Microorganisms

Parasitol Today, 1999 Aug, 15(8), 325 - 32
Antigen presentation by macrophages harboring intravesicular pathogens; Overath P et al.; Resting macrophages can be host cells for the replication of several protozoan parasites and bacteria . Upon activation, infected cells mobilize potent microbicidal mechanisms that eliminate the intracellular pathogen . This transition from a resting to an activated state is mediated by the interaction with specific T cells that recognize pathogen-derived peptides complexed to major histocompatibility complex (MHC) molecules at the surface of host cells . In this review, Peter Overath and Toni Aebischer discuss antigen presentation in infected macrophages from a cell biological point of view, a perspective that has important implications for the design of subunit vaccines.

J Neurol Neurosurg Psychiatry, 1999 Aug, 67(2), 239 - 42
Herpes simplex encephalitis after brain surgery: case report and review of the literature; Spuler A et al.; Intracranial infection after neurosurgical intervention most often is caused by bacteria . A rare case of fatal herpes simplex encephalitis after removal of a meningioma is described and similar cases reported in the literature are reviewed . Recent diagnostic tools, including detection of herpes viral DNA sequences by polymerase chain reaction, complement clinical suspicion and facilitate mandatory early diagnosis, because herpes encephalitis, without rapid initiation of treatment, may lead to severe disability or death.

Eur J Biochem, 1999 Jul, 263(2), 518 - 25
Cloning, expression and characterization of an A6-related protein; Rohwer A et al.; By interaction cloning (yeast two-hybrid system) using the catalytic domain of protein kinase Czeta (PKCzeta) as bait, we cloned a human full-length cDNA with 62% nucleotide homology to the A6 protein recently cloned and characterized by Beeler et al . {Beeler, J.F., LaRochelle, W.J., Chedid, M., Tronick, S.R . & Aaronson, S . A . (1994) Mol . Cell . Biol . 14, 982-988} . The deduced amino acid sequence (349 amino acids) of the A6-related protein (A6rp) contained potential actin-binding sites and ATP-binding sites . We also cloned the murine homolog of A6rp . Human A6rp was expressed in an in-vitro transcriptional/translational system with an apparent molecular mass of 40 kDa and as a glutathione S-transferase (GST) fusion protein in bacteria . A polyclonal anti-(A6rp) was raised in rabbits and used for the identification of A6rp by immunoblotting . A6rp was found to be expressed at the mRNA and the protein levels in all cells and tissues investigated . GST-A6rp was phosphorylated by PKCzeta but not significantly by other PKC isoenzymes . Moreover, it was phosphorylated by casein kinase 2 and most effectively by the tyrosine kinase Src . In contrast to GST-A6rp, GST-A6 was also phosphorylated by PKC isoforms other than PKCzeta and strongly by CK2, but just weakly by Src . In contrast to the results of Beeler et al . on beta-galactosidase-A6, we were unable to demonstrate autokinase activity or tyrosine phosphorylation of either GST-A6 or GST-A6rp . In accordance with the potential ATP-binding sites, both proteins were able to bind ATP.

Eur J Biochem, 1999 Jul, 263(2), 346 - 52
Cytochrome c-dependent methacrylate reductase from Geobacter sulfurreducens AM-1; Mikoulinskaia O et al.; Geobacter sulfurreducens AM-1 can use methacrylate as a terminal electron acceptor for anaerobic respiration . In this paper, we report on the purification and properties of the periplasmic methacrylate reductase, and show that the enzyme is dependent on the presence of a periplasmic cytochrome c (apparent K(m) = 0.12 microM) . The methacrylate reductase was found to be composed of only one polypeptide with an apparent molecular mass of 50 kDa and to contain, bound tightly but not covalently, 1 mol of FAD per mol . The N-terminal amino acid sequence showed sequence similarity to a periplasmic fumarate reductase from Shewanella putrefaciens . However, methacrylate reductase did not catalyze the reduction of fumarate . The periplasmic cytochrome c, which was also purified, had an apparent molecular mass of 30 kDa and contained approximately 4 mol of heme.mol(-1) . Cells of G . sulfurreducens AM-1 grown on acetate and methacrylate as an energy source were found to contain all the enzymes required for the oxidation of acetate to CO(2) via the citric acid cycle.

Glycobiology, 1999 Aug, 9(8), 787 - 95
Structural heterogeneity in the core oligosaccharide of the S-layer glycoprotein from Aneurinibacillus thermoaerophilus DSM 10155; Wugeditsch T et al.; The surface layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 has a total carbohydrate content of 15% (by mass), consisting of O-linked oligosaccharide chains . After proteolytic digestion of the S-layer glycoprotein byPronase E and subsequent purification of the digestion products by gel permeation chromatography, chromatofocusing and high-performance liquid chromatography two glycopeptide pools A and B with identical glycans and the repeating unit structure -->4)-alpha-l-Rha p -(1-->3)-beta-d- glycero -d- manno -Hep p -(1--> (Kosma et al., 1995b, Glycobiology, 5, 791-796) were obtained . Combined evidence from modified Edman-degradation in combination with liquid chromatography electrospray mass-spectrometry and nuclear magnetic resonance spectroscopy revealed that both glycopeptides contain equal amounts of the complete core structure alpha-l-Rha p -(1-->3)-alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and the truncated forms alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and beta-d-Gal p NAc-(1-->O)-Thr/Ser . All glycopeptides possessed the novel linkage types beta-d-Gal p NAc-(1-->O)-Thr/Ser . The different cores were substituted with varying numbers of disaccharide repeating units . By 300 MHz proton nuclear magnetic resonance spectroscopy the complete carbohydrate core structure of the fluorescently labeled glyco-peptide B was determined after Smith-degradation of its glycan chain . The NMR data confirmed and complemented the results of the mass spectroscopy experiments . Based on the S-layer glycopeptide structure, a pathway for its biosynthesis is suggested.

J Clin Virol, 1999 Jun, 13(1-2), 71 - 80
Rapid, phenotypic HIV-1 drug sensitivity assay for protease and reverse transcriptase inhibitors; Walter H et al.; BACKGROUND: Development of drug resistance is one of the major reasons for the failure of antiretroviral therapy of HIV-1 infection . Knowing the drug sensitivity-resistance profile of viruses present in a patient prior to treatment or change in treatment could help to optimize therapy . OBJECTIVE: Development of a rapid standardized phenotypic HIV-1 drug sensitivity assay for protease (PR) and reverse transcriptase (RT) inhibitors . DESIGN: The PR gene (codons 1-99) and the 5' part of the RT gene (codons 1-300) of HIV-1 is amplified from the plasma of infected individuals by RT-PCR and ligated into a proviral clone of HIV-1 containing a deletion of the PR gene and the 5' part of the RT gene . Bacteria are transformed with the ligation product and plasmid DNA is prepared from a library of transformed bacteria . The plasmid DNA is transfected into 293 T cells and recombinant virus is harvested from the supernatant of the transfected cells 2 days after transfection . The sensitivity of the recombinant virus is determined with the help of a sensitive indicator cell line . RESULTS: Recombinant viruses were generated with high efficiency . Determination of the drug sensitivity of the recombinant viruses with an indicator cell line was highly reproducible . The recombinant viruses accurately reflected the sensitivity-resistance profile of the parental viruses . The phenotypic drug sensitivity determined by this assay correlated well with the treatment history of patients . CONCLUSION: This assay system should allow rapid, high-throughput analyses of phenotypic HIV-1 drug sensitivity for PR and RT inhibitors . Due to the efficient generation of recombinant viruses, propagation of the recombinant viruses in cell culture is not required prior to the determination of the sensitivity of the recombinant viruses . The risk of selecting fitter non-resistant viruses due to culture conditions is minimized.

Int J Cancer, 1999 Aug 12, 82(4), 520 - 4
Role of Helicobacter pylori cagA(+) strains and specific host immune responses on the development of premalignant and malignant lesions in the gastric cardia; Peek RM Jr et al.; The incidence rates of gastric cardia and esophageal adenocarcinomas are increasing, but data suggest that carriage of cagA(+) Helicobacter pylori strains may protect against development of Barrett's esophagus and esophageal adenocarcinoma . Our aims were to examine the relationship between pre-malignant and malignant lesions in the gastric cardia and serum antibodies to H . pylori antigens in patients with and without complications of Barrett's esophagus . The prevalence of carditis was 40% in controls compared with 13% in patients with complicated or uncomplicated Barrett's esophagus and cardia adenocarcinoma (p < 0.001) . Cardia intestinal metaplasia (IM) and atrophy were present and concordant in 28% of controls but less frequent in patients with Barrett's alone or with dysplasia/adenocarcinoma (0% for each, p < 0.001) . Carriage of cagA(+) strains was present in 34% of patients with carditis and significantly associated with increased frequency and severity of cardia inflammation, IM, and atrophy but not with adenocarcinoma . IgA and HspA seropositivity were significantly increased in H . pylori-colonized patients with carditis compared to persons with normal cardia histology (p </= 0.005) but not in persons with esophageal disease or cardia adenocarcinoma . We conclude that carriage of cagA(+) H . pylori strains and induction of particular serological responses are significantly associated with marked histological findings in the gastric cardia but not with adenocarcinoma of either the gastric cardia or esophagus .

Mol Med, 1999 Mar, 5(3), 192 - 202
Gene expression pattern in human monocytes as a surrogate marker for systemic inflammatory response syndrome (SIRS); Wiegand G et al.; BACKGROUND: Systemic inflammatory response syndrome (SIRS) is a mild inflammatory episode which, in a minority of patients, may deteriorate into septic shock . In the mouse, injection of bacteria or bacterial endotoxin induces systemic inflammation through the activation of blood monocytes, which leads to lethal shock . A number of intervention strategies have been shown to prevent progression to shock in mouse model systems . However, recent clinical trials of a number of these therapeutic strategies in patients have been uniformly disappointing . In contrast to the situation in the mouse models, there may be many different ways to initiate systemic inflammation in patients and not all of them need necessarily involve activation of blood monocytes . If there is no unifying mechanism behind the induction of systemic inflammation in patients and no common rules governing its development, then it is unlikely that generally applicable therapeutic strategies will be found that can prevent progression into shock . MATERIALS AND METHODS: We used differential display to compare gene expression patterns in monocytes of recent-admission multi-trauma patients with clinically diagnosed SIRS to the patterns in monocytes of healthy controls . RESULTS: Of seven differentially displayed bands that were recovered and sequenced, five were associated with SIRS and two were preferentially expressed in the monocytes of healthy controls . CONCLUSION: The data show that monocytes of SIRS patients are in an activation state that is different from that of monocytes from the healthy controls, that monocytes from many individual patients share similar patterns of differentially expressed sequences, and that by this criterion, the multi-trauma SIRS patients are a remarkably coherent group.

Int J Parasitol, 1999 May, 29(5), 655 - 62
Characterisation of Fasciola hepatica cytochrome c peroxidase as an enzyme with potential antioxidant activity in vitro; Campos EG et al.; Cytochrome c peroxidase oxidises hydrogen peroxide using cytochrome c as the electron donor . This enzyme is found in yeast and bacteria and has been also described in the trematodes Fasciola hepatica and Schistosoma mansoni . Using partially purified cytochrome c peroxidase samples from Fasciola hepatica we evaluated its role as an antioxidant enzyme via the investigation of its ability to protect against oxidative damage to deoxyribose in vitro . A system containing FeIII-EDTA plus ascorbate was used to generate reactive oxygen species superoxide radical, H2O2 as well as the hydroxyl radical . Fasciola hepatica cytochrome c peroxidase effectively protected deoxyribose against oxidative damage in the presence of its substrate cytochrome c . This protection was proportional to the amount of enzyme added and occurred only in the presence of cytochrome c . Due to the low specific activity of the final partially purified sample the effects of ascorbate and calcium chloride on cytochrome c peroxidase were investigated . The activity of the partially purified enzyme was found to increase between 10 and 37% upon reduction with ascorbate . However, incubation of the partially purified enzyme with 1 mM calcium chloride did not have any effect on enzyme activity . Our results showed that Fasciola hepatica CcP can protect deoxyribose from oxidative damage in vitro by blocking the formation of the highly toxic hydroxyl radical (.OH) . We suggest that the capacity of CcP to inhibit .OH-formation, by efficiently removing H2O2 from the in vitro oxidative system, may extend the biological role of CcP in response to oxidative stress in Fasciola hepatica.

Biotechnol Bioeng, 1999 Sep 5, 64(5), 527 - 44
Quantitative structure-activity relationship (QSAR) analysis of surfactants influencing attachment of a Mycobacterium sp . to cellulose acetate and aromatic polyamide reverse osmosis membranes; Campbell P et al.; A series of 23 neutral, anionic, and zwitterionic surfactants were tested at a concentration of 0.1% wt/vol for their influence on attachment of a Mycobacterium sp . to cellulose acetate (CA) and polyamide (PA) reverse osmosis (RO) membranes . Four cell attachment bioassays were used: (1) semiconcurrent addition of surfactant and bacteria to RO coupons (standard assay); (2) surfactant pretreatment of RO membranes (membrane pretreatment assay); (3) surfactant treatment of adsorbed cells (detachment assay); and (4) surfactant pretreatment of mycobacteria (cell pretreatment assay) . Seventeen surfactants inhibited attachment to PA membranes, whereas 15 inhibited attachment to CA in standard assays and, in 13 cases, the same surfactant inhibited attachment to both PA and CA . Despite greater cell attachment to PA than CA, surfactants were typically more effective in the former membrane system . More surfactants were effective in impairing cell attachment than in promoting detachment and a number enhanced attachment in membrane pretreatment assays, suggesting surface modification of RO membranes . Cell pretreatment inhibited attachment to CA membranes, suggesting the bacterial surface was also a target for detergent activity . Multivariate regression and cluster analyses indicated that critical micellar concentration (CMC) was positively correlated with Mycobacterium attachment in CA and PA standard assays . Surfactant dipole moment and octanol/water partitioning (LogP) also contributed to detergent activity in the PA system, whereas dipole moment, molecular topology (i.e., connectivity indices), and charge properties influenced activity in the CA system . Influential variables in membrane pretreatment assays included the LogP, topology indices, and charge properties, whereas CMC played a diminished role . Surfactant dipole moment was most influential in CA membrane detachment assays . Increasing system ionic strength by LiBr addition strengthened inhibition of cell attachment to CA membranes by dodecylbenzene sulfonic acid (DBSA) and promoted DBSA adsorption to CA surfaces as indicated by attenuated total reflection Fourier-transform infrared spectrometry . Results indicate that inhibition of bacterial attachment to RO membranes may be maximized by manipulating surfactant molecular structure to optimize surface adsorption behavior .

FEBS Lett, 1999 Jun 18, 453(1-2), 6 - 10
New substrates of DNA polymerases; Victorova L et al.; Bis-(2'-deoxynucleoside) 5',5'-tetraphosphates and bis-(2'-deoxynucleoside) 5',5'-triphosphates were shown to be a new type of substrate for several DNA polymerases of human, bacterial and viral origin . Their substrate properties depend both on their structure and on the nature of the enzyme . They are incorporated by both termini in correspondence with the template nucleotide program in the active center . The results obtained support the mechanism of their direct incorporation rather than prior hydrolysis to dNTP . The highest activity of these compounds was observed for HIV reverse transcriptase . The probable biological significance of the reaction is discussed.

Arch Pharm Res, 1999 Jun, 22(3), 262 - 6
Preparation of dopamine transporter-specific antibodies using molecular cloned genes; Lee SY et al.; Dopamine transporter (DAT) plays the most important role in terminating the actions of dopamines released into the synaptic cleft . DAT is also the target of various psychotropic drugs such as cocaine and amphetamine . In this study we prepared DAT-specific antibodies using the 2nd extracellular loop of rat DAT as an antigen . The 2nd extracellular loop of the rat DAT was expressed in bacteria as a fusion protein with glutathione-S-transferase, and injected into rabbits to raise antibodies . Produced antibodies clearly recognized the rat DAT in ELISA, immunoblotting, and immunoprecipitation . As expected from the high sequence homology between the rat and human DAT, the antibodies raised for the rat DAT cross-reacted with the human DAT in the immunoblotting . Considering the specificity for DAT with wide range of applications such as ELISA, immunoblotting, and immunoprecipitation, these antibodies would be valuable tool for understanding the pharmacological actions of dopamine transporter and drug addiction.

Dis Aquat Organ, 1999 May 31, 36(3), 213 - 9
Environmental factors and chemical agents affecting the growth of the pathogenic marine ciliate Uronema nigricans; Crosbie PB et al.; The scuticociliate Uronema nigricans is an opportunistically parasitic marine ciliate known to cause disease in some aquacultural environments with epizootics documented from marine larval rearing systems, marine aquaria and in southern bluefin tuna Thunnus macoyii growout enclosures . This study examined growth responses of laboratory cultures of the ciliate and prey bacteria to variations in temperature and salinity, and the efficacy of potential chemotherapeutants for control of U . nigricans infections . Differences in ciliate growth responses were marginal at temperatures of 10 to 25 degrees C and at salinities between 15 and 35 ppt, though 3.5 ppt or less was lethal . Ciliates were found to be sensitive to fluctuations in bacterial densities, which may be a factor in the seasonal occurrence of the ciliate-related disease in tuna . Commonly used chemotherapeutants such as formalin, malachite green and hydrogen peroxide were all effective against the ciliate during in vitro trials.

J Virol, 1999 Aug, 73(8), 6626 - 33
Mapping of the feline calicivirus proteinase responsible for autocatalytic processing of the nonstructural polyprotein and identification of a stable proteinase-polymerase precursor protein; Sosnovtseva SA et al.; Expression of the region of the feline calicivirus (FCV) ORF1 encoded by nucleotides 3233 to 4054 in an in vitro rabbit reticulocyte system resulted in synthesis of an active proteinase that specifically processes the viral nonstructural polyprotein . Site-directed mutagenesis of the cysteine (Cys1193) residue in the putative active site of the proteinase abolished autocatalytic cleavage as well as cleavage of the viral capsid precursor, suggesting that this "3C-like" proteinase plays an important role in proteolytic processing during viral replication . Expression of the region encoding the C-terminal portion of the FCV ORF1 (amino acids 942 to 1761) in bacteria allowed direct N-terminal sequence analysis of the virus-specific polypeptides produced in this system . The results of these analyses indicate that the proteinase cleaves at amino acid residues E960-A961, E1071-S1072, E1345-T1346, and E1419-G1420; however, the cleavage efficiency is varied . The E1071-S1072 cleavage site defined the N terminus of a 692-amino-acid protein that contains sequences with similarity to the picornavirus 3C proteinase and 3D polymerase domains . Immunoprecipitation of radiolabeled proteins from FCV-infected feline kidney cells with serum raised against the FCV ORF1 C-terminal region showed that this "3CD-like" proteinase-polymerase precursor protein is apparently stable and accumulates in cells during infection.

J Bacteriol, 1999 Jul, 181(14), 4353 - 64
An additional regulatory gene for actinorhodin production in Streptomyces lividans involves a LysR-type transcriptional regulator; Martinez-Costa OH et al.; The sequence of a 4.8-kbp DNA fragment adjacent to the right-hand end of the actinorhodin biosynthetic (act) cluster downstream of actVB-orf6 from Streptomyces coelicolor A3(2) reveals six complete open reading frames, named orf7 to orf12 . The deduced amino acid sequences from orf7, orf10, and orf11 show significant similarities with the following products in the databases: a putative protein from the S . coelicolor SCP3 plasmid, LysR-type transcriptional regulators, and proteins belonging to the family of short-chain dehydrogenases/reductases, respectively . The deduced product of orf8 reveals low similarities with several methyltransferases from different sources, while orf9 and orf12 products show no similarities with other known proteins . Disruptions of orf10 and orf11 genes in S . coelicolor appear to have no significant effect on the production of actinorhodin . Nevertheless, disruption or deletion of orf10 in Streptomyces lividans causes actinorhodin overproduction . The introduction of extra copies of orf10 and orf11 genes in an S . coelicolor actIII mutant restores the ability to produce actinorhodin . Transcriptional analysis and DNA footprinting indicate that Orf10 represses its own transcription and regulates orf11 transcription, expression of which might require the presence of an unknown inducer . No DNA target for Orf10 protein was found within the act cluster.

J Bacteriol, 1999 Jul, 181(14), 4266 - 74
A mycobacterial extracytoplasmic sigma factor involved in survival following heat shock and oxidative stress; Fernandes ND et al.; Extracytoplasmic function (ECF) sigma factors are a heterogeneous group of alternative sigma factors that regulate gene expression in response to a variety of conditions, including stress . We previously characterized a mycobacterial ECF sigma factor, SigE, that contributes to survival following several distinct stresses . A gene encoding a closely related sigma factor, sigH, was cloned from Mycobacterium tuberculosis and Mycobacterium smegmatis . A single copy of this gene is present in these and other fast- and slow-growing mycobacteria, including M . fortuitum and M . avium . While the M . tuberculosis and M . smegmatis sigH genes encode highly similar proteins, there are multiple differences in adjacent genes . The single in vivo transcriptional start site identified in M . smegmatis and one of two identified in M . bovis BCG were found to have -35 promoter sequences that match the ECF-dependent -35 promoter consensus . Expression from these promoters was strongly induced by 50 degrees C heat shock . In comparison to the wild type, an M . smegmatis sigH mutant was found to be more susceptible to cumene hydroperoxide stress but to be similar in logarithmic growth, stationary-phase survival, and survival following several other stresses . Survival of an M . smegmatis sigH sigE double mutant was found to be markedly decreased following 53 degrees C heat shock and following exposure to cumene hydroperoxide . Expression of the second gene in the sigH operon is required for complementation of the sigH stress phenotypes . SigH is an alternative sigma factor that plays a role in the mycobacterial stress response.

J Bacteriol, 1999 Jul, 181(14), 4154 - 60
Eikenella corrodens phase variation involves a posttranslational event in pilus formation; Villar MT et al.; The human pathogen Eikenella corrodens synthesizes type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants . On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies . We are studying the molecular basis of this phase variation in the clinical isolate E . corrodens VA1 . A genomic fragment encoding the major type IV pilin was cloned from the S-phase variant of strain VA1 . Sequence analysis of the fragment revealed four tandemly arranged potential open reading frames (ORFs), designated pilA1, pilA2, pilB, and hagA . Both pilA1 and pilA2 predict a type IV pilin . The protein predicted by pilB shares sequence identity with the Dichelobacter nodosus FimB fimbrial assembly protein . The protein predicted by hagA resembles a hemagglutinin . The region containing these four ORFs was designated the pilA locus . DNA hybridization and sequence analysis showed that the pilA locus of an L-phase variant of strain VA1 was identical to that of the S-phase variant . An abundant pilA1 transcript initiating upstream of pilA1 and terminating at a predicted hairpin structure between pilA1 and pilA2 was detected by several assays, as was a less abundant read-through transcript encompassing pilA1, pilA2, and pilB . Transcription from the pilA locus was nearly indistinguishable between S- and L-phase variants . Electron microscopy and immunochemical analysis showed that S-phase variants synthesize, export, and assemble pilin into pili . In contrast, L-phase variants synthesize pilin but do not export and assemble it into pili . These data suggest that a posttranslational event, possibly involving an alteration in pilin export and assembly, is responsible for phase variation in E . corrodens.

Int J Biochem Cell Biol, 1999 May, 31(5), 545 - 9
CD14; Schutt C; The GPI-anchored 55 kDa glycoprotein CD14 is expressed on monocytes/macrophages and to a lesser extent on granulocytes . Engagement of CD14 by ligands like lipopolysaccharide, intact bacteria or apoptotic cells can result in either pro- or anti-inflammatory responses . Since the CD14 molecule does not have a membrane spanning domain it cannot transmit a signal into the cell . Some as yet unidentified accessory protein is thought to be involved . It will be important to clarify the signalling systems involved since they may provide a therapeutic target for sepsis intervention strategies.

Appl Biochem Biotechnol, 1999 Spring, 77-79, 585 - 93
Two-phase methanization of food wastes in pilot scale; Lee JP et al.; A 5 ton/d pilot scale two-phase anaerobic digester was constructed and tested to treat Korean food wastes in Anyang city near Seoul . The easily degradable presorted food waste was efficiently treated in the two-phase anaerobic digestion process . The waste contained in plastic bags was shredded and then screened for the removal of inert materials such as fabrics and plastics, and subsequently put into the two-stage reactors . Heavy and light inerts such as bones, shells, spoons, and plastic pieces were again removed by gravity differences . The residual organic component was effectively hydrolyzed and acidified in the first reactor with 5 d space time at pH of about 6.5 . The second, methanization reactor converted the acids into methane with pH between 7.4 and 7.8 . The space time for the second reactor was 15 d . The effluent from the second reactor was recycled to the first reactor to provide alkalinities . The process showed stable steady-state operation with the maximum organic loading rate of 7.9 kg volatile solid (VS)/m3/d and the volatile solid reduction efficiency of about 70% . The total of 3.6 tons presorted MSW containing 2.9 tons of food organic was treated to produce about 230 m3 of biogas with 70% (v/v) of methane and 80 kg of humus . This process is extended to full-scale treating 15 tons of food waste a day in Euiwang city and the produced biogas is utilized for the heating/cooling of adjacent buildings.

Immunol Rev, 1999 Apr, 168, 199 - 215
Human pathogen subversion of antigen presentation; Brodsky FM et al.; Many pathogens have co-evolved with their human hosts to develop strategies for immune evasion that involve disruption of the intracellular pathways by which antigens are bound by class I and class II molecules of the major histocompatibility complex (MHC) for presentation to T cells . Here the molecular events in these pathways are reviewed and pathogen interference is documented for viruses, extracellular and intracellular bacteria and intracellular parasites . In addition to a general review, data from our studies of adenovirus, Chlamydia trachomatis and Coxiella burnetii are summarized . Adenovirus E19 is the first viral gene product described that affects class I MHC molecule expression by two separate mechanisms, intracellular retention of the class I heavy chain by direct binding and by binding to the TAP transporter involved in class I peptide loading . Coxiella and Chlamydia both affect peptide presentation by class II MHC molecules as a result of their residence in endocytic compartments, although the properties of the parasitophorous vacuoles they form are quite different . These examples of active interference with antigen presentation by viral gene products and passive interference by rickettsiae and bacteria are typical of the strategies used by these different classes of pathogens, which need to evade different types of immune responses . Pathogen-host co-evolution is evident in these subversion tactics for which the pathogen crime seems tailored to fit the immune system punishment.

Immunogenetics, 1999 Aug, 49(9), 773 - 86
Origins of immunity: transcription factors and homologues of effector genes of the vertebrate immune system expressed in sea urchin coelomocytes; Pancer Z et al.; Echinoderms share common ancestry with the chordates within the deuterostome clade . Molecular features that are shared between their immune systems and that of mammals thus illuminate the basal genetic framework on which these immune systems have been constructed during evolution . The immune effector cells of sea urchins are the coelomocytes, whose primary function is protection against invasive marine pathogens; here we identify six genes expressed in coelomocytes, homologues of which are also expressed in cells of the mammalian immune system . Three coelomocyte genes reported here encode transcription factors . These are an NFKB homologue (SpNFKB); a GATA-2/3 homologue (SpGATAc); and a runt domain factor (SpRunt-1) . All three of these coelomocyte genes respond sharply to bacterial challenge: SpNFKB and SpRunt-1 genes are rapidly up-regulated, while transcripts of SpGATAc factor disappear within hours of injection of bacteria . Sham injection also activates SpNFKB and SpRunt, though with slower kinetics, but does not affect SpGATAc levels . Another gene, SpHS, encodes a protein related to the signal transduction intermediate HS1 of lymphoid cells . Two other newly discovered genes, SpSRCR1 and SpSRCR5, encode proteins featuring SRCR repeats . These genes are members of a complex family of SRCR genes all expressed specifically in coelomocytes . The SRCR repeats most closely resemble those of mammalian macrophage scavenger receptors . Remarkably, each individual sea urchin expresses a specific pattern of SRCR genes . Our results imply some shared immune functions and more generally, a shared regulatory architecture which underlies immune system gene expression in all deuterostomes . We conclude that the vertebrate immune system has evolved by inserting new genes into old gene regulatory networks dedicated to immunity.

J Mol Biol, 1999 Jul 23, 290(4), 881 - 902
Crystal structure of the oxidised and reduced acidic cytochrome c3from Desulfovibrio africanus; Norager S et al.; Unique among sulphate-reducing bacteria, Desulfovibrio africanus has two periplasmic tetraheme cytochromes c3, one with an acidic isoelectric point which exhibits an unusually low reactivity towards hydrogenase, and another with a basic isoelectric point which shows the usual cytochrome c3reactivity . The crystal structure of the oxidised acidic cytochrome c3of Desulfovibrio africanus (Dva.a) was solved by the multiple anomalous diffraction (MAD) method and refined to 1.6 A resolution . Its structure clearly belongs to the same family as the other known cytochromes c3, but with weak parentage with those of the Desulfovibrio genus and slightly closer to the cytochromes c3of Desulfomicrobium norvegicum . In Dva.a, one edge of heme I is completely exposed to the solvent and surrounded by a negatively charged protein surface . Heme I thus seems to play an important role in electron exchange, in addition to heme III or heme IV which are the electron exchange ports in the other cytochromes c3 . The function of Dva.a and the nature of its redox partners in the cell are thus very likely different.By alignment of the seven known 3D structures including Dva.a, it is shown that the structure which is most conserved in all cytochromes c3is the four-heme cluster itself . There is no conserved continuous protein structure which could explain the remarkable invariance of the four-heme cluster . On the contrary, the proximity of the heme edges is such that they interact directly by hydrophobic and van der Waals contacts . This direct interaction, which always involves a pyrrole CA-CB side-chain and its bound protein cysteine Sgammaatom, is probably the main origin of the four-heme cluster stability . The same kind of interaction is found in the chaining of the hemes in other multihemic redox proteins.The crystal structure of reduced Dva . a was solved at 1.9 A resolution . The comparison of the oxidised and reduced structures reveals changes in the positions of water molecules and polar residues which probably result from changes in the protonation state of amino acids and heme propionates . Water molecules are found closer to the hemes and to the iron atoms in the reduced than in the oxidised state . A global movement of a chain fragment in the vicinity of hemes III and IV is observed which result very likely from the electrostatic reorganization of the polypeptide chain induced by reduction .

J Mol Recognit, 1999 Mar-Apr, 12(2), 131 - 40
Unique single-domain antigen binding fragments derived from naturally occurring camel heavy-chain antibodies; Muyldermans S et al.; The humoral immune response of camels, dromedaries and llamas includes functional antibodies formed by two heavy chains and no light chains . The amino acid sequence of the variable domain of the naturally occurring heavy-chain antibodies reveals the necessary adaptations to compensate for the absence of the light chain . In contrast to the conventional antibodies, a large proportion of the heavy-chain antibodies acts as competitive enzyme inhibitors . Studies on the dromedary immunoglobulin genes start to shed light on the ontogeny of these heavy-chain antibodies . The presence of the heavy-chain antibodies and the possibility of immunizing a dromedary allows for the production of antigen binders consisting of a single domain only . These minimal antigen-binding fragments are well expressed in bacteria, bind the antigen with affinity in the nM range and are very stable . We expect that such camelid single domain antibodies will find their way into a number of biotechnological or medical applications . The structure of the camelid single domain is homologous to the human VH, however, the antigen-binding loop structures deviate fundamentally from the canonical structures described for human or mouse VHs . This has two additional advantages: (1) the camel or llama derived single domain antibodies might be an ideal scaffold for anti-idiotypic vaccinations; and (2) the development of smaller peptides or peptide mimetic drugs derived from of the antigen binding loops might be facilitated due to their less complex antigen binding site .

J Infect Dis, 1999 Aug, 180(2), 542 - 5
Increased nitric oxide in infective gastroenteritis; Herulf M et al.; Nitric oxide (NO) production is increased in several inflammatory disorders, although the role of this gas is not clear . The purpose of this study was to determine whether luminal NO in the intestine is increased in infective gastroenteritis . Rectal gas was sampled in 17 patients with gastroenteritis and 10 healthy volunteers, with balloon catheters made of 100% silicone and analyzed for NO by chemiluminescence . Plasma nitrate and nitrite levels were determined by capillary electrophoresis . Rectal NO was (mean+/-SEM) 9441+/-3126 parts per billion (ppb) in the patients and 74+/-13 ppb in controls (P<.0001) . There was no individual overlap . Plasma nitrite but not nitrate was significantly increased in patients compared with controls . These data indicate that luminal NO is greatly increased in gastroenteritis . The high levels of NO are easily measurable by rectal sampling, and measurement of luminal NO seems to be useful for evaluating local NO production in the gut in health and disease.

Proteins, 1999 Aug 1, 36(2), 157 - 74
Protein strain in blue copper proteins studied by free energy perturbations; De Kerpel JO et al.; Free energy perturbations have been performed on two blue copper proteins, plastocyanin and nitrite reductase . By changing the copper coordination geometry, force constants, and charges, we have estimated the maximum energy with which the proteins may distort the copper coordination sphere . By comparing this energy with the quantum chemical energy cost for the same perturbation on the isolated copper complex, various hypotheses about protein strain have been tested . The calculations show that the protein can only modify the copper-methionine bond length by a modest amount of energy-<5 kJ/mol-and they lend no support to the suggestion that the quite appreciable difference in the copper coordination geometry encountered in the two proteins is a result of the proteins enforcing different Cu-methionine bond lengths . On the contrary, this bond is very flexible, and neither the geometry nor the electronic structure change appreciably when the bond length is changed . Moreover, the proteins are rather indifferent to the length of this bond . Instead, the Cu(II) coordination geometries in the two proteins represent two distinct minima on the potential surface of the copper ligand sphere, characterized by different electronic structures, a tetragonal, mainly sigma-bonded, structure in nitrite reductase and a trigonal, pi-bonded, structure in plastocyanin . In vacuum, the structures have almost the same energy, and they are stabilized in the proteins by a combination of geometric and electrostatic interactions . Plastocyanin favors the bond lengths and electrostatics of the trigonal structure, whereas in nitrite reductase, the angles are the main discriminating factor . Proteins 1999;36:157-174 .

J Periodontol, 1999 Jun, 70(6), 668 - 78
Multi-layered periodontal pocket epithelium reconstituted in vitro: histology and cytokeratin profiles; Papaioannou W et al.; BACKGROUND: In order to study inter-individual differences in bacterial adhesion/invasion of periodontal tissues, an in vitro model for culturing multi-layered pocket epithelium without feeder layers or stromal equivalents (including the evaluation of their cytokeratin profiles) was developed . METHODS: Pocket epithelium was collected and grown until confluent in Falcon flasks using keratinocyte-serum free medium (KSFM), without a feeder layer . In the second passage, oral keratinocytes were re-grown in a 2 compartment system using either a clear polyester (transwell-clear {TCL}) or a collagen (transwell-col {TCO}) membrane as culture surface . After the first week, the calcium concentration was raised to 1.2 mM and in half the wells, the KSFM was supplemented with 10% fetal calf serum (FCS) . Histology and immunohistochemistry were performed after 1, 2, and 3 weeks of additional growth . RESULTS: In general, all conditions resulted in a structured epithelium consisting of 3 to 5 layers, but important differences were observed between the membrane types and between the media . CK4 was rarely and only lightly expressed while CK18 and 19 (characteristic of junctional epithelium) were very strongly expressed in the older (2 and 3 weeks) cultures . CK13 and 14 (characteristic of any stratifiable epithelial cell) also tended to increase over time; CK13 seemed to be stronger in KSFM with FCS while the contrary was true for CK14 . The multi-layer created by the combination TCL/KSFM + 10% FCS resembled a junctional epithelium most, while that grown on TCO without FCS mimicked the sulcular epithelium . CONCLUSIONS: It seems possible to create a histiotypic culture resembling either periodontal pocket or junctional epithelium without the use of stromal equivalents or feeder layers which make this approach more cumbersome . This multi-layered culture offers a model to investigate the permeability of pocket epithelium and the adhesion and penetration of bacteria under well-defined environmental conditions.

J Periodontol, 1999 Jun, 70(6), 632 - 45
One stage full- versus partial-mouth disinfection in the treatment of chronic adult or generalized early-onset periodontitis . I . Long-term clinical observations; Mongardini C et al.; BACKGROUND: A standard treatment strategy for periodontal infections often consists of 4 consecutive sessions of scaling and root planing (per quadrant, at 1- to 2-week intervals), without proper disinfection of the remaining intra-oral niches for periodontopathogens . This could theoretically lead to a reinfection of previously disinfected pockets by bacteria from an untreated region/niche . This study aimed to investigate, over an 8-month period, the clinical benefits of a one stage full-mouth disinfection in the control of severe periodontitis . METHODS: Sixteen patients with early-onset periodontitis and 24 patients with severe adult periodontitis were randomly assigned to test and control groups . The control group was scaled and root planed, per quadrant, at 2-week intervals and given standard oral hygiene instructions . A one stage full-mouth disinfection (test group) was sought by scaling and root planing the 4 quadrants within 24 hours in combination with the application of chlorhexidine to all intra-oral niches for periodontopathogens . Besides oral hygiene, the test group also rinsed twice daily with a 0.2% chlorhexidine solution and sprayed the tonsils with a 0.2% chlorhexidine spray, for 2 months . The plaque index, gingival index, probing depth, bleeding on probing, gingival recession, and clinical attachment level were recorded at baseline and at 1, 2, 4, and 8 months afterwards . RESULTS: The one stage full-mouth disinfection resulted, in comparison to the standard therapy, in a significant (P <0.001) additional probing depth reduction and gain in attachment up to 8 months . For initial pockets > or =7 mm, the "additional" probing depth reduction at the 8 month follow-up was 1.2 mm for single-rooted and 0.9 mm for multi-rooted teeth, with corresponding additional gains in attachment of 1.0 mm and 0.8 mm, respectively . The additional improvements were observed for all subgroups (adult periodontitis, generalized early-onset cases, smokers), with the largest differences in the non-smoking adult periodontitis patients . CONCLUSIONS: These findings suggest that a one stage full-mouth disinfection results in an improved clinical outcome for the treatment of chronic adult or early-onset periodontitis as compared to scaling and root planing per quadrant at 2-week intervals.

J Periodontol, 1999 Jun, 70(6), 618 - 25
Comparison of fluorescence microscopy and culture assays to quantitate adhesion of Porphyromonas gingivalis to mono- and multi-layered pocket epithelium cultures; Papaioannou W et al.; BACKGROUND: The present study compared 2 different methods (direct versus indirect evaluation) for the quantification of the adhesion of Porphyromonas gingivalis strains to in vitro cultured mono-layers of pocket epithelium . METHODS: The indirect culture viability assay (calculation of colony forming units) was compared to a direct microscopic evaluation using a novel fluorescent stain . The fluorescent kit was found to stain both bacteria and epithelial cells and enabled a differentiation between dead and living cells . RESULTS: Comparing the visual to the culture data, a high and significant correlation was found (Pearson's correlation = 0.75; P <0.001) . The adhesion capacity was in general higher for dead epithelial cells than for living cells (P <0.01) . Although comparable numbers of bacteria of 2 P . gingivalis strains (Pg 4 and Pg 5) were applied, Pg 4 showed a significantly lower adhesion capacity . This intra-strain variability was observed by the culture assay (2.3 x 10(6) versus 7.8 x 10(6)+/-2.7 x 10(6); P <0.01) and by the direct microscopy (P <0.01) for both live and dead epithelial cells . A second goal was to see whether there was a difference in the amount of bacterial adherence to mono- and multi-layers of in vitro cultured epithelium . No significant differences were found for the 5 examined P . gingivalis strains . However, interstrain differences in adhesion capacity were evident for both tissues . CONCLUSIONS: This study highlights the reproducibility of a direct microscopic evaluation of bacterial adhesion to in vitro cultured epithelial cells, and suggests both intrastrain (P . gingivalis) and inter-cell (live versus dead) variation in adhesion capacity . Studies are needed to determine the extent to which P . gingivalis strain variation is reflected in variation of other strains in humans.

J Oral Rehabil, 1999 Jun, 26(6), 453 - 8
Caries detector dyes--an in vitro assessment of some new compounds; Ansari G et al.; Previous studies have shown that the caries detector dyes, basic fuchsin and acid red, lack specificity . Accordingly, their clinical use can lead to the unnecessary removal of sound tissue . In the present study, the specificity of three further dyes, Carbolan Green, Coomassie Blue and Lissamine Blue was studied . Carious dentine was removed in vitro by means of rotary instruments until the cavities were deemed caries free by conventional clinical criteria . Experimental dyes were applied to the cavity floors, all of which became stained . Stained dentine was removed from half the cavity by means of a burr, the other half remaining as a control . Further stain was then applied and the procedure repeated until no further reduction of the staining of the cavity floor could be achieved . Light microscopy of ground sections of experimental teeth showed that sound tissue had been removed unnecessarily from the experimental half of the cavity due to the lack of specificity of these dyes . This lack of specificity of staining was similar to basic fuchsin and acid red . Only Carbolan Green showed possible differential staining between control and experimental sites, but this was not caries specific . If a clinically useful dye is to be developed, it would need to specifically stain either bacteria in infected dentine and/or the carious degradation products of dentine matrix.

J Clin Pathol, 1999 Feb, 52(2), 137 - 40
Sulphomucins favour adhesion of Helicobacter pylori to metaplastic gastric mucosa; Bravo JC et al.; AIMS: To assess the influence of sulphomucin secretion on Helicobacter pylori colonisation and adhesion to metaplastic gastric cells . METHODS: Gastric biopsies from 230 H pylori positive patients with intestinal metaplasia were analysed . Sulphated mucins and H pylori were visualised using a new technique combining high iron diamine-alcian blue mucin stains with the Steiner silver stain for the bacteria . RESULTS: Sulphomucin secretion anywhere in the mucosa and a histological diagnosis of dysplasia increase the risk of H pylori adhesion to metaplastic cells (odds ratios 19.9 and 4.3, respectively) . However, only 9.4% of cases showing sulphomucin secretion and 10.8% of cases with dysplasia had evidence of adhesion of H pylori bacteria to metaplastic cells . CONCLUSIONS: The findings suggest that H pylori may play a role in the advanced stages of carcinogenesis . It will be of interest to investigate if the relative small proportion of type III metaplasias that actually progress to carcinoma show persistence of H pylori.

Biochim Biophys Acta, 1999 Jul 9, 1439(1), 57 - 64
Functional analysis of genes from Streptomyces griseus involved in the synthesis of isorenieratene, a carotenoid with aromatic end groups, revealed a novel type of carotenoid desaturase; Krugel H et al.; The biosynthesis of the aromatic carotene isorenieratene is restricted to green photosynthetic bacteria and a few actinomycetes . Among them Streptomyces griseus has been used to study the genes involved in this pathway . Five genes out of seven of two adjacent operons in one cluster could be identified to be sufficient for the synthesis of isorenieratene . Stepwise deletions of these genes demonstrated their participation in phytoene synthesis, phytoene desaturation and lycopene cyclization . The novel gene crtU was assigned to encode a unique desaturase responsible for the conversion of beta-carotene via beta-isorenieratene to isorenieratene by a desaturation/methyltransferation mechanism . Sequence analysis of crtU revealed two conserved regions, one at the N-terminus and the other at the C-terminus of the protein which is universal to different types of carotene desaturases . In addition, the sequence comprises a motif typically found in methyltransferases . The deletion of the two remaining genes of the cluster left the carotenoid biosynthetic pathway unaffected.

J Mol Biol, 1999 Jul 16, 290(3), 627 - 38
The F plasmid centromere, sopC, is required for full repression of the sopAB operon; Yates P et al.; The SopB protein of the F plasmid has a dual role in the partition of F plasmid copies to daughter cells prior to division . It binds to the sopC centromere site to form the partition complex needed for stabilizing the plasmid, and it interacts with SopA to repress transcription of the sopAB operon, thus preventing the destabilization that results from excess SopB . We have isolated sop mutants by screening for unstable inheritance of mutagenized mini-F DNA . Four of the mutants resulted from different missense mutations in sopB . All four were deficient, to varying degrees, in autoregulation of Sop protein synthesis . The mutant proteins showed diminished capacity for reducing the linking number of mini-F and for destabilizing a plasmid carrying sopC, indicating that reduced ability to form a normal complex with sopC might underlie the autoregulation defect . Repression of the transcription of a sop promoter- lacZ fusion by SopA and SopB was strongly enhanced in the presence of sopC, in cis or in trans, and the enhancement was reduced or nullified when wild-type sopB was replaced by the mutant sopB alleles . A single 43 bp unit of sopC was almost as effective as sopC itself in enhancing repression . The results show that sopC is necessary for full repression of the sop promoter . They thus indicate a previously unsuspected role for this centromere site, and suggest that autoregulation and partition might normally be coordinated processes .

Genomics, 1999 Jul 1, 59(1), 102 - 4
Identification, sequence, and mapping of the mouse multiple PDZ domain protein gene, Mpdz; Simpson EH et al.; The PDZ domain gained its name from the three proteins that were first seen to have homology by virtue of these domains, the mammalian postsynaptic density protein, PSD-95, the Drosophila discs-large septate junction protein, DLG, and the mammalian epithelial tight-junction protein zona occludens, ZO-1 . Over 50 PDZ domain-containing genes have been recognized so far from almost any organism subjected to sequencing, including mammals, nematodes, yeast, plants, and bacteria . The domain consists of an approximately 90-amino-acid-residue unit, which is often repeated in the protein . The majority of residues form a conserved spatial structure while a few amino acids in critical positions confer protein binding specificity . A subgroup of PDZ domains have been shown to recognize a short carboxy-terminal amino acid motif, T/SXV (Ser/Thr-X-Val-COO-), where X is any amino acid . We have identified and completely sequenced a gene, Mpdz, that encodes a mouse protein containing 13 such domains . We have also mapped the gene to a series of overlapping deletions on mouse chromosome 4 and can therefore determine that its function is not essential for embryonic development or neonatal survival .

J Nutr, 1999 Jul, 129(7), 1315 - 8
10-Formyl-dihydrofolic acid is bioactive in human leukemia cells; Baggott JE et al.; The bioactivity of 10-formyl-7,8-dihydrofolic acid and 10-formyl-folic acid was determined in human leukemia (CCRF-CEM) cells grown in a folate-depleted medium containing methotrexate . Excess 10-formyl-7,8-dihydrofolic acid, (but not 10-formyl folic acid) supported the growth of these cells, but it was less potent than5-formyl-5,6,7,8-tetrahydrofolic acid (a control) . 10-formyl-7, 8-dihydrofolic acid (not 10-formyl folic acid) was active as substrate for aminoimidazole carboxamide ribotide transformylase and dihydrofolate reductase . This is the first experimental evidence that 10-formyl-7,8-dihydrofolic acid is a bioactive folate in mammalian cells . These experiments and several other lines of evidence in the literature suggest that 10-formyl-folic acid must be metabolized to bioactive folate by enteric bacteria before it can be utilized by the vertebrate host.

J Nat Prod, 1999 Jun, 62(6), 927 - 30
A new tyrosine kinase inhibitor from the marine brown alga Stypopodium zonale; Wessels M et al.; From the lipophilic extract of the marine brown alga Stypopodium zonale (Dictyotaceae) the new terpenoid compound stypoquinonic acid (1) together with the known compounds taondiol (2) and atomaric acid (3) were isolated . The structures of all isolates were determined from their spectroscopic data, including 1- and 2-dimensional NMR methods . The new compound, 1, and atomaric acid (3), showed inhibition of tyrosine kinase (p56lck).

Plant Mol Biol, 1999 May, 40(1), 91 - 7
The S7 ribosomal protein gene is truncated and overlaps a cytochrome c biogenesis gene in pea mitochondria; Zhuo D et al.; The pea mitochondrial genome contains a truncated rps7 gene lacking ca . 40 codons at its 5' terminus . This single-copy sequence is immediately downstream of and slightly overlapping an actively transcribed and edited reading frame of 744 bp (designated ccb248) homologous to the bacterial helC gene which encodes a subunit of the ABC-type heme transporter involved in cytochrome c biogenesis . This region of mitochondrial DNA appears recombinogenic, and the carboxy-termini of helC-type proteins are predicted to vary in sequence and length among plants . Sequences corresponding to the 5' coding region of rps7 were not detected elsewhere in the pea mitochondrial genome using wheat rps7 probes, and only a very short internal rps7 segment was observed in soybean mitochondrial DNA . The presence of rps7-homologous sequences in the nuclear genomes of pea and soybean is consistent with the recent transfer of a functional mitochondrial rps7 gene to the nucleus in certain plant lineages.

Plant Mol Biol, 1999 May, 40(1), 55 - 64
Arabidopsis thaliana 'extra-large GTP-binding protein' (AtXLG1): a new class of G-protein; Lee YR et al.; Heterotrimeric GTP-binding proteins, composed of alpha, beta, and gamma subunits, are involved in signal transduction pathways in animal and plant systems . In plants, physiological analyses implicate heterotrimeric G-proteins in ion channel regulation, light signaling, and hormone and pathogen responses . However, only one class of plant G alpha genes has been identified to date . We have cloned a novel gene, 'Arabidopsis thaliana extra-large GTP-binding protein' (AtXLG1) . AtXLG1 appears to be a member of a small gene family and is transcribed in all tissues assayed: roots, leaves, stems, flowers, and fruits . The conceptually translated protein from AtXLG1 is 99 kDa, twice as large as typical G alpha proteins . The carboxy-terminal half of the AtXLG1 protein has significant homology to animal and plant G alpha proteins . This region includes a GTP-binding domain, a predicted helical domain, and an aspartate/glutamate-rich loop, which are characteristics of G alpha's . Despite the absence of some of the amino acids implicated in GTP binding and hydrolysis by crystallographic and mutational analyses of mammalian G alpha's, recombinant AtXLG1 binds GTP with specificity . The amino-terminal region of AtXLG1 contains domains homologous to the bacterial TonB-box, which is involved in energy transduction between the inner and outer bacterial membranes, and to zinc-finger proteins . Given the unique structure of AtXLG1, it will be of interest to uncover its physiological functions.

Acta Crystallogr D Biol Crystallogr, 1999 Jul, 55 ( Pt 7), 1359 - 61
Crystallization and preliminary X-ray diffraction study of the endo-polygalacturonase from Fusarium moniliforme; Federici L et al.; Endo-polygalacturonases catalyze the fragmentation and solubilization of the homogalacturonan of the plant cell wall . These enzymes are extracellularly targeted glycoproteins produced by a number of organisms such as fungi, bacteria and plants, and are involved in both pathological and physiological processes . Single crystals of the endo-polygalacturonase from the phytopathogenic fungus Fusarium moniliforme were obtained by the vapour-diffusion method at 294 K . The starting material as well as the crystal consist of three forms with different degrees of glycosylation . The crystals belong to the orthorhombic space group P212121 and diffract to 1.9 A resolution on a synchrotron-radiation source under cryocooling conditions.

Acta Crystallogr D Biol Crystallogr, 1999 Jul, 55 ( Pt 7), 1273 - 90
Haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 refined at 1.15 A resolution; Ridder IS et al.; Crystals of the 35 kDa protein haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 diffract to 1.15 A resolution at cryogenic temperature using synchrotron radiation . Blocked anisotropic least-squares refinement with SHELXL gave a final conventional R factor of 10.51% for all reflections in the 15-1.15 A resolution range . The estimated r.m.s . errors of the model are 0.026 and 0.038 A for protein atoms and all atoms, respectively . The structure comprises all 310 amino acids, with 28 side chains and two peptide bonds in multiple conformations, two covalently linked Pb atoms, 601 water molecules, seven glycerol molecules, one sulfate ion and two chloride ions . Water molecules accounting for alternative solvent structure are modelled with a fixed occupancy of 0.5 . The structure is described in detail and compared with previously reported dehalogenase structures refined at 1.9-2.3 A resolution . An analysis of the protein's geometry and stereochemistry reveals eight mean values of bond lengths and angles which deviate significantly from the Engh & Huber parameters, a wide spread in the main-chain omega torsion angle around its ideal value of 180 (6) degrees and a role for C-HcO interactions in satisfying the hydrogen-bond acceptor capacity of main-chain carbonyl O atoms in the central beta-sheet.

Biochem J, 1999 Jul 15, 341 ( Pt 2), 395 - 400
Histidine-193 of rat glucosylceramide synthase resides in a UDP-glucose- and inhibitor (D-threo-1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol)-binding region: a biochemical and mutational study; Wu K et al.; Glucosylceramide synthase (GCS) catalyses the transfer of glucose from UDP-glucose (UDP-Glc) to ceramide to form glucosylceramide, the common precursor of most higher-order glycosphingolipids . Inhibition of GCS activity has been proposed as a possible target of chemotherapeutic agents for a number of diseases, including cancer . Design of new GCS inhibitors with desirable pharmaceutical properties is hampered by lack of knowledge of the secondary structure or catalytic mechanism of the GCS protein . Thus we cloned the rat homologue of GCS to begin studies to identify its catalytic regions . The histidine-modifying agent diethyl pyrocarbonate (DEPC) inhibited recombinant rat GCS expressed in bacteria; this inhibition was rapidly reversible by hydroxylamine and could be diminished by preincubation of GCS with UDP-Glc . These data suggest that DEPC acts on histidine residues within or near the UDP-Glc-binding site of GCS . Mutant proteins were expressed in which the eight histidine residues in GCS were individually replaced by other amino acids . H193A (His193-->Ala) and H193N (His193-->Asn) mutants were unaffected by 0.1 mM DEPC, a concentration that inhibited other histidine mutants and the wild-type enzyme by at least 60% . These results indicate that His193 is the primary target of DEPC and is at, or near, the UDP-Glc-binding site of GCS . His193 mutants were also insensitive to the GCS inhibitor d-threo-1-phenyl-2- decanoylamino-3-morpholinopropan-1-ol, at concentrations which inhibited the wild-type enzyme by >80% . These results have significance for both an understanding of the GCS active site and also for the possible design of new and specific inhibitors of GCS.

Biochem J, 1999 Jul 15, 341 ( Pt 2), 329 - 37
A shift in the equilibrium constant at the catalytic site of proton-translocating transhydrogenase: significance for a 'binding-change' mechanism; Venning JD et al.; In mitochondria and bacteria, transhydrogenase uses the transmembrane proton gradient (Deltap) to drive reduction of NADP+ by NADH . We have investigated the pre-steady-state kinetics of NADP+ reduction by acetylpyridine adenine dinucleotide (AcPdADH, an analogue of NADH) in complexes formed from the two, separately prepared, recombinant, peripheral subunits of the enzyme: the dI component, which binds NAD+ and NADH, and the dIII component, which binds NADP+ and NADPH . In the stopped-flow spectrophotometer the reaction proceeds as a single-turnover burst of hydride transfer to NADP+ on dIII before product NADPH release becomes limiting in steady state . The burst is biphasic . The results indicate that the fast phase represents direct hydride transfer from AcPdADH to NADP+ in dI:dIII complexes, and that the slow phase, which predominates when {dI}<{dIII}, corresponds to dissociation of the protein complexes during multiple turnovers of dI . Measurements on the amplitude of the burst, and on the apparent first-order rate constant of the fast phase, indicate that the equilibrium constant of the hydride-transfer step on the enzyme is shifted relative to that in solution . This has consequences for a model proposed earlier, in which Deltap is used, not at the hydride-transfer step, but to change the binding affinities of NADP+ and NADPH.

Biochem J, 1999 Jul 15, 341 ( Pt 2), 307 - 14
Reductive half-reaction of the H172Q mutant of trimethylamine dehydrogenase: evidence against a carbanion mechanism and assignment of kinetically influential ionizations in the enzyme-substrate complex; Basran J et al.; The reactions of wild-type trimethylamine dehydrogenase (TMADH) and of a His-172-->Gln (H172Q) mutant were studied by rapid-mixing stopped-flow spectroscopy over the pH range 6.0-10.5, to address the potential role of His-172 in abstracting a proton from the substrate in a 'carbanion' mechanism for C-H bond cleavage . The pH-dependence of the limiting rate for flavin reduction (klim) was studied as a function of pH for the wild-type enzyme with perdeuterated trimethylamine as substrate . The use of perdeuterated trimethylamine facilitated the unequivocal identification of two kinetically influential ionizations in the enzyme-substrate complex, with macroscopic pKa values of 6.5+/-0.2 and 8.4+/-0.1 . A plot of klim/Kd revealed a bell-shaped curve and two kinetically influential ionizations with macroscopic pKa values of 9.4+/-0.1 and 10.5+/-0.1 . Mutagenesis of His-172, a potential active-site base and a component of a novel Tyr-His-Asp triad in the active site of TMADH, revealed that the pKa of 8.4+/-0.1 for the wild-type enzyme-substrate complex represents ionization of the imidazolium side-chain of His-172 . H172Q TMADH retains catalytic competence throughout the pH range investigated . At pH 10.5, and in contrast with the wild-type enzyme, flavin reduction in H172Q TMADH is biphasic . The fast phase is dependent on the trimethylamine concentration and exhibits a kinetic isotope effect of about 3; C-H bond cleavage is thus partially rate-limiting . In contrast, the slow phase does not show hyperbolic dependence on substrate concentration, and the observed rate shows no dependence on isotope, revealing that C-H bond cleavage is not rate-limiting . The analysis of H172Q TMADH, together with data recently acquired for the Y169F mutant of TMADH, reveals that C-H bond breakage is not initiated via abstraction of a proton from the substrate by an active-site base . The transfer of reducing equivalents to flavin via a carbanion mechanism is therefore unlikely.

Arch Orthop Trauma Surg, 1999, 119(3-4), 236 - 40
Primary biomechanical influence of different sterilization methods on a freeze-dried bone-ligament transplant; Bettin D et al.; The transmission of bacteria and viruses in ligament transplants should be prevented by sterilization . In this study, the influence of two different methods on the mechanical properties of a freeze-dried medial collateral ligament was analyzed in sheep . Group I (n = 10) was treated with irradiation (26 kGy) and group II (n = 10) with ethyleneoxide . The mechanical properties changed in respect of the maximal load: group I (-29.9%; P < 0.05), group II (-7.7%), elongation: group I (-6.6%), group II (-0.3%), stress: group I (-20.1%), group II (-6.8%), strain: group I (-0.64%), group II (-0.3%), stiffness: group I (-10.2%), group II (-10.5%), energy: group I (-31.4%), group II (-6.9%) and elastic modulus: group I (-1.3%), group II (-5.0%) . The irradiation dose significantly reduced the maximal load, whereas ethyleneoxide sterilization resulted only in minor changes . Because of the potential cancerogenity of ethyleneoxide, a close monitoring of aeration times and its residuals are very essential . Further studies with lower irradiation doses of between 15 and 26 kGy seem to be justified.

Clin Diagn Lab Immunol, 1999 Jul, 6(4), 630 - 2
A novel enzyme-linked immunosorbent assay using the recombinant Actinobacillus pleuropneumoniae ApxII antigen for diagnosis of pleuropneumonia in pig herds; Leiner G et al.; For the surveillance of pig herds infected with porcine pleuropneumonia, an enzyme-linked immunosorbent assay (ELISA) using the recombinant Actinobacillus pleuropneumoniae ApxII protein as species- but not serotype-specific antigen was developed . Using this ELISA, 243 of 400 animals from 22 A . pleuropneumoniae-infected herds were classified as seropositive.

Clin Diagn Lab Immunol, 1999 Jul, 6(4), 558 - 66
Identification of Bartonella-specific immunodominant antigens recognized by the feline humoral immune system; Freeland RL et al.; The seroreactivities of both naturally and experimentally infected cats to Bartonella henselae was examined . Serum samples collected weekly from nine cats experimentally infected with B . henselae LSU16 were tested by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis . The magnitude and isotype of the antibody response were investigated by ELISA . Western blot analysis allowed the identification of at least 24 Bartonella-specific antigens recognized by the cats during infection . Antibody titers to specific antigens, as determined by Western blot analysis, ranged from 10 to 640 and varied among the different antibody-antigen interactions . Absorption of sera from an experimentally infected cat, using whole cells and cell lysates of various Bartonella species and other bacteria that commonly colonize cats, supported the identification of those Bartonella-specific antigens recognized by the experimentally infected cats . Furthermore, a number of possible species- and type-specific antigens were identified . Finally, sera obtained from cats at local animal shelters were screened for the presence of antibodies directed against the Bartonella-specific bands identified in the experimentally infected cats . A number of Bartonella-specific antigens have been identified to which strong antibody responses are generated in both experimentally and naturally infected cats, some of which may be useful in diagnosing species- and/or type-specific infections . In addition, the results from these experiments will lead to the development of monoclonal antibodies targeted against those genus-, species-, and type-specific antigens.

Indoor Air, 1999 Jun, 9(2), 134 - 8
Indoor air quality investigations at five classrooms; Lee SC et al.; Five classrooms, air-conditioned or naturally ventilated, at five different schools were chosen for comparison of indoor and outdoor air quality . Temperature, relative humidity (RH), carbon dioxide (CO2), sulphur dioxide (SO2), nitric oxide (NO), nitrogen dioxide (NO2), particulate matter with diameter less than 10 microns (PM10), formaldehyde (HCHO), and total bacteria counts were monitored at indoor and outdoor locations simultaneously . Respirable particulate matter was found to be the worst among parameters measured in this study . The indoor and outdoor average PM10 concentrations exceeded the Hong Kong standards, and the maximum indoor PM10 level was even at 472 micrograms/m3 . Air cleaners could be used in classrooms to reduce the high PM10 concentration . Indoor CO2 concentrations often exceeded 1,000 microliters/l indicating inadequate ventilation . Lowering the occupancy and increasing breaks between classes could alleviate the high CO2 concentrations . Though the maximum indoor CO2 level reached 5,900 microliters/l during class at one of the sites, CO2 concentrations were still at levels that pose no health threats.

Trends Microbiol, 1999 Jul, 7(7), 292 - 7
The cytolethal distending toxin family; Pickett CL et al.; Cytolethal distending toxins are produced by a small but diverse group of bacterial pathogens . This newly discovered toxin family can cause a variety of mammalian cells to become irreversibly blocked in the G2 phase of the cell cycle . How this novel effect is accomplished is unknown but the study of these fascinating toxins promises to reveal new methods of host-pathogen interaction.

Trends Microbiol, 1999 Jul, 7(7), 281 - 91
Variation and evolution of the citric-acid cycle: a genomic perspective; Huynen MA et al.; The presence of genes encoding enzymes involved in the citric-acid cycle has been studied in 19 completely sequenced genomes . In the majority of species, the cycle appears to be incomplete or absent . Several distinct, incomplete cycles reflect adaptations to different environments . Their distribution over the phylogenetic tree hints at precursors in the evolution of the citric-acid cycle.

Mutagenesis, 1999 Jul, 14(4), 433 - 6
Induction and prevention of micronuclei and chromosomal aberrations in cultured human lymphocytes exposed to the light of halogen tungsten lamps; D'Agostini F et al.; Previous studies have shown that the light emitted by halogen tungsten lamps contains UV radiation in the UV-A, UV-B and UV-C regions, induces mutations and irreparable DNA damage in bacteria, enhances the frequency of micronuclei in cultured human lymphocytes and is potently carcinogenic to the skin of hairless mice . The present study showed that the light emitted by an uncovered, traditional halogen lamp induces a significant, dose-related and time-related increase not only in micronuclei but also in chromosome-type aberrations, such as breaks, and even more in chromatid-type aberrations, such as isochromatid breaks, exchanges and isochromatid/chromatid interchanges, all including gaps or not, in cultured human lymphocytes . All these genotoxic effects were completely prevented by shielding the same lamp with a silica glass cover, blocking UV radiation . A new model of halogen lamp, having the quartz bulb treated in order to reduce the output of UV radiation, was considerably less genotoxic than the uncovered halogen lamp, yet induction of chromosomal alterations was observed at high illuminance levels.

J Mol Biol, 1999 Jul 9, 290(2), 471 - 9
Guiding a docking mode by phage display: selection of correlated mutations at the staphylokinase-plasmin interface; Jespers L et al.; During co-evolution of interacting proteins, functionally disruptive mutations on one side of the interface may be compensated by local amino acid changes on the other to restore binding affinity . This information can be useful for geometry-based docking approaches by reducing the translational and rotational space available to the proteins . Here, we demonstrate that correlated mutations at a protein-protein interface can be rapidly identified by selecting a phage-displayed library of a randomly mutated component of the complex for complementation of mutations that decreased binding in the interacting partner . This approach was used to deduce the binding mode of staphylokinase (Sak), a 15.5 kDa "indirect" plasminogen activator on microplasmin (microPli), the 28 kDa serine protease domain of plasmin . Biopanning indicated that residues Arg94 and Gly174 in microPli are located in close proximity to Glu75 and the Glu88:Ile128 pair in Sak, respectively . The coupled mutations Glu94<-->Lys75 reversed and Gly174<-->Lys88:Val128 introduced a salt bridge, whereby the binding affinities (with coupling energies of 1.8 to 2.3 kcal mol-1, respectively) and the plasminogen activation ability of the mutated complexes were partially restored . These findings suggested a unique docking mode of Sak at the western rim of the active-site cleft of microPli, that is in agreement with the structure of the Sak-microPli complex as recently derived by other methods .

Appl Environ Microbiol, 1999 Jul, 65(7), 3148 - 57
Nitrogen, carbon, and sulfur metabolism in natural thioploca samples
Otte S, Kuenen JG, Nielsen LP, Paerl HW, Zopfi J, Schulz HN, Teske A, Strotmann B, Gallardo VA, Jorgensen BB.
Filamentous sulfur bacteria of the genus Thioploca occur as dense mats on the continental shelf off the coast of Chile and Peru . Since little is known about their nitrogen, sulfur, and carbon metabolism, this study was undertaken to investigate their (eco)physiology . Thioploca is able to store internally high concentrations of sulfur globules and nitrate . It has been previously hypothesized that these large vacuolated bacteria can oxidize sulfide by reducing their internally stored nitrate . We examined this nitrate reduction by incubation experiments of washed Thioploca sheaths with trichomes in combination with 15N compounds and mass spectrometry and found that these Thioploca samples produce ammonium at a rate of 1 nmol min-1 mg of protein-1 . Controls showed no significant activity . Sulfate was shown to be the end product of sulfide oxidation and was observed at a rate of 2 to 3 nmol min-1 mg of protein-1 . The ammonium and sulfate production rates were not influenced by the addition of sulfide, suggesting that sulfide is first oxidized to elemental sulfur, and in a second independent step elemental sulfur is oxidized to sulfate . The average sulfide oxidation rate measured was 5 nmol min-1 mg of protein-1 and could be increased to 10.7 nmol min-1 mg of protein-1 after the trichomes were starved for 45 h . Incorporation of 14CO2 was at a rate of 0.4 to 0.8 nmol min-1 mg of protein-1, which is half the rate calculated from sulfide oxidation . {2-14C}acetate incorporation was 0.4 nmol min-1 mg of protein-1, which is equal to the CO2 fixation rate, and no 14CO2 production was detected . These results suggest that Thioploca species are facultative chemolithoautotrophs capable of mixotrophic growth . Microautoradiography confirmed that Thioploca cells assimilated the majority of the radiocarbon from {2-14C}acetate, with only a minor contribution by epibiontic bacteria present in the samples.

Dig Dis Sci, 1999 Jun, 44(6), 1202 - 7
Serum antibodies to Mycobacterium paratuberculosis in patients with Crohn's disease; Suenaga K et al.; Mycobacterium paratuberculosis has been suggested as a causative organism of Crohn's disease . Despite a long-term debate to prove this possibility, the role of this bacteria in the pathogenesis of Crohn's disease is still a subject of controversy . In the present study, serum antibodies (IgG, IgA, and IgM) to the protoplasmic antigen of M . paratuberculosis were quantified in patients with Crohn's disease and in control subjects by using an enzyme-linked immunosorbent assay whose specificity was increased by preabsorbing the sera with cell extracts of Mycobacterium phlei . As compared to normal controls (1/20; 0.062+/-0.022), a significant difference was seen in the antibody-positive prevalence rate and mean values of the serum IgG titer in patients with Crohn's disease (5/13; 0.102+/-0.039) (P < 0.05), but not in patients with ulcerative colitis (2/20; 0.065+/-0.035) and tuberculosis (0/4; 0.053+/-0.008) . No significant differences were seen in the antibody-positive prevalence rate and mean values of the serum IgA and IgM titers among the four study groups . These results indicate the unique immune response to M . paratuberculosis in patients with Crohn's disease, suggesting that this organism may play some role in the pathogenesis of Crohn's disease.

Dig Dis Sci, 1999 Jun, 44(6), 1189 - 95
Fatal ulcerative panenteritis following colectomy in a patient with ulcerative colitis; Annese V et al.; A 37-year-old man, previously submitted to colectomy for ulcerative pancolitis unresponsive to medical therapy, presented with nausea, vomiting, epigastric pain, and bloody diarrhea . An upper gastrointestinal endoscopy revealed mucosal friability, petechiae, and erosions throughout the duodenum, whereas prestomal ileum showed large ulcers and pseudopolyps . Histologically, a dense inflammation chiefly composed of lymphocytes and plasma cells with few neutrophils was detected . No bacteria, protozoa, and fungi could be detected . Despite intensive care, intra-1194 venous antibiotics and steroids, the patient died of diffuse intravascular coagulation and multiorgan failure . At post-mortem examination severe ulcerative lesions were observed scattered throughout the duodenum up to the distal ileum . The dramatic clinical presentation with fatal outcome, the widespread ulcers throughout the intestine, and the histological picture are peculiar features in our patient which can not be ascribed to any type of the ulcerative jejunoenteritis so far reported . Patients with pancolitis and diffuse ileal involvement do not necessarily have Crohn's disease but rather may have ulcerative colitis.

Indian J Public Health, 1998 Oct-Dec, 42(4), 131 - 2
Sterility testing of disposable syringes and needles marketed in Calcutta; Pal D et al.; Presterilized (disposable) syringes and needles were subjected to sterility testing for aerobic cultures . It was found that 56.3% of the samples were contaminated indicating failure of the sterilisation process . The implications of this could be far reaching and is discussed alongwith.

J Hosp Infect, 1999 Jun, 42(2), 125 - 33
Are most ICU infections really nosocomial? A prospective observational cohort study in mechanically ventilated patients; Silvestri L et al.; A prospective cohort study was undertaken with two end points: (i) to compare the 48 h time cut-off with the carrier state criterion for classifying infections, and (ii) to determine a time cut-off more in line with the carrier state concept . All patients admitted to the intensive care unit and expected to require mechanical ventilation for a period > or = 3 days were enrolled . Surveillance cultures of throat and rectum were obtained on admission and thereafter twice weekly to distinguish micro-organisms that were imported into the intensive care unit from those acquired during the stay in the unit . A total of 117 patients with median age of 61 years and median Simplified Acute Physiology Score II of 42, were included in the study . Of these patients, 48 (41%) developed a total of 74 infection episodes . Using the 48 h cut-off point, 80% of all infections were classified as ICU-acquired . According to the carrier state criterion, 44 infections (60%) were of primary endogenous development caused by micro-organisms imported into the intensive care unit . Seventeen secondary endogenous (23%) and 13 exogenous (17%) infections were caused by bacteria acquired in the unit . The carrier state classification allowed the transfer of 49% of infections from the ICU-acquired group into the import group . A time cut-off of nine days was found to identify ICU-acquired infections better than two days . These data suggest that monitoring of carriage of micro-organisms may be a more realistic approach to classify infections developing in the intensive care unit.

Appl Environ Microbiol, 1999 Jul, 65(7), 3248 - 50
Key physiology of anaerobic ammonium oxidation; Strous M et al.; The physiology of anaerobic ammonium oxidizing (anammox) aggregates grown in a sequencing batch reactor was investigated quantitatively . The physiological pH and temperature ranges were 6.7 to 8.3 and 20 to 43 degrees C, respectively . The affinity constants for the substrates ammonium and nitrite were each less than 0.1 mg of nitrogen per liter . The anammox process was completely inhibited by nitrite concentrations higher than 0.1 g of nitrogen per liter . Addition of trace amounts of either of the anammox intermediates (1 . 4 mg of nitrogen per liter of hydrazine or 0.7 mg of nitrogen per liter of hydroxylamine) restored activity completely.

Virology, 1999 Jul 5, 259(2), 274 - 85
Isolation and characterization of an oligomerization-negative mutant of HIV-1 integrase; Kalpana GV et al.; The yeast two-hybrid method was used to screen mutagenized DNAs to isolate a variant of the human immunodeficiency virus type 1 integrase (IN) that does not interact with the wild-type IN . The responsible mutation, leading to a single amino acid change (V260E) in the C-terminal domain of IN, blocks IN-IN multimerization but has only small effect on binding to a host interacting protein, INI1 (hSNF5) . Binding studies in vitro confirmed the defect in multimerization of the mutant IN . Biochemical analyses of the mutant IN enzyme expressed in bacteria detected only subtle changes in its properties, suggesting that the yeast system is a sensitive reporter of correct IN conformation . Mutant virus carrying the V260E substitution was blocked in replication at the time of DNA integration, consistent with IN multimerization being important for its activity in vivo .

J Mol Biol, 1999 Jul 2, 290(1), 137 - 48
DNA binding mediates conformational changes and metal ion coordination in the active site of PcrA helicase; Soultanas P et al.; Based upon the crystal structures of PcrA helicase, we have made and characterised mutations in a number of conserved helicase signature motifs around the ATPase active site . We have also determined structures of complexes of wild-type PcrA with ADPNP and of a mutant PcrA complexed with ADPNP and Mn2+ . The kinetic and structural data define roles for a number of different residues in and around the ATP binding site . More importantly, our results also show that there are two functionally distinct conformations of ATP in the active site . In one conformation, ATP is hydrolysed poorly whereas in the other (activated) conformation, ATP is hydrolysed much more rapidly . We propose a mechanism to explain how the stimulation of ATPase activity afforded by binding of single-stranded DNA stabilises the activated conformation favouring Mg2+binding and a consequent repositioning of the gamma-phosphate group which promotes ATP hydrolysis . A part of the associated conformational change in the protein forces the side-chain of K37 to vacate the Mg2+binding site, allowing the cation to bind and interact with ATP .

Biochemistry, 1999 Jun 22, 38(25), 8167 - 78
Secondary structure extensions in Pyrococcus furiosus ferredoxin destabilize the disulfide bond relative to that in other hyperthermostable ferredoxins . Global consequences for the disulfide orientational heterogeneity; Wang PL et al.; The single cubane cluster ferredoxin (Fd) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) possesses several unique properties when compared even to Fds from other hyperthermophilic archaea or bacteria . These include an equilibrium molecular heterogeneity, a six- to seven-residue increase in size, an Asp rather than the Cys as one cluster ligand, and a readily reducible disulfide bond . NMR assignments and determination of both secondary structure and tertiary contacts remote from the paramagnetic oxidized cluster of Pf 3Fe Fd with an intact disulfide bond reported previously (Teng Q., Zhou, Z . H., Smith, E . T., Busse, S . C., Howard, J . B . Adams, M . W . W., and La Mar, G . (1994) Biochemistry 33, 6316-6328) are extended here to the 4Fe oxidized cluster WT (1H and 15N) and D14C (1H only) Fds with an intact disulfide bond and to the 4Fe oxidized WT Fd (1H and 15N) with a cleaved disulfide bond . All forms are shown to possess a long (13-member) alpha-helix, two beta-sheets (one double-, one triple-stranded), and three turns outside the cluster vicinity, each with tertiary contacts among themselves as found in other Fds . While the same secondary structural elements, with similar tertiary contacts, are found in other hyperthermostable Fds, Pf Fd has two elements, the long helix and the triple-stranded beta-sheet, that exhibit extensions and form multiple tertiary contacts . All Pf Fd forms with an intact disulfide bond exhibit a dynamic equilibrium heterogeneity which is shown to modulate a hydrogen-bonding network in the hydrophobic core that radiates from the Cys21-Cys48 disulfide bond and encompasses residues Lys36, Val24, Cys21, and Cys17 and the majority of the long helix . The heterogeneity is attributed to population of the alternate S and R chiralities of the disulfide bond, each destabilized by steric interactions with the extended alpha-helix . Comparison of the chemical shifts and their temperature gradients reveals that the molecular structure of the protein with the less stable R disulfide resembles that of the Fd with a cleaved disulfide bond . Both cluster architecture (3Fe vs 4Fe) and ligand mutation (Cys for Asp14) leave the disulfide orientational heterogeneity largely unperturbed . It is concluded that the six- to seven-residue extension that results in a longer helix and larger beta-sheet in Pf Fd, relative to other hyperthermostable Fds, more likely serves to destabilize the disulfide bond, and hence make it more readily reducible, than to significantly increase protein thermostability.

Curr Microbiol, 1999 Jul, 39(1), 27 - 30
Presence of Na+-stimulated P-type ATPase in the membrane of a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum; Koyama N; It was found that a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum, possesses a membrane-bound ATPase, which was activated specifically by Na+ . The Na+-stimulated ATPase activity reached a maximum value at 200 mM NaCl . In the presence of 200 mM NaCl, the activity was drastically reduced by vanadate, a potent inhibitor of P-type ATPase, with a half-maximal inhibition at 1 microM . Incubation of the membranes with {gamma-32P}ATP followed by acidic lithium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the existence of two phosphorylated intermediates with apparent molecular masses of 60 and 100 kDa . Only phosphorylation of the 100-kDa polypeptide was inhibited by vanadate . The membrane extract containing Na+-stimulated ATPase, when reconstituted into soybean phospholipid vesicles, exhibited 22Na+ transport by the addition of ATP, which was inhibited by vanadate and gramicidin . It is likely that the Na+-stimulated ATPase belongs to P-type and is involved in Na+ transport.

Protein Sci, 1999 Jun, 8(6), 1232 - 40
The molecular structure of an unusual cytochrome c2 determined at 2.0 A; the cytochrome cH from Methylobacterium extorquens; Read J et al.; Cytochrome cH is the electron donor to the oxidase in methylotrophic bacteria . Its amino acid sequence suggests that it is a typical Class 1 cytochrome c, but some features of the sequence indicated that its structure might be of special interest . The structure of oxidized cytochrome cH has been solved to 2.0 A resolution by X-ray diffraction . It has the classical tertiary structure of the Class 1 cytochromes c but bears a closer gross resemblance to mitochondrial cytochrome c than to the bacterial cytochrome c2 . The left-hand side of the haem cleft is unique; in particular, it is highly hydrophobic, the usual water is absent, and the "conserved" Tyr67 is replaced by tryptophan . A number of features of the structure demonstrate that the usual hydrogen bonding network involving water in the haem channel is not essential and that other mechanisms may exist for modulation of redox potentials in this cytochrome.

FEMS Microbiol Lett, 1999 Jun 15, 175(2), 165 - 70
Regulation of the transcription of genes encoding different virulence factors in Helicobacter pylori by free iron; Szczebara F et al.; Since free iron possesses a poor solubility under physiologic conditions and thus becomes a limiting nutrient for growth, a shift from high- to low-iron environmental conditions is an important signal for bacteria to coordinate the regulation of gene expression . Here, we studied and compared the level of transcripts corresponding to the vacA (cytotoxin), ureA (urease), cagA (cytotoxin-associated antigen) and fur (ferric uptake regulator) genes of Helicobacter pylori, grown under iron-sufficient and iron-restricted conditions . A significant increase in the accumulation of vacA and fur transcripts was observed under iron-restricted conditions . This up-regulation by low levels of iron seems to be not directly regulated by Fur, and certainly requires other regulatory factors . No statistical difference was defined in the accumulation of cagA and ureA.

Microbiol Immunol, 1999, 43(4), 339 - 49
Proposal of Sphingomonas suberifaciens (van Bruggen, Jochimsen and Brown 1990) comb . nov., sphingomonas natatoria (Sly 1985) comb . nov., Sphingomonas ursincola (Yurkov et al . 1997) comb . nov., and emendation of the genus Sphingomonas; Yabuuchi E et al.; Based on the results of a phylogenetic analysis of 16S rRNA and the presence of sphingoglycolipid in cellular lipids of the type strains, transfer of "Rhizomonas" suberifaciens, Blastomonas natatoria and Erythromonas ursincola to the genus Sphingomonas as Sphingomonas suberifaciens (van Bruggen et al 1990) comb . nov., Sphingomonas natatoria (Sly 1985) comb . nov., and Sphingomonas ursincola (Yurkov et al 1997) comb . nov . are herein proposed together with the emendation of genus Sphingomonas . The type strain of S . suberifaciens is van Bruggen Cal=ATCC 49382=NCPPB 3629=IFO 15211=JCM 8521, that of S . natatoria is ATCC 35951 =DSM 3183=NCIMB 12085=JCM10396, and that of S . ursincola is DSM 9006= KR-99.

Baillieres Clin Endocrinol Metab, 1998 Dec, 12(4), 691 - 705
Experimental studies on lignans and cancer; Thompson LU; Mammalian lignans are produced from plant precursors such as secoisolariciresinol diglycoside (SDG) and matairesinol via the action of bacteria in the human or animal colon . While precursors are found in many plant foods, flaxseed is the richest source of SDG and was therefore used as a model to determine the anti-cancer effects of lignans . This paper reviews the experimental studies in animals and humans demonstrating the anti-cancer effects of flaxseed and its SDG as well as other studies relevant to the clinical use of lignans, such as those on their food sources, bio-availability and safety.

N J Med, 1999 Jun, 96(6), 25 - 7
A is for acne; Cornell DH; Acne is caused by hormones, androgen, and follicular keratinization, which leads to blocked pores, and P . acnes bacteria, which cause pustule form . Dermatologists report that acne treatments, like the skin of suffers, are clearly getting better . New treatments and the refinements of mainstay ones have changed the face of acne treatment . One advantage of the newer, more effective treatments is that patients now take lower doses of antibiotics for shorter periods of time.

Mamm Genome, 1999 Jul, 10(7), 706 - 9
A 5x genome coverage bovine BAC library: production, characterization, and distribution; Zhu B et al.; A bovine large-insert DNA library has been constructed in a Bacterial Artificial Chromosome (BAC) vector . The source DNA was derived from lymphocytes of a Jersey male . High-molecular-weight DNA fragments were produced by treatment with EcoRI/EcoRI methylase and cloned into the EcoRI site of pBACe3.6 . In total, 157,240 individual BACs have been picked into 384-well plates . Approximately 190 randomly chosen clones have been characterized by Pulsed Field Gel Electrophoresis (PFGE) and have an average insert size of 105 kb, suggesting library coverage representing 5-6 genome equivalents . The frequency of clones without inserts is 4% . The chromosomal location of 51 BACs was studied by FISH; 3 showed more than one signal, indicating a chimerism frequency of roughly 6% . Approximately 50% of the clones in the library contain Simple Repeat Sequences (microsatellites), and 4% of the clones contain centromeric repeats . Insert stability was assessed by restriction digestion of DNA prepared from 20 clones after serial culture for one and three nights . Only one clone showed any evidence of an altered restriction pattern . Clones from 360 x 384-well plates (138,240 colonies) were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones . Both membranes and superpools are available from the RZPD, Berlin . PCR 4-D superpools have been prepared from an additional 23,000 clones . The library has been screened for a total of 24 single-copy sequences; positive clones have been obtained in all cases.

J Bacteriol, 1999 Jul, 181(13), 3949 - 55
Interaction of Azospirillum lipoferum with wheat germ agglutinin stimulates nitrogen fixation; Karpati E et al.; In vitro, the nitrogen fixation capability of A . lipoferum is efficiently increased in the presence of wheat germ agglutinin (WGA) . A putative WGA-binding receptor, a 32-kDa protein, was detected in the cell capsule . The stimulatory effect required N-acetyl-D-glucosamine dimer (GlcNAcdi) terminated sugar side chains of the receptor and was dependent on the number of GlcNAcdi links involved in receptor-WGA interface . Binding to the primary sugar binding sites on WGA had a larger stimulatory effect than binding to the secondary sites . The WGA-receptor complex generated stimulus led to elevated transcription of the nifH and nifA genes and of the glnBA gene cluster but not of the glnA gene from its own promoter . There may well be a signalling cascade contributing to the regulation of nitrogen fixation.

Helicobacter, 1999 Jun, 4(2), 82 - 8
Differences among Helicobacter pylori strains isolated from three different populations and demonstrated by restriction enzyme analysis of an internal fragment of the conserved gene hpaA; Evans DG et al.; BACKGROUND: Our goal was to test the idea that Helicobacter pylori genotypes vary from one population to another . METHODS: Analysis of Sau3A and HinfI restriction fragment-length polymorphism (RFLP) in a 375-bp polymerase chain reaction amplicon of hpaA was used to compare 31 H . pylori isolates from a relatively small and genetically homogeneous population (Goteborg, Sweden) with those of large, genetically heterogeneous populations located in two different countries (50 isolates from Houston, TX, and 69 isolates from Minas Gerais, a state in the southeastern region of Brazil) . RESULTS: Five different Sau3A and three different HinfI restriction patterns were found; different combinations of these comprise 10 different RFLP types, I through X . The RFLP types found in the United States and Brazil collections were very similar, except for two Brazil isolates belonging to type VIII and five Brazil isolates belonging to type X, neither type found in the United States . The overall profile of H . pylori isolates from Sweden was remarkably different, with 18 of 31 (58%) having a new Sau3A restriction pattern, termed gS; 10 of these 18 isolates had HinfI restriction pattern E (RFLP type VIII), and 8 had HinfI restriction pattern F (RFLP type IX) . No isolates from Sweden belonged to RFLP type III or type X . CONCLUSIONS: RFLP typing of a 375-bp polymerase chain reaction-amplified DNA fragment of H . pylori hpaA revealed that H . pylori genotypes can and do vary from one population to another . We conclude that the unique RFLP profile shown by the group of H . pylori isolates from Goteborg is the result of a cohort effect in this relatively small, stable, genetically homogeneous population . Also, the overall similarity between RFLP profiles of the H . pylori isolates from Texas and Minas Gerais coincides with the fact that although geographically distanced, these populations are similar in being large, dynamic, and genetically heterogeneous.

Heredity, 1999 Jun, 82 ( Pt 6), 605 - 12
Low genetic diversity among pea aphid (Acyrthosiphon pisum) biotypes of different plant affiliation; Birkle LM et al.; Genetic diversity in the pea aphid Acyrthosiphon pisum was investigated by a restriction fragment length polymorphism (RFLP) analysis of three maternally inherited genomes (mitochondrial DNA and plasmids of the symbiotic bacteria Buchnera) . Twenty-nine parthenogenetic clones of three A . pisum biotypes, defined by their capacity to use the legume crops pea, alfalfa and red clover, respectively, were analysed, and a total of 67 restriction sites was scored . No restriction site variation in the mitochondrial genome was obtained, but length variation at two regions (the A + T-rich region and ND3-ND5 region) was noted . One aphid clone bore a variant HindIII restriction site in the Buchnera leucine plasmid (pAPEleu), and two clones were heteroplasmic for a 0.76-kb deletion in the Buchnera tryptophan plasmid (pAPEtrp) . Based on arthropod nucleotide substitution rates, it is proposed that the crop-feeding biotypes of A . pisum may have diversified within the last 100 000 years and possibly much more recently, since the advent of agriculture.

Aliment Pharmacol Ther, 1999 Jul, 13(7), 875 - 81
Effect of ranitidine bismuth citrate on the phospholipase A2 activity of Naja naja venom and Helicobacter pylori: a biochemical analysis; Ottlecz A et al.; BACKGROUND: Helicobacter pylori has become recognized as a fundamental pathogen in the development of gastritis and peptic ulcer disease . Bismuth compounds in combination with antibiotics are widely used to treat H . pylori associated peptic ulcer disease . METHODS: In this study we measured and analysed the inhibitory effect of ranitidine bismuth citrate (RBC, Pylorid, Tritec) on the activity and kinetics of phospholipase A2 (PLA2) (E.C.3.1.1.4) of commercial cobra (Naja naja) venom and H . pylori (French press lysates) using L-alpha-dipalmitoyl-(2{1-14C}palmitoyl)-phosphatidylcholine as substrate . RESULTS: Our data suggest that RBC might exert a dose-dependent uncompetitive inhibition on PLA2 activity of both H . pylori and Naja naja venom . the inhibitory effect of RBC on the PLA2 activity cannot be abolished by the optimal concentration of calcium (10 mM), indicating its mechanism to be unrelated to the displacement of calcium from the activation site of the enzyme . CONCLUSION: Our results suggest that one of the mechanisms by which bismuth compounds are therapeutically effective in the treatment of H . pylori associated gastritis is by inhibiting the activity of the degradative PLA2 enzyme secreted by H . pylori . As a consequence of the inhibitory action of RBC on PLA2 of the bacteria, the extracellular and/or intracellular phospholipid components of the gastric mucosal barrier are preserved.

Aliment Pharmacol Ther, 1999 Jun, 13(6), 753 - 60
Effects of ranitidine bismuth citrate on Helicobacter pylori motility, morphology and survival; Worku ML et al.; AIM: The effects of the anti-ulcer agents ranitidine bismuth citrate (RBC), ranitidine hydrochloride (R) and colloidal bismuth citrate (BC), on Helicobacter pylori motility, morphology and survival were examined to determine whether the clinical effectiveness of RBC might be linked to a specific action that inhibits bacterial motility . METHODS: H . pylori from patients with duodenal ulcer or non-ulcer dyspepsia were exposed to RBC and BC at bismuth concentrations ranging from 12.5 to 50 microg/mL, and R at ranitidine concentrations ranging from 12.5 to 50 microg/mL for a brief period (< 15 min), 6 h and 24 h . Bacterial motility was assessed with a Hobson BacTracker, bacterial morphology by transmission electron microscopy, and growth inhibition by counting colony-forming units . RESULTS: H . pylori motility was diminished with RBC and BC but not R . However, the effect of RBC was markedly greater than that of BC at each bismuth concentration and time of exposure tested: (i) brief exposure to RBC/bismuth 50 microg/mL but not to BC, resulted in a significant loss of motility without loss of viability or change in cell morphology, and (ii) bacteria were immobilized, and lost viability after exposure to RBC/bismuth 50 microg/mL for 24 h but not to BC . Morphological destruction caused by RBC differed from that by BC: after 24 h exposure to the highest concentration tested, cell fragmentation and flagella detachment occurred more frequently with BC than RBC, but the latter produced greater disruption of intracellular structures . CONCLUSIONS: RBC suppresses growth of H . pylori, and has a specific inhibitory effect on the bacterial motor mechanism . These pharmacological actions are likely to contribute to the clinical effectiveness of the agent.

J Biol Chem, 1999 Jul 2, 274(27), 19397 - 402
Mutational alterations in the homotetrameric chaperone SecB that implicate the structure as dimer of dimers; Muren EM et al.; Variant forms of SecB with substitutions of aminoacyl residues in the region from 74 to 80 were analyzed with respect to their ability to bind a physiological ligand, precursor galactose-binding protein, and to their oligomeric states . SecBL75Q and SecBE77K are tetramers with affinity for ligand indistinguishable from that of the wild-type SecB, and thus the export defect exhibited by strains producing these variants must result from an effect on interactions between SecB and other components . SecBF74I is tetrameric but binds ligand with a lower affinity . Substitutions at positions 76, 78, and 80 cause a shift in the equilibrium so that the SecB tetramer dissociates into dimers . We conclude that the tetramer is a dimer of dimers and that the residues Cys76, Val78, and Gln80 must be involved either directly or indirectly in forming the interface between dimers . These variant species are defective in binding ligand; however, because their oligomeric state is altered no conclusion can be drawn concerning the direct role of these residues in ligand binding.

J Antimicrob Chemother, 1999 May, 43(5), 615 - 23
Antimycobacterial activities of riminophenazines; Reddy VM et al.; Riminophenazines were specifically developed as drugs active against Mycobacterium tuberculosis but extensive research over several decades has shown that these compounds are also active against many other mycobacterial infections, particularly those caused by Mycobacterium leprae and the Mycobacterium avium complex (MAC) . Clofazimine, the lead compound in this series, is included in the regimens that are approved by the WHO for the treatment of leprosy and has contributed significantly to the control of that disease, particularly that caused by dapsone-resistant bacteria . Despite early problems, clofazimine has shown clinical efficacy in tuberculosis, in particular that caused by multiple drug resistant strains . Clofazimine does not induce resistance and also inhibits emergence of resistance to isoniazid in M . tuberculosis . The efficacy of clofazimine against MAC is more varied and the availability of better drugs has limited its use . Newer riminophenazines, such as B746 and B4157, not only showed increased anti-mycobacterial activity but also produced less skin pigmentation, which is the main drawback of this group of compounds . The most important virtues of riminophenazines, such as intracellular accumulation in mononuclear phagocytic cells, anti-inflammatory activity, a low incidence of drug resistance and slow metabolic elimination, make them attractive candidates for the treatment of mycobacterial infections . It is essential, however, to investigate the newer analogues clinically, while continuing the pursuit of alternate candidates that demonstrate higher anti-mycobacterial activity and lower rates of skin pigmentation.

J Clin Virol, 1999 May, 12(3), 211 - 9
A population-based prospective survey of newborn infants with suspected systemic infection: occurrence of sporadic enterovirus and adenovirus infections; Rosenlew M et al.; BACKGROUND: Enterovirus outbreaks are known to occur in neonatal wards and enteroviruses may cause community-acquired sepsis-like disease in the neonatal period . Less well is known their possible role in suspected systemic infections during the perinatal period . OBJECTIVES: To investigate the occurrence of enterovirus infections in neonatal patients suspected of systemic infection . STUDY DESIGN: A population-based prospective survey was organized in the hospitals of the Greater Helsinki Region during 13 months in 1993-94 . Criteria for enrollment included onset of symptoms before the age of 29 days and a decision, on clinical grounds, to take a blood culture for bacteria . Acute phase samples of blood, feces, nasopharyngeal swab, and cerebrospinal fluid, if available, were inoculated in monolayer cultures of four different cell lines . In addition, enterovirus infections were searched for using an enterovirus group-reacting IgM test . RESULTS: One hundred and thirty-seven patients had a sufficient number of specimens examined, and were thus evaluable . Most of the infants had the onset of the symptoms within a few days after birth . An enterovirus was isolated from four newborn infants (3%), while seven children (5%) were found to excrete adenovirus . Enteroviral antigen was detected in cell cultures inoculated with specimens from two additional infants . Virus-positive infants had no evidence of bacterial infection and did not show specific clinical signs or symptoms differentiating them from the rest of the study group . All enrolled infants recovered without sequelae . CONCLUSION: We conclude that sporadic viral infections may be common in neonatal patients with suspected systemic infection, and this should be taken into account when judging the etiology.

J Clin Periodontol, 1999 Jun, 26(6), 366 - 73
Extensive expression of TGF-beta1 in chronically-inflamed periodontal tissue; Steinsvoll S et al.; The host immune response in chronic marginal periodontitis (CMP) raised against bacteria colonizing the dentogingival area is modulated by cytokines . This study examines the distribution of the transforming growth factor-beta1 containing (TGF-beta1+) cells in formalin-fixed and paraffin-embedded gingival specimens from 11 patients with chronic marginal periodontitis and 7 persons with healthy gingiva . Inflamed periodontal tissue contained a 100-fold more TGF-beta1+ cells than healthy gingiva . Diverse morphological TGF-beta1+ cell types were discerned . Double immuno-enzymatic and -fluorescence staining revealed that TGF-beta1+ cells comprised 21-29% macrophages 2-3% T-cells, 3-9% B-cells, 34-35% neutrophilic granulocytes and 7-10% mast cells . The densities of all TGF-beta1+ cell types in CMP were strongly increased in the connective tissue adjacent to the pocket epithelium, in the lamina propria and adjacent to the oral epithelium . In lesions with extensive inflammation, expression was also marked in pocket epithelium . TGF-beta1 is an immunosuppressive cytokine that stimulates wound healing . Upregulation of the cytokine in inflamed gingiva may counterbalance for destructive gingival inflammatory responses that are simultaneously taking place in patients with CMP.

J Biomol NMR, 1999 May, 14(1), 71 - 4
Selective and extensive 13C labeling of a membrane protein for solid-state NMR investigations; Hong M et al.; The selective and extensive 13C labeling of mostly hydrophobic amino acid residues in a 25 kDa membrane protein, the colicin Ia channel domain, is reported . The novel 13C labeling approach takes advantage of the amino acid biosynthetic pathways in bacteria and suppresses the synthesis of the amino acid products of the citric acid cycle . The selectivity and extensiveness of labeling significantly simplify the solid-state NMR spectra, reduce line broadening, and should permit the simultaneous measurement of multiple structural constraints . We show the assignment of most 13C resonances to specific amino acid types based on the characteristic chemical shifts, the 13C labeling pattern, and the amino acid composition of the protein . The assignment is partly confirmed by a 2D homonuclear double-quantum-filter experiment under magic-angle spinning . The high sensitivity and spectral resolution attained with this 13C-labeling protocol, which is termed TEASE for ten-amino acid selective and extensive labeling, are demonstrated.

Gastroenterology, 1999 Jul, 117(1), 82 - 8
Isolation of a Helicobacter pylori protein, FldA, associated with mucosa-associated lymphoid tissue lymphoma of the stomach; Chang CS et al.; BACKGROUND & AIMS: The growth of gastric mucosa-associated lymphoid tissue lymphoma (MALToma) seems to depend on the stimulation of Helicobacter pylori . We attempted to identify specific antigen(s) from H . pylori strains associated with MALToma . Methods: Membranous and secreted proteins of H . pylori were compared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis by Western blot using sera from patients with MALToma . RESULTS: A 19-kilodalton protein was seen in all strains isolated from patients with MALToma but uncommonly in other strains . The protein was purified and sequenced . Amino acid sequence comparison showed it was an FldA homologue, a putative flavodoxin protein . DNA sequencing in 26 strains revealed that a nucleotide G insertion at position 481 of the fldA gene was more frequently observed in strains associated with MALToma than other strains (9/9 vs . 6/17; P = 0.002) . The mutation caused a short truncation . A recombinant protein with this truncation was expressed and tested . Sera of 12 (70.6%) of 17 patients with MALToma were positive for the antibody to the recombinant protein, and 7 (16.7%) of 42 control patients were positive (12/17 vs . 7/42; P < 0.0001) . CONCLUSIONS: Truncated FldA of H . pylori is associated with gastric MALToma . It may be involved in the pathogenesis of gastric MALToma . Antibody to this antigen could be used as a serological marker of the disease.

Science, 1999 Jun 25, 284(5423), 2124 - 9
Phylogenetic classification and the universal tree; Doolittle WF; From comparative analyses of the nucleotide sequences of genes encoding ribosomal RNAs and several proteins, molecular phylogeneticists have constructed a "universal tree of life," taking it as the basis for a "natural" hierarchical classification of all living things . Although confidence in some of the tree's early branches has recently been shaken, new approaches could still resolve many methodological uncertainties . More challenging is evidence that most archaeal and bacterial genomes (and the inferred ancestral eukaryotic nuclear genome) contain genes from multiple sources . If "chimerism" or "lateral gene transfer" cannot be dismissed as trivial in extent or limited to special categories of genes, then no hierarchical universal classification can be taken as natural . Molecular phylogeneticists will have failed to find the "true tree," not because their methods are inadequate or because they have chosen the wrong genes, but because the history of life cannot properly be represented as a tree . However, taxonomies based on molecular sequences will remain indispensable, and understanding of the evolutionary process will ultimately be enriched, not impoverished.

Chem Biol, 1999 Jul, 6(7), 441 - 9
Mutational and structural analyses of the regulatory protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath); Brandstetter H et al.; BACKGROUND: The soluble methane monooxygenase (sMMO) system in methanotrophic bacteria uses three protein components to catalyze the selective oxidation of methane to methanol . The coupling protein B (MMOB) both activates the carboxylate-bridged diiron center in the hydroxylase (MMOH) for substrate oxidation and couples the reaction to electron transfer from NADH through the sMMO reductase . Although the X-ray structure of the hydroxylase is known, little structural information is available regarding protein B . RESULTS: Wild-type protein B from Methylococcus capsulatus (Bath) is very susceptible to degradation . The triple mutant protein B, Gly10-->Ala, Gly13-->Gln, Gly16-->Ala is resistant to degradation . Analyzing wild-type and mutant forms of protein B using size exclusion chromatography and circular dichroism spectroscopy suggests that the amino terminus of MMOB (Ser1-Ala25) is responsible for the proteolytic sensitivity and unusual mobility of the protein . We used the stable triple glycine protein B mutant to generate an affinity column for the hydroxylase and investigated the interaction between MMOH and MMOB . These results suggest the interaction is dominated by hydrophobic contacts . CONCLUSIONS: A structural model is presented for protein B that explains both its proclivity for degradation and its anomalous behavior during size exclusion chromatography . The model is consistent with previously published biophysical data, including the NMR structure of the phenol hydroxylase regulatory protein P2 . Furthermore, this model allows for detailed and testable predictions about the structure of protein B and the role of proposed recognition sites for the hydroxylase.

Mol Phylogenet Evol, 1999 Jul, 12(2), 177 - 85
The structure and evolution of the ribosomal proteins encoded in the spc operon of the archaeon (Crenarchaeota) Sulfolobus acidocaldarius; Yang D et al.; The genes for nine ribosomal proteins, L24, L5, S14, S8, L6, L18, S5, L30, and L15, have been isolated and sequenced from the spc operon in the archaeon (Crenarchaeota) Sulfolobus acidocaldarius, and the putative amino acid sequence of the proteins coded by these genes has been determined . In addition, three other genes in the spc operon, coding for ribosomal proteins S4E, L32E, and L19E (equivalent to rat ribosomal proteins S4, L32, and L19), were sequenced and the structure of the putative proteins was determined . The order of the ribosomal protein genes in the spc operon of the Crenarchaeota kingdom of Archaea is identical to that present in the Euryarchaeota kingdom of Archaea and also identical to that found in bacteria, except for the genes for r-proteins S4E, L32E, and L19E, which are absent in bacteria . Although AUG is the initiation codon in most of the spc genes, GUG (val) and UUG (leu) are also used as initiation codons in S . acidocaldarius . Over 70% of the codons in the Sulfolobus spc operon have A or U in the third position, reflecting the low GC content of Sulfolobus DNA . Phylogenetic analysis indicated that the archaeal r-proteins are a sister group of their eucaryotic counterparts but did not resolve the question of whether the Archaea is monophyletic, as suggested by the L6P, L15P, and L18P trees, or the question of whether the Crenarchaeota is separate from the Euryarchaeota and closer to the Eucarya, as suggested by the S8P, S5P, and L24P trees . In the case of the three Sulfolobus r-proteins that do not have a counterpart in the bacterial ribosome (S4E, L32E, and L19E), the archaeal r-proteins showed substantial identity to their eucaryotic equivalents, but in all cases the archaeal proteins formed a separate group from the eucaryotic proteins .

Ecotoxicol Environ Saf, 1999 Jul, 43(3), 292 - 300
Toxicity and bioaccumulation of cadmium in marine protozoa communities; Fernandez-Leborans G et al.; The behavior of cadmium in a protozoan community was analyzed in order to obtain new data concerning the toxicity and bioaccumulation of this heavy metal . For this purpose, microcosms with different concentrations of the pollutant (without cadmium, 500 microg Cd.l-1 and 1000 microg Cd.l-1) were used . Protozoans bioaccumulated 8 . 74-283 microg Cd.g-1 dry weight, representing an accumulation capacity of 15.53-69.59 times more than that of bacteria . The addition of cadmium caused a significant reduction in protozoan density, whereas bacterial abundance was not affected .

Ecotoxicol Environ Saf, 1999 Jul, 43(3), 267 - 73
On the suitability of fiberglass reinforced polyester as building material for mesocosms; Berghahn R et al.; Gel- and topcoat surface layers on fiberglass {glass-reinforced plastic (GRP)} made of unsaturated resin based on isophthalic acid polyester and neopentyl glycol (ISO-NPG) were tested for leaching, ecotoxicity of water eluates, and abrasion by river sediments at a current speed of 0.5 m * s-1 . Leaching from topcoat tempered at low temperature was significant, whereas it was negligible from highly tempered gelcoat . Water eluates from both gel-and topcoat were nontoxic in routinely employed biotests (bacteria, algae, daphnids) . No abrasion by river sediments was detectable . Based on these results, GRP with gelcoat made of ISO-NPG is considered a suitable building material for mesocosms .

Scand J Infect Dis, 1999, 31(1), 63 - 8
Generalized mycobacterium genavense infection in HIV-infected patients: detection of the mycobacterium in hospital tap water; Hillebrand-Haverkort ME et al.; We describe 3 HIV-infected patients with disseminated M . genavense infection . The use of corticosteroids possibly favoured colonization and dissemination of atypical mycobacteria in these patients with low CD4 cell counts and may have masked symptoms of infection . The fact that these patients were treated with highly active antiretroviral therapy (HAART) together with antimycobacterial therapy may explain that 1 patient was free from mycobacteria 16 months after the end of specific treatment . Hospital tap water contained M . genavense at a concentration of >10 bacteria/l as examined by PCR . This species caused 12% of cases of non-tuberculous disseminated mycobacteriosis in HIV-infected patients at our hospital.

J Rheumatol, 1999 Jun, 26(6), 1338 - 46
A proposal for the classification of patients for clinical and experimental studies on reactive arthritis; Pacheco-Tena C et al.; OBJECTIVE: To propose classification criteria for patients entering clinical and basic studies on reactive arthritis (ReA) . METHODS: From a MEDLINE search of articles published between 1980 and 1996, we identified reports on HLA-B27 related ReA and Reiter's syndrome as study groups and analyzed the items that constituted the diagnostic, classification, and inclusion (or entry) criteria of patients . We developed disease categories that constituted our classification proposal . RESULTS: We reviewed 175 articles containing 110 study groups of patients with ReA and 94 with Reiter's syndrome . Most articles (89.7%) relied on arthritis for diagnosis, but only 48.0% relied on infection . Only 22.5% of articles used published criteria for diagnosis . Articles including a bacterial name to further describe a group of patients with ReA relied on cultures at the site of infection, serum antibodies, or both to confirm the diagnosis . There were inter/intra-group variations and overlapping of diagnostic criteria, at least 32 different terms referring to ReA or Reiter's syndrome, and 6 patterns of disease . According to these data, we propose 3 categories of disease for patients entering clinical and basic studies on ReA: probable ReA (2 subgroups); definite ReA triggered by bacteria (2 subgroups); and bacterial-associated undifferentiated oligoarthritis or spondyloarthropathy . CONCLUSION: This proposal provides a rationale for reducing the heterogeneity found in criteria for including patients with ReA in research and to facilitate scientific communication . In contrast to diagnostic criteria, this proposal does not restrict the study population to a minority of patients, but allows the investigator to include several forms of disease and to analyze results according to different categories.

Tidsskr Nor Laegeforen, 1999 May 10, 119(12), 1756 - 7
{Pneumatosis cystoides intestinalis}; Andresen SJ et al.; Patients with free intraperitoneal air usually undergo emergency surgery . Some of these patients will have no identifiable perforation, for instance those with pneumatosis cystoides intestinalis . This is a rare condition characterized by multiple intramural gas cysts in the gastrointestinal tract . The most common symptoms are meteorism, excessive flatulence, diarrhoea, abdominal pain, passage of mucus per rectum, or rectal bleeding . A case of pneumatosis cystoides intestinalsis is described . Plain abdominal radiographs showed distended bowel with free intraperitoneal air and intramural gas collections . At laparotomy, multiple intramural cysts were found, but no perforation or obstruction . The symptoms resolved after laparotomy, and the patient was discharged after a few days . The aetiology and pathogenesis of pneumatosis cystoides intestinalis are unknown, although deficient hydrogen metabolism and gasforming bacteria that penetrate the mucosal barrier may be involved . If needed, hyperbaric oxygen therapy is the treatment of choice . Surgery is indicated only in fulminant cases.

Pac Symp Biocomput . 1999;:542-53.
Characterisation of side-chain conformational preferences in a biologically active but unfolded protein; Mathieson SI et al.; A combination of experimental NMR 3J alpha beta coupling constant measurements and theoretical predictions from a statistical model for a random coil have been used to characterise the conformations of amino acid side-chains in an unfolded fibronectin binding protein . The statistical model uses the distribution of torsion angles in a data base of native folded protein structures to provide a description of the torsion angle populations of each residue in a random coil . For all but three of the residues studied a close agreement is observed between the experimental 3J alpha beta data and the model predictions (correlation coefficient 0.90; RMSD 0.70 Hz) . In these cases the populations about the chi 1 torsion angles in the conformational ensemble defining the fibronectin binding protein are well described by those present in the protein data base . For Phe 69, Asp 92 and Asp 105 however significant deviations are observed between the predictions and experimental data . Each of these side-chains is found to be involved in persistent non-random structural features arising from clustering of hydrophobic groups or interactions between charged side-chains . The analysis demonstrates the detailed insight that can be provided into conformationally disordered states by combining experimental and theoretical approaches.

Insect Mol Biol, 1999 May, 8(2), 185 - 91
Distribution and phylogeny of Wolbachia inducing thelytoky in Rhoditini and 'Aylacini' (Hymenoptera: Cynipidae); Plantard O et al.; Wolbachia are endosymbiotic bacteria responsible for thelytoky in several parasitoid hymenopteran genera . After finding these micro-organisms in some populations of Diplolepis spinosissimae (Hymenoptera: Cynipidae) where they are responsible for thelytoky through gamete duplication, we searched for Wolbachia spp . using specific PCR primers in nineteen other species of the Rhoditini tribe (rose gallwasps) and eight species of the 'Aylacini' tribe (gallwasps associated with herbaceous plants) . Wolbachia were found in twelve Rhoditini species and four 'Aylacini' species . The most infected species have very few males (spanandry) and the thelytoky of infected species/arrhenotoky of uninfected species is confirmed by previous research based on the sex of the offspring of virgin females . Phylogenetic analyses based on the partial Wolbachia ftsZ gene sequences indicate that some strains associated with closely related gallwasps are phylogenetically distant, suggesting that cynipids have been affected by several infection events . In contrast, the five infected European species of Diplolepis harbour the same strain of Wolbachia.

J Microbiol Methods, 1999 Jun, 36(3), 193 - 201
Comparison of API 20NE and Biolog GN identification systems assessed by techniques of multivariate analyses; Truu J et al.; The increasing use of commercial multitest systems for identification of environmental bacteria creates the problem of how to compare the identification results obtained from different systems . The limited use of species designations in such comparisons is caused by low usage of environmental bacteria in the development of commercial identification schemes . Two multivariate statistical methods, the Mantel's test and the co-inertia analysis, were applied to analyze data derived from the Biolog GN and the API 20NE systems of identification for 50 environmental bacterial strains . We found these two methods to be useful for revealing the relationship between the two sets of numerical taxonomic traits . Both of these methods showed that the distances according to the Biolog GN results between the studied strains were related to those derived from the API 20NE results, despite the differences in the test sets of the two systems . In addition, the co-inertia analysis allowed us to visualise the relationships between classifications of strains derived from the two identification systems and, simultaneously, to estimate the contribution of particular tests to the differentiation of bacterial strains.

Membr Cell Biol, 1998, 12(5), 593 - 608
Effect of deuteration and cryosolvents on the energy transduction in primary processes of photosynthesis; Gorokhov VV et al.; Effects of cryosolvents and D2O/H2O substitution on the reaction centres (RCs) isolated from photosynthetic bacteria were studied with respect to the role of intra-protein hydrogen bonds in the primary photosynthetic electron transfer . As a result of such treatment of RCs, the charge separation rate between the photoactive bacteriochlorophyll (P2 dimer) and bacteriopheophytin and the rate of electron transfer to the primary quinone slowed down . The energy migration rate from bacteriopheophytin (BPheM), inactive in electron transport, to P2 decreased as well . Although cryosolvents can shift the redox potential of the photoactive pigment, there is no direct correlation between the P2 potential and the effects of these modifying agents on the photosynthetic process in RCs occurring with participation of P2 . The removal of H subunit from the pigment-protein complex results in the pronounced weakening of the dimethyl sulfoxide modifying effects on the RC hydrogen bonds . The role of structural and dynamic state in the functioning of the photosynthetic bacterial RCs is analyzed . Relaxation processes in purple bacteria RCs accompanying the primary picosecond steps of energy transformation proceed with the participation of small proton-containing molecular groups in the immediate surroundings of electron transfer carriers . In this paper, we present results concerning mechanisms of primary photosynthetic steps, which were initiated by A . A . Krasnovsky and have been studied for several years at the Department of Biophysics . This paper is dedicated to the memory of our teacher Prof . A . A . Krasnovsky.

Chest, 1999 Jun, 115(6), 1641 - 5
Diagnosis of nosocomial pneumonia in cancer patients undergoing mechanical ventilation: a prospective comparison of the plugged telescoping catheter with the protected specimen brush; Casetta M et al.; STUDY OBJECTIVES: Quantitative culture of protected samples of lower respiratory tract secretions obtained by a fiberoptic protected specimen brush (PSB) is widely accepted for the diagnosis of ventilator-associated pneumonia (VAP), but this diagnostic procedure is time consuming, expensive, and may give rise to iatrogenic complications, especially in cancer patients who often present with thrombocytopenia . The plugged telescoping catheter (PTC) could be a satisfactory alternative to the PSB in this setting . The aim of the present study was to evaluate the interest of the PTC to diagnose VAP in ventilated cancer patients . DESIGN: A prospective observational study . SETTING: A 15-bed medical-surgical ICU in a comprehensive cancer center . PATIENTS AND INTERVENTIONS: Over a 9-month period, 42 patients suspected of having bacterial VAP during mechanical ventilation underwent 69 bronchial samplings: a blinded PTC and a fiberoptic PSB were performed successively in each case . A positive culture for both sampling procedures was defined as the recovery of > or = 10(3) cfu/mL of at least one potential pathogen . The PSB result was taken as the reference standard . MEASUREMENTS AND RESULTS: The overall agreement between the techniques was 87% (60/69) . PTC had a sensitivity of 67%, a specificity of 93%, a positive predictive value of 71%, and a negative predictive value of 91% . CONCLUSIONS: We conclude that the accuracy of the blinded PTC compares well with that of the PSB for the diagnosis of VAP in cancer patients . The sensitivity of the PTC observed herein, which is slightly lower than that described in previous studies, may be due to the blinded nature of the method: the indications for initial or secondary coupling with a directed sampling method in patients with suspicion of localized pneumonia remain to be determined.

Chest, 1999 Jun, 115(6), 1632 - 40
Decreasing catheter colonization through the use of an antiseptic-impregnated catheter: a continuous quality improvement project; Collin GR; STUDY OBJECTIVES: To evaluate the use of an antiseptic-impregnated (chlorhexidine and silver sulfadiazine) catheter for the prevention of catheter colonization and catheter-related bloodstream infection (CR-BSI) . Then, based on these findings, to implement changes in hospital policy and to assess their effect on a hospital service . DESIGN: Prospective, randomized, controlled (phase I); prospective, concurrent data collection (phase II) . SETTING: Tertiary referral hospital with level 1 trauma center . PATIENTS: Patients > 12 years of age with central venous catheters placed while they were in the emergency room, neurotrauma ICU, or medical/surgical ICU from May through December, 1995 (phase I) . All patients > 12 years of age on the trauma service admitted from November 16, 1996, through November 15, 1997 (phase II) . INTERVENTIONS: Randomization table determined whether the patient would receive an antiseptic-impregnated catheter (AIC) or nonimpregnated catheter (NIC) (phase I) . All removed or exchanged catheters were sent for semiquantitative culture . In phase II, only AICs were used; "length of time" and "fever" were discouraged as reasons for catheter exchange or removal; and only the tip was sent for culture . MEASUREMENTS AND RESULTS: In phase I, there were 139 catheters placed in 60 patients in the NIC group and 98 catheters placed in 55 patients in the AIC group . Two catheters (2.0/100 catheters) in the AIC group were found to be colonized, compared with 25 (18.0/100 catheters) in the NIC group (p = 0.001) . The catheter colonization rates were 2.27/1,000 catheter days (AIC) and 24.68/1,000 catheter days (NIC) (p < 0.001), while the CR-BSI rates were 1.14/1,000 catheter days (AIC) and 3.9.5/1,000 catheter days (NIC) (p = 0.31) . The reason for each catheter removal/exchange was noted, and only "positive blood culture" was statistically significant overall . The tip segment was found to be positive more often than the intracutaneous segment . In phase II, there were 213 AICs placed in 101 patients . The colonization rate was 3.8/100 catheters (4.52/1,000 catheter days), and CR-BSI rate was 1.0/100 catheters (0.6/1,000 catheter days) . The colonization rate for catheters left in place remained low for catheters left in place < 14 days (1.6/100 catheters) . Only 11% of catheters were exchanged/removed for reason of "fever," as compared with 23% in phase I . CONCLUSIONS: AICs significantly reduce the rate of central venous catheter colonization . In addition, the apparent protective effects of the catheter over time permit less frequent exchanges or removals of the catheters, decreasing both patient risk and hospital cost.

Yale J Biol Med, 1998 Mar-Apr, 71(2), 53 - 61
Helicobacter pylori and apoptosis; Moss SF; In an attempt to understand the diverse effects of infection with Helicobacter pylori on epithelial mucosal mass and consequent clinical outcome, the relationship between H . pylori infection and gastric epithelial cellular turnover has been investigated . Our results indicate that H . pylori increases epithelial cell proliferation and apoptosis in vivo, but that infection with bacteria of the cagA genotype leads to relatively more proliferation than apoptosis . This review explores the causes of the induction of apoptosis in gastric epithelial cells by H . pylori and the consequences of alterations in apoptosis to the maintenance of gastric mucosal homeostasis.

Lijec Vjesn, 1999 Jan-Feb, 121(1-2), 27 - 34
{Rickettsiales--modern findings}; Punda-Polic V; The term rickettsiae has as a rule encompassed the intracellular bacteria . Molecular studies brought new data to rickettsial taxonomy . Many new data on rickettsia have been accumulated over recent years, and a comparison of the newly discovered diseases with previously known rickettsioses, as well as with their agents is of interest . The number of "new" agents that have been discovered in the last few years is remarkable . This review discusses the current knowledge of bacterial species that historically belonged to the order Rickettsiales, as well as the role of these agents as human and animal pathogens.

Biochem J, 1999 Jul 1, 341 ( Pt 1), 147 - 55
The DmpA aminopeptidase from Ochrobactrum anthropi LMG7991 is the prototype of a new terminal nucleophile hydrolase family; Fanuel L et al.; The DmpA (d-aminopeptidase A) protein produced by Ochrobactrum anthropi hydrolyses p-nitroanilide derivatives of glycine and d-alanine more efficiently than that of l-alanine . When regular peptides are utilized as substrates, the enzyme behaves as an aminopeptidase with a preference for N-terminal residues in an l configuration, thus exemplifying an interesting case of stereospecificity reversal . The best-hydrolysed substrate is l-Ala-Gly-Gly, but tetra- and penta-peptides are also efficiently hydrolysed . The gene encodes a 375-residue precursor, but the active enzyme contains two polypeptides corresponding to residues 2-249 (alpha-subunit) and 250-375 (beta-subunit) of the precursor . Residues 249 and 250 are a Gly and a Ser respectively, and various substitutions performed by site-directed mutagenesis result in the production of an uncleaved and inactive protein . The N-terminal Ser residue of the beta-subunit is followed by a hydrophobic peptide, which is predicted to form a beta-strand structure . All these properties strongly suggest that DmpA is an N-terminal amidohydrolase . An exploration of the databases highlights the presence of a number of open reading frames encoding related proteins in various bacterial genomes . Thus DmpA is very probably the prototype of an original family of N-terminal hydrolases.

Biochem J, 1999 Jul 1, 341 ( Pt 1), 139 - 45
An aromatic, but not a basic, residue is involved in the toxicity of group-II phospholipase A2 neurotoxins; Pungercar J et al.; Ammodytoxins (Atxs) A, B and C are basic phospholipase A2s from Vipera ammodytes ammodytes snake venom, and they exhibit presynaptic toxicity . The most toxic is AtxA, followed by AtxC, its naturally occurring F124-->I/K128-->E mutant, which is 17 times less toxic . Two mutants of AtxA have been produced in bacteria and characterized . The specific enzymic activity of the K128-->E mutant on mixed phosphatidylcholine/Triton X-100 micelles is similar to that of the wild type . The K108-->N/K111-->N mutant, however, possesses 160% of the wild-type activity . Replacement of the two basic residues by uncharged, polar residues on the opposite side of the protein to the enzyme active site and interfacial adsorption surface results in increased enzymic activity at the water/lipid aggregate interface, due to a redistribution of electrostatic charge . The binding affinity of the double mutant for the specific acceptor in bovine brain was similar to that of AtxA, whereas the affinity of the single mutant was similar to that of AtxC, which was slightly weaker than that of AtxA . Interestingly, the substitution of any of these three basic surface residues did not significantly change the lethal potency of AtxA . Since the single mutant AtxA(K128-->E) is equivalent to the AtxC(I124-->F) mutant, this indicates that the residue at position 124 is important for presynaptic toxicity of Atxs . The more than 10-fold lower toxicity of AtxC, compared with AtxA, is a consequence of the substitution of Phe-124 (aromatic ring) with Ile (aliphatic chain) . Exposed aromatic residues in the C-terminal region may also be important for the neurotoxicity of other similar toxins.

J Neuroimmunol, 1999 Feb 1, 94(1-2), 204 - 11
Cloning the antibody response in humans with inflammatory CNS disease: isolation of measles virus-specific antibodies from phage display libraries of a subacute sclerosing panencephalitis brain; Burgoon MP et al.; We have developed a strategy to identify the disease-relevant antigens in a chronic inflammatory CNS disease exhibiting intrathecally expressed oligoclonal IgG . Using subacute sclerosing panencephalitis (SSPE), a chronic inflammatory measles virus infection of the brain as a model system, we constructed a phage display antibody Fab library from the amplified products of IgG expressed in the brain . Selection of the library against measles virus-infected cell lysates yielded four distinct Fabs which, by ELISA and by immunostaining, reacted specifically with measles virus-infected cells . Three Fabs immunoprecipitated a 72 kDa protein from infected cell cultures corresponding to the measles virus phosphoprotein . The fourth Fab immunoprecipitated and recognized by immunoblotting a 60 kDa protein corresponding to the measles virus nucleoprotein . The results demonstrate that functional antibodies from an inflammatory CNS disease can be expressed in bacteria and used to identify disease-relevant antigens . This approach could be applied to chronic inflammatory CNS diseases of unknown cause such as multiple sclerosis.

Nature, 1999 Jun 10, 399(6736), 541 - 8
Marine viruses and their biogeochemical and ecological effects; Fuhrman JA; Viruses are the most common biological agents in the sea, typically numbering ten billion per litre . They probably infect all organisms, can undergo rapid decay and replenishment, and influence many biogeochemical and ecological processes, including nutrient cycling, system respiration, particle size-distributions and sinking rates, bacterial and algal biodiversity and species distributions, algal bloom control, dimethyl sulphide formation and genetic transfer . Newly developed fluorescence and molecular techniques leave the field poised to make significant advances towards evaluating and quantifying such effects.

Bioessays, 1999 May, 21(5), 402 - 11
Eukaryotic DNA methylation as an evolutionary device; Colot V et al.; DNA methylation is catalyzed by a family of conserved DNA methyltransferases and is widespread among protists, plants, fungi and animals . It is however absent in some species and its genomic distribution varies among organisms . Sequence comparisons suggest that known and putative eukaryotic DNA methyltransferases fall into at least five structurally distinct subfamilies . Furthermore, it is now clear that DNA methylation can be involved in several functions, some of which may coexist within the same organism . It can inhibit transcription initiation, arrest transcript elongation, act as an imprinting signal, and suppress homologous recombination . On the basis of these observations, we argue that DNA methylation has been conserved during evolution because it provides unique possibilities for setting up functions of various types.

FEBS Lett, 1999 Jun 4, 452(1-2), 22 - 5
Extremophiles and their adaptation to hot environments; Stetter KO; Water-containing terrestrial, subterranean and submarine high temperature areas harbor a variety of hyperthermophilic bacteria and archaea which are able to grow optimally above 80 degrees C . Hyperthermophiles are adapted to hot environments by their physiological and nutritional requirements . As a consequence, cell components like proteins, nucleic acids and membranes have to be stable and even function best at temperatures around 100 degrees C . The chemolithoautotrophic archaeon Pyrolobus fumarii is able to grow at 113 degrees C and, therefore, represents the upper temperature border of life . For the first time, (vegetative) cultures of Pyrolobus and Pyrodictium are able to survive autoclaving.

FEBS Lett, 1999 Jun 4, 452(1-2), 11 - 5
Obligate intracellular parasites: Rickettsia prowazekii and Chlamydia trachomatis; Zomorodipour A et al.; Transitions to obligate intracellular parasitism have occurred at numerous times in the evolutionary past . The genome sequences of two obligate intracellular parasites, Rickettsia prowazekii and Chlamydia trachomatis, were published last year . A comparative analysis of these two genomes has revealed examples of reductive convergent evolution, such as a massive loss of genes involved in biosynthetic functions . In addition, both genomes were found to encode transport systems for ATP and ADP, not otherwise found in bacteria . Here, we discuss adaptations to intracellular habitats by comparing the information obtained from the recently published genome sequences of R . prowazekii and C . trachomatis.

Chem Biol, 1999 Jun, 6(6), 401 - 9
Novel mutant green fluorescent protein protease substrates reveal the activation of specific caspases during apoptosis; Mahajan NP et al.; BACKGROUND: The caspase-mediated proteolysis of many cellular proteins is a critical event during programmed cell death or apoptosis . It is important to determine which caspases are activated in mammalian cells, and where and when activation occurs, upon receipt of specific death stimuli . Such information would be useful in the design of strategies to regulate the activation of caspases during apoptosis . RESULTS: We developed two novel fluorescent substrates that were specifically cleaved by caspase-1 or caspase-3 . For in vitro studies, four-amino-acid recognition sequences, YVAD for caspase-1 and DEVD for caspase-3, were introduced between blue fluorescent protein (BFP) and green fluorescent protein (GFP), expressed in bacteria and purified . For in vivo studies, YVAD and DEVD were introduced between cyan fluorescent protein and yellow fluorescent protein, and expression was monitored in live mammalian cells . The proximity between fluorophores was determined using fluorescence resonance energy transfer . Purified substrates were cleaved following exposure to purified caspase-1 and caspase-3 . In Cos-7 cells, caspase-1 and caspase-3 substrates were cleaved upon induction of apoptosis with staurosporine, a protein-kinase inhibitor, whereas caspase-3 but not caspase-1 substrate was cleaved upon treatment of cells with the DNA-damaging agent mitomycin c . CONCLUSIONS: These substrates allow the spatial activation of specific members of the caspase family to be deciphered during the initiation and execution phase of programmed cell death, and allow activation of specific caspases to be monitored both in vivo and in vitro . This technology is also likely to be useful for high-throughput screening of reagents that modulate caspase activity.

Chem Biol, 1999 Jun, 6(6), R167 - 75
Histidine kinases and two-component signal transduction systems; Pirrung MC; The phosphorylation of histidine is the first step in many signal transduction cascades in bacteria, yeast and higher plants . The transfer of a very reactive phosphoryl group from phosphorylated histidine kinase to an acceptor is an essential step in many cellular signaling responses.

Arch Biochem Biophys, 1999 Jul 1, 367(1), 81 - 8
Phospholipase D activity of cytochrome P450 in human liver endoplasmic reticulum; Yun CH et al.; Phospholipase D (PLD) activity in mammalian liver endoplasmic reticulum (ER) has not been characterized . Purified human liver microsomal cytochromes P450 (P450)-P450 1A2 and P450 2E1-were shown to have appreciable PLD activity, hydrolyzing phosphatidylcholine but not other phospholipids, generating PA and choline . The activity was confirmed using recombinant and mutated human P450s expressed in bacteria . In human liver microsomes, immunoinhibition of PLD activity was observed with anti-P450 1A2 > anti-P450 2C > anti-P450 2E1 . Thus, P450 may act as a significant PLD in human liver ER and exert its biological effects by several mechanisms, including signaling functions and change of membrane properties .

Mol Cell Biol, 1999 Jul, 19(7), 4750 - 6
SDF-2 induction of terminal differentiation in Dictyostelium discoideum is mediated by the membrane-spanning sensor kinase DhkA; Wang N et al.; SDF-2 is a peptide released by prestalk cells during culmination that stimulates prespore cells to encapsulate . Genetic evidence indicates that the response is dependent on the dhkA gene . This gene encodes a member of the histidine kinase family of genes that functions in two-component signal transduction pathways . The sequence of the N-terminal half of DhkA predicts two hydrophobic domains separated by a 310-amino-acid loop that could bind a ligand . By inserting MYC6 epitopes into DhkA, we were able to show that the loop is extracellular while the catalytic domain is cytoplasmic . Cells expressing the MYC epitope in the extracellular domain of DhkA were found to respond only if induced with 100-fold-higher levels of SDF-2 than required to induce dhkA+ cells; however, they could be induced to sporulate by addition of antibodies specific to the MYC epitope . To examine the enzymatic activity of DhkA, we purified the catalytic domain following expression in bacteria and observed incorporation of labelled phosphate from ATP consistent with histidine autophosphorylation . Site-directed mutagenesis of histidine1395 to glutamine in the catalytic domain blocked autophosphorylation . Furthermore, genetic analyses showed that histidine1395 and the relay aspartate2075 of DhkA are both critical to its function but that another histidine kinase, DhkB, can partially compensate for the lack of DhkA activity . Sporulation is drastically reduced in double mutants lacking both DhkA and DhkB . Suppressor studies indicate that the cyclic AMP (cAMP) phosphodiesterase RegA and the cAMP-dependent protein kinase PKA act downstream of DhkA.

J Biol Chem, 1999 Jun 25, 274(26), 18644 - 50
Does prothymosin alpha affect the phosphorylation of elongation factor 2?
Enkemann SA, Pavur KS, Ryazanov AG, Berger SL.
Prothymosin alpha is a small, acidic, essential nuclear protein that plays a poorly defined role in the proliferation and survival of mammalian cells . Recently, Vega et al . proposed that exogenous prothymosin alpha can specifically increase the phosphorylation of eukaryotic elongation factor 2 (eEF-2) in extracts of NIH3T3 cells (Vega, F . V., Vidal, A., Hellman, U., Wernstedt, C., and Dominguez, F . (1998) J . Biol . Chem . 273, 10147-10152) . Using similar lysates prepared by four methods (detergent lysis, Dounce homogenization, digitonin permeabilization, and sonication) and three preparations of prothymosin alpha, one of which was purified by gentle means (the native protein, and a histidine-tagged recombinant prothymosin alpha expressed either in bacteria or in COS cells), we failed to find a response . A reconstituted system composed of eEF-2, recombinant eEF-2 kinase, calmodulin, and calcium was also unaffected by prothymosin alpha . However, unlike our optimized buffer, Vega's system included a phosphatase inhibitor, 50 mM fluoride, which when evaluated in our laboratories severely reduced phosphorylation of all species . Under these conditions, any procedure that decreases the effective fluoride concentration will relieve the inhibition and appear to activate . Our data do not support a direct relationship between the function of prothymosin alpha and the phosphorylation of eEF-2.

J Mol Biol, 1999 Jun 25, 289(5), 1423 - 33
Identification of the binding interfaces on CheY for two of its targets, the phosphatase CheZ and the flagellar switch protein fliM; McEvoy MM et al.; CheY is the response regulator protein serving as a phosphorylation-dependent switch in the bacterial chemotaxis signal transduction pathway . CheY has a number of proteins with which it interacts during the course of the signal transduction pathway . In the phosphorylated state, it interacts strongly with the phosphatase CheZ, and also the components of the flagellar motor switch complex, specifically with FliM . Previous work has characterized peptides consisting of small regions of CheZ and FliM which interact specifically with CheY . We have quantitatively measured the binding of these peptides to both unphosphorylated and phosphorylated CheY using fluorescence spectroscopy . There is a significant enhancement of the binding of these peptides to the phosphorylated form of CheY, suggesting that these peptides share much of the binding specificity of the intact targets of the phosphorylated form of CheY . We also have used modern nuclear magnetic resonance methods to characterize the sites of interaction of these peptides on CheY . We have found that the binding sites are overlapping and primarily consist of residues in the C-terminal portion of CheY . Both peptides affect the resonances of residues at the active site, indicating that the peptides may either bind directly at the active site or exert conformational influences that reach to the active site . The binding sites for the CheZ and FliM peptides also overlap with the previously characterized CheA binding interface . These results suggest that interaction with these three proteins of the signal transduction pathway are mutually exclusive . In addition, since these three proteins are sensitive to the phosphorylation state of CheY, it may be that the C-terminal region of CheY is most sensitive for the conformational changes occurring upon phosphorylation .

Microb Pathog, 1999 Jul, 27(1), 1 - 11
Investigation into the role of the response regulator NtrC in the metabolism and virulence of Brucella suis; Dorrell N et al.; During infection, Brucella species have to adapt to a range of different environments . Environmental sensing in bacteria often involves the concerted action of two-component regulatory systems consisting of sensor and response regulator components . In this study, we identified, cloned and sequenced four independent response regulator gene fragments from Brucella melitensis . One amplified gene fragment showed nearly 90% identity to the response regulator subfamily of NtrC transcriptional activators, and further analysis revealed the presence of an adjacent gene encoding the sensor protein NtrB . The NtrBC two-component regulatory system has been shown to play varying roles in nitrogen metabolism and potentially in virulence in other bacterial species . A B . suis ntrC isogenic mutant was constructed which showed no significant differences in growth rates compared to the wild-type strain when grown at different temperatures in vitro . However, the mutant exhibited a reduction in metabolic activity in the presence of many amino acids . The mutation did not affect survival or multiplication of B . suis in macrophages, but during the initial stages of infection in the murine brucellosis model, the ntrC mutant showed a reduced ability to multiply rapidly in splenic tissue .

J Med Primatol, 1999 Feb, 28(1), 11 - 8
Fatal outbreaks of proliferative enteritis caused by Lawsonia intracellularis in young colony-raised rhesus macaques; Klein EC et al.; Ten juvenile rhesus monkeys (Macaca mulatta) died acutely in two separate disease outbreaks . The animals had segmental thickening of the distal ileum with associated proliferative, rugous appearing mucosae . Microscopically, necrosis, exudative inflammation, mucosal ulceration, and crypt hyperplasia were present . Intracellular organisms were seen histochemically and ultrastructurally, and were confirmed to be Lawsonia intracellularis using a specific immunohistochemical method . Proliferative enteropathic conditions caused by L . intracellularis are reported in an ever-increasing range of hosts, suggesting that the infection may exist unrecognized in an even greater array of species, possibly including man.

J Dent Res, 1999 Jun, 78(6), 1238 - 44
Secretory immunoglobulin A heavy chain presents Galbeta1-3GalNAc binding structures for Actinomyces naeslundii genospecies 1; Bratt P et al.; Adherence of Actinomyces naeslundii ATCC 12104 to hydroxyapatite beads coated with protein fractions of parotid saliva, obtained by gel filtration on S-200 HR columns, showed GalNAcbeta1-3Galalpha-O-ethyl-inhibitable binding to high-molecular-weight proteins (Stromberg et al., 1992) . The present study investigates the nature of these high-molecular-weight binding proteins and determines their specific ability to mediate adherence to representative strains of Actinomyces species . Strain ATCC 12104 bound specifically in a lactose-inhibitable manner to the heavy chain of secretory immunoglobulin A (S-IgA), contained within a high-molecular-weight parotid protein fraction separated on SDS-PAGE and transferred to a solid membrane support . Lactose-inhibitable binding to the heavy chain of S-IgA from human colostrum was also demonstrated . Peanut agglutinin bound to the heavy chain of parotid and colostrum S-IgAs contained on solid support membranes, confirming the presence of Galbeta1-3GalNAc residues on these molecules . Both salivary and colostrum S-IgA aggregated with strain ATCC 12104 in a GalNAcbeta1-3Galalpha-O-ethyl-inhibitable fashion . Further separation of high-molecular-weight salivary proteins on S-500 HR columns showed GalNAcbeta1-3Galalpha-O-ethyl-inhibitable binding to both mucin- and S-IgA-containing fractions . The presence of S-IgA in salivary pellicles formed in vivo on teeth was demonstrated by Western blot analysis of pellicle extracts with anti-IgA antibodies . Among strains representing A . naeslundii genospecies 1 and 2 and A . odontolyticus, only those of genospecies 1 with a particular adherence profile showed efficient GalNAcbeta1-3Galalpha-O-ethyl-inhibitable binding to S-IgA . Thus, oligosaccharides on S-IgA may promote bacterial aggregation (or adherence) and provide a mechanism by which S-IgA can interact with bacteria without prior immunological challenge.

Ann Transplant, 1998, 3(4), 15 - 20
Pathways of antigen traffic from skin allotransplant to recipient lymph nodes--an evolutionarily developed route for initiation of rejection of foreign antigens; Olszewski WL et al.; Immune events developing in the bed of skin allograft and draining lymphatics and lymph nodes are probably a copy of what happens in skin after invasion by bacteria, viruses or fungi . The mechanism of local immune response developed in skin during the evolution and is highly conserved and efficient in elimination of foreign antigens . This is why the take of a skin allograft is so difficult and immunosuppressive measures applied after allogeneic skin transplantation remain so ineffective . Authors present their results of studies on the human skin immune humoral and cellular factors, underlining their specificities and differences compared with blood . They also analyze the role of non-immunological factors, such as tissue fluid formation and lymph flow in transportation of alloantigen to the lymph nodes . The migratory properties of immune cells are an indispensable factor in transfer of alloantigen to the lymph nodes . Understanding of the evolutionarily developed immune events in skin may allow to analyze the process of skin allograft rejection and can give hints for more effective immunosuppressive policy.

Clin Immunol, 1999 Jun, 91(3), 354 - 8
Neutrophils from Mycobacterium avium-infected mice produce TNF-alpha, IL-12, and IL-1 beta and have a putative role in early host response; Petrofsky M et al.; Recent evidence supports a role for neutrophils in the host defense against Mycobacterium avium . To determine whether the depletion of neutrophils has an effect on the outcome of infection in mice as determined by the number of bacteria in liver and spleen, we administered RB6-8C5 anti-neutrophil antibody intraperitoneally both early and late in the infection . Mice were then observed for 14 days and harvested . The number of viable bacteria in liver and spleen was determined . While administration of RB6-8C5 antibody early in infection resulted in a significant increase in the number of bacteria in organs when compared with mice receiving immunoglobulin control, administration of RB6-8C5 antibody late in infection (week 3) did not have an impact on the bacterial load in tissue . Infection of CD18 knockout mice (with impaired neutrophil function), however, did not show a significant enhancement of M . avium growth when compared with that of wild-type control mice . Neutrophils were found to produce increased amounts of TNF-alpha and IL-12 and IL-1 than control uninfected mice during the initial phase of infection, but not after 2 weeks following infection (although IL-1 beta levels continue elevated) . The results suggest that neutrophils may have a role in the early (innate) immune response against M . avium but it is only evident after acute depletion of neutrophils and not in mice with chronic neutrophil impairment.

J Mol Biol, 1999 Jun 18, 289(4), 777 - 84
Supercoiling-dependent site-specific binding of HU to naked Mu DNA; Kobryn K et al.; Using HU chemical nucleases to probe HU-DNA interactions, we report here for the first time site-specific binding of HU to naked DNA . An unique feature of this interaction is the absolute requirement for negative DNA supercoiling for detectable levels of site-specific DNA binding . The HU binding site is the Mu spacer between the L1 and L2 transposase binding sites . Our results suggest recognition of an altered DNA structure which is induced by DNA supercoiling . We propose that recruitment of HU to this naked DNA site induces the DNA bending required for productive synapsis and transpososome assembly . Implications of HU as a supercoiling sensor with a potential in vivo regulatory role are discussed . Finally, using HU nucleases we have also shown that non-specific DNA binding by HU is stimulated by increasing levels of supercoiling .

Rheumatology (Oxford), 1999 May, 38 Suppl 1, 24 - 32
Gastrointestinal toxicity of non-steroidal anti-inflammatory drugs: the effect of nimesulide compared with naproxen on the human gastrointestinal tract; Bjarnason I et al.; This overview includes theories and evaluation of non-steroidal anti-inflammatory drug (NSAID)-induced gastrointestinal toxicity . Factors in damage include microvascular aspects, neutrophil recruitment, mucosal prostaglandins, gastrointestinal secretions and bacteria . We have proposed an extensive simplified framework that includes an important local initiating effect which may involve NSAID accumulation, interaction with surface phospholipids, events that alter cellular ATP, and local/systemic effects of cyclooxygenase (COX) inhibition . COX-2-selective drugs are desirable not only because they spare COX-1 and so avoid gastrointestinal toxicity, but also because COX-2-selective agents are only weakly acidic and therefore avoid substantial accumulation in the gastric mucosa . Short-term endoscopy studies of NSAIDs are important initially to evaluate human gastroduodenal tolerability . They show that injury increases with the amount of NSAIDs even though the lowest therapeutic doses inhibit gastric COX almost completely, and that the more-acidic NSAIDs tend to cause greater gastric damage . Long-term endoscopy studies involve NSAID ingestion for at least 3 months . A main question is the extent to which the ulcers seen cause symptoms, substantial bleeding and/or perforation . Measurement of serious outcomes is thought by many to be the best assessment of gastrointestinal safety, but studies find marked variations even with the same drug . Damage to the small intestine by NSAIDs is even more frequent than to the upper gastrointestinal tract, but is difficult to evaluate . Conventional acidic NSAIDs increase the permeability of human small intestine, probably by a non-prostaglandin mechanism, but nimesulide does not do so, possibly because of its very weak acidity.

Laryngoscope, 1999 Jun, 109(6), 976 - 82
Establishment and characterization of human laryngeal squamous cell carcinoma cell lines; Ku JL et al.; OBJECTIVES: Six human laryngeal squamous cell carcinoma cell lines (SNU-46, -585, -899, -1066, -1076, -1214) established from Korean patients are reported . STUDY DESIGN: In vitro culture of six squamous cell carcinoma cell lines derived from primary tumors of the larynx . Description of the cell line phenotypes and determination of molecular characteristics . METHODS: Six laryngeal squamous cell carcinoma cell lines were cultured . The cell phenotypes, including the histopathology of the primary tumors and in vitro growth characteristics, were determined . Molecular characterization was also performed, including DNA fingerprinting analysis and abnormalities of p15, p16, p53, and TGF-betaRII genes by polymerase chain reaction-based single strand conformation polymorphism and sequencing analysis . RESULTS: All cell lines grew as adherent cells; five lines grew as monolayers and one other line grew as stratifying colonies . All lines showed 1) high viability (75%-92%) with various doubling times (36-96 h); 2) absence of Mycoplasma and other bacteria; and 3) genetic heterogeneity by DNA profile analysis . p53 Mutations were found in three lines and p16 mutations were observed in five cell lines . TGF-betaRII mutations were found in two lines: one line had frameshift mutation and another line had a missense mutation at the kinase domain . CONCLUSIONS: These newly established and characterized laryngeal squamous cell carcinoma cell lines will be useful for investigating the biologic characteristics of laryngeal cancer.

Mol Biol Evol, 1999 Jun, 16(6), 826 - 38
Molecular evolution of glutamate receptors: a primitive signaling mechanism that existed before plants and animals diverged; Chiu J et al.; We performed a genealogical analysis of the ionotropic glutamate receptor (iGluR) gene family, which includes the animal iGluRs and the newly isolated glutamate receptor-like genes (GLR) of plants discovered in Arabidopsis . Distance measures firmly placed the plant GLR genes within the iGluR clade as opposed to other ion channel clades and indicated that iGluRs may be a primitive signaling mechanism that predated the divergence of animals and plants . Moreover, phylogenetic analyses using both parsimony and neighbor joining indicated that the divergence of animal iGluRs and plant GLR genes predated the divergence of iGluR subtypes (NMDA vs . AMPA/KA) in animals . By estimating the congruence of the various glutamate receptor gene regions, we showed that the different functional domains, including the two ligand-binding domains and the transmembrane regions, have coevolved, suggesting that they assembled together before plants and animals diverged . Based on residue conservation and divergence as well as positions of residues with respect to functional domains of iGluR proteins, we attempted to examine structure-function relationships . This analysis defined M3 as the most highly conserved transmembrane domain and identified potential functionally important conserved residues whose function can be examined in future studies.

J Mol Evol, 1999 Jul, 49(1), 49 - 62
Two aspects of DNA base composition: G+C content and translation-coupled deviation from intra-strand rule of A = T and G = C; Sueoka N; The relative contribution of mutation and selection to the G+C content of DNA was analyzed in bacterial species having widely different G+C contents . The analysis used two methods that were developed previously . The first method was to plot the average G+C content of a set of nucleotides against the G+C content of the third codon position for each gene . This method was used to present the G+C distribution of the third codon position and to assess the relative neutrality of a set of nucleotides to that of the G+C content of the third codon position . The second method was to plot the intrastrand bias of the third codon position from Parity Rule 2 (PR2), where A = T and G = C . It was found that whereas intragenomic distributions of the DNA G+C content of these bacteria are narrow in the majority of species, in some species the G+C content of the minor class of genes distributes over wider ranges than the major class of genes . On the other hand, ubiquitous PR2 biases are amino acid specific and independent of the G+C content of DNA, so that when averaged over the amino acids, the biases are small and not correlated with the DNA G+C content . Therefore, translation coupled PR2-biases are unlikely to explain the wide range of G+C contents among different species . Considering all data available, it was concluded that the amino acid-specific PR2 bias has only a minor effect, if any, on the average G+C content . In addition, PR2 bias patterns of different species show phylogenetic relationships, and the pattern can be as a taxal fingerprint.

J Periodontol, 1999 May, 70(5), 485 - 9
The relationship between oral malodor, gingivitis, and periodontitis . A review; Ratcliff PA et al.; Volatile sulfur compounds (VSC) are a family of gases which are primarily responsible for halitosis, a condition in which objectionable odors are present in mouth air . Although most patients perceive this condition as primarily a cosmetic problem, an increasing volume of evidence is demonstrating that extremely low concentrations of many of these compounds are highly toxic to tissues . VSC may, therefore, play a role in the pathogenesis of inflammatory conditions such as periodontitis . Since these compounds result from bacterial putrefaction of protein, investigations have been conducted to determine whether specific bacteria are associated with odor production . Two members of this family, hydrogen sulfide (H2S) and methyl mercaptan (CH3SH), are primarily responsible for mouth odor . Although many bacteria produce H2S, the production of CH3SH, especially at high levels, is primarily restricted to periodontal pathogens . Direct exposure to either of these metabolites adversely affects protein synthesis by human gingival fibroblasts in culture . However, methyl mercaptan has the greatest effect . Other in vitro experiments have demonstrated that cells exposed to methyl mercaptan synthesize less collagen, degrade more collagen, and accumulate collagen precursors which are poorly cross-linked and susceptible to proteolysis . CH3SH also increases permeability of intact mucosa and stimulates production of cytokines which have been associated with periodontal disease . VSC, and in particular methyl mercaptan, are therefore capable of inducing deleterious changes in both the extracellular matrix and the local immune response of periodontal tissues to plaque antigens . This article reviews these data and emphasizes the potential importance of VSC in the transition of periodontal tissues from clinical health to gingivitis and then to periodontitis.

J Zoo Wildl Med, 1999 Mar, 30(1), 111 - 8
Fatal mycotic dermatitis in captive brown tree snakes (Boiga irregularis); Nichols DK et al.; Cutaneous fungal infections occurred in four captive brown tree snakes (Boiga irregularis) . The ventral scales were most commonly affected, and lesions began as areas of erythema and edema with vesicle formation, followed by development of caseous brown plaques . Lesions usually started where ventral scales overlapped and spread rapidly . All snakes died within 14 days after clinical signs were first noted . The deaths of three of the snakes were directly attributable to the cutaneous disease; the other snake died from renal failure and visceral gout, most likely induced by gentamicin therapy . Histologically, lesions consisted of epidermal hyperplasia and hyperkeratosis, with foci of epidermal necrosis, intraepidermal vesicle formation, and subacute inflammation of the underlying dermis . These lesions were associated with bacteria and numerous septate, branched fungal hyphae within the epidermis and overlying serocelluar crusts . Hyphae that penetrated through the superficial surface of the epidermis often formed terminal arthroconidia . The same species of fungus was isolated in pure culture from the skin of three snakes, but fungal cultures were not performed on samples from the fourth snake . The fungus has been identified as the Chrysosporium anamorph of Nannizziopsis vriesii based on its formation of solitary dermatophytelike aleurioconidia and alternate and fission arthroconidia . The source of the fungus in this outbreak was not determined; however, the warm, moist conditions under which the snakes were housed likely predisposed them to opportunistic cutaneous fungal infections.

Zhonghua Yi Xue Za Zhi (Taipei), 1999 Apr, 62(4), 203 - 8
Arylamine N-acetyltransferase: a possible promoter in Helicobacter pylori-related gastric carcinogenesis; Hung CF et al.; BACKGROUND: The hypothesis of an association between peptic ulcer and infection by Helicobacter pylori in the gastroduodenal tract was suggested by Marshall and Warren in 1984 . H pylori infection of the stomach is the most frequent infection in the world and exhibits an age-dependent increase . However, only a very small percentage of those infected develop gastric carcinoma, suggesting that H pylori acts as a cofactor in the pathogenesis of gastric carcinoma . N-Acetyltransferase (NAT) is expressed in uroepithelial cells and colon cytosol, while cytosolic acetyltransferase plays a critical role in susceptibility to arylamine-induced bladder and colon cancer . The presence of NAT activity in H pylori has yet to be determined . METHODS: NAT activity in H pylori from patients with peptic ulcer was studied using an acetyl coenzyme A (AcCoA) recycling assay and high-pressure liquid chromatography with p-aminobenzoic acid and aminofluorene substrates . RESULTS: The NAT activities from a number of H pylori samples were found to be 0.68 +/- 0.10 nmol/min/10(10) colony-forming units (CFUs) (intact bacteria); and 0.90 +/- 0.22 nmol/min/mg protein (cytosol) for the acetylation of 2-aminofluorene, and 0.63 +/- 0.06 nmol/min/10(10) CFUs (intact bacteria) and 0.72 +/- 0.24 nmol/min/mg protein (cytosol) for the acetylation of p-aminobenzoic acid . CONCLUSIONS: These studies show that H pylori has NAT activity, from which we speculate that the bioactivation of food-borne heterocyclic aromatic amines into genotoxic and carcinogenic products in the stomach is a possible promoter in the pathogenesis of gastric cancer.

J AOAC Int, 1999 May-Jun, 82(3), 669 - 75
Principles to guide international standard tests for liquid chemical germicides: a proposal; Miner N; This review discusses issues involved in developing standard tests for liquid chemical germicides and suggests some guiding principles to be considered for future development of harmonized international standard methods for testing disinfectants and sterilants . A published test method to measure sporicidal activity is used as an example of the implementation of these principles.

Brain Res Mol Brain Res, 1999 Jun 8, 69(2), 186 - 201
Production and characterization of an anti-serotonin 1A receptor antibody which detects functional 5-HT1A binding sites; Zhou FC et al.; We describe the production and characterization of a specific anti-5-HT1A receptor antibody made against a fusion protein consisting of glutathione-S-transferase (GST) coupled to a 75-amino acid sequence from the middle portion of the third intracellular loop (5-HT1A-m3i, serine253-arginine327) of the rat 5-HT1A receptor protein . This region was chosen to avoid putative phosphorylation and glycosylation sites and regions of known homology with other 5-HT receptors . Western blot analysis indicated that the polyclonal anti-5-HT1A-m3i antibody accurately recognized the fusion protein expressed in bacteria and labeled a prominent 67 kDa protein band in the hippocampus, cortex, brainstem, cerebellum and kidney with a density profile corresponding to the relative abundance of the 5-HT1A receptor in these tissues . No protein was detected in liver or muscle tissue preparations, and no protein bands were labeled in any of the above tissues following preabsorption of the antibody with the 5-HT1A-m3i fusion protein . Immunohistochemistry revealed prominent labeling in limbic structures including the hippocampus, amygdala, entorhinal cortex, and septum as well as in raphe nuclei . In the hippocampus, 5-HT1A-m3i labeling revealed a characteristic laminar pattern that coincided with that seen by autoradiographic binding of the 5-HT1A agonist {3H}-8-OH-DPAT in all strata of the hippocampal formation . In the dorsal and medial raphe nuclei, anti-5-HT1A-m3i antibodies labeled the somatodendritic membranes of 5-HT neurons, consistent with its role as an autoreceptor . The detailed matching of the anti-5-HT1A-m3i antibody with {3H}-8-OH-DPAT binding suggests that the antibody recognizes a functionally active form of the 5-HT1A receptor protein capable of binding 5-HT1A agonist ligands . These anti-5-HT1A antibodies may therefore be useful tools in localizing functional 5-HT1A receptors in specific regions of the brain as well as in studying the plasticity and ontogeny of the 5-HT1A receptor at the cellular and subcellular level .

Enferm Infecc Microbiol Clin, 1999 Apr, 17(4), 171 - 5
{Typification of strains of Helicobacter pylori by the detection of the cagA gene and subtypes of the vacA gene}; Gomez J et al.; BACKGROUND: Helicobacter pylori infection is probably the most common chronic bacterial infection in the world . The consequences of infection are pathologies like peptic ulcer, chronic gastritis, gastric cancer and gastric MALT lymphoma . The aim of this study was to detect the vacuolating cytotoxin gene (vacA) type, the cytotoxin-associated gen (cagA) of H . pylori isolates and study their association with the vacuolising activity . MATERIAL AND METHODS: Gastric biopsy specimens were obtained from dyspeptic patients . Isolates were further genotypically typed by PCR . RESULTS: All strains studied were vacA+ and 55% were cagA+ . All cytotoxic strains were cagA+, subtype vacA s1/m1 and corresponded to patients with peptic ulceration . The cagA- strains (11) corresponded to subtype s2/m2 . We didn't demonstrate vacuolising activity on subtypes s1/m2 and s2/m2 . CONCLUSIONS: A high genetic diversity exists among strains in our environment . The subset of bacteria, vacA s1/m1/cagA+, is associated with vacuolising activity in culture cells (tox+).

Int J Cancer, 1999 Jun 11, 81(6), 902 - 10
Establishment and characterization of 12 human colorectal-carcinoma cell lines; Oh JH et al.; In this article, we describe the characteristics of 12 human colorectal-carcinoma cell lines established from 6 primary tumors and 6 metastatic sites of 11 Korean colorectal-carcinoma patients, including the morphology in vivo and in vitro and mutations of K-ras2, p15, p16, p53, APC, beta-catenin, hMLH1 and hMSH2 genes in vitro . No lines were contaminated with Mycoplasma or bacteria . All lines were proven to be unique by DNA-fingerprinting analysis . All lines expressed the surface carcino-embryonic antigen and secreted it into the supernatant fluid . The morphological correlation between the original tumors and cultured cells suggested that the original tumors showing mucinous adenocarcinoma correlated with floating aggregates in culture, and degree of desmoplasia in the original tumor correlated with attached growth in culture . Five of the cell lines showed mutations in the K-ras2 gene, and 6 of the cell lines showed mutations in the p53 gene . The p15 gene was deleted in 2 cell lines, and the p16 gene was hypermethylated in 3 cell lines . The mutation of mismatch-repair genes (hMLH1 and hMSH2) was found in 4 lines, the APC gene and beta-catenin gene were mutated in 9 and 2 lines respectively . These well-characterized colorectal-cancer cell lines should serve as useful tools for investigating the biological characteristics of colorectal cancer.

J Virol, 1999 Jul, 73(7), 6177 - 81
A rhesus macaque rhadinovirus related to Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 encodes a functional homologue of interleukin-6; Kaleeba JA et al.; The rhesus rhadinovirus strain 17577 (RRV strain 17577) genome is essentially colinear with human herpesvirus 8 (HHV8)/Kaposi's sarcoma-associated herpesvirus (KSHV) and encodes several analogous open reading frames (ORFs), including the homologue of cellular interleukin-6 (IL-6) . To determine if the RRV IL-6-like ORF (RvIL-6) is biologically functional, it was expressed either transiently in COS-1 cells or purified from bacteria as a glutathione S-transferase (GST)-RvIL-6 fusion and analyzed by IL-6 bioassays . Utilizing the IL-6-dependent B9 cell line, we found that both forms of RvIL-6 supported cell proliferation in a dose-dependent manner . Moreover, antibodies specific to the IL-6 receptor (IL-6R) or the gp130 subunit were capable of blocking the stimulatory effects of RvIL-6 . Reciprocal titrations of GST-RvIL-6 against human recombinant IL-6 produced a more-than-additive stimulatory effect, suggesting that RvIL-6 does not inhibit but may instead potentiate normal cellular IL-6 signaling to B cells . These results demonstrate that RRV encodes an accessory protein with IL-6-like activity.

Arterioscler Thromb Vasc Biol, 1999 Jun, 19(6), 1456 - 69
SREBP-1 binds to multiple sites and transactivates the human ApoA-II promoter in vitro : SREBP-1 mutants defective in DNA binding or transcriptional activation repress ApoA-II promoter activity; Pissios P et al.; -Screening of an expression human liver cDNA library resulted in the isolation of several cDNA clones homologous to sterol regulatory element-binding protein-1 (SREBP-1) that recognize the regulatory element AIIAB and AIIK of the human apoA-II promoter . DNaseI footprinting of the apoA-II promoter using SREBP-1 (1 to 460) expressed in bacteria identified 5 overall protected regions designated AIIAB (-64 to -48), AIICD (-178 to -154), AIIDE (-352 to -332), AIIHI (-594 to -574), and AIIK (-760 to -743) . These regions contain inverted E-box palindromic or direct repeat motifs and bind SREBP-1 with different affinities . Transient cotransfection experiments in HepG2 cells showed that SREBP-1 transactivated the -911/29 apoA-II promoter 3.5-fold as well as truncated apoA-II promoter segments that contain 1, 2, 3, or 4 SREBP binding sites . Mutagenesis analysis showed that transactivation by SREBP was mainly affected by mutations in element AIIAB . Despite the strong transactivation of the apoA-II promoter by SREBP-1 we could not demonstrate significant changes on the endogenous apoA-II mRNA levels of HepG2 cells after cotransfection with SREBP-1 or in the presence or absence of cholesterol and 25-OH-cholesterol . An SREBP-1 mutant lacking the amino-terminal activation domain bound normally to its cognate sites and repressed the apoA-II promoter activity . Repression was also caused by specific amino acid substitutions of Leu, Val, or Gly for Lys359, which affected DNA binding . Repression by the DNA binding-deficient mutants was abolished by deletion of the amino-terminal activation domain (1 to 90) of SREBP-1 . Overall, the findings suggest that the wild-type SREBP-1 can bind and transactivate efficiently the apoA-II promoter in cell culture . SREBP-1 mutants lacking the activation domain bind to their cognate sites and directly repress the apoA-II promoter whereas mutants defective in DNA binding indirectly repress the apoA-II promoter activity, possibly by a squelching mechanism.

In Vivo, 1999 Mar-Apr, 13(2), 155 - 71
Prevention of oral diseases by polyphenols (review); Sakagami H et al.; This review summarizes the current data on the effects of natural products on various oral diseases, together with their basic biological activities . We have focused on polyphenols and their secondary metabolites, such as tannins, lignins and flavonoids, and their modulating factors, including saliva proline-rich proteins . These substances are found in a wide variety of plant sources such as vegetables, herbs, nuts and teas, and effectively reduce the incidence of carcinogenesis in the oral cavity, inhibit plaque growth and adsorption of oral bacteria, and inhibit the replication of various viruses . The mechanism of their action includes: the direct inactivation of the bacteria and viruses, inhibition of their replication enzymes, induction of apoptosis in tumor cells, stimulation of monocytes/macrophages to produce cytokines, and stimulation of myeloperoxidase-dependent iodination of neutrophiles . Polyphenols showed biphasic actions, acting as antioxidants at lower doses, but acting as prooxidants at higher doses . The development and progression of oral diseases might be modified not only by these natural products, but also by interaction with saliva, proline-rich proteins, antioxidants, metals and dental materials.

Am J Physiol, 1999 Jun, 276(6 Pt 1), G1461 - 72
IL-2-deficient mice raised under germfree conditions develop delayed mild focal intestinal inflammation; Schultz M et al.; Interleukin-2 (IL-2) amplifies immune stimuli and influences B cell differentiation . IL-2-deficient mice spontaneously develop intestinal inflammation if raised under specific pathogen-free (SPF) conditions . We quantitatively determined the aggressiveness and kinetics of gastrointestinal and hepatic inflammation in the presence or absence of viable bacteria in IL-2-deficient mice . Breeding colonies were maintained under SPF and germfree (GF) conditions . Intestinal tissues, serum, and mesenteric lymph nodes were obtained from mice at different ages for blind histological scoring, immunoglobulin measurements, mucosal T cell infiltration, and cytokine secretion . GF IL-2 -/- mice developed mild, focal, and nonlethal intestinal inflammation with delayed onset, whereas the more aggressive inflammation in SPF IL-2 -/- mice led to their death between 28 and 32 wk . Periportal hepatic inflammation was equal in the presence or absence of bacterial colonization . Intestinal immunoglobulin secretion decreased significantly by 13 wk of age in IL-2 -/- mice in both GF and SPF environments . In contrast to other genetically engineered rodents, IL-2 -/- mice develop mild focal gastrointestinal and active portal tract inflammation in the absence of viable bacteria.

Crit Care Med, 1999 May, 27(5), 923 - 8
Do the components of heat and moisture exchanger filters affect their humidifying efficacy and the incidence of nosocomial pneumonia?
Thomachot L, Vialet R, Arnaud S, Barberon B, Michel-Nguyen A, Martin C.
OBJECTIVES: To compare the efficiency of two heat and moisture exchange filters (HMEFs) of different compositions of the humidifying capacity and the rate of bronchial colonization and ventilator-associated pneumonia in patients in the intensive care unit (ICU) . DESIGN: Prospective, randomized study . SETTING: ICU of a university hospital . PATIENTS: All patients who required mechanical ventilation for 24 hrs or more during the study period . INTERVENTIONS: At admission to the ICU, patients were randomly assigned to one of two groups . In one group, the patients were ventilated with Humid-Vent Filter Light HMEF . The condensation surface was made of paper impregnated with CaCl2 . The filter membrane was made of polypropylene . In the other group, the patients were ventilated with the Clear ThermAl HMEF (Intersurgical, France) . The condensation surface was made of plastic foam impregnated with AlCl2 . The filter membrane was made of two polymer fibers (modacrylic and polypropylene) . In both groups, HMEFs were changed daily . MEASUREMENTS AND MAIN RESULTS: Seventy-seven patients were ventilated for 19+/-7 days with the Humid-Vent Filter Light HMEF and 63 patients for 17+/-6 days with the Clear ThermAl HMEF . Patients ventilated with the Humid-Vent Filter Light underwent 8.7+/-3.7 tracheal aspirations and 1.2+/-2.0 instillations per day and those with the Clear ThermAl, 8.2+/-3.9 and 1.5+/-2.4 per day, respectively (NS) . The abundance of tracheal secretions and the presence of blood and viscosity, as evaluated by semiquantitative scales, were similar in both groups . One episode of tracheal tube occlusion was observed with the Humid-Vent Filter Light HMEF and none with the other HMEF (NS) . Tracheal colonization was observed at a rate of 91% with the Humid-Vent Filter Light and 97% with the Clear ThermAl (NS) . The rate of ventilator-associated pneumonia was similar in both groups (35%) . Bacteria responsible for tracheal colonization and pneumonia were similar in both groups . CONCLUSIONS: Despite differences in their components, the two HMEFs that were tested achieved similar performances in terms of humidification and heating of inspired gases . Only one episode of endotracheal tube occlusion was detected, and very few patients (three in each group) had to be switched to an active heated humidifier . No difference was observed either in the rate of tracheal colonization or of ventilator-associated pneumonia . These data show that the Humid-Vent Filter Light and the Clear ThermAl HMEFs are suited for use with ICU patients.

J Biotechnol, 1999 Apr 15, 69(2-3), 183 - 90
A recombinant Fab neutralizes dengue virus in vitro; Thullier P et al.; A recombinant Fab that recognizes a neutralizing epitope located in the (296-400) region of protein E of dengue virus was obtained from cloned hybridoma cells secreting the mouse monoclonal antibody (mAb) 4E11 . The Fd and light chain antibody genes were amplified by polymerase chain reaction, cloned into the phagemid vector pMad, expressed in bacteria to produce Fab fragments and sequenced . The mAb 4E11, in particular its light chain complementary-determining regions, shared homologies with two other anti-viral mAbs . The affinity of the parental mAb and the cloned Fab to the MalE-E(296-400) fusion protein were shown to be of the same magnitude, i.e . nanomolar . Fab 4E11 neutralization capacity was found between 8 and 4-times or less lower than that of mAb 4E11, depending on serotypes, thus the Fab could have a smaller antiviral activity than the mAb in vitro.

FEMS Microbiol Lett, 1999 Jun 1, 175(1), 107 - 11
Colony formation by Helicobacter pylori after long-term incubation under anaerobic conditions; Yamaguchi H et al.; To evaluate the viability of Helicobacter pylori cultured under anaerobic conditions, H . pylori strain TK1029 was grown on blood agar in a microaerophilic environment at 37 degrees C for 4 days, and subsequently cultured under anaerobic conditions for 1 to 35 days . Colony formation by bacteria on blood agar plates cultured under anaerobic conditions was observed only for up to 4 days of microaerophilic incubation . By Gram staining, the morphological form of the bacteria was shown to be predominantly coccoid . However, bacteria cultured under anaerobic conditions for 15 to 35 days formed colonies on blood agar after pre-incubation of bacteria with PBS, but not without pre-incubation . These results suggest that H . pylori survives long-term culture under anaerobic conditions and that both pre-incubation in non-nutrient solution and high density of bacterial concentration might be important for recovery of H . pylori cultured for a prolonged time under anaerobic conditions.

FEMS Microbiol Lett, 1999 Jun 1, 175(1), 21 - 6
Development of a transposon mutagenesis system for Mycobacterium avium subsp . paratuberculosis; Harris NB et al.; Mycobacterium avium subspecies paratuberculosis, a slow-growing Mycobacterium, is the causative agent of Johne's disease . Although M . paratuberculosis is difficult to manipulate genetically, our laboratory has recently demonstrated the ability to introduce DNA into these bacteria by transformation and phage infection . In the current study we develop the first transposon mutagenesis system for M . paratuberculosis using the conditionally replicating mycobacteriophage phAE94 to introduce the mycobacterial transposon Tn5367 . Southern blotting and sequence analysis demonstrated that the transposon insertion sites are distributed relatively randomly throughout the M . paratuberculosis genome . We constructed a comprehensive bank of 5620 insertion mutants using this transposon . The transposition frequency obtained using this delivery system was 1.0 x 10(-6) transposition events per recipient cell . Auxotrophic mutants were observed in this library at a frequency of 0.3%.

Mol Microbiol, 1999 Jun, 32(5), 990 - 1001
Pore-forming activity is not sufficient for Legionella pneumophila phagosome trafficking and intracellular growth; Zuckman DM et al.; Bacterial pathogens often subvert eukaryotic cellular processes in order to establish a replicative niche and evade host immunity . Inhibition of phagosome lysosome fusion is a strategy used by several intracellular bacteria that grow within mammalian cells . It was shown recently that Legionella pneumophila possesses a cytolytic activity that results from the insertion of pores in the macrophage membrane upon contact, and that this activity requires the dot/icm gene products, which are necessary for intracellular growth and phagosome trafficking . Other bacteria that inhibit phagosome lysosome fusion, such as Mycobacterium tuberculosis, demonstrate similar cytolytic activities, which suggests that formation of pores in the phagosome membrane may account for the defects observed in phagosome trafficking . In this study, we identify a new class of L . pneumophila mutant that retains the pore-forming activity found in virulent bacteria, but is defective in phagosome lysosome fusion inhibition and intracellular growth . These data indicate that cytolytic activity is not sufficient for L . pneumophila-induced alterations in phagosome trafficking . Rather, the pore may be a vehicle that facilitates delivery of bacterial-derived effector molecules to the host cell cytoplasm.

Zentralbl Bakteriol, 1999 Apr, 289(2), 115 - 24
Phospholipid analogue distribution in Capnocytophaga; Drucker DB et al.; Polar lipids of nineteen previously characterised culture collection strains of Capnocytophaga were analysed using fast atom bombardment mass spectrometry (FAB MS) in negative mode . All strains examined had a major peak at m/z 241, consistent with the expected presence of the pentadecanoate anion . The most intense higher mass anions, consistent with expected presence of phospholipid molecular species, were as follows: m/z 574, 588, 618 and 662 which are consistent with presence of PE(24:2), PE(25:2), PE(27:1) and PE(30:0) respectively . Other anions putatively identified as phospholipid anions were: m/z 572, 578, 592, 602, 604, 616 and 720 consistent with presence of PE(24:3), PE(24:0), PE(25:0), PE(26:2), PE(26:1), PE(27:2) and OH-PE(33:0) . Capnocytophaga isolates share a distinctive phospholipid fingerprint which appears to lack the somewhat higher mass phospholipid analogues observed in related oral bacteria . Within the genus, the profiles obtained showed only quantitative differences which did not correlate with previous studies.

Bioorg Med Chem Lett, 1999 May 17, 9(10), 1443 - 6
Phosphofurylalanine, a stable analog of phosphohistidine; Schenkels C et al.; Phosphorylated histidine residues occur in a number of signal-transduction pathways in bacteria as well as in eukaryotes . Phosphohistidine is hydrolytically labile and therefore difficult to study, this by contrast to stable phosphoserine, phosphothreonine or phosphotyrosine . Here we report the design and enantioselective synthesis of (4'-phospho-2'-furyl)alanine 1, a non-hydrolyzable analog of 1-phosphohistidine . This novel amino-acid should be useful to synthesize peptides incorporating a stable analog phosphohistidine.

Proc Natl Acad Sci U S A, 1999 Jun 8, 96(12), 6808 - 13
Phytoremediation of methylmercury pollution: merB expression in Arabidopsis thaliana confers resistance to organomercurials; Bizily SP et al.; Methylmercury is an environmental toxicant that biomagnifies and causes severe neurological degeneration in animals . It is produced by bacteria in soils and sediments that have been contaminated with mercury . To explore the potential of plants to extract and detoxify this chemical, we engineered a model plant, Arabidopsis thaliana, to express a modified bacterial gene, merBpe, encoding organomercurial lyase (MerB) under control of a plant promoter . MerB catalyzes the protonolysis of the carbon---mercury bond, removing the organic ligand and releasing Hg(II), a less mobile mercury species . Transgenic plants expressing merBpe grew vigorously on a wide range of concentrations of monomethylmercuric chloride and phenylmercuric acetate . Plants lacking the merBpe gene were severely inhibited or died at the same organomercurial concentrations . Six independently isolated transgenic lines produced merBpe mRNA and MerB protein at levels that varied over a 10- to 15-fold range, and even the lowest levels of merBpe expression conferred resistance to organomercurials . Our work suggests that native macrophytes (e.g., trees, shrubs, grasses) engineered to express merBpe may be used to degrade methylmercury at polluted sites and sequester Hg(II) for later removal.

Curr Biol, 1999 Jun 3, 9(11), R400 - 2
Oxidative stress: Protein folding with a novel redox switch; Ruddock LW et al.; A novel cellular response to oxidative stress has been discovered, in which the activity of a molecular chaperone, Hsp33, is modulated by the environmental redox potential . This provides a rapid first defence mechanism against the potentially very harmful toxic effects of oxidative stress.

Curr Biol, 1999 Jun 3, 9(11), R416 - 8
Biological sensors: More than one way to sense oxygen; Pellequer JL et al.; Recently determined structures of the oxygen-sensing heme domain of the bacterial protein FixL have revealed a new binding environment and signal transduction mechanism for heme; they have also provided new insights into the diverse 'PAS' domain superfamily.

Annu Rev Immunol, 1999, 17, 467 - 522
The dynamics of T cell receptor signaling: complex orchestration and the key roles of tempo and cooperation; Germain RN et al.; T cells constantly sample their environment using receptors (TCR) that possess both a germline-encoded low affinity for major histocompatibility complex (MHC) molecules and a highly diverse set of CDR3 regions contributing to a range of affinities for specific peptides bound to these MHC molecules . The decision of a T cell "to sense and to respond" with proliferation and effector activity rather than "to sense, live on, but not respond" is dependent on TCR interaction with a low number of specific foreign peptide:MHC molecule complexes recognized simultaneously with abundant self peptide-containing complexes . Interaction with self-complexes alone, on the other hand, generates a signal for survival without a full activation response . Current models for how this distinction is achieved are largely based on translating differences in receptor affinity for foreign versus self ligands into intracellular signals that differ in quality, intensity, and/or duration . A variety of rate-dependent mechanisms involving assembly of molecular oligomers and enzymatic modification of proteins underlie this differential signaling . Recent advances have been made in measuring TCR:ligand interactions, in understanding the biochemical origin of distinct proximal and distal signaling events resulting from TCR binding to various ligands, and in appreciating the role of feedback pathways . This new information can be synthesized into a model of how self and foreign ligand recognition each evoke the proper responses from T cells, how these two classes of signaling events interact, and how pathologic responses may arise as a result of the underlying properties of the system . The principles of signal spreading and stochastic resonance incorporated into this model reveal a striking similarity in mechanisms of decision-making among T cells, neurons, and bacteria.

Am J Infect Control, 1999 Jun, 27(3), 258 - 61
Handwashing with soap or alcoholic solutions? A randomized clinical trial of its effectiveness; Zaragoza M et al.; BACKGROUND: The effectiveness of an alcoholic solution compared with the standard hygienic handwashing procedure during regular work in clinical wards and intensive care units of a large public university hospital in Barcelona was assessed . METHODS: A prospective, randomized clinical trial with crossover design, paired data, and blind evaluation was done . Eligible health care workers (HCWs) included permanent and temporary HCWs of wards and intensive care units . From each category, a random sample of persons was selected . HCWs were randomly assigned to regular handwashing (liquid soap and water) or handwashing with the alcoholic solution by using a crossover design . The number of colony-forming units on agar plates from hands printing in 3 different samples was counted . RESULTS: A total of 47 HCWs were included . The average reduction in the number of colony-forming units from samples before handwashing to samples after handwashing was 49.6% for soap and water and 88.2% for the alcoholic solution . When both methods were compared, the average number of colony-forming units recovered after the procedure showed a statistically significant difference in favor of the alcoholic solution (P <.001) . The alcoholic solution was well tolerated by HCWs . Overall acceptance rate was classified as "good" by 72% of HCWs after 2 weeks use . Of all HCWs included, 9.3% stated that the use of the alcoholic solution worsened minor pre-existing skin conditions . CONCLUSIONS: Although the regular use of hygienic soap and water handwashing procedures is the gold standard, the use of alcoholic solutions is effective and safe and deserves more attention, especially in situations in which the handwashing compliance rate is hampered by architectural problems (lack of sinks) or nursing work overload.

J Biol Chem, 1999 Jun 11, 274(24), 16933 - 9
A novel NDP-6-deoxyhexosyl-4-ulose reductase in the pathway for the synthesis of thymidine diphosphate-D-fucose; Yoshida Y et al.; The serotype-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans Y4 (serotype b) consists of D-fucose and L-rhamnose . Thymidine diphosphate (dTDP)-D-fucose is the activated nucleotide sugar form of D-fucose, which has been identified as a constituent of structural polysaccharides in only a few bacteria . In this paper, we show that three dTDP-D-fucose synthetic enzymes are encoded by genes in the gene cluster responsible for the synthesis of serotype b-specific polysaccharide in A . actinomycetemcomitans . The first and second steps of the dTDP-D-fucose synthetic pathway are catalyzed by D-glucose-1-phosphate thymidylyltransferase and dTDP-D-glucose 4,6-dehydratase, which are encoded by rmlA and rmlB in the gene cluster, respectively . These two reactions are common to the well studied dTDP-L-rhamnose synthetic pathway . However, the enzyme catalyzing the last step of the dTDP-D-fucose synthetic pathway has never been reported . We identified the fcd gene encoding a dTDP-4-keto-6-deoxy-D-glucose reductase . After purifying the three enzymes, their enzymatic activities were analyzed by reversed-phase high performance liquid chromatography . In addition, nuclear magnetic resonance analysis and gas-liquid chromatography analysis proved that the fcd gene product converts dTDP-4-keto-6-deoxy-D-glucose to dTDP-D-fucose . Moreover, kinetic analysis of the enzyme indicated that the Km values for dTDP-4-keto-6-deoxy-D-glucose and NADPH are 97.3 and 28.7 microM, respectively, and that the enzyme follows the sequential mechanism . This paper is the first report on the dTDP-D-fucose synthetic pathway and dTDP-4-keto-6-deoxy-D-glucose reductase.

Extremophiles, 1999 May, 3(2), 81 - 7
Polymer hydrolysis in a cold climate; Cummings SP et al.; In this review we discuss the activity of an ecologically significant group of psychrophilic bacteria, which are involved in the hydrolysis of plant cell wall polymers . Until now these organisms have been largely overlooked, despite the key role they play in releasing organic carbon fixed by primary producers in permanently cold environments such as Antarctica . This review details a specific group of plant cell wall polymer-degrading enzymes known as beta-glycanases . Studies on "cold" enzymes in general are in their infancy, but it has been shown that many exhibit structural and functional modifications that enable them to function at low temperature . beta-Glycanases in particular are intriguing because their substrates (cellulose and xylan) are very refractile, which may indicate that their "cold" modifications are pronounced . In addition, mesophilic beta-glycanases have been extensively studied and the current state of our knowledge is reviewed . This body of information can be exploited to enable meaningful comparative studies between mesophilic and psychrophilic beta-glycanases . The aim of such investigations is to obtain a deeper insight into those structural and functional modifications that enable these enzymes to function at low temperature and to examine the evolutionary relationship between mesophilic and psychrophilic beta-glycanases.

Arh Hig Rada Toksikol, 1998 Dec, 49(4), 371 - 8
Quality assurance in aquatic biology--a user's perspective; Hale PR; The essential elements of developing biological quality assurance systems are presented in terms of the 4M principles . These relate to methods, manpower, materials, and machines . The use of international or European standards is recommended where such standards exist . Users must be sure that these are appropriate to their specific needs as standardisation can require considerable compromise . Examples of limitations in international standards are given with reference to the coliform isolation by membrane filtration, Daphnia magna acute toxicity test and the luminescent bacteria test . The criteria for the selection and use of national methodologies are considered using macroinvertebrate, macrophytes, imposex, and the oyster embryo bioassay as examples . In recognising that the main resource in science is the skill, training, and dedication of the scientists themselves, the United Kingdom has developed a quality initiative aimed at best utilising the human resource, the so-called Investors in People (IIP) initiative . This contains the essential elements of any quality system: commitment, planning, action, and evaluation . Quality aspects of the materials and the machines used in biological analyses are briefly considered.

Int Endod J, 1999 Jan, 32(1), 40 - 8
Human teeth with periapical pathosis after overinstrumentation and overfilling of the root canals: a scanning electron microscopic study; Gutierrez JH et al.; AIM: The aim of this study was to determine whether overinstrumentation followed by immediate overfilling could be a potential risk in the treatment of infected root canals . METHODOLOGY: Thirty-five human teeth with infected root canals were overinstrumented and overfilled approximately 45 min after their extraction . The experimental teeth were enlarged up to size 40 and the overinstrumentation and overfilling were checked with the aid of a magnifying glass . The specimens were fixed in glutaraldehyde plus sodium cacodylate solution and prepared for scanning electron microscope examination . RESULTS: Bacteria were detected on the flute of the files and mostly at the root apices around the main foramen, remaining firmly attached to resorptive lacunae despite the fact that the apices had undergone great changes, including fracture or zipping . A control group consisting of 10 human teeth root canals containing vital pulps were also overinstrumented and overfilled . No bacteria were detected on the flutes of the files, at the apices or on the extruded master cone overfilling these samples . CONCLUSIONS: The high percentage of bacteria adhering to the resorptive lacunae or in the flutes of files used in overinstrumented human teeth with infected root canals carry a potential risk for postoperative pain, clinical discomfort and flare-ups . The hazards observed in these circumstances do not support the one-visit treatment of teeth having acute or chronic periapical abscesses.

Trends Microbiol, 1999 May, 7(5), 207 - 12
Families of arsenic transporters; Rosen BP; Bacterial arsenic resistance (ars) operons encode an arsenite-efflux system that can be a secondary carrier protein (ArsB) or an anion-translocating ATPase (ArsAB) . Yeasts extrude arsenite using Acr3p, a plasma membrane carrier protein, or sequester it in vacuoles as the glutathione conjugate using Ycf1p, an ABC transporter.

Mutat Res, 1999 May, 436(3), 263 - 83
Mutagenicity, carcinogenicity, and teratogenicity of acrylonitrile; Leonard A et al.; Acrylonitrile (AN) is an important intermediary for the synthesis of a variety of organic products, such as artificial fibres, household articles and resins . Although acute effects are the primary concern for an exposure to AN, potential genotoxic, carcinogenic and teratogenic risks of AN have to be taken seriously in view of the large number of workers employed in such industries and the world-wide population using products containing and possibly liberating AN . An understanding of the effect of acrylonitrile must be based on a characterization of its metabolism as well as of the resulting products and their genotoxic properties . Tests for mutagenicity in bacteria have in general been positive, those in plants and on unscheduled DNA synthesis doubtful, and those on chromosome aberrations in vivo negative . Wherever positive results had been obtained, metabolic activation of AN appeared to be a prerequisite . The extent to which such mutagenic effects are significant in man depends, however, also on the conditions of exposure . It appears from the limited data that the ultimate mutagenic factor(s), such as 2-cyanoethylene oxide, may have little opportunity to act under conditions where people are exposed because it is formed only in small amounts and is rapidly degraded . The carcinogenic action of AN has been evaluated by various agencies and ranged from 'reasonably be anticipated to be a human carcinogen' to 'cannot be excluded', the most recent evaluation being 'possibly carcinogenic to humans' . Animal data that confirm the carcinogenic potential of AN have certain limitations with respect to the choice of species, type of tumors and length of follow up . Epidemiological studies which sometimes, but not always, yielded positive results, encounter the usual difficulties of confounding factors in chemical industries . Exposure of workers to AN should continue to be carefully monitored, but AN would not have to be considered a cancer risk to the population provided limitations on releases from consumer products and guidelines on AN in water and air are enforced . AN is teratogenic in laboratory animals (rat, hamster) at high doses when foetal/embryonic (and maternal) toxicity already is manifest . Pregnant workers should not be exposed to AN . In view of the small concentrations generally encountered outside plants, women not professionally exposed would appear not to be at risk of teratogenic effects due to AN . Future research should concentrate on the elucidation of the different degradation pathways in man and on epidemiological studies in workers including pregnant women, assessing also, if possible, individual exposure by bio-monitoring .

Mutat Res, 1999 May, 436(3), 195 - 225
ICH-harmonised guidances on genotoxicity testing of pharmaceuticals: evolution, reasoning and impact; Muller L et al.; The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) has convened an expert working group which consisted of the authors of this paper and their respective committees, consulting groups and task forces . Two ICH guidances regarding genotoxicity testing have been issued: S2A, 'Guidance on Specific Aspects of Regulatory Genotoxicity Tests' and S2B, 'Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.' Together, these guidance documents now form the regulatory backbone for genotoxicity testing and assessment of pharmaceuticals in the European Union, Japan, and the USA . These guidances do not constitute a revolutionary new approach to genotoxicity testing and assessment, instead they are an evolution from preexisting regional guidelines, guidances and technical approaches . Both guidances describe a number of specific criteria as well as a general test philosophy in genotoxicity testing . Although these guidances were previously released within the participating regions in their respective regulatory communiques, to ensure their wider distribution and better understanding, the texts of the guidances are reproduced here in their entirety (see Appendix A) and the background for the recommendations are described . The establishment of a standard battery for genotoxicity testing of pharmaceuticals was one of the most important issues of the harmonisation effort . This battery currently consists of: (i) a test for gene mutation in bacteria, (ii) an in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells or an in vitro mouse lymphoma tk assay, (iii) an in vivo test for chromosomal damage using rodent hematopoietic cells . A major change in testing philosophy is the acceptance of the interchangeability of testing for chromosomal aberrations in mammalian cells and the mouse lymphoma tk assay . This agreement was reached on the basis of the extensive review of databases and newly generated experimental data which are in part described in this publication . The authors are fully aware of the fact that some of the recommendations given in these ICH guidances are transient in nature and that the dynamic qualities and ongoing evolution of genetic toxicology makes necessary a continuous maintenance process that would serve to update the guidance as necessary .

J Immunol, 1999 Jun 1, 162(11), 6784 - 91
Lysosomal accumulation and recycling of lipopolysaccharide to the cell surface of murine macrophages, an in vitro and in vivo study; Forestier C et al.; In this study, we detailed in a time-dependent manner the trafficking, the recycling, and the structural fate of Brucella abortus LPS in murine peritoneal macrophages by immunofluorescence, ELISA, and biochemical analyses . The intracellular pathway of B . abortus LPS, a nonclassical endotoxin, was investigated both in vivo after LPS injection in the peritoneal cavity of mice and in vitro after LPS incubation with macrophages . We also followed LPS trafficking after infection of macrophages with B . abortus strain 19 . After binding to the cell surface and internalization, Brucella LPS is routed from early endosomes to lysosomes with unusual slow kinetics . It accumulates there for at least 24 h . Later, LPS leaves lysosomes and reaches the macrophage cell surface . This recycling pathway is also observed for LPS released by Brucella S19 following in vitro infection . Indeed, by 72 h postinfection, bacteria are degraded by macrophages and LPS is located inside lysosomes dispersed at the cell periphery . From 72 h onward, LPS is gradually detected at the plasma membrane . In each case, the LPS present at the cell surface is found in large clusters with the O-chain facing the extracellular medium . Both the antigenicity and heterogenicity of the O-chain moiety are preserved during the intracellular trafficking . We demonstrate that LPS is not cleared by macrophages either in vitro or in vivo after 3 mo, exposing its immunogenic moiety toward the extracellular medium.

Helicobacter, 1999 Mar, 4(1), 7 - 16
In vitro aging of Helicobacter pylori: changes in morphology, intracellular composition and surface properties; Enroth H et al.; BACKGROUND: During the conversion from the bacillary into the coccoid form, Helicobacter pylori organisms are known to change extensively . The aim of this study was to determine some of the changes that occur regarding morphology, intracellular composition and surface properties during the aging of bacteria in vitro . MATERIALS AND METHODS: H . pylori from agar plate cultures of different ages was used in this study . The intracellular composition of the two morphological forms of the bacteria was tested by density centrifugation, DNA extraction and quantitative OD, mRNA and ATP measurements . Immunoblotting was used to observe changes in secreted/superficial protein patterns, and hydrophobicity measurements were used to observe changes in surface properties . RESULTS: All bacillary H . pylori organisms changed morphology gradually over 10 days of culture . Rods had a higher density than cocci; bacteria stored in PBS had the highest density and bacteria stored in water had the lowest . The quantitative DNA, RNA and ATP content were reduced in the aging bacteria . Fewer immunogenic proteins were expressed, and an increased surface hydrophobicity was observed in the older cultures . CONCLUSION: This study highlights several aspects of H . pylori aging in vitro and shows some of the differences that exist between bacillary and coccoid forms . This information is important for understanding the transmission and survival of H . pylori outside the human host, as the degradative changes in the intracellular composition and the surface properties shown here point to dead bacteria, and not to a viable but nonculturable form.

Dis Aquat Organ, 1999 Apr 15, 36(1), 37 - 44
Evaluation of a whole cell, p57- vaccine against Renibacterium salmoninarum; Piganelli JD et al.; A whole cell Renibacterium salmoninarum vaccine was developed using 37 degrees C heat treated cells that were subsequently formalin fixed; this treatment reduced bacterial hydrophobicity and cell associated p57 . Coho salmon Oncorhynchus kisutch were immunized with the p57- vaccine by either a combination of intraperitoneal (i.p.) and intramuscular (i.m.) injections or per os . In the first experiment, i.p./i.m . vaccination of coho salmon with p57- cells in Freund's Incomplete Adjuvant (FIA) conferred a statistically significant increase in mean time to death after the salmon were i.p . challenged with 4.1 x 10(6) colony forming units (cfu) of R . salmoninarum . There was no significant difference in response between fish immunized with R . salmoninarum cell surface extract in FIA and those immunized with extracellular protein (ECP) concentrated from culture supernatant in FIA . The i.p . challenge dose resulted in complete mortality of all fish by Day 43 . In a second experiment, fish were orally vaccinated with p57- R . salmoninarum cells encased in a pH protected, enteric-coated antigen microsphere (ECAM) . Fish were bath challenged with 4.2 x 10(6) cfu ml-1 on Day 0 and sampled at time points of 0 (pre-challenge), 50, 90, or 150 d immersion challenge . Vaccine efficacy was determined by monitoring the elaboration of p57 in the kidneys of vaccinated and control fish . Fish vaccinated orally demonstrated a significantly lower concentration of p57 (p < 0.01) at Day 150 post challenge compared to fish receiving ECAMs alone . Fish receiving p57 cells without ECAM coating also showed a significantly lower p57 level (p < 0.03) versus control . In contrast, fish injected intraperitoneally with the p57- cells or fish fed p57+ R . salmoninarum cells in ECAMs demonstrated no significant difference (p > 0.05) versus controls . In summary, these studies suggest the preliminary efficacy of 37 degrees C treatment of R . salmoninarum cells as an oral bacterial kidney disease vaccine.

Antimicrob Agents Chemother, 1999 Jun, 43(6), 1484 - 6
Subpopulations of Helicobacter pylori are responsible for discrepancies in the outcome of nitroimidazole susceptibility testing; van der Wouden EJ et al.; Metronidazole susceptibility testing by E test was compared to that by disk diffusion for 263 Helicobacter pylori isolates and to that by breakpoint agar dilution for 90 H . pylori isolates . In 5% and 6% of the cases, respectively, results were discrepant . For each of 52 clinical isolates an E test was performed on 10 separate colonies . Subpopulations of resistant and susceptible bacteria were found in five cases . From three isolates, each colony was subcultured and tested up to 10 times . All but 1 of 292 tests showed the same result . We conclude that the E test is reliable and that subpopulations are responsible for discordant results.

Arch Oral Biol, 1999 Apr, 44(4), 337 - 42
Induction of apoptosis in human gingival fibroblasts by a Porphyromonas gingivalis protease preparation; Wang PL et al.; Proteases produced by Porphyromonas gingivalis are believed to contribute to the pathogenesis of periodontal diseases . Here the cytotoxic effects of a purified preparation of a P . gingivalis protease with trypsin-like specificity were tested on human gingival fibroblasts in vitro . The active protease induced apoptotic cell death in the fibroblasts, as indicated by DNA fragmentation and the expression of 7A6 antigen . Thus, the production of proteases by periodontopathic bacteria could be an important factor in the induction of apoptosis of host cells in the aetiology of periodontal diseases.

Am J Trop Med Hyg, 1999 Apr, 60(4), 587 - 92
Serologic IgG response to urease in Helicobacter pylori-infected persons from Mexico; Leal-Herrera Y et al.; Helicobacter pylori urease is required to counteract acidity during colonization of the stomach, and has been suggested as a major immunodominant antigen . The aim of this study was to determine the anti-urease response in a representative national serologic survey in Mexico . The population surveyed included persons 1-90 years of age from all socioeconomic levels and geographic zones of the country . Helicobacter pylori status was determined by ELISA serology . The IgG anti-urease was studied by ELISA using a recombinant apoenzyme . We found that 2,930 of the 7,720 infected patients (38%) were seropositive for IgG urease . The rate of IgG anti-urease positivity increased with age; in children < 10 years old it was < 20% and in persons > 40 years old it was > 50% . Age and a region with a high level of development were risk factors for seropositivity, whereas gender, educational level, crowding, and socioeconomic level were not associated with seropositivity . In conclusion, in natural infection with H . pylori, the response to urease is poor, mainly during the first years of infection . This inconsistent immune response to the enzyme may favor persistence of infection . A vaccine eliciting a consistent anti-urease response might overcome immune evasion and enhance clearance of bacteria after exposure.

Medicina (B Aires), 1998, 58(6), 733 - 5
Acid fast filaments in stool samples from an AIDS patient; Bava J et al.; The presence of filamentous bacteria morphologically similar to Nocardia in a fresh stool sample from an AIDS patient with pulmonary nocardiosis is here reported . The material was submitted to our laboratory for a parasitologic examination and was stained by the Kinyoun method, revealing numerous delicate, irregularly stained, branching acid-fast filaments . Nocardia asteroides had been isolated from sputum samples of this patient . The patient was a 32 year-old HIV+ female admitted to our center on June 1997 because of productive cough, right-sided thoracic pain and weight loss . Chest X rays showed the presence of right superior lobe excavated pneumonia . This was the first time we had observed filamentous bacteria similar to Nocardia in a stool sample submitted to parasitologic examination . For similar cases, and when its presence was not detected in other specimens collected from the same patient, intestinal endoscopy and biopsy should be performed for eventual lesions and smear examination repeated with Kinyoun stain and cultures for Nocardia.

J Biol Chem, 1999 Jun 4, 274(23), 16003 - 9
Zinc coordination and substrate catalysis within the neuropeptide processing enzyme endopeptidase EC 3.4.24.15 . Identification of active site histidine and glutamate residues; Cummins PM et al.; Endopeptidase EC 3.4.24.15 (EP24.15) is a zinc metalloendopeptidase that is broadly distributed within the brain, pituitary, and gonads . Its substrate specificity includes a number of physiologically important neuropeptides such as neurotensin, bradykinin, and gonadotropin-releasing hormone, the principal regulatory peptide for reproduction . In studying the structure and function of EP24.15, we have employed in vitro mutagenesis and subsequent protein expression to genetically dissect the enzyme and allow us to glean insight into the mechanism of substrate binding and catalysis . Comparison of the sequence of EP24.15 with bacterial homologues previously solved by x-ray crystallography and used as models for mammalian metalloendopeptidases, indicates conserved residues . The active site of EP24.15 exhibits an HEXXH motif, a common feature of zinc metalloenzymes . Mutations have confirmed the importance, for binding and catalysis, of the residues (His473, Glu474, and His477) within this motif . A third putative metal ligand, presumed to coordinate directly to the active site zinc ion in concert with His473 and His477, has been identified as Glu502 . Conservative alterations to these residues drastically reduces enzymatic activity against both a putative physiological substrate and a synthetic quenched fluorescent substrate as well as binding of the specific active site-directed inhibitor, N-{1-(RS)-carboxy-3-phenylpropyl}-Ala-Ala-Tyr-p-aminobenzoate, the binding of which we have shown to be dependent upon the presence, and possibly coordination, of the active site zinc ion . These studies contribute to a more complete understanding of the catalytic mechanism of EP24.15 and will aid in rational design of inhibitors and pharmacological agents for this class of enzymes.

Appl Environ Microbiol, 1999 Jun, 65(6), 2789 - 93
Characterization of the meta-cleavage compound hydrolase gene involved in degradation of the lignin-related biphenyl structure by Sphingomonas paucimobilis SYK-6; Peng X et al.; Sphingomonas paucimobilis SYK-6 has the ability to transform a lignin-related biphenyl compound, 2,2'-dihydroxy-3,3'-dimethoxy-5, 5'-dicarboxybiphenyl (DDVA), to 5-carboxyvanillic acid (5CVA) via 2, 2',3-trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl (OH-DDVA) . In the 4.9-kb HindIII fragment containing the OH-DDVA meta-cleavage dioxygenase gene (ligZ), we found a novel hydrolase gene (ligY) responsible for the conversion of the meta-cleavage compound of OH-DDVA to 5CVA . Incorporation of 18O from H218O into 5CVA indicated there was a hydrolytic conversion of the OH-DDVA meta-cleavage compound to 5CVA . LigY exhibited hydrolase activity only toward the meta-cleavage compound of OH-DDVA, suggesting its restricted substrate specificity.

Appl Environ Microbiol, 1999 Jun, 65(6), 2409 - 17
Flow cytometric analysis of 5-cyano-2,3-ditolyl tetrazolium chloride activity of marine bacterioplankton in dilution cultures; Sieracki ME et al.; The respiratory activity of marine bacteria is an important indication of the ecological functioning of these organisms in marine ecosystems . The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) is reduced intracellularly in respiring cells to an insoluble, fluorescent precipitate . This product is detectable and quantifiable by flow cytometry in individual cells . We describe here an evaluation of flow cytometry for measuring CTC activity in natural assemblages of marine bacteria growing in dilution cultures . We found that more CTC-positive cells are detected by flow cytometry than by visual epifluorescence microscopy . Samples can be stored refrigerated or frozen in liquid nitrogen for at least 4 weeks without a significant loss of total cells, CTC-positive cells, or CTC fluorescence . Cytometry still may not detect all active cells, however, since the dimmest fluorescing cells are not clearly separated from background noise . Reduction of CTC is very fast in most active cells, and the number of active cells reaches 80% of the maximum number within 2 to 10 min . The proportion of active cells is correlated with the growth rate, while the amount of fluorescence per cell varies inversely with the growth rate . The CTC reduction kinetics in assemblages bubbled with nitrogen and in assemblages bubbled with air to vary the oxygen availability were the same, suggesting that CTC can effectively compete with oxygen for reducing power . A nonbubbled control, however, contained more CTC-positive cells, and the amount of fluorescence per cell was greater . Activity may have been reduced by bubble-induced turbulence . Addition of an artificial reducing agent, sodium dithionite, after CTC incubation and fixation resulted in a greater number of positive cells but did not "activate" a majority of the cells . This indicated that some of the negative cells actually transported CTC across their cell membranes but did not reduce it to a detectable level . Automated analysis by flow cytometry allows workers to study single-cell variability in marine bacterioplankton activity and changes in activity on a small temporal or spatial scale.

Electrophoresis, 1999 Apr-May, 20(4-5), 977 - 83
Mapping and identification of HeLa cell proteins separated by immobilized pH-gradient two-dimensional gel electrophoresis and construction of a two-dimensional polyacrylamide gel electrophoresis database; Shaw AC et al.; The HeLa cell line, a human adenocarcinoma, is used in many research fields, since it can be infected with a wide range of viruses and intracellular bacteria . Therefore, the mapping of HeLa cell proteins is useful for the investigation of parasite host cell interactions . Because of the recent improvements of two-dimensional gel electrophoresis with immobilized pH gradients (IPG) compared to isoelectric focusing with carrier ampholytes, a highly reproducible method for examining global changes in HeLa cell protein expression due to different stimuli is now available . Therefore, we have initiated the mapping of {35S}methionine/cysteine-labeled HeLa cell proteins with the 2-D PAGE (IPG)-system, using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and N-terminal sequencing for protein identification . To date 21 proteins have been identified and mapped . In order to make these and future data accessible for interlaboratory comparison, we constructed a 2-D PAGE database on the World Wide Web.

Cell Motil Cytoskeleton, 1999, 43(1), 72 - 81
Centrin-like filaments in the cytopharyngeal apparatus of the ciliates Nassula and Furgasonia: evidence for a relationship with microtubular structures; Vigues B et al.; The cytopharyngeal apparatus in the Nassulinid ciliates Nassula and Furgasonia is a highly specialized microtubular/filamentous organelle designed for ingestion of organisms such as filamentous bacteria . From studies on living cells, it was previously shown that this organelle, also called "feeding basket," guides the filamentous bacteria and manipulates them to some extent during the early steps of ingestion . This results in a complex sequence of movements where the basket is successively dilated and constricted in its upper part . Whereas some of these movements (dilation) seem to be intrinsic to the microtubular components of the basket, others (constriction) are believed to be mediated by contractile filamentous structures {Tucker, 1968: J . Cell Sci . 3:493-514} . In this study, we have used antibodies raised against ciliate centrins to demonstrate these proteins by Western blot and immunocytochemical methods in Nassula and Furgasonia . In both ciliates, a 20-kDa centrin immunoanalog was localized in the upper (contractile) part of the cytopharyngeal apparatus . Immunoelectron microscopy revealed that cytopharyngeal centrin is engaged in filamentous material, forming a sphincter-like structure possibly involved in the movements of contraction . Interestingly, physical links were noted between filaments labeled for centrin and cytopharyngeal microtubules . The mechanistic implications of these findings are discussed.

Cell Motil Cytoskeleton, 1999, 43(1), 1 - 9
Microtubule severing; Quarmby LM et al.; The regulation of microtubule stability by severing of the polymer along its length is a newly appreciated and potentially important mechanism for controlling microtubule function . Microtubule severing occurs in living cells, but direct observation of this event is infrequent . The paucity of direct observations leave open to question the significance of regulated microtubule severing in the control of microtubule organization . Nevertheless, several lines of evidence suggest that microtubule severing is an important cellular activity . First, the ATP-dependent microtubule-severing activity of katanin is well documented . Katanin is found in most cell types and is enriched at MTOCs . Although it is possible that katanin does not sever microtubules in vivo, this seems unlikely . Second, a physiological event, deflagellation, has been shown to depend on microtubule severing . The deflagellation system of Chlamydomonas has provided a genetic approach to the problem of microtubule severing . The FA genes are essential for the regulated severing of axonemal microtubules during deflagellation, but whether these genes define new severing proteins or whether they are important for katanin activity remains to be determined . Microtubule severing is a relatively new area of investigation and there are still many more questions than answers . It is anticipated that the recent cloning of katanin and the introduction of a genetic model system will soon lead to significant breakthroughs in this problem.

J Orthop Trauma, 1999 May, 13(4), 241 - 6
Anatomy of the distal knee joint and pyarthrosis following external fixation; Hyman J et al.; OBJECTIVE: To determine the limits of the distal synovial reflection of the human knee joint . SPECIMENS: Six paired knees studied by magnetic resonance imaging (MRI), fluoroscopic arthrography, and gross dissection . The right knees of five patients with chronic idiopathic knee effusions were studied by MRI . Cadaveric knees were injected with saline prior to MRI . The joint capsules were dissected to visualize local anatomy and check for capsular tears . In each modality (MRI, fluoroscopy, and dissection), the most distal extent of knee synovial fluid was measured . RESULTS: The right versus left agreement for paired specimens was generally two to three millimeters . Some specimens showed asymmetric capsular reflection . Medial fluid was identified at distances greater than forty-nine millimeters from the subchondral bone in seven knees and less than fifteen millimeters in four knees (range 0 to 70 millimeters, mean thirty-three millimeters) . Laterally, the range was ten to thirty-five millimeters (mean twenty-three millimeters) . In six of the twelve cadaveric specimens, there was evidence of a communication between the knee joint and the proximal tibiofibularjoint . In the knees of volunteers, joint fluid tracked medially to a range of ten to fifty millimeters and laterally to a range of six to fifteen millimeters, with means of twenty-six and eleven millimeters, respectively . The knees of the volunteers had no evidence of tibiofibular joint communication with the knee . CONCLUSION: Insertion of external fixation pins within sixty to seventy millimeters of the proximal articular surface of the tibia is associated with a high probability of synovial penetration and possibly provides a conduit for the introduction of bacteria, which may be etiologic in iatrogenic pyarthrosis.

FEMS Immunol Med Microbiol, 1999 May, 24(1), 105 - 14
Tissue culture adherence and haemagglutination characteristics of Moraxella (Branhamella) catarrhalis; Fitzgerald M et al.; The haemagglutination and tissue culture adherence properties of 20 isolates of Moraxella catarrhalis obtained from the sputum of elderly patients with lower respiratory tract infections were compared with those of 20 isolates of M . catarrhalis obtained from the nasopharynx of elderly persons colonised by the organism . Eighty percent of isolates from the infected group as opposed to 5% of isolates from the colonised group haemagglutinated human erythrocytes (P < 0.001), indicating that the haemagglutinin might be a marker of pathogenicity for M . catarrhalis . There was a significant difference in the adherence to HEp-2 cells of isolates from the infected group in comparison to isolates from the colonised group (P = 0.03) . Haemagglutination and tissue culture adherence properties were unrelated, indicating that separate adhesin systems are involved . The adherence of M . catarrhalis to HEp-2 cells was unaffected following pronase and trypsin treatment, however, sodium periodate pre-treatment of the bacteria significantly reduced the tissue culture adherence index, indicating that the adhesin by which the bacteria bind to HEp-2 cells may have a carbohydrate moiety . Transmission electron microscopy studies revealed that adherence of M . catarrhalis to HEp-2 cells was mediated by trypsin-resistant 'tack-/spicule-like' structures protruding from the surface of the bacteria.

FEMS Microbiol Lett, 1999 May 15, 174(2), 321 - 6
Mycoplasma cavipharyngis and Mycoplasma fastidiosum, the closest relatives to Eperythrozoon spp . and Haemobartonella spp; Johansson KE et al.; The 16S rRNA gene sequences of Mycoplasma cavipharyngis and Mycoplasma fastidiosum have been determined . Phylogenetic analysis showed that these species formed a new cluster within the so-called pneumoniae group of the mollicutes (class Mollicutes) . This cluster will be referred to as the M . fastidiosum cluster . Interestingly, the M . fastidiosum cluster formed a sister lineage to the haemotrophic bacteria . Eperythrozoon spp . and Haemobartonella spp . The two latter genera, formerly classified as rickettsias, formed a stable phylogenetic entity in the tree as judged from branch lengths, bootstrap values and sequence signatures . Thus, the members of the M . fastidiosum cluster are the closest known relatives to the haemotrophic bacteria . Our data strongly support that the haemotrophic bacteria should be reclassified to reflect their actual phylogenetic affiliation.

FEMS Microbiol Lett, 1999 May 15, 174(2), 225 - 9
Early colonization of the rat upper respiratory tract by temperature modulated Bordetella bronchiseptica; Brockmeier SL; The ability of nonmodulated Bvg+ phase cultures, temperature modulated Bvg- phase cultures, and a Bvg- phase-locked mutant of Bordetella bronchiseptica to colonize the rat upper respiratory tract was investigated . Initially, greater numbers of the temperature modulated Bvg- phase bacteria adhered to the nasal cavity of the rats . The temperature modulated Bvg- phase bacteria appeared to be quickly cleared to levels lower than the Bvg+ phase bacteria by 4 h after inoculation and stayed lower until 48 h after inoculation when colonization levels were equal to the Bvg+ phase bacteria . The level of colonization with the Bvg- phase-locked mutant of B . bronchiseptica was lower than both the nonmodulated Bvg+ phase and temperature modulated Bvg- phase cultures and declined over time during the experiment . These findings suggest that there may be increased adherence from an environmental phase to ensure bacteria survive initial clearance mechanisms until the switch to the Bvg+ phase occurs.

Proc Natl Acad Sci U S A, 1999 May 25, 96(11), 6285 - 90
Convergent evolution of Trichomonas vaginalis lactate dehydrogenase from malate dehydrogenase; Wu G et al.; Lactate dehydrogenase (LDH) is present in the amitochondriate parasitic protist Trichomonas vaginalis and some but not all other trichomonad species . The derived amino acid sequence of T . vaginalis LDH (TvLDH) was found to be more closely related to the cytosolic malate dehydrogenase (MDH) of the same species than to any other LDH . A key difference between the two T . vaginalis sequences was that Arg91 of MDH, known to be important in coordinating the C-4 carboxyl of oxalacetate/malate, was replaced by Leu91 in LDH . The change Leu91Arg by site-directed mutagenesis converted TvLDH into an MDH . The reverse single amino acid change Arg91Leu in TvMDH, however, gave a product with no measurable LDH activity . Phylogenetic reconstructions indicate that TvLDH arose from an MDH relatively recently.

Blood, 1999 Jun 1, 93(11), 3876 - 84
Interleukin-2-activated rat natural killer cells express inducible nitric oxide synthase that contributes to cytotoxic function and interferon-gamma production; Cifone MG et al.; Natural killer (NK) cells are large granular lymphocytes capable of destroying cells infected by virus or bacteria and susceptible tumor cells without prior sensitization and restriction by major histocompatability complex (MHC) antigens . Their cytotoxic activity could be strongly enhanced by interleukin-2 (IL-2) . Previous findings, even if obtained with indirect experimental approaches, have suggested a possible involvement of the inducible nitric oxide (iNOS) pathway in the NK-mediated target cell killing . The aim of the present study was first to directly examine the induction of iNOS in IL-2-activated rat NK cells isolated from peripheral blood (PB-NK) or spleen (S-NK), and second to investigate the involvement of the iNOS-derived NO in the cytotoxic function of these cells . Our findings clearly indicate the induction of iNOS expression in IL-2-activated PB-NK and S-NK cells, as evaluated either at mRNA and protein levels . Accordingly, significantly high levels of iNOS activity were shown, as detected by the L-arginine to L-citrulline conversion in appropriate assay conditions . The consequent NO generation appears to partially account for NK cell-mediated DNA fragmentation and lysis of sensitive tumor target cells . In fact, functional inhibition of iNOS through specific inhibitors, as well as the almost complete abrogation of its expression through a specific iNOS mRNA oligodeoxynucleotide antisense, significantly reduced the lytic activity of IL-2-activated NK cells . Moreover, IL-2-induced interferon-gamma production appears also to be dependent, at least in part, on iNOS induction.

Curr Biol, 1999 May 20, 9(10), R371 - 3
Evolutionary genetics: The economics of mutation; Rainey PB; The presence of mutator genotypes in populations of bacteria may be favoured by selection because they produce rare beneficial mutations and thereby increase the rate of adaptive evolution . Recent work, however, shows that the relationship between mutation rates and adaptive evolution is more complicated.

Infect Immun, 1999 Jun, 67(6), 3146 - 50
Stability of erp loci during Borrelia burgdorferi infection: recombination is not required for chronic infection of immunocompetent mice; El Hage N et al.; Borrelia burgdorferi can persistently infect mammals despite their production of antibodies directed against bacterial proteins, including the Erp lipoproteins . We sequenced erp loci of bacteria reisolated from laboratory mice after 1 year of infection and found them to be identical to those of the inoculant bacteria . We conclude that recombination of erp genes is not essential for chronic mammalian infection.

Infect Immun, 1999 Jun, 67(6), 3108 - 11
Host defense against Mycobacterium avium does not have an absolute requirement for major histocompatibility complex class I-restricted T cells; Bermudez LE et al.; The role of CD8(+) T cells was evaluated in a mouse model of disseminated Mycobacterium avium infection . C57BL/6J and C57BL/6Jbeta2-/- (beta2-/-) mice were infected intravenously, and the number of viable bacteria in each liver and spleen was determined . No significant difference between the number of bacteria in the two strains of mice was observed at 2, 4, 6, and 8 weeks after infection . Histopathological examination of granulomas from C57BL/6J and beta2-/- mice did not show any difference either in the number of organisms per granuloma or in the size of the granulomas . Investigation of the cytokine profile in the spleen demonstrated that the beta2-/- strain of mice produced a significantly lower amount of gamma interferon at 8 weeks after infection and significantly increased concentrations of tumor necrosis factor alpha compared with that from the wild-type mouse . Interleukin-12 and transforming growth factor beta1 levels did not differ between the two strains of mice at 2, 4, 6, and 8 weeks . Although previous work had found that host response against Mycobacterium tuberculosis involves major histocompatibility complex class I-restricted T cells, our results indicate that chronic deficiency of CD8(+) T cells does not lead to a different expression of the disease and that if CD8(+) T cells are involved in the host response, their function can be assumed by other immune cells.

Infect Immun, 1999 Jun, 67(6), 3073 - 81
Chitinase secretion by encysting Entamoeba invadens and transfected Entamoeba histolytica trophozoites: localization of secretory vesicles, endoplasmic reticulum, and Golgi apparatus; Ghosh SK et al.; Entamoeba histolytica, the protozoan parasite that phagocytoses bacteria and host cells, has a vesicle/vacuole-filled cytosol like that of macrophages . In contrast, the infectious cyst form has four nuclei and a chitin wall . Here, anti-chitinase antibodies identified hundreds of small secretory vesicles in encysting E . invadens parasites and in E . histolytica trophozoites overexpressing chitinase under an actin gene promoter . Abundant small secretory vesicles were also identified with antibodies to the surface antigen Ariel and with a fluorescent substrate of cysteine proteinases . Removal of an N-terminal signal sequence directed chitinase to the cytosol . Addition of a C-terminal KDEL peptide, identified on amebic BiP, retained chitinase in a putative endoplasmic reticulum, which was composed of a few vesicles of mixed sizes . A putative Golgi apparatus, which was Brefeldin A sensitive and composed of a few large, perinuclear vesicles, was identified with antibodies to ADP-ribosylating factor and to epsilon-COP . We conclude that the amebic secretory pathway is similar to those of other eukaryotic cells, even if its appearance is somewhat different.

Infect Immun, 1999 Jun, 67(6), 2867 - 73
Persistence and protective efficacy of a Mycobacterium tuberculosis auxotroph vaccine; Jackson M et al.; New vaccines against tuberculosis are urgently required because of the impressive incidence of this disease worldwide and the highly variable protective efficacy of the current vaccine . The possibility of creating new live vaccines by the rational attenuation of strains from the Mycobacterium tuberculosis complex was investigated . Two auxotrophic mutants of M . tuberculosis and M . bovis BCG were constructed by disruption of one of their purine biosynthetic genes . These mutants appeared unable to multiply in vitro within mouse bone-marrow derived macrophages . They were also attenuated in vivo in the mouse and guinea pig animal models . In guinea pigs, the two mutants induced strong delayed-type hypersensitivity response to purified protein derivative . In a preliminary experiment, the two mutants were compared to the BCG vaccine for their protective efficacy in a challenge against aerosolized virulent M . tuberculosis in the guinea pig model . Both mutants conferred some level of protection . These experiments demonstrate that the rational attenuation of M . tuberculosis could lead to the design of new candidate live vaccines against tuberculosis.

Inflamm Bowel Dis, 1999 May, 5(2), 92 - 7
The importance of ileocaecal integrity in the arthritic complications of Crohn's disease; Orchard TR et al.; Experiments in animal models have suggested a role for bacterial overgrowth in the caecum in the pathogenesis of extracolonic inflammation in IBD . The aim of this study was to identify patients with Crohn's disease who have undergone ileocaecal resection and to compare the incidence of new arthritic complications in these patients with those who have never undergone surgery . Patients who had undergone surgery were identified by case note review . The date and nature of surgery were noted . The occurrence of new joint complications (Type 1 and 2 peripheral arthropathy and AS) was noted in patients who had undergone ileocaecal resection and in patients who had never undergone surgery . In the surgery group the timing in relation to surgery was determined . The groups were compared using Kaplan-Meier survival curves and the Logrank test . One hundred sixty-four patients who had undergone ileocaecal resection and 221 patients who had never undergone surgery for Crohn's disease were studied . The rate of development of arthritic complications in patients presurgery and in the nonsurgical group was identical . However few arthritic complications occurred postoperatively . There were highly significant differences between the nonsurgical group and the postsurgical group (p = 0.0001) and between patients presurgery and postsurgery (p = 0.0006) . New arthritic complications are less common in Crohn's disease after resection of the ileocaecal area . This would be consistent with the hypothesis that luminal bacteria in this region are important in the pathogenesis of these complications.

FEBS Lett, 1999 Apr 23, 449(2-3), 159 - 64
Constitutive and nitrogen-regulated promoters of the petH gene encoding ferredoxin:NADP+ reductase in the heterocyst-forming cyanobacterium Anabaena sp; Valladares A et al.; Determination of the putative transcription start points of the petH gene encoding ferredoxin:NADP+ reductase in the heterocyst-forming cyanobacteria Anabaena sp . PCC 7119 and PCC 7120 showed that this gene is transcribed from two promoters, one constitutively used under different conditions of nitrogen nutrition and the other one used in cells subjected to nitrogen stepdown and in nitrogen-fixing filaments . The latter promoter, whose use was NtcA-dependent but HetR-independent, was functional in heterocysts . The N-control transcriptional regulator NtcA was observed to bind in vitro to this promoter . For the sake of comparison, the transcription start points of the nifHDK operon in strain PCC 7120 and binding of NtcA to the nifHDK promoter were also examined.

Intern Med, 1999 Mar, 38(3), 240 - 3
Helicobacter heilmannii associated erosive gastritis; Yamamoto T et al.; The spiral bacteria, Helicobacter heilmannii (H . heilmannii), distinct from Helicobacter pylori (H . pylori), was found in the gastric mucosa of a 71-year-old man without clinical symptoms . The endoscopic examination revealed erosive gastritis . Rapid urease test from the antral specimen was positive, but both culture and immunohistological staining for H . pylori were negative . Touch smear cytology showed tightly spiral bacteria, which were consistent with H . heilmannii . At the second endoscopy after medication regimen for eradication of H . pylori, inflammation was decreased and the rapid urease test was negative . The second cytology showed no evidence of H . heilmannii . Anti-H . pylori therapy may be a useful medication for H . heilmannii.

Pol Merkuriusz Lek, 1999 Feb, 6(32), 107 - 9
{Ehrlichiosis: a tick-born infection}; Krupa W et al.; Ehrlichiosis is the potentially life-threating infection . It is caused by obligate intracellular bacteria . The clinical presentations are fever, headache, myalgia, malaise, nausea, vomiting and other nonspecyfic symptoms . Some patients develop neurologic symptoms and signs . The are two distinct forms of human ehrlichiosis: human monocytic ehrlichiosis /HME/--cased by Ehrlichia chaffeensis that infects mononuclear phagocytes and human granulocytic ehrlichiosis /HGE/--caused by E . species closely related to E . phagocytophyla and E . equi and infects granulocytes . Successful treatment of these infections may depend on proper diagnosis . Appropiate diagnostic tests are still not available . This diagnisis should be considered in febrile patients with tick bites.

Glycobiology, 1999 Jun, 9(6), 527 - 32
Rat liver contains age-regulated cytosolic 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid (Kdn); Campanero-Rhodes MA et al.; Kdn (3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid), a unique deaminated member of the sialic acid family, has emerged as a new building block of glycoconjugates from a wide variety of organisms, ranging from bacteria to mammals . In particular, the presence of Kdn has been demonstrated in different rat organs and tissues, but not in liver . Here we report on the detection and quantitation of Kdn in rat liver and on its variations with postnatal development and aging . We have previously established the optimal conditions for derivatization of Kdn with 1,2-diamino-4, 5-methylene-dioxybenzene (DMB), and detection by reverse-phase HPLC . Analysis of whole liver homogenates and different subcellular fractions reveals that Kdn is fundamentally present in the cytosolic fraction as nucleotide precursor . The expression of Kdn, Neu5Gc, and Neu5Ac changes unevenly with age . While the content of Neu5Ac, the major species, and Neu5Gc decreases to a different extent from newborn to old animals, Kdn content decreases from newborn to trace amounts in adult rats and increases again with aging . Thus, expression of Kdn, Neu5Gc, and Neu5Ac appears to be independently regulated.

J Biol Chem, 1999 May 28, 274(22), 15473 - 9
The compact conformation of fibronectin is determined by intramolecular ionic interactions; Johnson KJ et al.; Fibronectin exists in a compact or extended conformation, depending upon environmental pH and salt concentration . Using recombinant fragments expressed in bacteria and baculovirus, we determined the domains responsible for producing fibronectin's compact conformation . Our velocity and equilibrium sedimentation data show that FN2-14 (a protein containing FN-III domains 2 through 14) forms dimers in low salt . Experiments with smaller fragments indicates that the compact conformation is produced by binding of FN12-14 of one subunit to FN2-3 of the other subunit in the dimer . The binding is weakened at higher salt concentrations, implying an electrostatic interaction . Furthermore, segment FN7-14+A, which contains the alternatively spliced A domain between FN11 and 12, forms dimers, whereas FN7-14 without A does not . Segment FN12-14+A also forms dimers, but the isolated A domain does not . These data imply an association of domain A with FN12-14, and the presence of A may favor an open conformation by competing with FN2-3 for binding to FN12-14.

Int J Legal Med, 1999, 112(3), 201 - 3
Saliva from cheese bite yields DNA profile of burglar: a case report; Sweet D et al.; Physical evidence in the form of a high quality bite mark was discovered on a piece of yellow cheese found at the scene of a crime . The cheese had been frozen by police for 10 days after recovery and before submission to the laboratory for testing . The double swab technique was used to collect DNA samples . A sample of the suspect's blood was obtained . Using PCR-based DNA typing at ten STR loci, (Profiler Plus, Perkin Elmer-Applied Biosystems) it was determined that the DNA from the cheese originated from the suspect . This case illustrates the importance of a) always considering human bite marks as both physical and biological evidence, and b) attempting DNA recovery in any case in which minute traces of saliva may be present, even in situations involving bacteria-rich foods.

Drug Metab Rev, 1999 May, 31(2), 523 - 44
Merits and limitations of recombinant models for the study of human P450-mediated drug metabolism and toxicity: an intralaboratory comparison; Friedberg T et al.; A wide variety of pharmacological and toxicological properties of drugs are determined by cytochrome P450-mediated metabolism . Characterization of these pathways and of the P450 isoenzymes involved constitutes an essential part of drug development . Similarly, because P450s are catalyzing the toxication and detoxication of environmental pollutants, an understanding of these reactions facilitates risk assessment in environmental toxicology . Recently, a variety of recombinant expression systems has been employed to study the role of human P450s in these reactions . These include insect, bacterial, yeast, and mammalian models . As these were developed and characterized by different laboratories, evaluation of their merits and limitations is inherently difficult . To resolve this problem, we have established and characterized the latter three systems and present the key results here . In general, the catalytic properties of P450 isozymes in the various models were rather similar . However, taking technical considerations into account as well as the high level of functional expression of P450s achieved in bacteria make this system ideally suited for drug metabolism research, including the generation of milligram quantities of metabolites for structural determinations . For toxicological studies, however, expression of P450s in mammalian cells was most appropriate . This is exemplified here by studies into the role of human P450s in the activation and inactivation of chemotherapeutic drugs.

Biochem J, 1999 Jun 1, 340 ( Pt 2), 433 - 8
Involvement of Arg-328, Arg-334 and Arg-342 of DnaA protein in the functional interaction with acidic phospholipids; Yamaguchi Y et al.; We reported previously that three basic amino acids (Arg-360, Arg-364 and Lys-372) of DnaA protein are essential for its functional interaction with cardiolipin . In this study, we examined the effect of mutation of some basic amino acids in a potential amphipathic helix (from Lys-327 to Ile-345) of DnaA protein on this interaction . ATP binding to the mutant DnaA protein, in which Arg-328, Arg-334 and Arg-342 were changed to acidic amino acids, was less inhibited by cardiolipin than that of the wild-type protein, as was the case for mutant DnaA protein with mutations of Arg-360, Arg-364 and Lys-372 . A mutant DnaA protein with mutations of all six basic amino acids showed the most resistance to the inhibition of ATP binding by cardiolipin . These results suggest that Arg-328, Arg-334 and Arg-342, like Arg-360, Arg-364 and Lys-372, are also involved in the functional interaction between DnaA protein and acidic phospholipids.

Eur J Gastroenterol Hepatol, 1999 Mar, 11(3), 219 - 21
Postgastrectomy pancreatic malabsorption: is there a case for intervention?
Griffiths A, Taylor RH.
More patients are surviving surgery for gastric carcinoma than ever before . The possible causes of malabsorption following total gastrectomy are multifactorial, and pancreatic insufficiency has been proposed as one mechanism . Other contributing factors include loss of gastric reservoir, rapid small bowel transit, small bowel bacterial overgrowth and the type of operation performed . Although pancreatic exocrine insufficiency has been demonstrated after gastrectomy, the key question remains whether pancreatic enzyme supplements offer any substantial clinical benefit to the patient . The evidence to date does not support the routine use of pancreatic enzyme supplementation after gastrectomy.

Vet Pathol, 1999 May, 36(3), 249 - 52
A case of intestinal Mycobacterium simiae infection in an SIV-infected immunosuppressed rhesus monkey; Didier A et al.; Although Mycobacterium simiae was identified and classified more than three decades ago, only a few cases are mentioned in the current literature . After experimental simian immunodeficiency virus infection, a 9-year-old female rhesus monkey (Macaca mulatta) developed progressive immunosuppression and gastrointestinal disease very similar to the clinical and pathomorphologic features of Johne's disease, which is caused by M . paratuberculosis . Acid-fast-positive bacteria reacted immunohistochemically with antibodies against M . paratuberculosis and M . bovis but were not useful for differentiation because of a high degree of cross-reactivity . In contrast to immunohistochemistry and histopathology, biochemical methods and cycle sequencing analysis of the 16S ribosomal RNA identified M . simiae as the disease-causing pathogen . This case demonstrates the importance of molecular biological methods for the diagnosis of M . simiae infection in monkeys.

Hum Mol Genet, 1999 Jun, 8(6), 1025 - 37
Large genomic duplicons map to sites of instability in the Prader-Willi/Angelman syndrome chromosome region (15q11-q13); Christian SL et al.; The most common etiology for Prader-Willi syndrome and Angelman syndrome is de novo interstitial deletion of chromosome 15q11-q13 . Deletions and other recurrent rearrangements of this region involve four common 'hotspots' for breakage, termed breakpoints 1-4 (BP1-BP4) . Construction of an approximately 4 Mb YAC contig of this region identified multiple sequence tagged sites (STSs) present at both BP2 and BP3, suggestive of a genomic duplication event . Interphase FISH studies demonstrated three to five copies on 15q11-q13, one copy on 16p11.1-p11.2 and one copy on 15q24 in normal controls, while analysis on two Class I deletion patients showed loss of approximately three signals at 15q11-q13 on one homolog . Multiple FISH signals were also observed at regions orthologous to both human chromosomes 15 and 16 in non-human primates, including Old World monkeys, suggesting that duplication of this region may have occurred approximately 20 million years ago . A BAC/PAC contig for the duplicated genomic segment (duplicon) demonstrated a size of approximately 400 kb . Surprisingly, the duplicon was found to contain at least seven different expressed sequence tags representing multiple genes/pseudogenes . Sequence comparison of STSs amplified from YAC clones uniquely mapped to BP2 or BP3 showed two different copies of the duplicon within BP3, while BP2 comprised a single copy . The orientation of BP2 and BP3 are inverted relative to each other, whereas the two copies within BP3 are in tandem . The presence of large duplicated segments on chromosome 15q11-q13 provides a mechanism for homologous unequal recombination events that may mediate the frequent rearrangements observed for this chromosome.

Can J Gastroenterol, 1999 Apr, 13(3), 237 - 41
Immunomodulation of helicobacter infection; Croitoru K; Helicobacter pylori leads to a chronic infection in humans that is associated with gastric inflammation and a vigorous immune response . Despite the humoral and cellular responses that can be detected in both human and animal models of helicobacter infection, the immune response fails to eliminate the organism . Eradication failure may be due to the niche in which H pylori confines itself, well away from direct contact with elements of the immune system . Alternatively, the general tendency of the intestinal immune response to down- regulate reactivity to noninvasive luminal bacteria also may contribute to the failure to eliminate helicobacter infection . Results of vaccine studies in mouse models indicate that modulating the helper T cell response from a T helper cell type 1 to a T helper cell type 2 response likely is required for the prevention and elimination of helicobacter infection . Understanding the mechanisms by which the immune response controls bacterial infections will allow for the design of novel strategies of immune modulation and the development of vaccines for both the treatment and prevention of H pylori.

J Leukoc Biol, 1999 May, 65(5), 658 - 64
Increased neutrophil adherence and adhesion molecule mRNA expression in endothelial cells during selenium deficiency; Maddox JF et al.; Leukocyte aggregation and activation on endothelial cells (EC) are important preliminary events in leukocyte migration into tissue and subsequent inflammation . Thus, an increase in leukocyte adherence has the potential to affect inflammatory disease outcome . Selenium (Se) is an integral part of the antioxidant enzyme glutathione peroxidase (GSH-Px) and plays an important role in the maintenance of the redox state of a cell . Se supplementation in the bovine has been shown to improve the outcome of acute mastitis caused by coliform bacteria, in part by enhancing the speed of neutrophil migration into the affected mammary gland . However, the mechanisms by which Se modulates neutrophil migration have not been elucidated . Therefore, an in vitro model of Se deficiency in primary bovine mammary artery EC was used to examine the impact of Se status on the adhesive properties of EC . The effect of Se on functional activities was examined by measuring neutrophil adherence to Se-deficient and Se-supplemented EC . Se-deficient EC showed significantly enhanced neutrophil adherence when stimulated with tumor necrosis factor alpha (TNF-alpha) for 4 or 24 h, interleukin-1 for 12 h, or H2O2 for 20 min (P < 0.05) . To determine the mechanisms underlying these changes in neutrophil adherence, the expression of EC adhesion molecules, ICAM-1, E-selectin, and P-selectin were examined at the molecular level by a competitive reverse transcription-polymerase chain reaction . Results revealed higher mRNA expression for E-selectin and ICAM-1 in Se-deficient EC stimulated with TNF-alpha for 3 and 6 h, and greater expression of P-selectin mRNA in Se-supplemented EC with 3-h TNF-alpha stimulation . These studies provide new information to establish the role of Se nutrition in the initiation of leukocyte adherence to endothelium.

Crit Care Clin, 1999 Apr, 15(2), 387 - 414
Serious waterborne and wilderness infections; Greenberg SB; Serious waterborne and wilderness infections are common and usually treatable if diagnosed early . The differential diagnosis for these infections requires a careful and thorough history and physical examination . Common clinical presentations include acute febrile illnesses, altered mental status, diarrhea, or pneumonia . Pathogens causing serious infections include bacteria, fungi, viruses, and protozoa . Epidemiologic help can be obtained from local or state health departments as well as the Centers for Disease Control.

Hokkaido Igaku Zasshi, 1999 Jan, 74(1), 67 - 76
{Analysis of immunological mechanisms in the pathogenesis of generalized pustular psoriasis}; Kawashima T; Generalized pustular psoriasis is one of life-threatening skin diseases which represents sudden onset of severe systemic symptoms such as high fever and chill as well as burning erythema and aseptic pustules over the entire skin . Although the precise mechanism of this rare disease is unknown, several lines of clinical and experimental observations have suggested that certain immunological mechanisms play important roles in the pathogenesis of this disease . In this study, in order to attain better understanding of the immunological events involved in generalized pustular psoriasis, several in vivo and in vitro immunological experiments have been performed . The results obtained are as follows: 1) sera from patients contained high amount of inflammatory cytokines, 2) peripheral blood mononuclear cells(PBMC) showed high proliferative responses to bacteria-derived super antigens, 3) PBMC from patients produced a large amount of cytokines when stimulated by mitogens in vitro, 4) endothelial cells in the lesional skins of patients exhibited enhanced expression of adhesion molecules, and 5) the expressions of these adhesion molecules on human endothelial cells were differently regulated by several cytokines . These results suggest that the activation of peripheral blood mononuclear cells by bacteria-derived super antigens followed by cytokine production, and the induction of the adhesion molecules expressions on the endothelial cells are important immunological events in the forming the characteristic clinical symptoms of generalized pustular psoriasis.

Klin Khir, 1999, (2), 28 - 30
{The use of sorbent "Sillard" in comprehensive treatment of patients with hemophilia and purulent-inflammatory complications}; Sukhovii MV et al.; An applicational usage of a siliconcontaining sorpent "Sillard" in the complex of treatment of patients with hemophilia, suffering inflammatory-purulent complications, had promoted the shortening of the wound clearance duration by 2-3 days and of the patient stationary treatment--by 3-4 days.

Toxicol Sci, 1999 Mar, 48(1), 38 - 50
Biological regulation of receptor-hormone complex concentrations in relation to dose-response assessments for endocrine-active compounds; Andersen ME et al.; Some endocrine-active compounds (EACs) act as agonists or antagonists of specific hormones and may interfere with cellular control processes that regulate gene transcription . Many mechanisms controlling gene expression are universal to organisms ranging from unicellular bacteria to more complex plants and animals . One mechanism, coordinated control of batteries of gene products, is critical in adaptation of bacteria to new environments and for development and tissue differentiation in multi-cellular organisms . To coordinately activate sets of genes, all living organisms have devised molecular modules to permit transitions, or switching, between different functional states over a small range of hormone concentration, and other modules to stabilize the new state through homeostatic interactions . Both switching and homeostasis are regulated by controlling concentrations of hormone-receptor complexes . Molecular control processes for switching and homeostasis are inherently nonlinear and often utilize autoregulatory feedback loops . Among the biological processes contributing to switching phenomena are receptor autoinduction, induction of enzymes for ligand synthesis, mRNA stabilization/activation, and receptor polymerization . This paper discusses a variety of molecular switches found in animal species, devises simple quantitative models illustrating roles of specific molecular interactions in creating switching modules, and outlines the impact of these switching processes and other feedback loops for risk assessments with EACs . Quantitative simulation modeling of these switching mechanisms made it apparent that highly nonlinear dose-response curves for hormones and EACs readily arise from interactions of several linear processes acting in concert on a common control point . These nonlinear mechanisms involve amplification of response, rather than multimeric molecular interactions as in conventional Hill relationships.

Aviakosm Ekolog Med, 1999, 33(1), 10 - 6
{Biological life support systems: investigations on board of orbital complex "Mir"}; Sychev VN et al.; From 1989 till 1998 twelve experiments were performed by Bulgarian, Russian, Slovak, and US researchers and engineers on the effects of space flight on the model of ecosystem "algae-fishes-bacteria", and ontogenesis of birds (Japanese quail) and higher plants . For the first time several viable chicks were hatched and passed the whole cycle of their embryonic development in the MIR microgravity . The length of the plant ontogenetic cycle as a whole and its specific stages appeared to be same as on Earth . Seeds of Brassica rapa gathered and planted in greenhouse Svet on board MIR yielded robust shoots . Photosynthesis and dark respiration of plants growing in spaceflight were successfully measured.

Int Immunol, 1999 May, 11(5), 745 - 51
Construction and binding analysis of recombinant single-chain TCR derived from tumor-infiltrating lymphocytes and a cytotoxic T lymphocyte clone directed against MAGE-1; Lake DF et al.; The TCR is responsible for the specificity of cytotoxic T lymphocytes (CTL) by recognizing peptides presented in the context of MHC . By producing recombinant soluble TCR, it is possible to study this interaction at the molecular level . We generated single-chain TCR (scTCR) from tumor infiltrating lymphocytes (TIL) and one CTL clone directed against melanoma-associated antigen (MAGE)-1 . Sixty-eight day anti-MAGE-1 TIL and one cloned anti-MAGE-1 CTL were analyzed by PCR for their Valpha and Vbeta gene usage . The TIL population showed a restriction in Valpha and Vbeta usage with only Valpha4 and Valpha9 and Vbeta2 and Vbeta7 expressed . The anti-MAGE-1 CTL clone demonstrated absolute restriction with only Valpha12 and Vbeta1 expressed . DNA sequence analysis was performed on all V regions . For the TIL, each possible Valpha-Vbeta combination (i.e . Valpha4-Vbeta2, Valpha9-Vbeta2, Valpha4-Vbeta7 and Valpha9-Vbeta7) was constructed as a distinct scTCR and the recombinant proteins expressed in bacteria . From the anti-MAGE-1 TIL, Valpha4-Vbeta2 scTCR demonstrated binding activity to HLA-A1(+) cells pulsed with MAGE-1 peptide . Results obtained from screening a panel of our scTCR constructs on HLA-A1(+) cells pulsed with MAGE-1 peptide or irrelevant peptide demonstrated that Vbeta2 plays a significant role in binding to the MAGE-1 peptide . Amino acid alignment analysis showed that each Vbeta sequence is distinctly different from the others . These findings demonstrate that soluble TCR in single-chain format have binding activity . Furthermore, the results indicate that in TCR, like antibodies, one chain may contribute a dominant portion of the binding activity.

J Biol Chem, 1999 May 21, 274(21), 15271 - 7
Activation of an MDM2-specific caspase by p53 in the absence of apoptosis; Pochampally R et al.; Cells undergoing p53-mediated apoptosis activate caspase 3-like activities, resulting in the cleavage of the MDM2 oncoprotein and other apoptotic substrates such as poly(ADP-ribose) polymerase . To investigate the mechanism of p53-mediated apoptosis and to determine whether cleavage of MDM2 has a potential role in regulating p53, we examined caspase activation and cleavage of MDM2 in a cell line undergoing p53-mediated growth arrest and delayed apoptosis . We found that in H1299 cells expressing a temperature-sensitive human p53, a distinct caspase activity specific for the MDM2 cleavage site DVPD is induced by p53 prior to the onset of apoptosis and loss of viability . This is accompanied by the cleavage of MDM2 but not the apoptotic substrate poly(ADP-ribose) polymerase . The cleaved MDM2 loses the ability to promote p53 degradation and may potentially function in a dominant-negative fashion to stabilize p53 . These results suggest that p53 activation may induce a positive feedback effect by cleavage of MDM2 through a unique caspase.

Biochem Biophys Res Commun, 1999 May 10, 258(2), 256 - 9
The GM2 activator protein, a novel inhibitor of platelet-activating factor; Rigat B et al.; The GM2 activator protein is required as a substrate-specific cofactor for beta-hexosaminidase A to hydrolyze GM2 ganglioside . The GM2 activator protein reversibly binds and solubilizes individual GM2 ganglioside molecules, making them available as substrate . Although GM2 ganglioside is the strongest binding ligand for the activator protein, it can also bind and transport between membranes a series of other glycolipids, even at neutral pH . Biosynthetic studies have shown that a large portion of newly synthesized GM2 activator molecules are not targeted to the lysosome, but are secreted and can then be recaptured by other cells through a carbohydrate independent mechanism . Thus, the GM2 activator protein may have other in vivo functions . We found that the GM2 activator protein can inhibit, through specific binding, the ability of platelet activating factor (PAF) to stimulate the release of intracellular Ca2+ pools by human neutrophils . PAF is a biologically potent phosphoacylglycerol . Inhibitors for PAF's role in the pathogenesis of inflammatory bowel disease and asthma have been sought as potential therapeutic agents . The inherent stability and protease resistance of the small, monomeric GM2 activator protein, coupled with the ability to produce large quantities of the functional protein in transformed bacteria, suggest it may serve as such an agent .

J Virol Methods, 1999 Apr, 79(1), 97 - 111
Techniques for the evaluation of nucleic acid amplification technology performance with specimens containing interfering substances: efficacy of boom methodology for extraction of HIV-1 RNA; Witt DJ et al.; Accurate HIV-1 RNA quantitation with nucleic acid amplification assays (NAAA) is partly dependent on overall assay design to ensure proper and reproducible functioning in the presence of endogenous interfering substances present in a clinical specimen, or exogenous interfering substances introduced as a result of specimen collection or handling . This study tested various methods of evaluating interfering substances that could potentially affect the outcome of HIV-1 RNA amplification in a NAAA . Clinical specimens from HIV-1 seronegative subjects containing various endogenous interferents were evaluated with and without an HIV-1 RNA spike to assess recovery and specificity, respectively, with a non-PCR NAAA (NASBA HIV-1 RNA QT, Organon Teknika) that incorporates Boom methodology for nucleic acid extraction . Additional specimens were prepared to simulate various circumstances that might occur during specimen preparation to result in the introduction of exogenous interferents . A retrovirus reverse transcriptase inhibitor, zidovudine (AZT), was added to plasma specimens prior to testing . NAAA results obtained with 127 total clinical specimens, 10 bacterially contaminated specimens, 5 platelet enriched specimens, 5 AZT specimens, and 30 anticoagulated specimens were consistent with the expected outcomes in the presence and absence of the HIV-1 RNA spike, giving an assay specificity of 100% . The spiked HIV-1 RNA copies in the clinical specimens reported by the assay were 99% of the copies reported for a positive index control (normal plasma plus HIV-1 RNA spike) . Compared to the amplification levels of the three internal assay calibrators obtained for normal plasma controls, no differences in the amplification levels of the calibrators for each type of specimen were observed . This result indicated that the interferents examined did not affect adversely assay function . Addition of known PCR interferents (hemoglobin and heparin) and AZT to isolated HIV-1 RNA resulted in a substantial reduction of amplification and invalid results, whereas no inhibition was observed when these interferents were added to the test system prior to isolation; these results directly demonstrate the efficient removal of such interferents during the NASBA HIV-1 RNA QT isolation procedure . The several approaches to investigate interference described in this study may be utilized for the evaluation of other assays using nucleic acid amplification technology.

J Anim Sci, 1999 Apr, 77(4), 1016 - 25
Effects of ruminal administration of supplemental degradable intake protein and starch on utilization of low-quality warm-season grass hay by beef steers; Olson KC et al.; Hereford x Angus steers were used in a 13-treatment, four-period, incomplete Latin square design to examine the effects of starch and degradable intake protein (DIP) supplements on forage utilization and ruminal function . Steers were given ad libitum access to low-quality hay (4.9% CP) and were not supplemented (NS) or received different amounts of starch (cornstarch grits; 0, .15, and .3% of initial BW) and DIP (Na-caseinate; .03, .06, .09, and .12% of initial BW) administered via ruminal fistulae in a 3 x 4 factorial arrangement of treatments . Supplemented steers consumed more (P < .01) forage OM, total OM, NDF, and digestible OM (DOM) than NS steers . Forage OM, total OM, NDF, and DOM intakes increased linearly (P < .01) as the amount of supplemental DIP increased . The addition of starch to supplements linearly decreased ( P < .01) the intake of forage OM, NDF, and DOM . The digestion of DM, OM, and NDF increased linearly (P < .01) with supplemental DIP and decreased linearly (P < or = .06) with supplemental starch . Particulate and liquid passages generally increased with DIP; however, starch level influenced the nature of the response (P = .03 and .06, respectively) . Similarly, ruminal acid detergent-insoluble ash content generally decreased as starch increased, but the effect was dependent on DIP level (P < .01) . Supplementation increased (P < .01) ruminal NH3 and total VFA and decreased (P < .01) ruminal pH relative to NS . All treatments supported average pH values in a range (6.3 to 6.7) unlikely to inhibit fibrolytic bacteria . Ruminal NH3 concentration increased quadratically (P = .03) with DIP and decreased linearly (P = .02) with starch . As DIP increased, total VFA concentration increased linearly (P = .02) . Providing supplemental DIP to steers fed low-quality forage increased OM intake and digestion, whereas addition of starch to supplements decreased forage intake and digestion.

Sex Transm Dis, 1982 Jan-Mar, 9(1), 1 - 8
The microaerophilic nature of Treponema pallidum: enhanced survival and incorporation of tritiated adenine under microaerobic conditions in the presence or absence of reducing compounds; Cover WH et al.; Treponema pallidum, although sensitive to atmospheric concentrations of O2, requires low levels of O2 for optimal survival and metabolic activity . The addition of 0.0125-0.2 mg/ml of sodium metabisulfite to a basal medium consisting of Eagle's minimal essential medium and 50% fresh, heat-inactivated normal rabbit serum was found to have an effect similar to that of dithiothreitol in extending the survival of T . pallidum (Nichols strain) under 3% O2 . Detailed analysis of the effect of O2 tension revealed that 50% motility was retained longest at atmospheric O2 concentrations of 1-5%, whether or not dithiothreitol or sodium metabisulfite were present . Concentrations of O2 of 3-10% were optimal for nucleic acid synthesis, as determined by {3H}adenine incorporation during the first 24 hr of incubation . Sodium metabisulfite was less effective than dithiothreitol in stimulating nucleic acid synthesis . Neither sodium metabisulfite nor dithiothreitol at their effective concentrations had any effect on levels of dissolved O2 . During incubation under 3% O2, motility was maintained at > 50% for 15 days and virulence for at least 13 days by dilution of the treponemal suspensions every three days with fresh medium containing sodium metabisulfite . The optimal retention of motility and nucleic acid synthesis under microaerobic conditions in the absence of reducing compounds provides further evidence that T . pallidum is a microaerophilic organism.

Cell Calcium, 1999 Feb, 25(2), 143 - 52
Adenine-nucleotide binding sites on the inositol 1,4,5-trisphosphate receptor bind caffeine, but not adenophostin A or cyclic ADP-ribose; Maes K et al.; Binding of ATP to the inositol 1,4,5-trisphosphate receptor (IP3R) results in a more pronounced Ca2+ release in the presence of inositol 1,4,5-trisphosphate (IP3) . We have expressed the cDNAs encoding two putative adenine-nucleotide binding sites of the neuronal form of IP3R-1 as glutathione S-transferase (GST)-fusion proteins in bacteria . Specific {alpha-32P}ATP binding was observed for the two GST-fusion proteins, representing aa 1710-1850 and aa 1944-2040 of IP3R-1 . The ATP-binding sites in both fusion proteins had the same nucleotide specificity as found for the intact IP3R (ATP > ADP > AMP > GTP) . Smaller GST-fusion proteins (aa 1745-1792 and aa 2005-2023) displayed a much weaker ATP-binding activity . CoA, which also potentiated IP3-induced Ca2+ release in A7r5 cells, interacted with the ATP-binding sites on the fusion proteins . Such interaction was not observed for 1,N6-etheno CoA and 3'-dephospho-CoA, which are much less effective in potentiating IP3-induced Ca2+ release . Since the adenine-containing compounds adenophostin A, caffeine and cyclic ADP-ribose modulate IP3-induced Ca2+ release, a possible effect of these compounds on the ATP-binding sites was examined . ATP stimulated adenophostin A- and IP3-induced Ca2+ release in A7r5 cells with an EC50 of respectively 21 and 20 microM . Also the threshold concentration of ATP for stimulating the release was similar for the two agonists . Adenophostin A (100 microM) and cyclic ADP-ribose (100 microM) were ineffective in displacing {alpha-32P}ATP from the binding sites of both GST-fusion proteins . Caffeine (50 mM), however, inhibited {alpha-32P}ATP binding to both fusion proteins by more than 50% . These data provide evidence for a direct interaction of caffeine but not of adenophostin A or cyclic ADP-ribose with the adenine-nucleotide binding sites of the IP3R.

Wei Sheng Yan Jiu, 1997 Mar, 26(2), 126 - 9
{The method to remove nitrite from tap water by tea}; Lu M et al.; Drinking water (tap water) is polluted in pipelines by bacteria after long distance transportation . The water contains nitrite (NO2-) which is potentially harmful to human health . The nitrite concentrations range from 0.10 to 2.0 mg/L . Our experiment proved that NO2- could not be removed by boiling, but could be removed by tea . As a natural antioxidant, tea contains several antioxidants, such as ascorbic acid and catechins, which removed NO2- from tap water effectively.

Wei Sheng Yan Jiu, 1997 Mar, 26(2), 90 - 3
{A study on the corrosion of desalinated drinking water from electrodialysis process and its control}; Li S et al.; The stability index IR of desalinated drinking water from electrodialysis process was more than 9.0 . Its corrosion rate P reached 0.59 mm/a . This is the main cause of "Red Water" and severe corrosion in network of pipes of water supply in an oil field . A large number of iron bacteria and sulfur bacteria in water are the biological causes of tubercles and block up . But the P becomes less than 0.05 mm/a, when the water is remineralized . It is an effective measurement to control the pipe corrosion and to provide good quality drinking water.

Nucleic Acids Res, 1999 Jun 1, 27(11), 2369 - 76
Alignment of whole genomes; Delcher AL et al.; A new system for aligning whole genome sequences is described . Using an efficient data structure called a suffix tree, the system is able to rapidly align sequences containing millions of nucleotides . Its use is demonstrated on two strains of Mycoplasma tuberculosis, on two less similar species of Mycoplasma bacteria and on two syntenic sequences from human chromosome 12 and mouse chromosome 6 . In each case it found an alignment of the input sequences, using between 30 s and 2 min of computation time . From the system output, information on single nucleotide changes, translocations and homologous genes can easily be extracted . Use of the algorithm should facilitate analysis of syntenic chromosomal regions, strain-to-strain comparisons, evolutionary comparisons and genomic duplications.

J Clin Microbiol, 1999 Jun, 37(6), 2077 - 9
Infection of laboratory mice with the human granulocytic ehrlichiosis agent does not induce antibodies to diagnostically significant Borrelia burgdorferi antigens; Bunnell JE et al.; Laboratory diagnosis of Borrelia burgdorferi is routinely made by an enzyme-linked immunosorbent assay, with positive results confirmed by Western blot analysis . Concern has been raised that false-positive diagnoses may be made on the basis of serologic cross-reactivity with antibodies directed against other bacterial pathogens, in particular the agent of human granulocytic ehrlichiosis (HGE) . The present study made use of a mouse model to ascertain the validity of these concerns . Two different strains of mice were inoculated with the HGE agent and assayed for production of polyclonal and monoclonal antibodies to antigens of both of these bacteria . Infection of mice with the HGE agent does not induce diagnostically significant B . burgdorferi serologic cross-reactions.

J Clin Microbiol, 1999 Jun, 37(6), 1994 - 8
Differentiation of Helicobacter pylori isolates based on lectin binding of cell extracts in an agglutination assay; Hynes SO et al.; Plant and animal lectins with various carbohydrate specificities were used to type 35 Irish clinical isolates of Helicobacter pylori and the type strain NCTC 11637 in a microtiter plate assay . Initially, a panel of eight lectins with the indicated primary specificities were used: Anguilla anguilla (AAA), Lotus tetragonolobus (Lotus A), and Ulex europaeus I (UEA I), specific for alpha-L-fucose; Solanum tuberosum (STA) and Triticum vulgaris (WGA), specific for beta-N-acetylglucosamine; Glycine max (SBA), specific for beta-N-acetylgalactosamine; Erythrina cristagali (ECA), specific for beta-galactose and beta-N-acetylgalactosamine; and Lens culinaris (LCA), specific for alpha-mannose and alpha-glucose . Three of the lectins (SBA, STA, and LCA) were not useful in aiding in strain discrimination . An optimized panel of five lectins (AAA, ECA, Lotus A, UEA I, and WGA) grouped all 36 strains tested into eight lectin reaction patterns . For optimal typing, pretreatment by washing bacteria with a low-pH buffer to allow protein release, followed by proteolytic degradation to eliminate autoagglutination, was used . Lectin types of treated samples were stable and reproducible . No strain proved to be untypeable by this system . Electrophoretic and immunoblotting analyses of lipopolysaccharides (LPSs) indicated that the lectins interact primarily, but not solely, with the O side chain of H . pylori LPS.

Gut, 1999 Jun, 44(6), 886 - 8
Mesalazine (5-aminosalicylic acid) induced chronic hepatitis; Deltenre P et al.; BACKGROUND: Treatment of ulcerative colitis or Crohn's disease with sulphasalazine causes several adverse effects, including hepatitis . Sulphasalazine is cleaved by colonic bacteria into 5-aminosalicylic acid and sulphapyridine . Received wisdom was that 5-aminosalicylic acid was topically active, whereas sulphapyridine was absorbed and caused immunoallergic side effects . Mesalazine, a slow release formulation of 5-aminosalicylic acid, was expected to be a safe alternative . However, several cases of acute hepatitis have been reported . CASE REPORT: A 65 year old man had increased liver enzymes, anti-nuclear and anti-smooth muscle autoantibodies and IgG levels, and lesions of chronic hepatitis after 21 months of mesalazine treatment . Although liver dysfunction had been identified eight months earlier, simvastatin rather than mesalazine had been withdrawn, without any improvement . In contrast, liver enzyme and IgG levels became normal and autoantibodies disappeared after discontinuation of mesalazine administration . CONCLUSION: Contrary to initial expectations, mesalazine can cause most of the sulphasalazine induced adverse effects, and hepatic side effects may be almost as frequent . When liver dysfunction occurs, mesalazine administration should be discontinued to avoid the development of chronic hepatitis and liver fibrosis.

Arthritis Rheum, 1999 May, 42(5), 942 - 7
Different humoral immune response to Chlamydia trachomatis major outer membrane protein variable domains I and IV in Chlamydia-infected patients with or without reactive arthritis; Bas S et al.; OBJECTIVE: The possibility that some bacterial-specific factor(s) may play a role in triggering Chlamydia trachomatis reactive arthritis was investigated . METHODS: Since the variable domains of the major outer membrane protein (MOMP) contain the serovar-determining epitopes of C . trachomatis, the ability of serum IgG to recognize peptides mimicking these epitopes was determined in 2 groups of infected patients, one with and the other without reactive arthritis . Because asymptomatic C . trachomatis infections are frequent, and nonspecific reactions due to inflammation could be observed, this study was also performed with samples from healthy blood donors and from patients with inflammatory arthritis unrelated to C . trachomatis infection . RESULTS: A predominant reactivity against peptides duplicating the J serovar-specific epitopes was only observed in the group of patients with reactive arthritis . For positive samples, differences between the two groups of C . trachomatis-infected patients were clearly observed . The mean numbers of positive responses obtained for each of the 7 peptides of the MOMP domain I or each of the 8 peptides of the MOMP domain IV were significantly higher for samples from patients with reactive arthritis (4.7 and 6) than for those from patients with only C . trachomatis urogenital infection (1.3 and 2.9) . CONCLUSION: Patients with reactive arthritis had a pattern of reactivities that was compatible with infection by several serotypes of bacteria . Repeated exposures to C . trachomatis might therefore be involved in the development of the disease.

Zhongguo Zhong Xi Yi Jie He Za Zhi, 1997 Aug, 17(8), 484 - 6
{Experimental study on effect of zhuyu tongfu mixture on anti-enterogenous infection caused by endotoxin}; Yang N et al.; OBJECTIVE: To evaluate the effect of Zhuyu Tongfu mixture (ZYTFM) in curing enterogenus infection . METHODS: Experimental model of enterogenous infection in mice caused by endotoxin was used . The positive translocation trate (PTR) and mumber of viable bacteria in viscera and serum level of superoxide dismutase (SOD) were measured before and after ZYTFM treatment . RESULTS: The PTR and number of viable bacteria in liver, spleen and mesocolon in the ZYTFM group were much lower than those in the model group and the placebo group (P < 0.01) . Whereas the SOD level of the ZYTFM group was significantly higher than that in the latter two groups (P < 0.05-0.01) . Pathological examination displayed that ZYTFM could markedly alleviate the mucosal damage of small intestine . CONCLUSION: ZYTFM has an obvious curative effect on enterogenous infections induced by endotoxin.

Zhonghua Liu Xing Bing Xue Za Zhi, 1998 Apr, 19(2), 72 - 4
{An epidemiologic analysis of brucellosis in Liaoning province}; Sun G et al.; In order to understand the increasing incidence rate of Brucellosis, the relationship between the epidemic trend and physical conditions of humans, data regarding Brucellosis in Liaoning province, in 1990-1996 was analysed . Results showed that the major cause which led to the Brucellosis recurrence was the importation of sheep named short-tail cold-resistant . The distribution of disease was widely spread . However, most endemic foci were being expanded . The spots in Tieling and Fushun area were merged and became one endemic area . Brucellosis maily involved in the farmer's family that raise sheep while pregnancy make women at reprodactive age susceptible to the disease . The incidence rate in city and rural areas were significantly different despite that they had the same environment that bacteria-carrier sheep were raised.

Zhonghua Liu Xing Bing Xue Za Zhi, 1998 Apr, 19(2), 69 - 71
{Analysis on the data regarding brucellosis at the National Monitoring Center}; Zhang S et al.; Surveillance on brucellosis was carried out in 15 major monitoring places in 14 provinces from 1990 to 1996 . The result indicated that in a few monitoring places the epidemic situation was stable but in many monitoring places outbreak had occurred in one or two monitoring places . Major source of infection was from infected sheep and cattle while in some old epidemic areas the pastoral area was active, which kept animals continuously excrete bacteria, making the potential of outbreak brucellosis exist . A tendency showed that the epidemic area was shifting to the farming and stockbreeding and agricultural areas . A comprehensive strategy including effective management and control the circulation of causative agent, needs to be developed.

Trends Genet, 1999 May, 15(5), 166 - 8
A surfeit of RAD51-like genes?
Thacker J.
The process of homologous recombination appears enigmatic: it creates genetic diversity but also provides an important way of repairing DNA damage without errors . The recent cloning and functional analysis of eukaryotic recombination genes suggests that at least part of the intricate mechanism involved is conserved from bacteria to humans, but is also yielding some surprises.

Curr Opin Microbiol, 1999 Apr, 2(2), 159 - 65
The sigmaE and Cpx regulatory pathways: overlapping but distinct envelope stress responses; Raivio TL et al.; The Cpx and sigmaE extracytoplasmic stress responses sense and respond to misfolded proteins in the bacterial envelope . Recent studies have highlighted differences between these regulatory pathways in terms of activating signals, mechanisms of signal transduction and the nature of the responses . Cumulatively, the findings suggest distinct physiological roles for these partially overlapping envelope stress responses . The sigmaE pathway is essential for survival and is primarily responsible for monitoring and responding to alterations in outer membrane protein folding . Mounting evidence suggests that the Cpx regulon may have been adapted to ensure properly timed expression and assembly of adhesive organelles.

Curr Opin Genet Dev, 1999 Apr, 9(2), 132 - 9
RNA polymerase II as a control panel for multiple coactivator complexes; Hampsey M et al.; 1999 marks the 30th anniversary of the reported discovery of sigma factor and the bacterial RNA polymerase holoenzyme . In 1994, an RNA polymerase II complex was discovered in yeast - mammalian complexes were subsequently identified . Recent developments regarding the composition and function of RNA polymerase II complexes suggest, however, that the concept of the holoenzyme, as defined in bacteria, might not be relevant to eukaryotes.

J Endod, 1999 Mar, 25(3), 151 - 4
A histopathological study of the effects of pulsed Nd:YAG laser irradiation on infected root canals in dogs; Koba K et al.; The effects of pulsed Nd:YAG laser irradiation during root canal treatment of infected teeth were investigated histopathologically in dogs . One hundred and eight teeth with a single root in 15 healthy adult beagle strain dogs were used in this study . After inducing infected teeth, each root canal was shaped with up to at least a #40 K-file; then, after coating with black ink, the canal was irradiated using the following parameters: 1 W, 30 pps, for 1 and 2 s, and 2 W, 30 pps, for 2 s . The degree of inflammation of the periapical regions at 2, 4, and 8 wk was examined histopathologically by light microscopy . Inflammation of the periapical regions in the laser-treated groups was significantly less than that in the control group at 4 and 8 wk (p < 0.05) . These results suggest that pulsed Nd:YAG laser is useful for root canal treatment of infected teeth, if appropriate parameters are selected.

Biochim Biophys Acta, 1999 May 14, 1445(2), 185 - 95
Metabolic diversity in myxobacteria: identification of the myxalamid and the stigmatellin biosynthetic gene cluster of Stigmatella aurantiaca Sg a15 and a combined polyketide-(poly)peptide gene cluster from the epothilone producing strain Sorangium cellulosum So ce90; Beyer S et al.; Myxobacterial strains producing polyketides (PKs) assumed to be biosynthesized by a type I polyketide synthase (PKS) were analysed . Myxobacteria also produce a variety of polypeptides (PP) and PKs with incorporated amino acids ('mixed PK-PP') . In order to be able to identify the biosynthetic gene clusters for these metabolites a PCR based approach has been developed to clone ketosynthase (KS) domains of PKS genes from these organisms . Conserved regions of peptide synthetases of the non-ribosomal type (NRPS) were also amplified via PCR . KS fragments from Stigmatella aurantiaca Sg a15 were used for chromosomal gene inactivation experiments resulting in a series of mutants including such that were unable to produce stigmatellins and myxalamids . A NRPS fragment and PKS fragments from Sorangium cellulosum So ce90 were used to identify cosmids hybridizing with both types of probes from a genomic library . Both a NRPS and a PKS fragment were cloned and sequenced from a relatively short restriction fragment of one of these cosmids . The method described here should be very useful to clone and identify PKS, NRPS and mixed PKS-NRPS from myxobacteria in general and thereby open opportunities to use the biochemical diversity of these bacteria for genetic engineering and combinatorial biosynthesis.

Mol Microbiol, 1999 May, 32(3), 505 - 17
Alteration of the synthesis of the Clp ATP-dependent protease affects morphological and physiological differentiation in Streptomyces; de Crecy-Lagard V et al.; The genes of Streptomyces coelicolor A3(2) encoding catalytic subunits (ClpP) and regulatory subunits (ClpX and ClpC) of the ATP-dependent protease family Clp were cloned, mapped and characterized . S . coelicolor contains at least two clpP genes, clpP1 and clpP2, located in tandem upstream from the clpX gene, and at least two unlinked clpC genes . Disruption of the clpP1 gene in S . lividans and S . coelicolor blocks differentiation at the substrate mycelium step . Overexpression of clpP1 and clpP2 accelerates aerial mycelium formation in S . lividans, S . albus and S . coelicolor . Overproduction of ClpX accelerates actinorhodin production in S . coelicolor and activates its production in S . lividans.

Biochemistry, 1999 May 11, 38(19), 6335 - 45
Unusual NMR, EPR, and Mössbauer properties of Chromatium vinosum 2{4Fe-4S} ferredoxin; Kyritsis P et al.; The ferredoxin from Chromatium vinosum (CvFd) exhibits sequence and structure peculiarities . Its two Fe4S4(SCys)4 clusters have unusually low potential transitions that have been unambiguously assigned here through NMR, EPR, and Mossbauer spectroscopy in combination with site-directed mutagenesis . The {4Fe-4S}2+/1+ cluster (cluster II) whose coordination sphere includes a two-turn loop between cysteines 40 and 49 was reduced by dithionite with an E degrees ' of -460 mV . Its S = 1/2 EPR signal was fast relaxing and severely broadened by g-strain, and its Mossbauer spectra were broad and unresolved . These spectroscopic features were sensitive to small perturbations of the coordination environment, and they were associated with the particular structural elements of CvFd, including the two-turn loop between two ligands and the C-terminal alpha-helix . Bulk reduction of cluster I (E degrees ' = -660 mV) was not possible for spectroscopic studies, but the full reduction of the protein was achieved by replacing valine 13 with glycine due to an approximately 60 mV positive shift of the potential . At low temperatures, the EPR spectrum of the fully reduced protein was typical of two interacting S = 1/2 {4Fe-4S}1+ centers, but because the electronic relaxation of cluster I is much slower than that of cluster II, the resolved signal of cluster I was observed at temperatures above 20 K . Contact-shifted NMR resonances of beta-CH2 protons were detected in all combinations of redox states . These results establish that electron transfer reactions involving CvFd are quantitatively different from similar reactions in isopotential 2{4Fe-4S} ferredoxins . However, the reduced clusters of CvFd have electronic distributions that are similar to those of clusters coordinated by the CysIxxCysIIxxCysIII.CysIVP sequence motif found in other ferredoxins with different biochemical properties . In all these cases, the electron added to the oxidized clusters is mainly accommodated in the pair of iron ions coordinated by CysII and CysIV.

Biochemistry, 1999 May 11, 38(19), 6178 - 86
Probing the mechanism of C-H activation: oxidation of methylcubane by soluble methane monooxygenase from Methylosinus trichosporium OB3b; Jin Y et al.; The soluble form of methane monooxygenase (MMO) isolated from methanotrophic bacteria catalyzes the O2-dependent conversion of methane to methanol, as well as the adventitious oxidation of many other hydrocarbons . In past studies, it was reported that the oxidation reaction of methylcubane, a radical clock substrate, catalyzed by MMO from Methylococcus capsulatus (Bath) gave only cubylmethanol as the product rather than methylcubanol(s) or rearranged products characteristic of a radical formed on the methyl group {Choi, S.-Y., Eaton, P . E., Hollenberg, P . F., Liu, K . E., Lippard, S . J., Newcomb, M., Putt, D . A., Upadhyaya, S . P., and Xiong, Y . (1996) J . Am . Chem . Soc . 118, 6547-6555} . Such a substrate radical intermediate would be expected if the mechanism of MMO involves hydrogen atom abstraction as indicated by many previous mechanistic studies . Here it is shown that the reaction of methylcubane with the reconstituted MMO system from Methylosinus trichosporium OB3b yields both cubylmethanol and methylcubanols, with methyl hydroxylation favored over cubyl hydroxylation . This unexpected regioselectivity indicates steric effects on the reaction in agreement with past product distribution studies . In addition, the apparent majority product of the reaction is tentatively assigned as one of the possible rearranged products for this radical probe, on the basis of gas chromatography and mass spectrometry data . This result suggests the formation of a radical intermediate in the reaction, thus supporting a radical-based mechanism for this form of MMO.

Rev Gastroenterol Mex, 1998 Oct-Dec, 63(4), 187 - 97
{Relation of Helicobacter pylori and the stomach operated on for peptic ulcer}; Hurtado-Andrade H; OBJECTIVE: To review the present information about the relation of Helicobacter pylori with the operated stomach for peptic ulcer . BACKGROUND: The frequency of elective surgical operations for peptic ulcer has decreased in the last years as a result of the therapeutic efficacy of modern drugs while urgent operations have increased . Although the association of peptic ulcer and Helicobacter pylori is well known, the effects of this bacteria on the operated stomach and the effects of the operated stomach on the Helicobacter pylori are not clear . RESULTS: It seems to be that Helicobacter pylori may not be related with peptic ulcer complications that require surgical treatment . It may cause gastritis and mucosal atrophy on the operated patient, it seems not to provoke recurrent ulcer and it's relation with postoperative gastric cancer is not clear . Non-complete vagotomy favors the colonization by Helicobacter pylori and this is lower in patients that have been submitted to operations that cause biliary reflux (gastrectomy Billroth I, gastrectomy Billroth II, pyloroplasty or gastroenterostomy) . CONCLUSIONS: Helicobacter pylori may cause alterations in the operated stomach and the operated stomach may favor Helicobacter pylori colonization or, on the contrary, cause its suppression . New studies are needed in order to know with precision the relationship of this bacteria with the operated stomach and to determine the possible usefulness of postoperative eradication treatment.

Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 803 - 13
Identification of nine species of the Chlamydiaceae using PCR-RFLP; Everett KD et al.; The family Chlamydiaceae contains two genera and nine species . Rapid and easy identification of these species is essential for taxonomic, epidemiological and clinical determinations . Currently, DNA sequence analysis is the only accepted method that decisively distinguishes all nine species . In this study, a simple and rapid PCR-RFLP procedure was developed by which laboratory-cultured chlamydial specimens could be identified . To accomplish this, conserved oligonucleotide primers and restriction sites were deduced from 16S and 23S rRNA sequence data from > 50 chlamydial strains representing all nine species . DNA from 25 previously characterized chlamydial strains were tested with these primers and restriction enzymes . All nine chlamydial species were reliably distinguished in the tests . The procedure was optimized by adjusting the annealing temperature using both a standard and a heat-activated DNA polymerase to reduce mismatch PCR amplification of mycoplasmas and other bacteria . The result was that a PCR method for species identification of chlamydial isolates and for distinguishing mycoplasmas and chlamydiae was created . This method can be used to rapidly identify known species of the family Chlamydiaceae.

Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 689 - 96
Phylogenetic diversity, polyamine pattern and DNA base composition of members of the order Planctomycetales; Griepenburg U et al.; The 16S rDNA sequences of 20 novel isolates of members of the order Planctomycetales were compared to those of the type strains of described planctomycete species and 22 planctomycete isolates for which the 16S rDNA sequences had been previously determined . The novel isolates could be assigned to several phylogenetically broad groups, four of which are defined by the genera Gemmata, Isosphaera, Planctomyces and Pirellula . To evaluate polyamines as a chemotaxonomic marker within this order, the polyamine pool was determined for six planctomycete reference species and for 20 planctomycete isolates . All analysed members of the order Planctomycetales contained significant amounts of polyamines . sym-Homospermidine (HSPD) is present in all strains except Planctomyces limnophilus and related strains, which had high amounts of putrescine (PUT) as the dominant polyamine component . The distribution of PUT, HSPD and spermidine reflects the phylogenetic diversity within the Planctomycetales as closely related representatives of the phylogenetic groups defined by described species and novel isolates exhibit similar polyamine patterns . Determination of the DNA base composition revealed G + C contents of > 60 mol% for members of Gemmata and Isosphaera whereas, except for two isolates, strains which are phylogenetically associated with Planctomyces and Pirellula had G + C contents of 51-57 mol%.

Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 663 - 9
Assignment of Centers for Disease Control group IVc-2 to the genus Ralstonia as Ralstonia paucula sp . nov; Vandamme P et al.; An integrated genotypic and phenotypic analysis of 12 Centers for Disease Control (CDC) group IVc-2 strains revealed that this taxon represents a novel species belonging to the genus Ralstonia . Comparative 16S rDNA sequence analysis allocated a representative CDC group IVc-2 strain to the Ralstonia branch of the beta subclass of the Proteobacteria . DNA-DNA hybridizations did not detect significant binding levels towards any presently known Ralstonia species, including Ralstonia pickettii . Its DNA base ratio is between 65 and 67 mol% . The name Ralstonia paucula sp . nov . is proposed, with strain LMG 3244 (= CDC E6793), isolated from a human respiratory tract, as the type strain . R . paucula can be differentiated from other Ralstonia species by whole-cell protein analysis, amplified rDNA restriction analysis and a variety of classical biochemical tests . Strains have been isolated from various human clinical and environmental sources.

Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 567 - 76
Phylogenetic relationships among members of the Comamonadaceae, and description of Delftia acidovorans (den Dooren de Jong 1926 and Tamaoka et al . 1987) gen . nov., comb . nov; Wen A et al.; The phylogenetic relationships among members of the family Comamonadaceae and several unclassified strains were studied by direct sequencing of their PCR-amplified 16S rRNA genes . Based on the 16S rRNA gene sequence analysis, members of the family formed a coherent group . The closest relatives are species of the Rubrivivax sub-group: Leptothrix discophora, Ideonella dechloratans and Rubrivivax gelatinosus . The genus Hydrogenophaga formed two subclusters, as did the species of Acidovorax, whereas the five species of the genus {Aquaspirillum} were polyphyletic . Comamonas acidovorans was phylogenetically distant from the type species of Comamonas, Comamonas terrigena . On the basis of this work and previous studies, Comamonas acidovorans is removed from the genus Comamonas and renamed as Delftia acidovorans gen . nov., comb . nov . Descriptions of the new genus Delftia and of the type species Delftia acidovorans, for which the type strain is ATCC 15668T, are presented.

Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 545 - 56
Characterization of the anaerobic propionate-degrading syntrophs Smithella propionica gen . nov., sp . nov . and Syntrophobacter wolinii; Liu Y et al.; A strain of anaerobic, syntrophic, propionate-oxidizing bacteria, strain LYPT (= OCM 661T; T = type strain), was isolated and proposed as representative of a new genus and new species, Smithella propionica gen . nov., sp . nov . The strain was enriched from an anaerobic digestor and isolated . Initial isolation was as a monoxenic propionate-degrading co-culture containing Methanospirillum hungateii JF-1T as an H2- and formate-using partner . Later, an axenic culture was obtained by using crotonate as the catabolic substrate . The previously described propionate-degrading syntrophs of the genus Syntrophobacter also grow in co-culture with methanogens such as Methanospirillum hungateii, forming acetate, CO2 and methane from propionate . However, Smithella propionica differs by producing less methane and more acetate; in addition, it forms small amounts of butyrate . Smithella propionica and Syntrophobacter wolinii grew within similar ranges of pH, temperature and salinity, but they differed significantly in substrate ranges and catabolic products . Unlike Syntrophobacter wolinii, Smithella propionica grew axenically on crotonate, although very slowly . Co-cultures of Smithella propionica grew on propionate, and grew slowly on crotonate or butyrate . Syntrophobacter wolinii and Syntrophobacter pfennigii grow on propionate plus sulfate, whereas Smithella propionica did not . Comparisons of 16S rDNA genes indicated that Smithella propionica is most closely related to Syntrophus, and is more distantly related to Syntrophobacter.

Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 449 - 57
Roseateles depolymerans gen . nov., sp . nov., a new bacteriochlorophyll a-containing obligate aerobe belonging to the beta-subclass of the Proteobacteria; Suyama T et al.; Strains 61AT (T = type strain) and 61B2, the first bacteriochlorophyll (BChl) a-containing obligate aerobes to be classified in the beta-subclass of the Proteobacteria, were isolated from river water . The strains were originally isolated as degraders of poly(hexamethylene carbonate) (PHC) . The organisms can utilize PHC and some other biodegradable plastics . The strains grow only under aerobic conditions . Good production of BChl a and caroterioid pigments is achieved on PHC agar plates and an equivalent production is observed under oligotrophic conditions on agar medium . Spectrometric results suggest that BChl a is present in light-harvesting complex I and the photochemical reaction centre . The main carotenoids are spirilloxanthin and its precursors . Analysis of the 16S rRNA gene sequence indicated that the phylogenetic positions of the two strains are similar to each other and that their closest relatives are the genera Rubrivivax, ideonella and Leptothrix with similarities of 96.3, 96.2 and 96.1%, respectively . The cells are motile, straight rods and contain poly-beta-hydroxybutyrate granules . Ubiquinone-8 is the predominant quinone . Vitamins are not required for growth . The G + C content of genomic DNA is 66.2-66.3 mol% . Genetic and phenotypic features suggest that the strains represent a new genus in the beta-subclass which is evenly distant from known genera . Consequently, the name Roseateles depolymerans gen . nov., sp . nov . is proposed for the strains; the type strain of Roseateles depolymerans is strain 61AT (= DSM 11813T).

Immunol Rev, 1999 Feb, 167, 327 - 37
Major histocompatibility complex class I genes in primates: co-evolution with pathogens; Vogel TU et al.; The major histocompatibility complex (MHC) is the most polymorphic genetic system known, playing a central role in the cellular immune response to pathogens . The relationship between the MHC of humans and non-human primates has increased our understanding of MHC evolution and how polymorphism of this gene family may have been generated . We will review MHC class I evolution in great apes and Old World and New World primates and discuss new data from the simian immunodeficiency virus/rhesus monkey animal model that demonstrate the role of MHC class I alleles in selecting for new populations of viruses . This suggests that certain pathogens co-evolve with the MHC class I molecules they encounter in a population.

Dig Dis Sci, 1999 May, 44(5), 868 - 75
Development of Helicobacter pylori infection model in BALB/c mice with domestic cagA-positive and -negative strains in Taiwan; Sheu BS et al.; We aimed to develop an H . pylori-infected mouse model using clinically stored strains in Taiwan and to test whether development of H . pylori infection in an in vivo animal model is related to the status of the cagA gene . A total of 100 male BALB/c mice, 6-8 weeks old, including 80 in the experimental group and 20 in the control group, were used . Two clinically stored H . pylori isolates, a cagA-positive and a cagA-negative strain, were selected to induce the H . pylori infection in half (N = 40) of the mice in the experimental group . Bacterial isolates of 0.8 x 10(9) CFU/ml were orally inoculated in each mouse of the experimental group for three consecutive days . Ten mice in the control group were sacrificed to confirm the initial absence of H . pylori . Eight weeks after inoculation of the experimental group and no inoculation of the remaining 10 mice of the control group, each mouse was killed . Gastrectomy was then performed for rapid urease test (CLOtest) and histology . In the control group, none of 20 mice had positive results from the CLOtest or histology . In contrast, excluding eight of 80 mice that died before the eighth week, 90.3% (65/72) of the mice challenged with H . pylori showed persistent presence of H . pylori by histology . The severity of gastritis at the eighth week was more evident in H . pylori-infected mice than in control and noninfected mice (P < 0.05) . Although gastritis was more severe in mice inoculated with the cagA-positive strain than with the cagA-negative strain, the rates of H . pylori infection in mice were not different between cagA-positive and -negative strains (91.4% vs 89.2%, P > 0.05) . In summary, stored strains of H . pylori can be applied to induce an infection model in BALB/c mice . The less virulent cagA-negative strain can induce H . pylori infection in mice as effectively as the cagA-positive strain . The high prevalence of cagA-positive strains in Taiwanese patients may be related to factors other than only the cagA gene of the bacteria.

Am J Gastroenterol, 1999 May, 94(5), 1214 - 7
Citric acid as the test meal for the 13C-urea breath test; Graham DY et al.; OBJECTIVE: Test meals are used in the urea breath test to slow gastric emptying and to increase the area of contact with the substrate . Recently, citric acid has been suggested as an improved liquid test meal . The mechanism is unknown and could act by delaying gastric emptying, decreasing the pH at the site of the bacteria, or both . Our aim was to evaluate the effects of citric acid test meals on urea hydrolysis in vivo, to identify the possible mechanism for enhanced urea hydrolysis, and to identify the minimum effective dose . METHODS: We compared the U.S . commercial 13C-urea breath test with four liquid test meals (200 ml of water) consisting of citric acid, ascorbic acid, sodium citrate, and glucose polymer and also after the subcutaneous administration of pentagastrin . We studied healthy volunteers with and without proven H . pylori infection (by serology and histology) . 13C-urea was administered orally simultaneously with the liquid test meals or immediately after the pudding had been ingested . Breath samples were taken before and after oral administration of the 13C-urea . RESULTS: A dose response in urease activity was evident as the amount of citric acid was increased from 1 to 4 g . Citric acid at 1, 2, or 4 g produced significant increases in breath 13CO2 activity, compared with the commercial pudding (p < 0.05) . Ascorbic acid (p = 0.053), subcutaneous pentagastrin (to lower pH) (p = 0.199), and glucose polymer (p = 0.03) (to delay gastric emptying) all approximately doubled breath 13CO2, compared with the commercial kit . Nevertheless, the increases were all significantly less than with the 4 g citric acid test meal . CONCLUSIONS: The data are consistent with the marked effect of citric acid on gastric emptying and, possibly, distribution of the urea within the stomach being largely responsible for the enhanced urease activity with citric acid test meals . It should be possible to use a low dose of citric acid (e.g., 1 g per 200 ml) to enhance the simplicity and palatability of the test.

FEMS Microbiol Lett, 1999 May 1, 174(1), 143 - 9
The 40- and 90-kDa membrane proteins (ORF6 gene product) of Mycoplasma pneumoniae are responsible for the tip structure formation and P1 (adhesin) association with the Triton shell; Layh-Schmitt G et al.; After Triton X-100 treatment of Mycoplasma pneumoniae cells, a portion of the adhesin P1 (transmembrane protein) proved to remain tightly associated with the Triton insoluble material (Triton shell) as shown previously by several authors . However, the spontaneous loss of two cytadherence-associated membrane proteins of 90 and 40 kDa (gene product of the open reading frame 6 of the P1 operon) in a hemadsorption-negative mutant, designated M5, resulted in a 100% release of the P1 protein into the Triton phase and in the lack of the characteristic tip-like attachment organelle of M . pneumoniae indicating an essential role of the open reading frame 6 gene product in tip structure formation.

FEMS Microbiol Lett, 1999 May 1, 174(1), 33 - 9
Functional analysis of the roles of FliQ and FlhB in flagellar expression in Helicobacter pylori; Foynes S et al.; Expression of the two Helicobacter pylori flagellin proteins FlaA and FlaB is required for full motility and persistent infection of the gastric mucosa . The mechanisms and regulation of the biosynthesis and export of flagella in H . pylori are still poorly understood . Scrutiny of the H . pylori 26695 genome sequence revealed homologues of FliQ and FlhB . The roles of the fliQ and flhB genes in H . pylori were investigated by the construction and characterisation of defined isogenic mutants . The results indicate that these genes are involved in the flagellar expression, adhesion to and colonisation of the gastric mucosa.

J Wildl Dis, 1999 Apr, 35(2), 266 - 74
Infections of granulocytic ehrlichiae and Borrelia burgdorferi in white-tailed deer in Connecticut; Magnarelli LA et al.; Serum or whole blood samples, obtained from 141 white-tailed deer (Odocoileus virginianus) in Connecticut (USA) during 1980, 1991, and 1996, were analyzed to detect past or current infections of Ehrlichia phagocytophila genogroup organisms and Borrelia burgdorferi . When the BDS or NCH-1 strains of granulocytic ehrlichiae were used separately in indirect fluorescent antibody (IFA) staining methods, antibody positivity rates varied from 25 to 64% in 1991 and 1996, respectively . All 50 sera tested from 1980 collections were negative . Although percentages of sera with B . burgdorferi antibodies, as detected by an enzyme-linked immunosorbent assay, also differed (23 to 53%), there were coexisting antibodies to both bacteria in 20 (49%) of 41 sera . In tests on specificity, 19 deer sera with ehrlichial antibodies also were tested by IFA staining procedures for Anaplasma marginale antibodies; one serum with a titer of 1:5,120 to ehrlichial antigen reacted to A . marginale antigen at a serum dilution of 1:320 . In parallel analyses of 69 sera, results of Western blot analyses for ehrlichial infections in deer were concordant (72% agreement) with those of IFA staining methods containing ehrlichial antigen . All positive immunoblots showed bands to peptides of the NCH-1 strain of the human granulocytic ehrlichiosis (HGE) agent having molecular masses of about 44, 105, or 110 kDa . In polymerase chain reaction (PCR) studies of blood samples from 63 deer, 11 (18%) specimens were positive for 16S ribosomal DNA of an Ehrlichia phagocytophila genogroup organism, whereas 23 (37%) samples were positive for the DNA of the 44 kDa gene of the HGE agent . White-tailed deer are exposed to different tick-borne bacteria in areas where Ixodes scapularis ticks are abundant and may, in some instances, have had concurrent infections.

Br J Haematol, 1999 Apr, 105(1), 40 - 9
Expansion and differentiation of haemopoietic progenitor cells on endothelialized hydroxyapatite under static conditions; Conrad V et al.; We have analysed the potential of an endothelialized hydroxyapatite matrix (HAP), a synthetic bone substitute, as a cellularized support for the expansion of haemopoietic progenitor cells . Scanning electron microscopy (SEM) showed that the endothelial cells (EC) tended to form a monolayer fitting closely to the matrix, and that the progenitors adhered to the EC layer . Endothelialized HAP supported the proliferation and differentiation of the progenitors with the addition of the exogenous cytokines IL-1, and IL-3 . The expanded cell population essentially belonged to the myeloid and monocytic lineages, with a smaller percentage of the megakaryocyte lineage . In comparative experiments CD34+ progenitors were expanded on endothelialized tissue culture flasks, and a significant higher viability of the expanded cells was found with the endothelialized HAP . A high percentage (approx . 40%) of mature granulocytes was generated in accordance with the presence of differentiating cytokines such as G-CSF and GM-CSF at high concentrations in the coculture medium . Other cytokines that could be detected were IL-6, M-CSF, SCF, flt3-ligand . More than 50% of the expanded cell population was able to phagocytose bacteria and to generate an oxydative burst . These data indicate that cellularized HAP may be a useful matrix for stromal cell-based expansion systems.

Gene, 1999 Apr 29, 231(1-2), 87 - 93
Duplicate genes for Fe-containing superoxide dismutase in Streptomyces coelicolor A3(2); Chung HJ et al.; Streptomyces coelicolor Muller contains two types of superoxide dismutase (SOD) containing Ni (encoded by sodN) or Fe (encoded by sodF) . Unlike a single species of Fe-containing SOD in Muller strain, multiple forms of FeSODs were detected in S . coelicolor A3(2) strain by activity staining and Western blot analysis . Genomic Southern hybridization suggested the presence of at least two copies of the sodF-like gene in A3(2) . Two different genes for FeSOD (sodF1 and sodF2) were isolated from the phage library of A3(2) genome . The nucleotide sequence of the sodF1 coding region was identical with the unique sodF gene from Muller while that of sodF2 shared 88% identity . The gene products of sodF1 and sodF2 were identified by activity staining and immunoblot analysis . Expression from the sodF1 gene was repressed by nickel as sensitively as Muller sodF, suggesting the presence of Ni-responsive regulatory site within the region shared by the two genes . Among 12 other Streptomyces species examined, only S . fradiae contained two FeSOD-like polypeptides . We postulate that the additional copy of the sodF gene (sodF2) was provided by the horizontal transfer from remotely related bacteria.

Gene, 1999 Apr 29, 231(1-2), 67 - 75
The DNA puff BhB10-1 gene encodes a glycine-rich protein secreted by the late stage larval salivary glands of Bradysia hygida; Fontes AM et al.; We present the molecular characterization of a gene of Bradysia hygida DNA puff B10 whose temporal expression in the salivary gland correlates with the puff expansion . The transcription unit of this gene, named BhB10-1, was mapped in a 2-kb EcoRI genomic fragment that is amplified in the salivary gland of late fourth instar larvae . Its 1.3-kb transcript undergoes poly-A tail shortening during development, indicating that post-transcriptional controls as well as transcription activation are involved in the temporal regulation of the BhB10-1 gene . Analysis of the deduced amino acid sequence from the cDNA indicates that the BhB10-1 protein is a glycine-rich secretory protein . A BhB10-1-fusion protein expressed in bacteria was used to raise polyclonal antibodies . Using an immunopurified antibody, we identified the product of the DNA puff BhB10-1 gene as a 23-kDa polypeptide that is produced mainly by the salivary gland regions S1 and S3 and is present in the saliva of late larvae . This is the first direct identification of a protein encoded by a DNA puff amplified gene.

Biochemistry, 1999 May 4, 38(18), 5872 - 7
Specific binding of integrin alpha v beta 3 to the fibrinogen gamma and alpha E chain C-terminal domains; Yokoyama K et al.; Integrin alpha v beta 3, a widely distributed fibrinogen receptor, recognizes the RGD572-574 motif in the alpha chain of human fibrinogen . However, this motif is not conserved in other species, nor is it required for alpha v beta 3-mediated fibrin clot retraction, suggesting that fibrinogen may have other alpha v beta 3 binding sites . Fibrinogen has conserved C-terminal domains in its alpha (E variant), beta, and gamma chains (designated alpha EC, beta C, and gamma C, respectively), but their function in cell adhesion is not known, except that alpha IIb beta 3, a platelet fibrinogen receptor, binds to the gamma C HHLGGAKQAGDV400-411 sequence . Here we used mammalian cells expressing recombinant alpha v beta 3 to show that recombinant alpha EC and gamma C domains expressed in bacteria specifically bind to alpha v beta 3 . Interaction between alpha v beta 3 and gamma C or alpha EC is blocked by LM609, a function-blocking anti-alpha v beta 3 mAb, and by RGD peptides . alpha v beta 3 does not require the HHLGGAKQAGDV400-411 sequence of gamma C for binding, and alpha EC does not have such a sequence, indicating that the alpha v beta 3 binding sites are distinct from those of alpha IIb beta 3 . A small fragment of gamma C (residues 148-226) supports alpha v beta 3 adhesion, suggesting that an alpha v beta 3 binding site is located within the gamma chain 148-226 region . We have reported that the CYDMKTTC sequence of beta 3 is responsible for the ligand specificity of alpha v beta 3 . gamma C and alpha EC do not bind to wild-type alpha v beta 1, but do bind to the alpha v beta 1 mutant (alpha v beta 1-3-1), in which the CYDMKTTC sequence of beta 3 is substituted for the corresponding beta 1 sequence CTSEQNC . This suggests that gamma C and alpha EC contain determinants for fibrinogen's specificity to alpha v beta 3 . These results suggest that fibrinogen has potentially significant novel alpha v beta 3 binding sites in gamma C and alpha EC.

Wound Repair Regen, 1999 Mar-Apr, 7(2), 97 - 105
Effects of chronic wound fluid on the bioactivity of platelet-derived growth factor in serum-free medium and its direct effect on fibroblast growth; He C et al.; The fate of biologically active proteins applied to chronic wounds is almost totally unknown . Growth factors may be degraded by proteases, which are produced by both inflammatory and skin cells and by resident bacteria . However, there has been little work on the effect of chronic wound fluid on the activity of growth factors . A bioassay method has been chosen to examine the effect of incubation of platelet-derived growth factor with chronic wound fluid from leg ulcers on the in vitro growth of human dermal fibroblasts . Human dermal fibroblasts were cultured in serum-free medium, and a dose-response curve for proliferation in response to platelet-derived growth factor was obtained . Wound fluid was collected under occlusive dressings from five patients with chronic leg ulcers . Platelet-derived growth factor was incubated with chronic wound fluid at 37 degrees C for 4 hours, and the reactions arrested by snap freezing . The resultant solutions were tested for their ability to promote fibroblast proliferation . A colorimetric assay was used to monitor changes in the platelet-derived growth factor mitogenicity . The results showed that, in our standard culture conditions, chronic wound fluid always stimulated fibroblast proliferation, and, in most cases, incubation of platelet-derived growth factor with chronic wound fluid increased the stimulation compared with that produced by platelet-derived growth factor or chronic wound fluid alone.

Mol Microbiol, 1999 Apr, 32(2), 379 - 91
Cell cycle-dependent degradation of a flagellar motor component requires a novel-type response regulator; Aldridge P et al.; The poles of each Caulobacter crescentus cell undergo morphological development as a function of the cell cycle . A single flagellum assembled at one pole during the asymmetric cell division is later ejected and replaced by a newly synthesized stalk when the motile swarmer progeny differentiates into a sessile stalked cell . The removal of the flagellum during the swarmer-to-stalked cell transition coincides with the degradation of the FliF flagellar anchor protein . We report here that the cell cycle-dependent turnover of FliF does not require the structural components of the flagellum itself, arguing that it is the initial event leading to the ejection of the flagellum . Analysis of a polar development mutant, pleD, revealed that the pleD gene was required for efficient removal of FliF and for ejection of the flagellar structure during the swarmer-to-stalked cell transition . The PleD requirement for FliF degradation was also not dependent on the presence of any part of the flagellar structure . In addition, only 25% of the cells were able to synthesize a stalk during cell differentiation when PleD was absent . The pleD gene codes for a member of the response regulator family with a novel C-terminal regulatory domain . Mutational analysis confirmed that a highly conserved motif in the PleD C-terminal domain is essential to promote both FliF degradation and stalk biogenesis during cell differentiation . Signalling through the C-terminal domain of PleD is thus required for C . crescentus polar development . A second gene, fliL, was shown to be required for efficient turnover of FliF, but not for stalk biogenesis . The possible roles of PleD and FliL in C . crescentus polar development are discussed.

Bioorg Med Chem Lett, 1999 Apr 5, 9(7), 949 - 52
Investigation of new potent KDO-8-phosphate synthetase inhibitors; Coutrot P et al.; A total synthesis of (Z,E)-D-Glucophosphoenolpyruvate and its carboxylic ester derivatives is described in four or five steps from 2,3:5,6-Di-O-isopropylidene-4-O-t-butydimethylsilyl-D-glucose.

Mol Cell, 1999 Apr, 3(4), 495 - 504
The mechanism of intrinsic transcription termination; Gusarov I et al.; In bacteria, an intrinsic transcription termination signal appears in RNA as a hairpin followed by approximately eight uridines (U stretch) at the 3' terminus . This signal leads to rapid dissociation of the ternary elongation complex (TEC) into RNA, DNA, and an RNA polymerase . We demonstrate that the hairpin inactivates and then destabilizes TEC by weakening interactions in the RNA-DNA hybrid-binding site and the RNA-binding site that hold TEC together . Formation of the hairpin is restricted to the moment when TEC reaches the point of termination and depends upon melting of four to five hybrid base pairs that follow the hairpin's stem . The U stretch-induced pausing at the point of termination is crucial, providing additional time for hairpin formation . These results explain the mechanism of termination and aid in understanding of how cellular factors modulate this process.

Microbiol Immunol, 1999, 43(2), 107 - 13
The effects of S-carboxymethylcysteine and N-acetylcysteine on the adherence of Moraxella catarrhalis to human pharyngeal epithelial cells; Zheng CH et al.; We investigated the effects of two mucoregulating drugs, S-carboxymethylcysteine (S-CMC) and N-acetylcysteine (NAC), on the attachment of Moraxella catarrhalis (M . catarrhalis) to pharyngeal epithelial cells . The attachment of M . catarrhalis decreased (33-57%) significantly (P<0.01) in a dose-dependent manner in cells treated with mucoregulating drugs as compared to the control . There was a significant (P<0.01) decrease (35-45%) in the attachment of M . catarrhalis to pharyngeal cells after oral administration of S-CMC . By electron microscopic observation, it was found that there was a fine, granular, electron-dense, ruthenium red-positive layer on the surface of pharyngeal epithelial cells; this layer was absent on cell surfaces treated with mucoregulating drugs . Possibly, this layer contained the portion of M . catarrhalis receptor which is responsible for the attachment of this bacteria to pharyngeal epithelial cells . From the above results, it may be concluded that one of the mechanisms of mucoregulating drugs to decrease the episode of respiratory infections in patients with chronic respiratory diseases is by inhibiting the attachment of bacteria to the upper respiratory tract.

Hepatogastroenterology, 1999 Jan-Feb, 46(25), 543 - 8
Cardiac biopsy of stomach may improve the detection of H . pylori after dual therapy; Sheu BS et al.; BACKGROUND/AIMS: To determine whether gastric cardia biopsy may improve the detection of Helicobacter pylori (H . pylori) before and after eradication therapy . METHODOLOGY: A total of 150 dyspeptic patients with H . pylori infection completing a 2-week course of dual therapy (amoxicillin plus omeprazole) were studied . Endoscopy was carried out at the initial stage and 4 weeks after the completion of dual therapy . During each endoscopy, gastric biopsies were sampled in order from cardia, lower body, and antrum and stored separately to survey the distribution of H . pylori by histology . RESULTS: Before treatment, 88% (132/150) of the study cases had H . pylori found in antrum and 3.3% (5/150) of cases presented with bacteria only in cardia . After treatment, 38 cases had failure of dual therapy . The detection rates of H . pylori by biopsies without cardia decreased after the dual therapy (by antrum only: 88% to 60.5%, p < 0.05; antrum and body: 96.7% to 81.6%, p < 0.05) . In contrast, the incidence of patients with only cardia involvement by H . pylori significantly increased from 3.3% (5/150) before to 18.4% (7/38) after treatment (p < 0.01) . Among the 7 patients with H . pylori only in cardia after dual therapy, 3 cases had recurrent dyspepsia during follow-up because of no further anti-H . pylori therapy . Two of these 3 cases disclosed diffuse bacterial involvement in antrum and body besides cardia; the last case later had a positive result of urea breath test . CONCLUSIONS: Biopsy obtained from gastric cardia can improve the detection rate of H . pylori especially after dual therapy, which encounters antibiotics with possible sanctuary sites here . Thus, it will be useful to prevent over diagnosis of H . pylori eradication.

Am J Respir Crit Care Med, 1999 May, 159(5 Pt 1), 1533 - 40
Bronchoalveolar cell profiles in children with asthma, infantile wheeze, chronic cough, or cystic fibrosis; Marguet C et al.; Differential cell counts of bronchoalveolar lavage (BAL) have been reported in normal children but few data on cellular profiles in bronchial diseases in childhood are available . We determined the BAL cell profiles of 72 children divided into 5 groups: asthma (n = 14), chronic cough (n = 12), infantile wheeze (n = 26), cystic fibrosis (n = 10), and control (n = 10) . The highest total cell, eosinophil, and neutrophil counts were found in children with cystic fibrosis . The cell profile of children with chronic cough was similar to that of control children . Asthma and infantile wheeze were characterized by a high median ratio of eosinophils (3%) and neutrophils (12%), respectively . In both diseases, epithelial shedding was suggested by an elevated epithelial cell count, 13.5 and 12%, respectively . Lymphocyte subset analysis showed a higher proportion of CD8 cells (58 versus 40%) and therefore a lower CD4/CD8 ratio (0.266 versus 0 . 455) in children with asthma compared with infantile wheezers (p = 0 . 02) . Irrespective of the presence or absence of radiological abnormalities, a proportion of neutrophils > 10%, was found in one-third of the children with asthma and in half of the infantile wheezers, and was related to symptom severity . We suggest that neutrophil-mediated inflammation, with or without bacterial infection, may contribute to symptoms of asthma in childhood . Chronic cough, however, is not associated with the cell profiles suggestive of asthma and in isolation should not be treated with prophylactic antiasthma drugs.

J Immunol, 1999 May 1, 162(9), 5238 - 46
Synergism for cytokine-mediated disease during concurrent endotoxin and viral challenges: roles for NK and T cell IFN-gamma production; Nguyen KB et al.; Viral infections in humans or mice can result in increased sensitivity to challenges with bacteria, bacterial products, or cytokine administration . During lymphocytic choriomeningitis virus infections, mice are more sensitive to the lethal effects of bacterial endotoxin LPS, and in the experiments reported here, were observed at up to 10-fold lower doses in infected than in uninfected mice . The mechanisms responsible for heightened susceptibility under these conditions were evaluated . Kinetic studies demonstrated that virus-infected mice had 3- to 50-fold increases over uninfected mice in peak serum TNF, IL-12, and IFN-gamma levels after LPS administration . All three cytokines contributed to lethality during dual challenge, because neutralization of any one of the factors protected from death . Production of TNF was not dependent on either NK or T cells . In contrast, these populations were the predominant sources of IFN-gamma, as determined by lack of detectable IFN-gamma production in NK and T cell-deficient mice and by intracellular cytokine expression in the cell subsets . Concordant with the demonstrations that both cell populations produced IFN-gamma and that this factor was critical for lethality, removal of either subset alone was not sufficient to protect mice from death resulting from dual challenges . Increased resistance required absence of both cell subsets . Taken together, the data show that during viral infections, the normally protective immune responses can profoundly modify reactions to secondary heterologous challenges, to result in dysregulated cytokine expression and consequent heightened detrimental effects.

J Biomater Sci Polym Ed, 1999, 10(4), 469 - 82
Effect of sterilization on the physical and structural characteristics of polyhydroxyoctanoate (PHO); Marois Y et al.; The present study examined the potential applicability of poly(beta-hydroxy octanoate) (PHO), a bacterial polyester, as a candidate for biomaterial applications, by investigating the effect of sterilization on the physical and structural characteristics of PHO . PHO-cast films were sterilized by either ethylene oxide (EO) gas at 38 degrees C or gamma radiation (2.5 Mrad) in air at room temperature . The physical characteristics of the EO and gamma-sterilized PHO were determined by scanning electron microscopy (SEM) and tensile strength analyses . In addition, various analytical methods were used to detect modifications in the chemical and morphological structure of PHO, namely, electron spectroscopy for chemical analysis (ESCA), Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), wide angle X-ray diffraction (WAXD), and size exclusion chromatography (SEC) . The results show that EO sterilization did not modify the chemical and physical characteristics of PHO, however, significant modifications in both the structural and tensile properties were observed with gamma-sterilized PHO . These changes accounted for decreases in both the weight average, number average and melting temperature, and increases in the heat of fusion and tensile strength . No residual EO was detected following sterilization as revealed by head-space chromatography . The physical and structural properties of PHO were shown to be well preserved following EO sterilization, whereas gamma radiation caused random chain scission and physical cross-linking, a frequent phenomenon observed with organic polymers.

Artif Organs, 1999 Apr, 23(4), 303 - 9
The cleaning and disinfecting of hemodialysis equipment using electrolyzed strong acid aqueous solution; Tanaka N et al.; In general, sodium hypochlorite, formalin, and Dialox (Teijin Gambro Medical, Ltd., Tokyo, Japan {main ingredients: H2O2, CH3CHOOOH, CH3COOH, H2O}) are used to clean and disinfect hemodialysis pipelines . In this study, the suitability of electrolyzed strong acid aqueous solution (ESAAS), which has attracted considerable interest in Japan because of its strong disinfecting properties, was examined . The crossover method was used to investigate the effectiveness of ESAAS in disinfecting the dialysis pipelines in comparison to that of sodium hypochlorite (200 ppm) used alternately with 1% acetic acid . The number of bacteria and the concentration of endotoxin (Et) were measured over an approximately 3 year period, starting in September 1994 . Until then, 200 ppm sodium hypochlorite had been used alternately with 1% acetic acid, and the contamination of the pipeline had been marked . However, after switching to the ESAAS disinfection method, the dialysis pipelines very rapidly became cleaner . Therefore, the decision to develop an automated ESAAS cleaning system for long-term use was made . During the development period, the original disinfectants (200 ppm sodium hypochlorite used alternately with 1% acetic acid) were used as a stopgap . After confirmation of its performance and safety, the automated ESAAS cleaning system was introduced . To find out whether the decrease in bacteria secondarily caused a decrease in the Et concentration or whether the ESAAS directly inactivated the Et, an in vitro experiment was carried out . Highly concentrated Et, which had been left in the reverse osmosis (RO) drainage pipeline, was used as a sample to investigate the effects of ESAAS on Et at various concentrations and temperatures and on the recovery test . The results showed that ESAAS directly inactivated Et . This paper reports the results of the crossover test . The results of parallel tests carried out over an approximately 4 year period have already been reported . No significant problems occurred in the dialysis . The automated ESAAS cleaning system that was developed proved to be more economical than the conventional disinfecting method.

Clin Diagn Lab Immunol, 1999 May, 6(3), 377 - 82
Helicobacter pylori heat shock protein A: serologic responses and genetic diversity; Ng EK et al.; Helicobacter pylori synthesizes an unusual GroES homolog, heat shock protein A (HspA) . The present study was aimed at an assessment of the serological response to HspA in a group of Chinese patients with defined gastroduodenal pathologies and determination of whether diversity is present in the nucleotide sequences encoding HspA in isolates from these patients . Serum samples collected from 154 patients who had an upper gastrointestinal pathology and the presence of H . pylori defined by biopsy were tested for an immunoglobulin G (IgG) serologic response to H . pylori HspA by an enzyme linked immunosorbant assay . HspA-encoding nucleotide sequences in H . pylori isolates from 14 patients (7 seropositive and 7 seronegative for HspA) were analyzed by PCR and direct sequencing of the PCR products . The sequencing results were compared to those of 48 isolates from other parts of the world . Of the 154 known H . pylori-positive patients, 54 (35.1%) were seropositive for HspA . The A domain (GroES homology) of HspA was highly conserved in the 14 isolates tested . Although the B domain (metal-binding site unique to H . pylori) resembled that in the known major variant, particular amino acid substitutions allowed definition of an HspA variant associated with isolates from East Asia . There were no associations between patient characteristics and HspA seropositivity or amino acid sequences . We confirmed in this study that the clinical outcomes of H . pylori infection are not related to HspA antigenicity or to sequence variation . However, B-domain sequence variation may be a marker for the study of the genetic diversity of H . pylori strains of different geographic origins.

Intern Med, 1999 Feb, 38(2), 97 - 101
Pathogenesis of reactive arthritis; Yu DT; Reactive arthritis is a member of the spondyloarthropathy . Bacteria which cause reactive arthritis infect the mucosal surfaces . Either the whole bacteria or their fragments are subsequently carried to the joints inside which are induced a TH1 lymphocyte response in which oligoclonal T lymphocytes as well peptide-specific CD8+ T lymphocytes participate . Human lymphocyte antigen (HLA)-B27 is a predisposing gene . Besides being determinants for the CD8+ T lymphocyte response it can also modify the response of other cells to the invasive bacteria . This would lead to alteration of the fate of the bacteria as well as release of arthritis-causing cytokines.

FEBS Lett, 1999 Apr 16, 449(1), 88 - 92
Identification, molecular cloning, and characterization of subunit 11 of the human 26S proteasome; Hoffman L et al.; We sequenced five peptides from subunit 11 (S11), a 43 kDa protein of the human 26S proteasome, and used this information to clone its cDNA . The S11 cDNA encodes a 376 amino acid protein with a pI of 5.6 and a molecular mass of 42.9 kDa . Translation of S11 RNA in the presence of {35S}methionine produces a radiolabeled protein that co-migrates with S11 of the human 26S proteasome on SDS-PAGE . Polyclonal antiserum made against recombinant S11 recognizes a protein of the same size in extracts of bacteria expressing S11 and in purified 26S proteasomes from human red blood cells or rabbit reticulocytes . The S11 sequence does not contain motifs that suggest a biological function . S11 is, however, the human homolog of Rpn9, a recently identified subunit of the yeast 26S proteasome.

FASEB J, 1999 May, 13(8), 913 - 22
Interruption of transmembrane signaling as a novel antisecretory strategy to treat enterotoxigenic diarrhea; Zhang W et al.; Bacteria that produce heat-stable enterotoxins (STs), a leading cause of secretory diarrhea, are a major cause of morbidity and mortality worldwide . ST stimulates guanylyl cyclase C (GCC) and accumulation of intracellular cyclic GMP ({cGMP}i), which opens the cystic fibrosis transmembrane conductance regulator (CFTR)-related chloride channel, triggering intestinal secretion . Although the signaling cascade mediating ST-induced diarrhea is well characterized, antisecretory therapy targeting this pathway has not been developed . 2-ChloroATP (2ClATP) and its cell-permeant precursor, 2-chloroadenosine (2ClAdo), disrupt ST-dependent signaling in intestinal cells . However, whether the ability to disrupt guanylyl cyclase signaling translates into effective antisecretory therapy remains untested . In this study, the efficacy of 2ClAdo to prevent ST-induced water secretion by human intestinal cells was examined . In Caco-2 human intestinal cells, ST increased {cGMP}i, induced a chloride current, and stimulated net basolateral-to-apical water secretion . This effect on chloride current and water secretion was mimicked by the cell-permeant analog of cGMP, 8-bromo-cGMP . Treatment of Caco-2 cells with 2ClAdo prevented ST-induced increases in {cGMP}i, chloride current and water secretion . Inhibition of the downstream consequences of ST-GCC interaction reflects proximal disruption of cGMP production because 8-bromo-cGMP stimulated chloride current and water secretion in 2ClAdo-treated cells . Thus, this study demonstrates that disruption of guanylyl cyclase signaling is an effective strategy for antisecretory therapy and provides the basis for developing mechanism-based treatments for enterotoxigenic diarrhea.

Appl Environ Microbiol, 1999 May, 65(5), 2222 - 9
Differentiation of methanosaeta concilii and methanosarcina barkeri in anaerobic mesophilic granular sludge by fluorescent In situ hybridization and confocal scanning laser microscopy
Rocheleau S, Greer CW, Lawrence JR, Cantin C, Laramee L, Guiot SR.
Oligonucleotide probes, designed from genes coding for 16S rRNA, were developed to differentiate Methanosaeta concilii, Methanosarcina barkeri, and mesophilic methanogens . All M . concilii oligonucleotide probes (designated MS1, MS2, and MS5) hybridized specifically with the target DNA, but MS5 was the most specific M . concilii oligonucleotide probe . Methanosarcina barkeri oligonucleotide probes (designated MB1, MB3, and MB4) hybridized with different Methanosarcina species . The MB4 probe specifically detected Methanosarcina barkeri, and the MB3 probe detected the presence of all mesophilic Methanosarcina species . These new oligonucleotide probes facilitated the identification, localization, and quantification of the specific relative abundance of M . concilii and Methanosarcina barkeri, which play important roles in methanogenesis . The combined use of fluorescent in situ hybridization with confocal scanning laser microscopy demonstrated that anaerobic granule topography depends on granule origin and feeding . Protein-fed granules showed no layered structure with a random distribution of M . concilii . In contrast, a layered structure developed in methanol-enriched granules, where M . barkeri growth was induced in an outer layer . This outer layer was followed by a layer composed of M . concilii, with an inner core of M . concilii and other bacteria.

Appl Environ Microbiol, 1999 May, 65(5), 1949 - 58
Viral lysis and bacterivory during a phytoplankton bloom in a coastal water microcosm
Guixa-Boixereu N, Lysnes K, Pedros-Alio C.
The relative importance of viral lysis and bacterivory as causes of bacterial mortality were estimated . A laboratory experiment was carried out to check the kind of control that viruses could exert over the bacterial assemblage in a non-steady-state situation . Virus-like particles (VLP) were determined by using three methods of counting (DAPI {4',6-diamidino-2-phenylindole} staining, YOPRO staining, and transmission electron microscopy) . Virus counts increased from the beginning until the end of the experiment . However, different methods produced significantly different results . DAPI-stained VLP yielded the lowest numbers, while YOPRO-stained VLP yielded the highest numbers . Bacteria reached the maximal abundance at 122 h (3 x 10(7) bacteria ml-1), after the peak of chlorophyll a (80 mug liter-1) . Phototrophic nanoflagellates followed the same pattern as for chlorophyll a . Heterotrophic nanoflagellates showed oscillations in abundance throughout the experiment . The specific bacterial growth rate increased until 168 h (2.6 day-1) . The bacterivory rate reached the maximal value at 96 hours (0.9 day-1) . Bacterial mortality due to viral infection was measured by using two approaches: measuring the percentage of visibly infected bacteria (%VIB) and measuring the viral decay rates (VDR), which were estimated with cyanide . The %VIB was always lower than 1% during the experiment . VDR were used to estimate viral production . Viral production increased 1 order of magnitude during the experiment (from 10(6) to 10(7) VLP ml-1 h-1) . The percentage of heterotrophic bacterial production consumed by bacterivores was higher than 60% during the first 4 days of the experiment; afterwards, this percentage was lower than 10% . The percentage of heterotrophic bacterial production lysed by viruses as assessed by the VDR reached the highest values at the beginning (100%) and at the end (50%) of the experiment . Comparing both sources of mortality at each stage of the bloom, bacterivory was found to be higher than viral lysis at days 2 and 4, and viral lysis was higher than bacterivory at days 7 and 9 . A balance between bacterial losses and bacterial production was calculated for each sampling interval . At intervals of 0 to 2 and 2 to 4 days, viral lysis and bacterivory accounted for all the bacterial losses . At intervals of 4 to 7 and 7 to 9 days, bacterial losses were not balanced by the sources of mortality measured . At these time points, bacterial abundance was about 20 times higher than the expected value if viral lysis and bacterivory had been the only factors causing bacterial mortality . In conclusion, mortality caused by viruses can be more important than bacterivory under non-steady-state conditions.

Appl Environ Microbiol, 1999 May, 65(5), 1930 - 5
Selective isolation and distribution of sporichthya strains in soil
Suzuki Si, Okuda T, Komatsubara S.
A simplified enrichment method in which centrifugation is used for selective isolation of Sporichthya strains from soil is described . Gellan gum plus 2 mM CaCl2 stimulated growth of Sporichthya polymorpha KCC A0089 so that this organism was readily recognized on an isolation plate . High yields of motile spores were obtained by using a flooding solution containing 0.1% skim milk in 10 mM MOPS (morpholinepropanesulfonic acid) (pH 8.0) and then incubating the preparation at 27 degrees C for 60 min and centrifuging it at 1,000 x g for 10 min . Dry heat treatment at 80 degrees C for 60 min increased the ratio of Sporichthya colonies to nonfilamentous bacteria on a gellan gum plate . Since S . polymorpha was sensitive to 14 antibiotics, including nalidixic acid, addition of these antibiotics was not suitable for isolating Sporichthya strains . Our isolates were identified as Sporichthya strains on the basis of their morphological and chemotaxonomic characteristics . By combining the techniques described above, we isolated a number of Sporichthya strains from 21 soil samples, which were collected in Belgium, France, India, Japan, Papua New Guinea, Spain, Taiwan, the United Kingdom, and the United States . Sporichthya strains are widely distributed in the world . To our knowledge, this is the first time that Sporichthya strains have been isolated from locations other than the United States or Japan.

EDTNA ERCA J, 1998 Oct-Dec, 24(4), 32 - 5
Three year prospective follow up of the peritoneal access; Reyero A et al.; Peritoneal access-related complications were prospectively studied in 54 peritoneal catheters placed in 49 patients between January 1994 and December 96 in a single centre . There were no perioperative complications . Five (9%) catheters were removed because of catheter-derived complications (3 outflow obstruction, 2 leak), 4 (7.4%) because of peritonitis and 1 was spontaneously extruded . Complications included 4 (7.4%) migrations with outflow obstruction, 6 (11%) leaks and 27 episodes of exit site infection occurring in 16 (30%) catheters . Six catheters suffered more than one infection . Exit site infection in the first month after catheter placement is a risk factor for multiple exit site infections by different bacteria and for catheter removal.

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