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 (s