI-deletion strains (ureI-ve) . Addition of urea at pH 5.5 to the coculture resulted first in elevation of bacterial periplasmic pH, followed by an increase of medium pH and then pH in AGS cells . No change in periplasmic pH occurred in ureI-deletion mutants, which also induced a slower increase in the pH of the medium . Pretreatment of the mutant bacteria with the detergent C(12)E(8) before adding urea resulted in rapid elevation of bacterial cytoplasmic pH and medium pH . UreI-dependent NH(3) generation by intrabacterial urease buffers the bacterial periplasm, enabling acid resistance at the low urea concentrations found in gastric juice . Perfusion of AGS cells with urea-containing medium from coculture at pH 5.5 did not elevate pH(in) or {Ca(2+)}(in), unless the conditioned medium was first neutralized to elevate the NH(3)/NH(4)(+) ratio . Therefore, cellular effects of intrabacterial ammonia generation under acidic conditions are indirect and not through a type IV secretory complex . The pH(in) and {Ca(2+)}(in) elevation that causes the NH(3)/NH(4)(+) ratio to increase after neutralization of infected gastric juice may contribute to the gastritis seen with H . pylori infection.

J Biol Chem, 2000 Jul 28, 275(30), 22704 - 12
Cysteine-scanning mutagenesis of transmembrane segments 4 and 5 of the Tn10-encoded metal-tetracycline/H+ antiporter reveals a permeability barrier in the middle of a transmembrane water-filled channel; Iwaki S et al.; Cysteine-scanning mutants as to putative transmembrane segments 4 and 5 and the flanking regions of Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) were constructed . All mutants were normally expressed . Among the 57 mutants (L99C to I155C), nine conserved arginine-, aspartate-, and glycine-replaced ones exhibited greatly reduced tetracycline resistance and almost no transport activity, and five conserved glycine- and proline-replaced mutants exhibited greatly reduced tetracycline transport activity in inverted membrane vesicles despite their high or moderate drug resistance . All other cysteine-scanning mutants retained normal drug resistance and normal tetracycline transport activity except for the L142C and I143C mutants . The transmembrane (TM) regions TM4 and TM5 were determined to comprise 20 amino acid residues, Leu-99 to Ile-118, and 17 amino acid residues, Ala-136 to Ala-152, respectively, on the basis of N-{(14)C}ethylmaleimide ({(14)C}NEM) reactivity . The NEM reactivity patterns of the TM4 and TM5 mutants were quite different from each other . TM4 could be divided into two halves, that is, a NEM nonreactive periplasmic half and a periodically reactive cytoplasmic half, indicating that TM4 is tilted toward a water-filled transmembrane channel and that only its cytoplasmic half faces the channel . On the other hand, NEM-reactive mutations were observed periodically (every two residues) along the whole length of TM5 . A permeability barrier for a membrane-impermeable sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, was present in the middle of TM5 between Leu-142 and Gly-145, whereas all the NEM-reactive mutants as to TM4 were not accessible to 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, indicating that the channel-facing side of TM4 is located inside the permeability barrier . Tetracycline protected the G141C mutant from the NEM binding, whereas the other mutants in TM4 and TM5 were not protected by tetracycline.

Plant J, 2000 Jul, 23(2), 267 - 78
Cell-type-specific calcium responses to drought, salt and cold in the Arabidopsis root; Kiegle E et al.; Little is known about the signalling processes involved in the response of roots to abiotic stresses . The Arabidopsis root is a model system of root anatomy with a simple architecture and is amenable to genetic manipulation . Although it is known that the root responds to cold, drought and salt stress with increases in cytoplasmic free calcium, there is currently no information about the role(s) of the functionally diverse cell types that comprise the root . Transgenic Arabidopsis with enhancer-trapped GAL4 expression in specific cell types was used to target the calcium reporting protein, aequorin, fused to a modified yellow fluorescent protein (YFP) . The luminescence output of targeted aequorin enabled in vivo measurement of changes in cytosolic free calcium concentrations ({Ca2+}cyt) in specific cell types during acute cold, osmotic and salt stresses . In response to an acute cold stress, all cell types tested as well as plants constitutively expressing aequorin displayed rapid {Ca2+}cyt peaks . However, there were significant quantitative differences between different cell types in terms of their response to cold stress, osmotic stress (440 mM mannitol) and salt stress (220 mM NaCl), implying specific roles for certain cell types in the detection and/or response to these stimuli . In response to osmotic and salt stress, the endodermis and pericycle displayed prolonged oscillations in cytosolic calcium that were distinct from the responses of the other cell types tested . Targeted expression of aequorin circumvented the technical difficulties involved in fluorescent dye injection as well as the lack of cell specificity of constitutively expressed aequorin, and revealed a new level of complexity in root calcium signalling.

Immunology, 2000 Jul, 100(3), 364 - 9
Comparative analysis of integrin expression on monocyte-derived macrophages and monocyte-derived dendritic cells; Ammon C et al.; Both macrophages (MAC) and dendritic cells (DC) are members of the mononuclear phagocyte system (MPS) with monocytes (MO) as common precursor cells . Cells of the MPS are able to take up, process and present antigens to T lymphocytes, thereby inducing a primary or secondary immune response . Adhesion molecules are of crucial importance for the interaction of antigen-presenting cells with immune cells, especially T lymphocytes . By representational difference analysis, we identified CD49c (VLA-3), a member of the beta1-integrin family of adhesion receptors, as differentiation-associated antigen in MO-derived MAC . In contrast, MO-derived DC did not express CD49c mRNA . These data prompted us to compare the integrin expression pattern of MAC and DC . Both cell types showed a low expression of the alpha-chains of the beta1-integrins CD49a, CD49b, CD49d and CD49e, whereas a marked difference was observed for CD49c and CD49f . Expression of both integrins increased during MO to MAC differentiation, but was not detectable on DC . In parallel the beta1-chain (CD29) was clearly up-regulated during MO to MAC differentiation but was only weakly expressed on DC . On the other hand, the beta2-integrins CD11a, CD11b, CD11c and CD18 were all expressed on MAC and DC . Beside their role in cell-cell interaction and adhesion, beta2-integrins are also known as possible binding molecules for bacteria and lipopolysaccharide (LPS), especially for high LPS concentrations . Therefore we investigated the LPS response of MAC versus DC in terms of tumour necrosis factor-alpha (TNF-alpha) release . DC were less responsive to low doses of LPS, which can easily be explained by the very low CD14 expression on DC compared for MAC . In contrast, the TNF-alpha response was comparable to MAC when DC were stimulated with high LPS concentrations . Our results show a specific, differentiation-dependent pattern of beta1- and beta2-integrin expression on in vitro-generated MAC and DC . We suggest that the high expression of CD11/CD18 on DC could be involved in the LPS binding of DC . As LPS is not only an activation but also a differentiation stimulus for DC, the expression of CD11/CD18 on DC may be important for the successful maturation of DC and thereby the initiation of a primary immune response.

J Thorac Imaging, 2000 Jul, 15(3), 173 - 9
Pulmonary nodules and masses after lung and heart-lung transplantation; Schulman LL et al.; The authors assess clinical and radiographic findings of pulmonary nodules and masses after lung and heart-lung transplantation . One hundred and fifty nine patients who survived at least 3 months after lung and heart-lung transplantation were followed by serial chest radiographs for a median of 27 months . Single or multiple lung nodules or masses were noted at chest radiography in 15 (9.4%) of 159 patients . Imaging findings and causes of these nodules and masses were reviewed retrospectively . Infection was found in 10 (6%) of 159 patients . Specific pathogens (11 pathogens in 10 patients) were Aspergillus (n = 4), Mycobacteria (n = 4), and other bacteria (n = 3) . Noninfectious causes were found in 5 (3%) of patients and included B-cell lymphoma (n = 2), bronchogenic carcinoma (n = 2), and pulmonary infarcts (n = 1) . Nodules and masses appeared a median of 11 months after transplantation (range: 0.2 to 36 months) . Five patients (33%) had single lesions; the other 10 (67%) patients had multiple lesions (range 2 to 50) . Aspergillus lesions were most commonly located in the upper lobes, were cavitary in three of four patients, and all were fatal . Nodules and masses arose in the transplanted lung in 12 (80%) of the patients, and in the native lung in 3 (20%) of the patients (2 bronchogenic carcinoma, 1 M . tuberculosis simulating bronchogenic carcinoma) . Nodules and masses detected by chest radiography are not uncommon (9.4%) after lung and heart-lung transplantation . Infections are more common than noninfectious causes of posttransplant nodules and masses . Specific clinical and imaging characteristics may provide clues to etiology.

J Neurogenet, 1999 Nov, 13(3), 157 - 80
A phenylalanine hydroxylase gene from the nematode C . elegans is expressed in the hypodermis; Loer CM et al.; We have identified an aromatic amino acid hydroxylase gene from the nematode C . elegans that likely encodes the worm phenylalanine hydroxylase (PheH) . The predicted amino acid sequence is most similar to that of other PheH and TrpH proteins . Reporter gene fusions and staining with an antibody to mammalian PheH indicate the gene is expressed in hypodermal cells . A fusion protein expressed in bacteria can convert phenylalanine to tyrosine, and, to a lesser extent, tryptophan to 5-hydroxytryptophan . We hypothesize that the protein is necessary to produce additional tyrosine for protein cross-linking in the nematode cuticle.

PDA J Pharm Sci Technol, 2000 May-Jun, 54(3), 264 - 76
Disinfection with flash lamps; Wekhof A; The analysis of published and new data on a pulsed disinfection is presented . It allows the conclusion that the pulsed disinfection mechanism includes both germicidal action of UVC light and a rupture of bacteria due to thermal stress, caused primarily by all UV components of the light pulse . The role of simultaneous cooling and heating of bacteria during a flash lamp pulse has been estimated and a direct detection of such a mechanism is proposed . It is suggested that an optimum pulsed light source for disinfection must have as much percentile content of a broad UV spectrum and a high peak power as is technically justified . Two new applications for pharmaceutics and medicine are suggested.

Am J Ophthalmol, 2000 Jun, 129(6), 800 - 1
Nd:YAG laser corneal disruption as adjuvant treatment for infectious crystalline keratopathy; Daneshvar H et al.; PURPOSE: To report on the use of the Nd:YAG laser corneal disruption in the treatment of infectious crystalline keratopathy . METHOD: Case report . A 52-year-old woman with infectious crystalline keratopathy unresponsive to topical antibiotics was treated with an Nd:YAG laser to the intrastromal crystals . RESULTS: After Nd:YAG laser treatment, the infiltrate completely cleared within 4 weeks . CONCLUSIONS: Nd:YAG laser treatment may be effective in disrupting the protective glycocalyx matrix within the intrastromal crystals, rendering the bacteria susceptible to topical antibiotics . This treatment should be considered for patients with infectious crystalline keratopathy clinically resistant to topical antibiotics.

Science, 2000 Aug 4, 289(5480), 765 - 8
CikA, a bacteriophytochrome that resets the cyanobacterial circadian clock; Schmitz O et al.; The circadian oscillator of the cyanobacterium Synechococcus elongatus, like those in eukaryotes, is entrained by environmental cues . Inactivation of the gene cikA (circadian input kinase) shortens the circadian period of gene expression rhythms in S . elongatus by approximately 2 hours, changes the phasing of a subset of rhythms, and nearly abolishes resetting of phase by a pulse of darkness . The CikA protein sequence reveals that it is a divergent bacteriophytochrome with characteristic histidine protein kinase motifs and a cryptic response regulator motif . CikA is likely a key component of a pathway that provides environmental input to the circadian oscillator in S . elongatus.

Science, 2000 Aug 4, 289(5480), 751 - 4
Aggregation-based crystal growth and microstructure development in natural iron oxyhydroxide biomineralization products; Banfield JF et al.; Crystals are generally considered to grow by attachment of ions to inorganic surfaces or organic templates . High-resolution transmission electron microscopy of biomineralization products of iron-oxidizing bacteria revealed an alternative coarsening mechanism in which adjacent 2- to 3-nanometer particles aggregate and rotate so their structures adopt parallel orientations in three dimensions . Crystal growth is accomplished by eliminating water molecules at interfaces and forming iron-oxygen bonds . Self-assembly occurs at multiple sites, leading to a coarser, polycrystalline material . Point defects (from surface-adsorbed impurities), dislocations, and slabs of structurally distinct material are created as a consequence of this growth mechanism and can dramatically impact subsequent reactivity.

J Mol Biol, 2000 Aug 4, 301(1), 157 - 71
Crystallographic and energetic analysis of binding of selected anions to the yellow variants of green fluorescent protein; Wachter RM et al.; The fluorescence emission of yellow fluorescent proteins (YFPs) has been shown to respond rapidly and reversibly to changes in the concentration of some small anions such as halides; this allows for the use of YFPs as genetically encodable Cl(-) sensors that may be targeted to specific organelles in living cells . Fluorescence is suppressed due to protonation of the chromophore upon anion binding, with a stronger level of interaction at low pH values . At pH 6.0, the apparent dissociation constant (K(app)) for Cl(-) is 32 mM for YFP and 22 mM for YFP-H148Q, whereas at pH 7.5, K(app) is 777 mM and 154 mM, respectively . In the cytosol, YFP-H148Q appears most promising as a halide sensor due to its high degree of sensitivity towards I(-) (K(app)=23 mM at pH 7.5) . To aid in the design of variants with improved levels of specificity and affinity for Cl(-), we solved apo and I(-)-bound crystal structures of YFP-H148Q to 2.1 A resolution . The halide-binding site is found near van der Waals contact with the chromophore imidazolinone oxygen atom, in a small buried cavity adjacent to Arg96, which provides electrostatic stabilization . The halide ion is hydrogen bonded to the phenol group of T203Y, consistent with a mutational analysis that indicates that T203Y is indispensible for tight binding . A series of conformational changes occurs in the amphiphilic site upon anion binding, which appear to be propagated to the beta-bulge region around residue 148 on the protein surface . Anion binding raises the chromophore pK(a) values, since delocalization of the phenolate negative charge over the chromophore skeleton is suppressed . Extraction of microscopic binding constants for the linked equilibrium between anion and proton binding indicates that anion selectivity by YFP is related to hydration forces . Specific suggestions to improve Cl(-) binding to YFP-H148Q based on size and hydration energy are proposed .

Membr Cell Biol, 2000, 13(4), 485 - 501
On the influence of pigment-protein interactions on energy transfer processes in photosynthetic membrane structures . 3 . The FMO complex of Chlorobium tepidum at high pressure; Klevanik AV; The low-temperature absorption spectra of the Chlorobium tepidum FMO bacteriochlorophyll-protein complex at various pressures have been calculated within the framework of mini-exciton theory . The dependences of the Qy transition energies of the monomeric pigments on pressure have been found by means of functional minimization . This functional includes the parameters of both theoretical and experimental absorption spectra at low temperatures and various pressures . The dependences obtained are compared with those derived for the exciton transition energies, which have been obtained by deconvoluting absorption spectra with seven Gaussian components at each pressure . The pressure increase has been shown to result in the increased coupling energy between both the pigment molecules themselves and pigments and amino acid residues . The pigment molecules capable of binding histidines and water molecules have been shown to have the greatest and smallest responses to increased pressure, respectively . The couplings of Bchl molecules with the surrounding amino acid residues have been shown to change both the exciton delocalization index and the exciton distribution between the pigment molecules within the protein subunit; the increased pressure does not change these parameters significantly.

Aust Dent J, 2000 Jun, 45(2), 76 - 82
Preventive dentistry for the general dental practitioner; Walsh LJ; Although the prevalence of dental caries has decreased gradually in the past three decades in the Australian population, dental caries remains the primary reason for tooth loss in Australia . At the community level, total health expenditure on the treatment of dental caries (and its consequences) is substantial . Accordingly, caries prevention is an important focus at both the individual and community levels . This paper outlines the principles upon which modern caries prevention is based and stresses the importance of manipulating the oral environment (in terms of salivary parameters, ions, pH and the oral flora) as a major strategy for effective long-term caries prevention . Practical advice is provided on the correct home use of preventive agents, including chewing gums, chlorhexidine, fluorides, bicarbonate rinses and phosphopeptides.

Gene, 2000 Jul 25, 253(1), 77 - 85
Identification of genes in the genome of the archaeon Methanosarcina mazeii that code for homologs of nuclear eukaryotic molecules involved in RNA processing; Hickey AJ et al.; A 2.6kb fragment of chromosomal DNA from the archaeon Methanosarcina mazeii was sequenced and analyzed, and it was found to contain coding regions for three proteins that were 321, 234, and 193 amino acids (aa) in length . Homologs of the 321-aa protein were found in all archaeal genomes examined, but not in eukaryotic or bacterial genomes, with one exception in the latter . The protein with 234aa (named PrpM) was most similar to the putative protein Prp31p from Methanobacterium thermoautotrophicum, while the 193-aa protein (named FibM) was identified as an archaeal fibrillarin homolog . Prp and fibrillarin proteins are involved in RNA processing in eukaryotes, but their functions in archaea are not yet understood . The M . mazeii PrpM was also similar to three proteins from Saccharomyces cerevisiae: Prp31p, Nop56p, and Nop58p . Prp31p is a pre-mRNA processing protein, while Nop56p and Nop58p are involved in rRNA processing and interact with fibrillarin . No homologs of either protein were found in bacteria . The archaeal fibrillarin was shorter than its eukaryotic counterpart because it lacked the N-terminal glycine-arginine-rich (GAR) domain, present in most eukaryal homologs . The archaeal prp and fibrillarin gene homologs were found adjacent to each other, whereas in eukarya these genes are on separate chromosomes . Sequence signatures typical of the eukaryal molecules were identified in the M . mazeii and the other archaeal molecules studied . The close proximity of the prp and fib genes raises the possibility of a Prp-fibrillarin interaction in archaea.

Vet Microbiol, 2000 Sep 15, 76(1), 81 - 90
Attenuation and immunogenicity of a Brucella abortus htrA cycL double mutant in cattle; Edmonds M et al.; PHE1 is a htrA cycL double gene deletion mutant of virulent Brucella abortus strain 2308 (S2308) which has previously been evaluated in the murine and caprine models of bovine brucellosis . This report describes the results of studies conducted with this mutant in the natural bovine host . Six sexually mature, non-gravid heifers were inoculated via the conjunctival sac with 1 x 10(10) colony forming units (CFU) of either the parental S2308 or the htrA cycL gene deletion mutant, PHE1 . At 4, 7 and 11 days post-inoculation, PHE1 was found to colonize the bovine host at lower levels than S2308 . In a second experiment, eight heifers in mid-gestation were infected with 1 x 10(7) CFU of either strain via the conjunctival sac . The virulent S2308 caused abortions or weak calves in 4/4 cows, while all four cows infected with PHE1 had healthy calves . Furthermore, PHE1 exhibited decreased resistance to killing by cultured bovine neutrophils and macrophages compared to the parental strain . These studies demonstrate that the B . abortus htrA cycL gene deletion mutant PHE1 is highly attenuated in the bovine host when compared to the virulent parental S2308.

Genetics, 2000 Aug, 155(4), 1535 - 42
Genetic analysis of the role of Pol II holoenzyme components in repression by the Cyc8-Tup1 corepressor in yeast; Lee M et al.; The Cyc8-Tup1 corepressor complex is targeted to promoters by pathway-specific DNA-binding repressors, thereby inhibiting the transcription of specific classes of genes . Genetic screens have identified mutations in a variety of Pol II holoenzyme components (Srb8, Srb9, Srb10, Srb11, Sin4, Rgr1, Rox3, and Hrs1) and in the N-terminal tails of histones H3 and H4 that weaken repression by Cyc8-Tup1 . Here, we analyze the effect of individual and multiple mutations in many of these components on transcriptional repression of natural promoters that are regulated by Cyc8-Tup1 . In all cases tested, individual mutations have a very modest effect on SUC2 RNA levels and no detectable effect on levels of ANB1, MFA2, and RNR2 . Furthermore, multiple mutations within the Srb components, between Srbs and Sin4, and between Srbs and histone tails affect Cyc8-Tup1 repression to the same modest extent as the individual mutations . These results argue that the weak effects of the various mutations on repression by Cyc8-Tup1 are not due to redundancy among components of the Pol II machinery, and they argue against a simple redundancy between the holoenzyme and chromatin pathways . In addition, phenotypic analysis indicates that, although Srbs8-11 are indistinguishable with respect to Cyc8-Tup1 repression, the individual Srbs are functionally distinct in other respects . Genetic interactions among srb mutations imply that a balance between the activities of Srb8 + Srb10 and Srb11 is important for normal cell growth.

Appl Environ Microbiol, 2000 Aug, 66(8), 3650 - 3
Thiosulfate disproportionation by Desulfotomaculum thermobenzoicum; Jackson BE et al.; Desulfotomaculum thermobenzoicum, but not Desulfotomaculum nigrificans, Desulfotomaculum ruminis, or Desulfosporosinus orientis, grew by disproportionation of thiosulfate, forming stoichiometric amounts of sulfate and sulfide; sulfite was not disproportionated . The addition of acetate enhanced growth and thiosulfate disproportionation by D . thermobenzoicum compared to those observed with thiosulfate alone.

Appl Environ Microbiol, 2000 Aug, 66(8), 3241 - 8
Three-year study to assess human enteric viruses in shellfish; Le Guyader F et al.; The main pathogenic enteric viruses able to persist in the environment, such as hepatitis A virus (HAV), Norwalk-like virus (NLV), enterovirus (EV), rotavirus (RV), and astrovirus (AV), were detected by reverse transcription-PCR and hybridization in shellfish during a 3-year study . Oyster samples (n = 108), occasionally containing bacteria, were less frequently contaminated, showing positivity for AV (17%), NLV (23%), EV (19%), and RV (27%), whereas mussel samples, collected in areas routinely impacted by human sewage, were more highly contaminated: AV (50%), HAV (13%), NLV (35%), EV (45%), and RV (52%) . Sequences obtained from HAV and NLV amplicons showed a great variety of strains, especially for NLV (strains close to Mexico, Snow Mountain Agent, or Norwalk virus) . Viral contamination was mainly observed during winter months, although there were some seasonal differences among the viruses . This first study of virus detection over a fairly long period of time suggests that routine analysis of shellfish by a molecular technique is feasible.

Allergy, 2000, 55 Suppl 61, 42 - 5
Respiratory infections and asthma; Micillo E et al.; Clinical and experimental evidence suggests an important role for respiratory infections in the development of asthma attacks . Viral upper respiratory infections have been associated with 80% of asthma exacerbations in children and 50% of all asthma episodes in adults . Human rhinovirus has been implicated as the principal virus associated with asthma episodes . Separate studies indicate that atypical bacteria such as Chlamydia pneumoniae and Mycoplasma pneumoniae may precipitate asthma symptoms . Although not completely clarified, the intricate pathogenetic mechanisms by which viral infections promote asthma attacks have been extensively investigated in recent years . By contrast, it has not yet been established whether atypical bacterial infections are an epiphenomenon or a pathogenic event in asthma.

Fungal Genet Biol, 2000 Mar, 29(2), 61 - 71
pH regulation of gene expression in fungi; Denison SH; A system for the regulation of gene expression by ambient (extracellular) pH was first identified in Aspergillus nidulans . This system consists of the products of the pacC and palA, B, C, F, H, and I genes . pacC encodes a zinc finger transcription factor and these pal genes encode components of an ambient pH signal transduction pathway . pH regulatory systems have also been identified in other fungi . Components of these regulatory systems are homologous to those in A . nidulans . This review describes the pH regulatory system in A . nidulans and the history of this research and how it relates to other systems . pH regulation in bacteria and animal cells is also briefly discussed.

Nature, 2000 Jul 20, 406(6793), 315 - 8
A tertiary interaction that links active-site domains to the 5' splice site of a group II intron; Boudvillain M et al.; Group II introns are self-splicing RNAs that are commonly found in the genes of plants, fungi, yeast and bacteria . Little is known about the tertiary structure of group II introns, which are among the largest natural ribozymes . The most conserved region of the intron is domain 5 (D5), which, together with domain 1 (D1), is required for all reactions catalysed by the intron . Despite the importance of D5, its spatial relationship and tertiary contacts to other active-site constituents have remained obscure . Furthermore, D5 has never been placed directly at a site of catalysis by the intron . Here we show that a set of tertiary interactions (lambda-lambda') links catalytically essential regions of D5 and D1, creating the framework for an active-site and anchoring it at the 5' splice site . Highly conserved elements similar to components of the lambda-lambda' interaction are found in the eukaryotic spliceosome.

Med Microbiol Immunol (Berl), 2000 Jun, 188(4), 161 - 8
Site-specific invasion of the basal ganglia by Nocardia asteroides GUH-2; Beaman BL et al.; Nocardia asteroides GUH-2 (GUH-2) invades the nigrostriatal region of the brain in mice {15} . Selective dopaminergic neuronal dropout in the substantia nigra results in parkinsonian changes characterized by movement disorders responsive to L-dopa {15} . This is the only reported example of an experimental bacterial model for parkinsonism . Following i.v . inoculation of GUH-2 into the non-human primate Macaca fasicularius, the nocardiae preferentially invaded and grew within the basal ganglia (substantia nigra, caudate, putamen, and globus pallidus) often without inducing apparent inflammation . Reduced, limited growth of nocardiae occurred in the white matter of the cerebral cortex, medulla, and hippocampus, whereas neither significant adherence to nor growth within the meninges was observed . Twenty-four hours after injection, nocardial cells were found within capillary endothelial cells, the basal lamina, neurons, astroglia and in axonal extensions . The bacteria, in endothelial cells, were surrounded by a unit membrane, but in the basal lamina they appeared to be free and not membrane bound . After the organisms passed into the brain parenchyma, the nocardiae once again became surrounded by membrane, often being encapsulated by numerous layers with the innermost layer tightly adherence to the bacterial surface . There was a propensity for nocardial growth within and along myelinated axons, either with or without disruption to the surrounding myelin sheath . There was electron microscopic evidence that the nocardiae induced a neurodegenerative response especially in the substantia nigra region . Thus, the early interactions of GUH-2 within the primate brain appeared to be similar to those reported in the mouse.

Dtsch Tierarztl Wochenschr, 2000 Jun, 107(6), 221 - 7
{Effect of progesterone on the immune system in consideration of bovine placental retention}; Scheibl P et al.; Effects of progesterone on the immune system of different species are presented by a literature study . Elevated progesterone concentrations in blood result in more severe course and longer persistence of bacteria in infectious diseases and decrease of the clearance of antibody-coated erythrocytes in vivo . Progesterone suppresses specific components of the immune system and natural killer (NK) cell activity while it has a mainly positive influence on other nonspecific components . It suppresses blastogenesis and cytotoxicity of lymphocytes and increases synthesis of asymmetric antibodies without effector function to block fetal antigens . Progesterone increases the concentration of leucocytes in blood, random migration and chemotactic ability, synthesis of reactive oxygen intermediates (ROI) and antibody-dependent cellular cytotoxicity of polymorphonuclear leucocytes (PMN) and expression of complement receptors, synthesis of ROI and phagocytosis of peritoneal macrophages . In the uterus mainly immunosuppressive effects of progesterone are described (decreased migration of PMN into the gravid uterus, persistence of bacteria or transplants, synthesis of immunosuppressive uterine milk proteins, decreased random migration of PMN) except for an increased content of IgA in the secretory products indicating a significance of progesterone in the pathogenesis of endometritis mainly in the cow and bitch . No correlation could be found between blood concentrations of progesterone and placental retention in cattle . Progesterone takes part in avoiding immunologic aggression of the maternal organism against the semiallogenic conceptus by suppressing specific components of the immune system and NK cell activity . Positive effects on nonspecific components of the immune system could be regarded as compensatory mechanisms to maintain the efficiency of the complete immune system.

Arerugi, 2000 Jun, 49(6), 463 - 71
{A relationship between a psychosomatic and a skin condition in patients with atopic dermatitis}; Hariya T et al.; Atopic dermatitis (AD) has been clinically well-known to be frequently exacerbated by some psychosomatic stress . In this study, we examined a relationship between a psychosomatic and a skin condition in patients with AD . Visual analogue scale (VAS) for the grade of general physical condition, mental stress and others were reported daily for themselves, while skin physiological parameters, resident skin bacteria and a psychological questionnaire (POMS) were measured every 2 weeks . It was observed that tense-anxious and depressive scores of POMS tended to correlate with skin conductance, a skin clinical score and a number of total skin bacteria and others . These results suggest that changes of psychosomatic condition reciprocally correlate with the exacerbation and improvement of skin symptom in patients with AD.

Zhonghua Yi Xue Za Zhi, 1998 Jul, 78(7), 494 - 7
{Development of mouse and rat model of Helicobacter pylori infection}; Zeng Z et al.; OBJECTIVE: To develop different rodent model of H . pylori infection as to meet different requirement for different purpose of study . METHODS: 40 two-grade Wistar rats, 40 two-grade C57BL/6 mice and 40 SPF BALB/c mice were randomly divided into two groups: experimental and control groups . Animals in the experimental group were inoculated orally Hp strain (Sydney Strain 1, SS1), 0.4 ml of inoculum per mouse, 1.5 ml per rat (10(9) organisms/ml) five times for a week . In 4, 8, 12 and 24 weeks after the last bacteria inoculum, 5 animals in the experimental and control groups were sacrificed respectively . Histology and Hp colonization were assessed by HE staining, Gimesa staining, Urease test, and Hp culture . RESULTS: In 4 weeks, in all experimental animals bacteria were clearly visible at antrum and body, but the number of Hp colonization varied according to the animal strain . Heavy colonization was seen in C57BL/6 in antrum and body, and in BALB/c and Wistar colonization was located mainly at antrum, less at body, which tended to increase over the experiment time, especially in Wistar . Hp was negative in the controls . All animals had no inflammatory changes in 4 weeks, however, in 8, 12 and 24 weeks in Balb/c . Wistar and C57BL/6 of the experimental group, mild to moderate chronic active gastritis was observed in antrum and body, which increased in severity over time, atrophy gastritis was still not seen in 24 weeks . In contrast, in the controls, no remarkable changes were detected . CONCLUSIONS: SS1 Hp can colonize in the glandular stomach mucosa of BALB/c, C57BL/6 mice and Wistar rats and leads to chronic active gastritis in long-term study . SS1 mouse and rat model is adaptable for animal experimentation of H . pylori including vaccine studies, screening for novel therapeutics and investigation of mechanisms of pathogenesis.

Aust Vet J, 2000 Jul, 78(7), 472 - 7
Effect of the microclimate on horses during international air transportation in an enclosed container; Thornton J; OBJECTIVE: To determine if the microclimate is detrimental to horses during international air transportation in an enclosed container . PROCEDURE: On each of two 12 h and two 24 h flights three horses were transported in an enclosed container designed to prevent exposure to insect vectors . Heart rates were monitored throughout and blood samples were collected periodically . Air in the container was sampled for bacteria and fungal spores and the temperature and relative humidity were recorded inside and outside the container periodically during the flight . On the two 12 h flights similar observations were made on three horses transported in regular open containers, which were used as controls . RESULTS: Heart rates during the flights reflected any agitation of the horses . Agitation was only mild and generally associated with take-off and landing . There were no changes in haematological or blood biochemical values that suggested any detrimental effects of the flights . The temperature in the Airstable was relatively constant during each flight (means ranged from 18.7 to 23.4 degrees C) and was significantly warmer than in the cargo hold (range 13.9 to 18.3 degrees C) . Relative humidity fluctuated more widely and reflected the ambient humidity during airport stops . The numbers of bacteria and fungal spores in the Airstable air varied during the flights but were of no apparent significance to the horses' health . CONCLUSION: The Airstable proved a convenient means to transport horses on international flights and caused no discernible ill effects on the horses studied.

J Clin Microbiol, 2000 Aug, 38(8), 2943 - 8
Characterization of Bartonella clarridgeiae flagellin (FlaA) and detection of antiflagellin antibodies in patients with lymphadenopathy; Sander A et al.; Cat scratch disease (CSD) is a frequent clinical outcome of Bartonella henselae infection in humans . Recently, two case reports indicated Bartonella clarridgeiae as an additional causative agent of CSD . Both pathogens have been isolated from domestic cats, which are considered to be their natural reservoir . B . clarridgeiae and B . henselae can be distinguished phenotypically by the presence or absence of flagella, respectively . Separation of the protein content of purified flagella of B . clarridgeiae by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis indicated that the flagellar filament is mainly composed of a polypeptide with a mass of 41 kDa . N-terminal sequencing of 20 amino acids of this protein revealed a perfect match to the N-terminal sequence of flagellin (FlaA) as deduced from the sequence of the flaA gene cloned from B . clarridgeiae . The flagellin of B . clarridgeiae is closely related to flagellins of Bartonella bacilliformis and several Bartonella-related bacteria . Since flagellar proteins are often immunodominant antigens, we investigated whether antibodies specific for the FlaA protein of B . clarridgeiae are found in patients with CSD or lymphadenopathy . Immunoblotting with 724 sera of patients suffering from lymphadenopathy and 100 healthy controls indicated specific FlaA antibodies in 3.9% of the patients' sera but in none of the controls . B . clarridgeiae FlaA is thus antigenic and expressed in vivo, providing a valuable tool for serological testing . Our results further indicate that B . clarridgeiae might be a possible etiologic agent of CSD or lymphadenopathy . However, it remains to be clarified whether antibodies to the FlaA protein of B . clarridgeiae are a useful indicator of acute infection.

Crit Care Med, 2000 Jul, 28(7), 2567 - 72
Inhibition of p38 mitogen activate kinase attenuates the severity of pancreatitis-induced adult respiratory distress syndrome; Denham W et al.; OBJECTIVE: Adult respiratory distress syndrome (ARDS) is responsible for a significant portion of the morbidity and mortality during severe acute pancreatitis . Because inflammatory mediators such as tumor necrosis factor (TNF)-alpha and nitric oxide (NO) produced within the lungs have been implicated in sepsis-induced ARDS, we aimed to determine the role of these mediators in pancreatitis-induced ARDS using a model whereby ascites from animals with pancreatitis is transferred to otherwise healthy animals resulting in pulmonary injury . DESIGN: Prospective, randomized, controlled trial . SETTING: Research laboratory at a university medical school . SUBJECTS: Pathogen-free Sprague-Dawley rats weighing 225-250 g . INTERVENTIONS: Sterile, endotoxin- and cytokine-free pancreatic ascites tested for interleukin (IL)-1beta , TNF-alpha, interferon-gamma, and IL-6 was obtained from rats 18 hrs after the induction of severe, acute pancreatitis . Ascites was subsequently administered intravenously (20 mL/kg) to healthy rats . Sham animals were administered intravenous saline . Healthy animals administered intravenous ascites were randomized to receive a single intraperitoneal injection of the p38 mitogen activated kinase inhibitor CNI-1493 (1 mg/kg) or vehicle . MEASUREMENTS: Pulmonary injury was assessed at 24 hrs by histology and leukocyte and protein concentrations via bronchoalveolar lavage . Pulmonary TNF-alpha protein was detected by immunohistochemistry . Serum nitrite, as a measure of NO production, was measured utilizing the Griess reaction . MAIN RESULTS: After the intravenous administration of pancreatic ascites, the number of leukocytes and the protein concentration within the bronchoalveolar fluid were increased and pulmonary histology was worsened consistent with acute lung injury (all p < .001 vs . sham) . Each of these variables of pulmonary injury was lessened in animals receiving CNI-1493 and intravenous ascites (p < .05 vs . vehicle) . Pulmonary TNF-alpha protein and serum nitrites were decreased with the administration of CNI-1493 (p < .005 vs . vehicle) . CONCLUSIONS: A component of pancreatic ascites other than endotoxin, bacteria, or cytokines (IL-1beta, TNF, interferon-gamma, or IL-6) is capable of inducing ARDS in healthy animals . Inhibition of p38 mitogen activated kinase decreases the pulmonary injury through attenuated production of TNF-alpha and NO suggesting a primary role for these mediators in pancreatitis-induced ARDS.

J Gastroenterol Hepatol, 2000 Jun, 15(6), 617 - 21
Modified rapid urease test for Helicobacter pylori detection in relation to an immunohistochemical stain; Tokunaga Y et al.; BACKGROUND: The rapid urease test and touch cytology have been used for the rapid detection of Helicobacter pylori infection . Recently, a modified rapid urease (MRU) test, which provides results in 20 min has been available on a commercial basis . To date, few reports have evaluated the accuracy of this test . This study evaluated the sensitivity, specificity, and accuracy of the MRU test and touch cytology to detect H . pylori in relation to the density of H . pylori infection determined semi-quantitatively by using immunohistochemical stains . METHODS: Biopsy specimens obtained from a total of 60 patients who underwent endoscopy for evaluation of gastroduodenal diseases were studied by using the MRU test, Giemsa stain for touch smear tissue and histological methods . An immunohistochemical stain was used as a standard, and the density of H . pylori infection was graded according to the number of individual bacteria seen as follows: grade 0 = 0; grade 1+ = 1-9; grade 2+ = 10-29; grade 3+ = 30-99; grade 4+ > or = 100 . The severity of gastritis was evaluated histologically in each specimen and compared with the density of H . pylori infection . RESULTS: The MRU test had an overall sensitivity of 73%, specificity of 100% and accuracy of 85% . The Giemsa stain had a sensitivity of 91%, specificity of 100% and accuracy of 95%.The sensitivities of the MRU test and Giemsa stain decreased in mild H . pylori infection . In the MRU test, the sensitivity was 47% when the density of H . pylori infection was 1+, while 80-100% sensitivities were obtained when the densities of infection were > or = 2+ . With the Giemsa stain, the sensitivity was 80% when the density was 1+, while the sensitivity increased to 100% when the densities were > or = 2+ . The severity of gastritis determined by the Rauws scores showed a positive correlation with the density of H . pylori infection as evaluated by immunohistochemical staining . CONCLUSIONS: The MRU test had high sensitivity and specificity for moderate to severe H . pylori infection, but it may result in false-negative results in tests for mild infection . As the MRU test has the advantages of shorter incubation times and low cost, a combination of the MRU test and the Giemsa stain for touch cytology may be the most time- and cost-efficient tests in a clinical setting for the diagnosis of H . pylori infection.

Nihon Kokyuki Gakkai Zasshi, 2000 May, 38(5), 398 - 402
{Squamous cell type lung cancer that produced granulocyte colony-stimulating factor}; Hirokawa R et al.; A 73-year-old man was hospitalized because of weight loss and fever . Laboratory data showed marked leukocytosis (21,200/mm3), granulocytosis (89.7%), thrombocytosis (47.8 x 10(4)/mm3), increased CRP (15.8 mg/dl), and increased SCC (5.0 ng/ml) . Chest X-ray films demonstrated a mass shadow in the right upper lung field . Chest computed tomographic scans revealed a mass shadow 58 mm in diameter with mediastinal pleural invasion in the right S1 . Right upper lobectomy and dissection of regional lymph nodes was performed under a diagnosis of lung cancer (squamous cell carcinoma, T3 N0 M0 stage IIB) with concomitant infection . Serum G-CSF was 234 pg/ml pre-operatively and 68.8 pg/ml postoperatively . The cytoplasm of tumor cells stained positively with anti-recombinant human G-CSF monoclonal antibody . No general bacteria or mycobacteria were detected within the specimen . Postoperatively, the patient's white blood cell count, platelet count, and CRP level soon decreased, and the fever disappeared . We diagnosed the disease as G-CSF-producing squamous cell type lung cancer.

Parazitologiia, 2000 May-Jun, 34(3), 234 - 40
{An electron microscopic study of Borrelia in the body of the female ixodid tick Ixodes persulcatus}; Amosova LI; Borrelia burgdorferi s . lato in naturally infected females of tick Ixodes persulcatus were examined by transmission electron microscopy . The Borreliae were found in midgut and ovary . Location and ultrastructure of bacteria indicate extracellular migration through the midgut epithelium as a preferential way . In gonad, the borreliae intracellular situate in ovarian epithelium and oocytes before and at the beginning of vitellogenesis . The demonstration of numerous spirochetes in the oocytes provides the support for transovarial transmission of the agent . Two morphological types of borreliae were observed.

Pediatrics . 2000 Aug;106(2):E27.
Kawasaki disease: A brief history; Burns JC et al.; Tomisaku Kawasaki published the first English-language report of 50 patients with Kawasaki disease (KD) in 1974 . Since that time, KD has become the leading cause of acquired heart disease among children in North America and Japan . Although an infectious agent is suspected, the cause remains unknown . However, significant progress has been made toward understanding the natural history of the disease and therapeutic interventions have been developed that halt the immune-mediated destruction of the arterial wall . We present a brief history of KD, review progress in research on the disease, and suggest avenues for future study . Kawasaki saw his first case of KD in January 1961 and published his first report in Japanese in 1967 . Whether cases existed in Japan before that time is currently under study . The most significant controversy in the 1960s in Japan was whether the rash and fever sign/symptom complex described by Kawasaki was connected to subsequent cardiac complications in a number of cases . Pathologist Noboru Tanaka and pediatrician Takajiro Yamamoto disputed the early assertion of Kawasaki that KD was a self-limited illness with no sequelae . This controversy was resolved in 1970 when the first Japanese nationwide survey of KD documented 10 autopsy cases of sudden cardiac death after KD . By the time of the first English-language publication by Kawasaki in 1974, the link between KD and coronary artery vasculitis was well-established . KD was independently recognized as a new and distinct condition in the early 1970s by pediatricians Marian Melish and Raquel Hicks at the University of Hawaii . In 1973, at the same Hawaiian hospital, pathologist Eunice Larson, in consultation with Benjamin Landing at Los Angeles Children's Hospital, retrospectively diagnosed a 1971 autopsy case as KD . The similarity between KD and infantile periarteritis nodosa (IPN) was apparent to these pathologists, as it had been to Tanaka earlier . What remains unknown is the reason for the simultaneous recognition of this disease around the world in the 1960s and 1970s . There are several possible explanations . KD may have been a new disease that emerged in Japan and emanated to the Western World through Hawaii, where the disease is prevalent among Asian children . Alternatively, KD and IPN may be part of the spectrum of the same disease and clinically mild KD masqueraded as other diseases, such as scarlet fever in the preantibiotic era . Case reports of IPN from Western Europe extend back to at least the 19th century, but, thus far, cases of IPN have not been discovered in Japan before World War II . Perhaps the factors responsible for KD were introduced into Japan after the World War II and then reemerged in a more virulent form that subsequently spread through the industrialized Western world . It is also possible that improvements in health care and, in particular, the use of antibiotics to treat infections caused by organisms including toxin-producing bacteria reduced the burden of rash/fever illness and allowed KD to be recognized as a distinct clinical entity . Itsuzo Shigematsu, Hiroshi Yanagawa, and colleagues have conducted 14 nationwide surveys in Japan . These have indicated that: 1) KD occurred initially in nationwide epidemics but now occurs in regional outbreaks; 2) there are approximately 5,000 to 6,000 new cases each year; 3) current estimates of incidence rates are 120 to 150 cases per 100,000 children <5 years old; 4) KD is 1.5 times more common in males and 85% of cases occur in children <5 years old; and 5) the recurrence rate is low (4%) . In 1978, David Morens at the Centers for Disease Control and Prevention published a case definition based on Kawasaki's original criteria . The Centers for Disease Control and Prevention developed a computerized database in 1984, and a passive reporting system currently exists in 22 states . Regional investigations and national surveys suggest an annual incidence of 4 to 15 cases per 100 000 children <5 years o

Dtsch Med Wochenschr, 2000 Jun 23, 125(25-26), 789 - 93
{The value of bronchoalveolar lavage in the clarifying of HIV-associated lung diseases}; Ersch J et al.; BACKGROUND AND OBJECTIVE: Examination of sputum is the first diagnostic step in the evaluation of HIV-infected patients with pulmonary complications . We were interested in the indications and the diagnostic value of subsequent bronchoscopy and bronchoalveolar lavage (BAL) and especially the importance of so-called atypical bacteria . PATIENTS AND METHODS: We analysed 59 consecutive BALs performed in HIV positive patients with pulmonary symptoms (48 men, 11 women, age 22-70 years, 36 in stage C) . Culture for Legionella pneumophila and PCR analysis for Mycoplasma pneumoniae and Chlamydia pneumoniae were done in all cases . RESULTS: In 50 patients (85%) there was a clear indication for performing a BAL because either an infectious agent could no be detected on sputum examination (n = 36), or the patient's sputum production was insufficient (n = 14) . In 30 (60%) of these cases bronchoscopy and BAL were able to establish a diagnosis . The most frequent agent was Pneumocystis carinii (n = 12) . In contrast, BAL was able to establish coinfection in patients with positive sputum samples in one case only . So-called atypical bacteria were not found . CONCLUSION: In patients with a clear indication, especially a non-diagnostic sputum examination, BAL plays an important role in the work-up of HIV-associated pulmonary disease . The search for so-called atypical bacteria yields unsatisfactory results.

Immunol Today, 2000 Aug, 21(8), 371 - 8
Natural and designer binding sites made by phage display technology; Hoogenboom HR et al.; In the past decade, the drive to develop completely human antibodies for human therapy has led to the development of phage display technology . This technology is able to deliver the ultimate in antibody engineering, that is, high-affinity fully human antibodies to any antigen of choice . Here, this application of phage display technology is reviewed, and the many other antibody-engineering avenues this technology offers are highlighted.

Immunol Today, 2000 Aug, 21(8), 364 - 70
Of mice and men: hybridoma and recombinant antibodies; Little M et al.; Thousands of mouse monoclonal antibodies have been produced from hybridomas over the past 25 years . The same technique can now be used to clone human antibodies from transgenic mice . Full-length antibodies and recombinant fragments engineered for various diagnostic and therapeutic applications can be obtained in reasonably large amounts after expression in mammalian cells, milk and plants.

MMWR Recomm Rep, 2000 Jul 14, 49(RR-8), 3 - 17
Compendium of measures to control Chlamydia psittaci infection among humans (psittacosis) and pet birds (avian chlamydiosis), 2000 . Centers for Disease Control and Prevention; Reduced numbers of Langerhans cells and increased HLA-DR expression in keratinocytes in the oral gingival epithelium of HIV-infected patients with periodontitis; Department of Oral Biology, University of Oslo, NorwayBACKGROUND: HIV-seropositive (HIV+) patients become increasingly susceptible to periodontal diseases as HIV infection proceeds . We have previously shown that HIV+ patients with chronic marginal periodontitis (CMP) have remarkably increased numbers of gingival plasma cells in the connective tissue underlying the oral gingival epithelium, but depressed specific serum IgG levels towards periodontopathogenic bacteria . Langerhans cells (LC) and keratinocytes (KC) are antigen-presenting cells that are important in promoting immune responses . METHOD: In this study we examined, by means of immunofluorescence, the distribution and numbers of LC and activated KC in biopsies taken from inflamed periodontal sites in HIV+ and HIV patients with CMP . RESULTS: In the pocket epithelium in both patient groups, basal layer KC expressed HLA-DR molecules . In the oral gingival epithelium of HIV+ patients, basal layer KC also expressed HLA-DR molecules and numbers of LC were decreased as compared with HIV persons . CONCLUSION: The findings suggest that the oral gingiva in HIV+ patients may be affected by inflammation.

J Clin Periodontol, 2000 Jul, 27(7), 489 - 93
Fc(alpha) receptor I (CD89) on neutrophils in periodontal lesions; Yuan ZN et al.; AIMS: In this study, we have examined the occurrence of FcalphaRI-bearing cells in gingival tissue, gingival fluid and blood, in search for possible roles of IgA and FcalphaRI in periodontal lesions . METHODS: Gingival biopsies from inflamed and healthy sites were obtained from patients with chronic marginal periodontitis . Sections of inflamed gingiva were examined by immunofluorescence techniques and compared to sections from healthy sites . Smears were made from blood and gingival crevicular fluid and similarly studied . RESULTS: Dense infiltrates of neutrophils with strong expression of FcalphaRI (and FcgammaRIII) were found in connective tissue and epithelium of the apical part of periodontal pockets from diseased sites . In contrast, only few such cells were found in healthy gingiva from the same patients . Neutrophils in gingival fluid, tissue and blood expressed FcalphaRI with similar intensity, whereas the expression of FcgammaRIII was significantly decreased in gingival crevicular fluid . Considerable numbers of bacteria from gingival plaque were found to be covered by IgA . CONCLUSION: It is suggested that FcalphaRI on neutrophils may play an important role in elimination of IgA-opsonized bacteria, both in periodontal tissue and the adjacent pockets.

J Periodontol, 2000 Jun, 71(6), 948 - 54
Occurrence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia in progressive adult periodontitis; Lopez NJ; BACKGROUND: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia are the major periodontal bacteria species in most forms of progressive periodontitis in Scandinavia and the United States . The occurrence of periodontal pathogens appears to be different in subjects of different ethnic origin, and geographical factors may influence the distribution of these species . METHODS: The occurrence of A . actinomycetemcomitans, P . gingivalis, and P . intermedia was determined using a DNA probe in progressive adult periodontitis in Chileans . Sixty patients (mean age 43.6 +/- 8 years) who had not previously received any type of periodontal therapy were selected . Bleeding on probing, probing depth, and clinical attachment level measurements were made with an automated probe . Patients were monitored at 2-month intervals until at least 2 sites exhibited > or =2 mm attachment loss . Two subgingival plaque samples from active sites were taken in 56 subjects and matched with 2 plaque samples from inactive sites in the same individuals . RESULTS: P . gingivalis was found in 75% of active sites and in 59.7% of inactive sites in 96% of the patients (P = 0.022) . P . gingivalis at high levels of detection was significantly more frequent in active sites (48.2%) than in inactive sites (31.2%) (P = 0.014) . A . actinomycetemcomitans was detected in 6.25% of active sites and in 12.5% of inactive sites in 11.6% of patients . P . intermedia was found in 33% of patients and at a significantly higher proportion in active sites (49.1%) than in inactive sites (30.3%) (P = 0.006) . There was a significantly higher proportion of inactive sites (34.8%) than active sites (19.6%) without any of the 3 pathogens (P = 0.016) . Bleeding on probing was significantly more associated with active sites with high levels of P . gingivalis and with active sites with P . intermedia than with inactive sites . CONCLUSIONS: A high prevalence of P . gingivalis and P . intermedia was found in adult periodontitis, and the occurrence of these bacteria appears to be higher in Chileans than in other populations . No apparent association exists between A . actinomycetemcomitans and progressive adult periodontitis in Chileans.

J Periodontol, 2000 Jun, 71(6), 929 - 39
Interleukin-8 and granulocyte elastase in gingival crevicular fluid in relation to periodontopathogens in untreated adult periodontitis; Jin L et al.; BACKGROUND: This study aimed to determine the relationships among interleukin (IL)-8 and granulocyte elastase levels in gingival crevicular fluid (GCF) and the concomitant presence of periodontopathogens in untreated adult periodontitis . METHODS: GCF and subgingival plaque samples were collected from 16 patients with untreated adult periodontitis and 10 healthy control subjects . IL-8 levels were determined by enzyme-linked immunosorbent assay (ELISA) . Granulocyte elastase was analyzed with a neutrophilic granulocyte-specific, low molecular weight and chromogenic substrate, L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide, and the maximal rate of elastase activity (MR-EA) was calculated . Five DNA probes were used to detect the presence of A . actinomycetemcomitans (A.a.), B . forsythus (B.f.), P . gingivalis (P.g.), P . intermedia (P.i.), and T . denticola (T.d.) . RESULTS: Lower IL-8 concentrations and higher granulocyte elastase activities were found in patients than in healthy controls as well as in diseased conditions co-infected with B.f., P.g., P.i., and T.d . as compared to healthy conditions without the target species (P <0.05) . IL-8 concentrations were positively correlated with MR-EA levels in the periodontitis conditions co-infected with B.f., P.g., P.i., and T.d . (P <0.05) . A wide range of IL-8 concentrations was found among 15 patients when the periodontitis condition was characterized by co-infection with B.f., P.g., P.i., and T.d . MR-EA levels in the high IL-8 group of subjects were significantly higher than those in the low IL-8 group of subjects (P <0.01) . CONCLUSIONS: The present study shows that the local host-bacteria interactions in untreated periodontitis are diverse in terms of the intensity of inflammatory responses measured by IL-8-related granulocyte elastase activity in GCF . This might reflect different phases of the inflammatory response due to shifts in host-bacteria interactions and therefore be indicative of a range of periodontal disease activity levels.

Methods Enzymol, 2000, 322, 325 - 45
Production of recombinant TRAIL and TRAIL receptor: Fc chimeric proteins; Schneider P; The tumor necrosis factor (TNF)/TNF receptor (TNFR) families of ligands and receptors are implicated in a variety of physiological and pathological processes and regulate cellular functions as diverse as proliferation, differentiation, and death . Recombinant forms of these ligands and receptors can act to agonize or antagonize these functions and are therefore useful for laboratory studies and may have clinical applications . A protocol is presented for the expression and purification of dimeric soluble receptors fused to the Fc portion of human IgG1 and of soluble, N-terminally Flag-tagged ligands . Soluble recombinant proteins are easier to handle than membrane-bound proteins and the use of tags greatly facilitates their detection and purification . In addition, some tags may provide enhanced biological activity to the recombinant proteins (mainly by oligomerization and stabilization effects) and facilitate their functional characterization . Expression in bacterial (for selected ligands) and eukaryotic expression systems (for ligands and receptors) was performed using M15 pREP4 bacteria and human embryonic kidney 293 cells, respectively . The yield of purified protein is about 1 mg/liter for the mammalian expression system and several milligrams per liter for the bacterial expression system . Protocols are given for a specific ligand-receptor pair, namely TRAIL (Apo-2L) and TRAIL receptor 2 (DR5), but can be applied to other ligands and receptors of the TNF family.

FEMS Microbiol Lett, 2000 Aug 1, 189(1), 55 - 9
Hemin-dependent growth and hemin binding of Bartonella henselae; Sander A et al.; Bartonella henselae causes cat-scratch disease and bacillary angiomatosis peliosis . The bacteria reside in erythrocytes of asymptomatic cats, which represent the natural reservoir for this pathogen . B . henselae is usually grown on blood-enriched media . Growth experiments on Brucella medium without blood demonstrated that heme compounds are essential for the growth of B . henselae and can completely substitute the addition of blood components . The heme precursor protoporphyrin IX alone, or in combination with FeCl(2) or FeCl(3), as well as transferrin or lactoferrin did not support growth, indicating that B . henselae cannot synthesize heme itself . Hemin supported growth even when free iron was chelated, indicating that hemin is also used as an iron source . Binding assays showed that hemin starvation increased the binding capacity of B . henselae for hemin, providing evidence that the bacteria carry a specific hemin uptake system, which might be regulated by hemin.

Biochim Biophys Acta, 2000 Jul 26, 1475(3), 383 - 9
N-glycosylation site occupancy of rat alpha-1,3-fucosyltransferase IV and the effect of glycosylation on enzymatic activity; Baboval T et al.; All mammalian alpha-1,3-fucosyltransferases (Fuc-Ts) so far characterized have potential N-glycosylation sites, but the role of these sites in enzymatic activity or localization has not been investigated . When one member of this family, rFuc-TIV, is expressed in bacteria, the unglycosylated form of rFuc-TIV has no detectable enzymatic activity . The two potential N-glycosylation sites of rFuc-TIV were mutated to determine site occupancy and the effect of site occupancy on enzyme activity and targeting of this enzyme . Results obtained with singly mutated forms of rFuc-TIV indicate that both sites are occupied in mammalian cells . Lack of glycosylation at sites 117-119, 218-220, or both of these sites, decreased enzyme activity to approximately 64%, 5% or 1%, respectively, of that seen in the unmutated enzyme . These results show that N-glycosylation is necessary for optimal enzyme activity, with glycosylation at site 218-220 playing the major role . However, N-glycosylation does not appear to affect the major intracellular location of the enzyme, as immunocytochemistry reveals the same perinuclear pattern of staining for the unglycosylated mutants as is seen for the wild-type rFuc-TIV in transfected cells.

FEMS Microbiol Lett, 2000 Jul 15, 188(2), 129 - 33
Phospholipase A secreted by Legionella pneumophila destroys alveolar surfactant phospholipids; Flieger A et al.; Destruction of alveolar surfactant phospholipids by bacterial phospholipases is suggested to be a major virulence factor involved in bacterial pneumonia . Since Legionella pneumophila secretes phospholipase A, we analyzed phospholipid degradation in natural bovine surfactant by L . pneumophila . Phospholipids were reduced in amount after incubation with bacteria or culture supernatant of L . pneumophila serogroup 6 . Free fatty acids and lysophosphatidylcholine were formed, the latter is known to be highly cytotoxic . Surface tension of surfactant as determined by pulsating bubble surfactometer increased significantly compared to the control . Phospholipase A activity seems to be a powerful agent of legionellae in causing lung disease.

Biochemistry, 2000 Aug 1, 39(30), 8870 - 7
Oligomerization of mouse alpha 1-syntrophin and self-association of its pleckstrin homology domain 1 containing sequences; Oak SA et al.; Syntrophins are known to self-associate to form oligomers . Mouse alpha 1-syntrophin sequences were produced as chimeric fusion proteins in bacteria and were found to also oligomerize and in a micromolar Ca(2+)-dependent manner . The oligomerization was localized to the N-terminal pleckstrin homology domain (PH1) or adjacent sequences; the second, C-terminal PH2 domain did not show oligomerization . PH1 was found to self-associate, and calmodulin or Ca(2+)-chelating agents such as ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) could effectively prevent this oligomerization . A single calmodulin bound per syntrophin to cause inhibition of the precipitation . Since calmodulin inhibited syntrophin oligomerization in the presence or absence of Ca(2+), Ca(2+) binding to syntrophin is responsible for the inhibition by EGTA of syntrophin oligomerization.

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