Microorganism

J Lipid Res, 1999 May, 40(5), 881 - 92
Localization of adipocyte long-chain fatty acyl-CoA synthetase at the plasma membrane; Gargiulo CE et al.; Long-chain fatty acyl-CoA synthetase (FACS) catalyzes esterification of long-chain fatty acids (LCFAs) with coenzyme A (CoA), the first step in fatty acid metabolism . FACS has been shown to play a role in LCFA import into bacteria and implicated to function in mammalian cell LCFA import . In the present study, we demonstrate that FACS overexpression in fibroblasts increases LCFA uptake, and overexpression of both FACS and the fatty acid transport protein (FATP) have synergistic effects on LCFA uptake . To explore how FACS contributes to LCFA import, we examined the subcellular location of this enzyme in 3T3-L1 adipocytes which natively express this protein and which efficiently take up LCFAs . We demonstrate for the first time that FACS is an integral membrane protein . Subcellular fractionation of adipocytes by differential density centrifugation reveals immunoreactive and enzymatically active FACS in several membrane fractions, including the plasma membrane . Immunofluorescence studies on adipocyte plasma membrane lawns confirm that FACS resides at the plasma membrane of adipocytes, where it co-distributes with FATP . Taken together, our data support a model in which imported LCFAs are immediately esterified at the plasma membrane upon uptake, and in which FATP and FACS function coordinately to facilitate LCFA movement across the plasma membrane of mammalian cells.

J Biol Chem, 1999 May 7, 274(19), 13147 - 54
The reaction of trimethylamine dehydrogenase with trimethylamine; Jang MH et al.; The reductive half-reaction of trimethylamine dehydrogenase with its physiological substrate trimethylamine has been examined by stopped-flow spectroscopy over the pH range 6.0-11.0, with attention focusing on the fastest of the three kinetic phases of the reaction, the flavin reduction/substrate oxidation process . As in previous work with the slow substrate diethylmethylamine, the reaction is found to consist of three well resolved kinetic phases . The observed rate constant for the fast phase exhibits hyperbolic dependence on the substrate concentration with an extrapolated limiting rate constant (klim) greater than 1000 s-1 at pH above 8.5, 10 degrees C . The kinetic parameter klim/Kd for the fast phase exhibits a bell-shaped pH dependence, with two pKa values of 9.3 +/- 0.1 and 10 . 0 +/- 0.1 attributed to a basic residue in the enzyme active site and the ionization of the free substrate, respectively . The sigmoidal pH profile for klim gives a single pKa value of 7.1 +/- 0 . 2 . The observed rate constants for both the intermediate and slow phases are found to decrease as the substrate concentration is increased . The steady-state kinetic behavior of trimethylamine dehydrogenase with trimethylamine has also been examined, and is found to be adequately described without invoking a second, inhibitory substrate-binding site . The present results demonstrate that: (a) substrate must be protonated in order to bind to the enzyme; (b) an ionization group on the enzyme is involved in substrate binding; (c) an active site general base is involved, but not strictly required, in the oxidation of substrate; (d) the fast phase of the reaction with native enzyme is considerably faster than observed with enzyme isolated from Methylophilus methylotrophus that has been grown up on dimethylamine; and (e) a discrete inhibitory substrate-binding site is not required to account for excess substrate inhibition, the kinetic behavior of trimethylamine dehydrogenase can be readily explained in the context of the known properties of the enzyme.

J Biol Chem, 1999 May 7, 274(19), 13127 - 32
Characterization and cloning of celR, a transcriptional regulator of cellulase genes from Thermomonospora fusca; Spiridonov NA et al.; CelR, a protein that regulates transcription of cellulase genes in Thermomonospora fusca (Actinomycetaceae) was purified to homogeneity . A 6-kilobase NotI-SacI fragment of T . fusca DNA containing the celR gene was cloned into Esherichia coli and sequenced . The celR gene encodes a 340-residue polypeptide that is highly homologous to members of the GalR-LacI family of bacterial transcriptional regulators . CelR specifically binds to a 14-base pair inverted repeat, which has sequence similarity to the binding sites of other family members . This site is present in regions upstream of all six cellulase genes in T . fusca . The binding of CelR to the celE promoter is inhibited specifically by low concentrations of cellobiose (0.2-0.5 mM), the major end product of cellulases . The other sugars tested did not affect binding at equivalent or 50-fold higher concentrations . The results suggest that CelR may act as a repressor, and that the mechanism of induction involves a direct interaction of CelR with cellobiose.

Ned Tijdschr Geneeskd, 1999 Feb 6, 143(6), 306 - 8
{Children hospitalized with acute gastroenteritis . II . No relation between clinical symptoms and causative agents isolated from feces}; Tjon A Ten WE; OBJECTIVE: To determine if it is possible to predict a bacterial cause in children with gastroenteritis on the basis of clinical features and if the number of stool cultures can be reduced . DESIGN: Retrospective . METHOD: The results of the stool cultures of 227 children admitted with acute gastroenteritis to the department of Paediatrics of Sint Joseph Hospital, Veldhoven, the Netherlands, in the period 1992-1996 were related by review of medical records to clinical symptoms and blood tests . The diagnostic values of the various parameters were calculated . RESULTS: Rotavirus was identified in 40% of the faeces of the children, other viruses in 10% and bacteria in 8% . In 95% the bacterial pathogen was identified in the first stool sample . The diagnostic value of the investigated parameters was low . CONCLUSION: It is not possible on the basis of clinical parameters to predict a bacterial gastroenteritis . The number of stool samples may, however, be reduced if the stools of each child with acute diarrhoea are first investigated for rotavirus . Only if this test is negative are further investigations required . The number of bacterial cultures can be limited to one stool sample.

Anaesthesiol Reanim, 1999, 24(1), 4 - 12
{Acute failure of the intestinal barrier--pathophysiology, diagnosis, prophylaxis and therapy}; Hachenberg T et al.; The gut not only serves as a main target for the detrimental effects of stress during and after surgery, but may also promote the development of multiple organ failure after different types of severe shock . According to a current hypothesis, an impaired intestinal barrier function is associated with a decreased separation of intraluminal bacteria and toxins and systemic circulation, which may induce sepsis and multiple organ failure . Hypoperfusion during shock, reperfusion injury of the splanchnic mucosa, alterations of the micro-ecology of the gut and immunologic and hormonal disturbances are important underlying pathophysiological mechanisms . Various therapeutic concepts have been proposed such as improvement of splanchnic perfusion, nutritive and metabolic treatment by means of immunomodulating nutrients, parenteral substitution of glutamine, early onset of enteral nutrition, normalization of gut motility and selective decontamination of the gut . However, no clinical study to date could clearly demonstrate a key role of the gut in the pathogenesis of sepsis and multiple organ failure . Likewise, the efficacy of different prophylactic and therapeutic procedures remain to be studied . An aggressive treatment of shock and avoidance of microcirculatory disturbances are of principal importance for prophylaxis of multiple organ failure.

J Prosthet Dent, 1999 May, 81(5), 597 - 609
Mechanical properties of dental luting cements; Li ZC et al.; STATEMENT OF PROBLEM: Dental luting cements fail by microcrack formation and bacterial ingress or by gross failure and crown dislodgment . Both of these failure modes are related to mechanical properties and deformation . PURPOSE: This study evaluated those mechanical properties of cements . METHODS AND MATERIAL . Elastic modulus for 8 representative cements (zinc phosphate, polycarboxylate, glass ionomer, encapsulated glass ionomer, resin-modified glass ionomer, resin composite, and adhesive resin composite) was measured by using a nondestructive technique and evaluated for cement type and storage time (1 hour, 1 day, 1 week, 1 month, 1 year) by 2-way ANOVA (P <.05) . Compressive properties (proportional limit, resilience, and toughness), ultimate strengths (compressive, diametral tensile, and flexural), and flexural toughness were determined and evaluated by 2-way ANOVA for 2 crosshead testing rates (5 and 0.5 mm/min) and cement type (P <.05) . RESULTS: Cements varied with respect to elastic moduli, compressive proportional limit, compressive resilience, compressive strength, compressive toughness, diametral tensile strength, flexural strength, and flexural toughness . Storage time affected the elastic moduli of different materials in different ways . Elastic moduli of polycarboxylate and glass ionomer cements increased over time, whereas the other materials changed little after the first day . Crosshead rate only significantly affected compressive proportional limit and resilience . CONCLUSIONS: Luting cements differed considerably with respect to mechanical properties.

Biochemistry, 1999 Apr 27, 38(17), 5651 - 8
Proteasome activator 11S REG or PA28: recombinant REG alpha/REG beta hetero-oligomers are heptamers; Zhang Z et al.; The proteasome activator 11S REG or PA28 is a conical molecule composed of two homologous subunits, REG alpha and REG beta . Recombinant REG alpha forms a heptamer, whereas recombinant REG beta is a monomer . When mixed with REG beta, a monomeric REG alpha mutant (N50Y) forms an active hetero-oligomer in which the molar ratio of REG beta to REG alpha(N50Y) is close to 1.3 . This apparent stoichiometry is consistent with the REG alpha(N50Y)/REG beta hetero-oligomer being a heptamer composed of three alpha and four beta subunits . Chemical cross-linking of the alpha/beta oligomers revealed the presence of REG alpha-REG beta and REG beta-REG beta dimers, but REG alpha-REG alpha dimers were not detected . The mass of the REG alpha(N50Y)/REG beta hetero-oligomer determined by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) is 194 871 +/- 40 Da in good agreement with the theoretical mass of 194 856 Da for an alpha 3 beta 4 heptamer . Hexamers were not observed in the mass spectrum . For wild-type REG subunits coexpressed in bacteria cells at an apparent beta/alpha molar ratio of approximately 1.2, the resulting hetero-oligomers observed by ESI-TOF MS were again predominantly alpha 3 beta 4 heptamers, with trace amounts of alpha 4 beta heptamers also present . On the other hand, the mass spectrum contained a mixture of alpha 7, alpha 6 beta 1, alpha 5 beta 2, and alpha 4 beta 3 heptamers when the REG beta/REG alpha ratio was 0.1 . Thus, formation of heptamers is an intrinsic property of recombinant REG alpha and REG beta subunits . On the basis of these results, we propose that 11S REG purified directly from eukaryotic cells is also heptameric, likely alpha 3 beta 4 or a mixture of alpha 3 beta 4 and alpha 4 beta 3 species.

Biochemistry, 1999 Apr 27, 38(17), 5596 - 602
Pleckstrin homology domain 1 of mouse alpha 1-syntrophin binds phosphatidylinositol 4,5-bisphosphate; Chockalingam PS et al.; Mouse alpha 1-syntrophin sequences were produced as chimeric fusion proteins in bacteria and found to bind phosphatidylinositol 4, 5-bisphosphate (PtdIns4,5P2) . Half-maximal binding occurred at 1.9 microM PtdIns4,5P2 and when 1.2 PtdIns4,5P2 were added per syntrophin . Binding was specific for PtdIns4,5P2 and did not occur with six other tested lipids including the similar phosphatidylinositol 4-phosphate . Binding was localized to the N-terminal pleckstrin homology domain (PH1); the second, C-terminal PH2 domain did not bind lipids . Key residues in PtdIns4,5P2 binding to a PH domain were found to be conserved in alpha-syntrophins' PH1 domains and absent in PH2 domains, suggesting a molecular basis for binding.

Microbiology, 1999 Apr, 145 ( Pt 4), 935 - 47
Identification of a novel nutrient-deprivation-induced Sinorhizobium meliloti gene (hmgA) involved in the degradation of tyrosine; Milcamps A et al.; Sinorhizobium meliloti strain N4 carries a Tn5luxAB insertion in a gene which is induced by nitrogen and carbon deprivation as well as in the presence of tyrosine . The Tn5luxAB-tagged locus was found to share significant similarity with the human hmgA gene and the corresponding Aspergillus nidulans gene, encoding the enzyme homogentisate dioxygenase, which is involved in the degradation of tyrosine . Extended DNA sequence analysis of the tagged locus revealed the presence of several ORFs, including one encoding a polypeptide sharing a high degree of similarity with human and fungal maleylacetoacetate isomerases . Strain N4 was found to be unable to use tyrosine as carbon source, to lack homogentisate dioxygenase activity, to produce a melanin-like pigment and to be affected in stationary-phase survival . This is believed to be the first report of a hmgA-homologous gene in bacteria.

J Chromatogr B Biomed Sci Appl, 1999 Mar 19, 724(2), 265 - 74
Formation and identification of azaarene transformation products from aquatic invertebrate and algal metabolism; de Voogt P et al.; The metabolism of two azaarenes, viz . acridine and phenanthridine, by aquatic organisms was studied in short-term and chronic laboratory tests . The identity of metabolites observed in the test waters was investigated with different analytical methods, including HPLC, GC and hyphenated LC- or GC-MS . The Zebra mussel (Dreissena polymorpha), one green alga species (Selenastrum capricornutum) and periphyton or bacteria transformed acridine into 9{10H}-acridinone . Phenanthridine was transformed into 5{6H}-phenanthridinone by midge (Chironomus riparius) larvae . The findings indicate that closely related isomers may undergo species-specific biotransformation . It was concluded that keto-metabolites are major products in the aquatic fate of benzoquinolines, which may be overlooked in the risk assessment of parent compounds . This study illustrates the typical problems with, as well as the potency of, chromatographic methods in the elucidation of metabolic routes of organic contaminants.

Oral Microbiol Immunol, 1999 Apr, 14(2), 117 - 21
Restriction fragment-length polymorphism analysis of 16S ribosomal RNA genes amplified by polymerase chain reaction for rapid identification of cultivable oral treponemes; Sato T et al.; Although oral treponemes are among the most frequently found bacteria in periodontal pockets, identification of these organisms can be difficult . In this study, restriction fragment-length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified 16S ribosomal RNA genes (16S rRNA gene PCR-RFLP) was used to generate restriction profiles of reference strains of oral treponemes including Treponema denticola, Treponema socranskii, Treponema vincentii . Treponema pectinovorum and Treponema medium as well as for Treponema phagedenis and Treponema pailidum and five treponeme strains isolated from human peridontal pockets . Before RFLP analysis, the 16S rRNA gene sequences were obtained from the GenBank database, and the analysis of the theoretical banding patterns for HpaII suggested good species discrimination . 16S rRNA gene sequences were amplified from isolated genomic DNA samples by PCR with spirochete-specific primers . The PCR products were then purified and characterized by single digestion with restriction endonuclease HpaII, and this allowed discrimination between the respective reference strains . Five clinical isolates, four T . denticola and one T . socranskii, were assigned on the basic of their restriction profiles by digestion with HpaII . 16S rRNA gene PCR-RFLP using HpaII is a rapid and reliable method for differentiation of cultivable oral treponemes.

Oral Microbiol Immunol, 1999 Apr, 14(2), 109 - 16
Binding of the periodontal pathogen Actinobacillus actinomycetemcomitans to extracellular matrix proteins; Mintz KP et al.; The interaction of Actinobacillus actinomycetemcomitans, an important pathogen implicated in juvenile and adult periodontitis, with collagenous and noncollagenous proteins of the extracellular matrix was investigated . A . actinomycetemcomitans SUNY 465 bound to immobilized type I, II, III and V but not type IV collagen . Binding to immobilized collagen was saturable and concentration dependent . This interaction could not be inhibited by soluble collagen, suggesting that binding was dependent on a specific collagen conformation . Bacteria grown anaerobically exhibited decreased collagen-binding activity as compared with organisms grown acrobically . Bacterial outer membrane proteins were essential for binding to collagen . A actinomycetemcomitans SUNY 465 also bound to immobilized fibronectin . In contrast, bacteria did not bind to fibrinogen, bone sialoprotein, alpha 2-HS glycoprotein or albumin . The mechanism of the interaction with fibronectin was more complex, possibly involving both protein and nonproteinaceous components . The majority of other A . actinomycetemcomitans strains tested bound to extracellular matrix proteins in a manner similar to SUNY 465 but with minor variation . These results demonstrate that A . actinomycetemcomitans binds to proteins found in connective tissue . The interaction with extracellular matrix proteins may contribute to the virulence of this pathogen at oral and extraoral sites of infection.

Nucleic Acids Res, 1999 May 15, 27(10), 2063 - 71
Comparative sequence analysis of tmRNA; Zwieb C et al.; Minimal secondary structures of the bacterial and plastid tmRNAs were derived by comparative analyses of 50 aligned tmRNA sequences . The structures include 12 helices and four pseudoknots and are refinements of earlier versions, but include only those base pairs for which there is comparative evidence . Described are the conserved and variable features of the tmRNAs from a wide phylogenetic spectrum, the structural properties specific to the bacterial subgroups and preliminary 3-dimensional models from the pseudoknotted regions.

Ophthalmic Surg Lasers, 1999 Apr, 30(4), 295 - 8
A study of the incidence of culture-positive endophthalmitis after cataract surgery in an ambulatory care center; Bohigian GM; BACKGROUND AND OBJECTIVE: To determine the incidence of culture-positive endophthalmitis after cataract surgery in an ambulatory surgical care center and to analyze the effectiveness of povidoneiodine solution in the preoperative preparation in preventing culture-positive endophthalmitis . PATIENTS AND METHODS: A retrospective series of 19,269 consecutive cases of cataract extraction with lens implantation over 12 years in an ambulatory care center was reviewed . RESULTS: Nine cases of culture-positive endophthalmitis occurred, for an incidence of 0.05% . The initial 4,740 cases (1985-1989) were performed without the use of povidone-iodine; the following 14,529 cases (1990-1996) were done using povidone-iodine . The incidence of culture-positive endophthalmitis was 0.08% and 0.03%, respectively . CONCLUSION: The incidence of culture-positive endophthalmitis in this series was very low . The use of 5% povidone-iodine, topically, appeared to be beneficial in reducing the incidence of culture-positive endophthalmitis after cataract extraction (P=0.24), but was not statistically significant in this retrospective series . Evaluation and methods to prevent endophthalmitis are difficult in retrospective clinical studies due to multiple variables and the rarity of this complication.

Monaldi Arch Chest Dis, 1999 Feb, 54(1), 22 - 37
Mechanisms of ventilation-induced lung injury: physiological rationale to prevent it; Verbrugge SJ et al.; It is being increasingly realized that modes of mechanical ventilation that result in end-inspiratory alveolar overstretching and/or repeated alveolar collapse and re-expansion disturb the normal fluid balance across the alveolocapillary membrane . The effects of this include disturbance of the integrity of the endothelium and epithelium and impairment of the surfactant system and are similar to those seen in acute respiratory distress syndrome (ARDS) . There is now also evidence that these modes of mechanical ventilation may result in the translocation of bacteria from the lungs into the bloodstream and the release of inflammatory mediators from the lung tissue into the systemic circulation . It may thus be speculated that mechanical ventilation may contribute to the development of multiple organ failure (MOF) . Therefore, during mechanical ventilation, alveolar overstretching and the repeated collapse and re-expansion of alveoli should be prevented by ventilation modes that open up the lung and keep the lung open and ventilate with the smallest possible pressure amplitude . For the future, monitoring techniques should be developed that can evaluate, on-line, whether or not these therapeutic directives are being achieved.

Acta Neurol Belg, 1999 Mar, 99(1), 57 - 60
Nitric oxide synthase expression in neurogenic bladder disease: a pilot study; De Ridder D et al.; Neurogenic bladders are susceptible to bladder cancer development, especially in the case of chronic indwelling catheters . The classic carcinogenesis theory involves the formation of carcinogenic nitrosamines by bacteria due to chronic infection . We designed a pilot study to evaluate the expression of the of Nitric Oxide Synthase (NOS) isoforms in bladder tissue to study the role of the endogenous formation of NO (Nitric Oxide) in neurogenic bladders . Immunohistochemistry was performed on bladder biopsies from neurogenic, normal, and obstructed bladders . The neurogenic bladder had a higher expression of endothelial NOS (eNOS), but especially of neuronal NOS (nNOS) . Besides, this extra-neuronal expression of nNOS by urothelium and interstitial cells was observed . This study proves the important role of endogenous formation of NO by suburothelial nerves but also by urothelium and interstitial cells . This overexpression could possibly be a factor in the higher incidence of bladder cancer in neurogenic bladders . On the other hand, it shows the plasticity of the NO pathways in these cases and raises important research questions concerning the physiological role of these changes.

J Bacteriol, 1999 May, 181(9), 2797 - 801
Requirement of NifX and other nif proteins for in vitro biosynthesis of the iron-molybdenum cofactor of nitrogenase; Shah VK et al.; The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1 . In vitro synthesis of FeMo-co has been established, and the reaction requires an ATP-regenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH) . The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of Azotobacter vinelandii defective in the biosynthesis of cofactor or an extract of a mutant strain complemented with the purified missing component . Surprisingly, the in vitro synthesis of FeMo-co with only purified components failed to generate significant FeMo-co, suggesting the requirement for one or more other components . Complementation of these assays with extracts of various mutant strains demonstrated that NifX has a role in synthesis of FeMo-co . In vitro synthesis of FeMo-co with purified components is stimulated approximately threefold by purified NifX . Complementation of these assays with extracts of A . vinelandii DJ42 . 48 (DeltanifENX DeltavnfE) results in a 12- to 15-fold stimulation of in vitro FeMo-co synthesis activity . These data also demonstrate that apart from the NifX some other component(s) is required for the cofactor synthesis . The in vitro synthesis of FeMo-co with purified components has allowed the detection, purification, and identification of an additional component(s) required for the synthesis of cofactor.

Biochim Biophys Acta, 1999 Apr 21, 1411(1), 206 - 13
Characterization of the photoreduction of the secondary quinone QB in the photosynthetic reaction center from rhodobacter capsulatus with FTIR spectroscopy
Nabedryk E.
The photoreduction of the secondary quinone acceptor QB in reaction centers (RCs) of the photosynthetic bacteria Rhodobacter (Rb.) capsulatus has been investigated by light-induced FTIR difference spectroscopy in 1H2O and 2H2O . The Q-B/QB FTIR spectra reflect reorganization of the protein upon electron transfer, changes of protonation state of carboxylic acid groups, and (semi)quinone-protein interactions . As expected from the conservation of most of the amino acids near QB in the RCs from Rb . capsulatus and Rb . sphaeroides, several protein and quinone IR bands are common to both spectra, e.g., the 1728 cm-1 band is assigned to proton uptake by a carboxylic acid residue, most probably Glu L212 as previously proposed for Rb . sphaeroides RCs . However, noticeable changes are observed at 1709 cm-1 (deprotonation of a Glu or Asp residue), 1674 and 1659 cm-1 (side chain and/or backbone), around 1540 cm-1 (amide II), and in the semiquinone absorption range . This FTIR study demonstrates that the environment of the secondary quinone in Rb . capsulatus is close but not identical to that in Rb . sphaeroides suggesting slight differences in the structural organization of side chains and/or ordered water molecules near QB.

Vaccine, 1999 Apr 9, 17(15-16), 1846 - 57
Immunogenicity of peptides derived from a fibronectin-binding protein of S . aureus expressed on two different plant viruses; Brennan FR et al.; The D2 peptide derived from an S . aureus fibronectin-binding protein (FnBP) was expressed on the surface of the icosahedral cowpea mosaic virus (amino acids 1-30 of D2) or on the rod-shaped potato virus X (amino acids 1-38 of D2), termed CPMV-MAST1 and PVX-MAST8, respectively . Mice and rats were immunized subcutaneously with CPMV-MAST1 and mice with PVX-MAST8 in adjuvant and high titres of FnBP-specific antibody were obtained . The mouse IgG was predominantly of the IgG2a and IgG2b isotypes, which strongly bound complement component C1q, suggesting a TH1-bias in the peptide-specific responses . Sera from mice and rats immunized with CPMV-MAST1 and from mice immunized with PVX-MAST8 were shown to completely inhibit the binding of fibronectin to immobilised recombinant FnBP and rat sera against CPMV-MAST1 were able to block adherence of S . aureus to fibronectin . These studies demonstrate that the D2 peptide is highly immunogenic when expressed on 2 different plant viruses and highlight the potential of plant virus-based vaccines to protect against S . aureus infections.

Microbiology, 1999 Mar, 145 ( Pt 3), 743 - 53
Organization and expression of nitrogen-fixation genes in the aerobic nitrogen-fixing unicellular cyanobacterium Synechococcus sp . strain RF-1; Huang TC et al.; Sixteen nif and 'nif-associated' genes (expressed only under conditions of nitrogen fixation) in Synechococcus sp . strain RF-1 have been cloned and sequenced . All of the nif and nif-associated genes identified in Synechococcus RF-1 were arranged in a continuous cluster spanning approximately 18 kb and containing seven operons . The nifH operon (nifH-nifD-nifK) has been reported previously . nifB, fdxN, nifS, nifU and nifP were found to be located upstream of the nifH operon . nifB-fdxN-nifS-nifU were expressed as an operon . A nifP-like gene was found to be located just upstream of nifB . nifE, nifN, nifX, nifW and the nif-associated hesA, hesB and 'fdx' were found to be located downstream from nifK . The genes located downstream from nifK are arranged nifE-nifN-nifX-orf-nifW-hesA-hesB-'+ ++fdx' and span approximately 7 kb . The function of the ORF situated between nifX and nifW is not known . However, it was identified as a counterpart of ORF-2 in Anabaena sp . strain PCC 7120 based on the deduced amino acid sequence . Northern hybridization and primer extension analysis indicated that the nif and nif-associated genes are organized in nifE-nifN, nifX-orf, nifW-hesA-hesB and 'fdx'-containing operons, respectively . According to the results of this study and previous reports, the genes are expressed in a rhythmic pattern with peaks during the dark phase when the culture is grown in a 12 h light/12 h dark regimen . The rhythm persisted after the culture was transferred to continuous illumination.

Int J Biochem Cell Biol, 1999 Jan, 31(1), 139 - 49
Translational regulation by Y-box transcription factor: involvement of the major mRNA-associated protein, p50; Evdokimova VM et al.; p50, the major core protein of messenger ribonucleoprotein particles (mRNPs), is a universal protein found exclusively in association with different mRNA species in the cytoplasm of somatic mammalian cells . Furthermore, p50 is the most abundant and tightly bound protein within both inactive mRNPs and active mRNPs derived from polysomes, although the latter contain a lower level of p50 . Recent experiments have revealed that, depending on the p50 to mRNA ratio, p50 may either act as a repressor or an activator of protein synthesis . On the other hand, p50 exhibits about 98% amino acid sequence identity to mammalian transcription factors that bind specifically to Y-box containing DNA . Thus, it is a counterpart of the Y-box binding proteins which are found in bacteria, plants and animals, exhibiting multiple biological activities ranging from transcriptional regulation of a wide variety of genes to 'masking' mRNA activity in germinal cells . This review summarizes our current knowledge of p50 structure and function . It also discusses the biological roles of p50 and related proteins in gene expression and describes the likely mechanisms of their action.

Avian Dis, 1999 Jan-Mar, 43(1), 98 - 105
Detection of avian adenovirus by polymerase chain reaction; Xie Z et al.; An avian adenovirus-specific polymerase chain reaction was developed . The origin of primers was from the DNA sequence data of the chicken embryo lethal orphan avian adenovirus virus genome . An avian adenovirus-specific 421-bp DNA product was amplified by these primers from group I of adenovirus containing 12 serotypes and serotypes of adenovirus from group II and group III . The adenovirus-specific DNA product was also amplified from the 19 field isolates of avian adenoviruses but not from the mammalian adenovirus and other avian pathogenic viruses and bacteria . As little as 1 fg of avian adenovirus DNA was detected by gel electrophoresis and Southern blot analysis.

Avian Dis, 1999 Jan-Mar, 43(1), 1 - 7
Investigations on different Ornithobacterium rhinotracheale "ORT" isolates; Hafez HM et al.; The aim of the present investigation was to determine the antigenic relationship between different Ornithobacterium rhinotracheale (ORT) isolates and to serotype field isolates obtained from turkey and chickens . Different antigen extractions (heat-stable, proteinase K-stable {lipopolysaccharide}, and sodium dodecyl sulfate {SDS} extractions) were prepared from each serotype (A, B, C, D, E, and G) as well as from 21 ORT field isolates and examined in agar gel precipitation (AGP) and enzyme-linked immunosorbent assay (ELISA) tests . The field isolates were cultured from turkey (16 isolates) and chicken (5 isolates) flocks showing respiratory manifestations . Monospecific reactions were obtained with heat-stable as well as proteinase K-stable antigens prepared from serotypes A, C, D, E, and G in AGP tests . On the other hand, with the same antigen preparations from a strain of serotype B in AGP tests, cross-reactions with antisera prepared against serotypes A and E could be detected . The cross-reactions were observed mostly between 48 and 72 hr . In applications of SDS-antigen preparations in AGP tests, cross-reactions between all serotypes except serotype C were detected between 24 and 72 hr . Testing all antigen preparation in ELISA, different cross-reactions were observed and the evaluation of the results is very difficult . Serotyping of the field isolates in AGP tests by using heat-extracted antigens showed after 24 hr that 10 out of 16 isolates from turkey belonged to serotype B, five to serotype A, and one to serotype E . Results obtained after 48-72 hr revealed cross-reactions between serotype B and E in 11 cases and between A and B in two cases . All five isolates obtained from chicken reacted after 24 hr only with serum against serotype A . After 48-72 hr, two isolates showed cross-reaction with antiserum against serotype B . Similar results were obtained with proteinase K-stable antigen.

Gene, 1999 Apr 16, 230(2), 187 - 95
Characterization of a maize heat-shock protein 101 gene, HSP101, encoding a ClpB/Hsp100 protein homologue; Nieto-Sotelo J et al.; Heat shock protein 101 (HSP101) cDNA and genomic clones from maize have been isolated . The structure of maize HSP101 reveals the presence of six exons interrupted by five introns . Maize HSP101 contains a predicted open reading frame that translates into a 912-aa sequence with a mass of 101kDa . Initiation of transcription was mapped 146 bases upstream of the AUG codon . Five heat shock element (HSE) boxes were found within the proximal 289 bases of the promoter region . Southern blot analysis of genomic DNA indicates that the maize genome contains only one copy of HSP101 . A protein sequence comparison showed that maize Hsp101 belongs to the heat shock 100kDa and caseino-lytic protease B protein family (Hsp100/ClpB) that plays important roles in bacteria and yeast in the survival to extremely high temperatures and the control of proteolysis . Accumulation of HSP101 mRNA was strong under heat shock conditions, but not detectable after cold or osmotic stress treatments or by exogenous application of ABA . The analysis of the predicted supersecondary structure of maize Hsp101 showed that a coiled-coil located in the middle region of the protein is evolutionarily conserved in all members of the Clp A, B and C subfamilies . It is proposed that these supersecondary structures may have important roles in Clp function.

Mutat Res, 1999 Apr 6, 425(2), 213 - 24
Damage to mitochondrial DNA induced by the quinolone Bay y 3118 in embryonic turkey liver; Enzmann H et al.; Quinolones are a class of antibiotics that induce damage to and loss of DNA from bacteria . The structural organization of bacterial DNA is more similar to eukaryotic mitochondrial DNA (mtDNA) than to eukaryotic chromosomal or nuclear DNA (nDNA) . Antibiotics affecting the bacterial genome may therefore preferentially damage mtDNA rather than nDNA . We investigated the effect of a quinolone on mtDNA in avian embryonic hepatocytes in ovo . The quinolone Bay y 3118 (1-cyclopropyl-7-(2,8-diazabicyclo{4.3.0}non-8-yl) 6-fluoro-8-chloro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid hydrochloride, chemical structure see Bremm et al . {K.D . Bremm, U . Petersen, K.G . Metzger, R . Endermann, In vitro evaluation of Bay-y 3118, a new full-spectrum fluoroquinolone, Chemotherapy 38 (1992) 376-387} was injected into fertilized turkey eggs 8 days before hatching at doses of 1, 3, 10 and 30 mg per egg . The embryos were removed from the eggs after 4 days and liver samples were shock frozen . Mitochondrial DNA was purified from samples of the embryonic liver . The integrity of mtDNA was investigated by electrophoresis on agarose gels with native mtDNA and with ribonuclease-treated mtDNA . Fluorescent staining of the electrophoresis gels allows the densitometric quantification of the mtDNA of the regular band at 16 kilobases (kb) and the amount of DNA fragments of irregular size (smear) . The genotoxic nitrosamine nitrosodiethylamine (NDEA) has previously been shown to reduce the content of mtDNA of the regular size of 16 kb and to induce the occurrence of smaller fragments of mtDNA {H . Enzmann, C . Kuhlem, E . Loser, P . Bannasch, Damage to mitochondrial DNA induced by the hepatocarcinogen, diethylnitrosamine in ovo, Mutation Res . 329 (1995) 113-120} . After exposure to 10 and 30 mg Bay y 3118, a dose-dependent induction of damage to the mtDNA was found, whereas exposure to 3 and 1 mg showed no effect . NDEA (25 mg) was used as positive control . Testing chemical compounds in the in ovo model is a simple and rapid approach for investigations on chemically induced alterations of mtDNA .

Biochim Biophys Acta, 1999 Apr 21, 1411(1), 134 - 41
The unusual iron sulfur composition of the Acidianus ambivalens succinate dehydrogenase complex; Gomes CM et al.; The succinate dehydrogenase complex of the thermoacidophilic archaeon Acidianus ambivalens was investigated kinetically and by EPR spectroscopy in its most intact form, i.e., membrane bound . Here it is shown that this respiratory complex has an unusual iron-sulfur cluster composition in respect to that of the canonical succinate dehydrogenases known . The spectroscopic studies show that center S3, the succinate responsive {3Fe-4S}1+/0 cluster of succinate dehydrogenases, is not present in membranes prepared from aerobically grown A . ambivalens, nor in partially purified complex fractions . On the other hand, EPR features associated to the remaining centers, clusters S1 ({2Fe-2S}1+/2+) and S2 ({4Fe-4S}2+/1+), could be observed . Similar findings were made in other archaea, namely Acidianus infernus and Sulfolobus solfataricus . Kinetic investigations showed that the A . ambivalens enzyme is reversible, capable of operating as a fumarate reductase - a required activity if this obligate autotroph performs CO2 fixation via a reductive citric acid cycle . Sequencing of the sdh operon confirmed the spectroscopic data . Center S3 ({3Fe-4S}) is indeed replaced by a second {4Fe-4S} center, by incorporation of an additional cysteine, at the cysteine cluster binding motif (CxxYxxCxxxC-->CxxCxxCxxxC) . Genomic analysis shows that genes encoding for succinate dehydrogenases similar to the ones here outlined are also present in bacteria, which may indicate a novel family of succinate/fumarate oxidoreductases, spread among the Archaea and Bacteria domains.

Blood, 1999 May 1, 93(9), 3140 - 7
Prevention of transfusion-associated graft-versus-host disease by photochemical treatment; Grass JA et al.; Photochemical treatment (PCT) with the psoralen S-59 and long wavelength ultraviolet light (UVA) inactivates high titers of contaminating viruses, bacteria, and leukocytes in human platelet concentrates . The present study evaluated the efficacy of PCT to prevent transfusion-associated graft-versus-host disease (TA-GVHD) in vivo using a well-characterized parent to F1 murine transfusion model . Recipient mice in four treatment groups were transfused with 10(8) splenic leukocytes . (1) Control group mice received syngeneic splenic leukocyte transfusions; (2) GVHD group mice received untreated allogeneic splenic leukocytes; (3) gamma radiation group mice received gamma irradiated (2,500 cGy) allogeneic splenic leukocytes; and (4) PCT group mice received allogeneic splenic leukocytes treated with 150 micromol/L S-59 and 2.1 J/cm2 UVA . Multiple biological and clinical parameters were used to monitor the development of TA-GVHD in recipient mice over a 10-week posttransfusion observation period: peripheral blood cell levels, spleen size, engraftment by donor T cells, thymic cellularity, clinical signs of TA-GVHD (weight loss, activity, posture, fur texture, skin integrity), and histologic lesions of liver, spleen, bone marrow, and skin . Mice in the control group remained healthy and free of detectable disease . Mice in the GVHD group developed clinical and histological lesions of TA-GVHD, including pancytopenia, marked splenomegaly, wasting, engraftment with donor derived T cells, and thymic hypoplasia . In contrast, mice transfused with splenic leukocytes treated with (2,500 cGy) gamma radiation or 150 micromol/L S-59 and 2.1 J/cm2 UVA remained healthy and did not develop detectable TA-GVHD . Using an in vitro T-cell proliferation assay, greater than 10(5.1) murine T cells were inactivated by PCT . Therefore, in addition to inactivating high levels of pathogenic viruses and bacteria in PC, these data indicate that PCT is an effective alternative to gamma irradiation for prevention of TA-GVHD.

J Vasc Res, 1999 Mar-Apr, 36(2), 83 - 90
The emergence of physiological genomics; Cowley AW Jr; 'Physiological genomics' represents a research paradigm shift emerging to define the functions of tens of thousands of newly discovered genes which are expected to emerge from the sequencing of the human genome and other model organisms . Genomic tools, which will allow a higher efficiency of identification of gene function, are being developed at remarkable speed . This article discusses some of the genomic and bioinformatic tools currently available or under development to provide the infrastructure for mapping and identification of gene function in simple organisms (bacteria, zebrafish, fly, worm) and complex mammalian organisms (mouse and rat) . The problems facing the scientific community in the implementation of this functional approach are discussed as it is now evident that new technological and organizational infrastructures are emerging to link genes to overall function of whole organisms.

Eur J Biochem, 1999 Apr, 261(2), 475 - 80
A methenyl tetrahydromethanopterin cyclohydrolase and a methenyl tetrahydrofolate cyclohydrolase in Methylobacterium extorquens AM1; Pomper BK et al.; Recently it was found that Methylobacterium extorquens AM1 contains both tetrahydromethanopterin (H4MPT) and tetrahydrofolate (H4F) as carriers of C1 units . In this paper we report that the aerobic methylotroph contains a methenyl H4MPT cyclohydrolase (0.9 U x mg-1 cell extract protein) and a methenyl H4F cyclohydrolase (0.23 U x mg-1) . Both enzymes, which were specific for their substrates, were purified and characterized and the encoding genes identified via the N-terminal amino acid sequence . The purified methenyl H4MPT cyclohydrolase with a specific activity of 630 U x mg-1 (Vmax = 1500 U x mg-1; Km = 30 microm) was found to be composed of two identical subunits of molecular mass 33 kDa . Its sequence was approximately 40% identical to that of methenyl H4MPT cyclohydrolases from methanogenic archaea . The methenyl H4F cyclohydrolase with a specific activity of 100 U x mg-1 (Vmax = 330 U x mg-1; Km = 80 microm) was found to be composed of two identical subunits of molecular mass 22 kDa . Its sequence was not similar to that of methenyl H4MPT cyclohydrolases or to that of other methenyl H4F cyclohydrolases . Based on the specific activities in cell extract and from the growth properties of insertion mutants it is suggested that the methenyl H4MPT cyclohydrolase might have a catabolic, and the methenyl-H4F cyclohydrolase an anabolic function in the C1-unit metabolism of M . extorquens AM1.

Eur J Biochem, 1999 Apr, 261(2), 459 - 67
NMR studies of subunit c of the ATP synthase from Propionigenium modestum in dodecylsulphate micelles; Matthey U et al.; The structure of the Na+, Li+ or H+-binding c subunit of the ATP synthase from Propionigenium modestum was studied by NMR . Subunit c in dodecylsulphate micelles consists of four alpha-helical segments, I-IV, that are connected by short linker peptides with non-regular secondary structures . We propose that helices I (V4-I26) and IV (I69-V85) are membrane-spanning structures, and that helices II and III and the intervening hydrophilic loop are located in the cytoplasm . The Na+-binding residues Q32, E65 and S66 are located in the I-->II and III-->IV helix connections, probably near the membrane surface on the cytoplasmic side.

FEBS Lett, 1999 Mar 26, 447(2-3), 131 - 4
A second mechanism of respiratory control; Kadenbach B et al.; According to the chemosmotic hypothesis, ATP is synthesized in mitochondria, bacteria and chloroplasts via the proton motive force delta p, the energy-rich intermediate of electron transport and photosynthetic phosphorylation . The general applicability of the chemosmotic hypothesis, however, was disputed until present . In particular the relationship between the rate of respiration and delta p in mitochondria was found variable, depending on the experimental conditions . Recently, a new mechanism of respiratory control was found, based on binding of ATP or ADP to subunit IV of cytochrome c oxidase, which is independent of delta p and could explain many previous results contradicting the chemosmotic hypothesis.

Biochimie, 1999 Jan-Feb, 81(1-2), 139 - 46
Effect of DNA lesions on transcription elongation; Tornaletti S et al.; Some types of damage to cellular DNA have been shown to interfere with the essential transactions of replication and transcription . Not only may the translocation of the polymerase be arrested at the site of the lesion but the bound protein may encumber recognition of the lesion by repair enzymes . In the case of transcription a subpathway of excision repair, termed transcription-coupled repair (TCR) has been shown to operate on lesions in the transcribed strands of expressed genes in bacteria, yeast, mammalian cells and a number of other organisms . Certain genes in mammalian cells (e.g., CSA and CSB) have been uniquely implicated in TCR while others (e.g., XPC-HR23 and XPE) have been shown to operate in the global genomic pathway of nucleotide excision repair, but not in TCR . In order to understand the mechanism of TCR it is important to learn how an RNA polymerase elongation complex interacts with a damaged DNA template . That relationship is explored for different lesions and different RNA polymerase systems in this article.

Bull Soc Pathol Exot, 1999 Feb, 92(1), 6 - 8
{Prevalence of anti-Chlamydia pneumoniae antibodies in preadolescent children in Congo}; Kabeya BK et al.; Chlamydia pneumoniae (CPn) is recognised as a common cause of both upper and lower respiratory tract infections . Seroepidemiological studies seem to indicate a world-wide distribution of this organism . In order to evaluate the prevalence of antibodies to CPn in a healthy pediatric population, we measured anti-C . pneumoniae antibodies in a group of 253 infants without respiratory tract infections, aged from 1 to 12 years . Sera were obtained from children seen at immunization clinics and schools in Lubumbashi (Congo) . Antibodies to CPn were evaluated using micro-immunofluorescence assay . IgG antibody to CPn in a titre 16 was considered as positive . The antibody prevalence was found to be 25.7% . This prevalence was 6.2% in children aged from 1 to 6 years, and 37.8% in children aged from 7 to 12 years . It was less than 10% under five years, increasing to 50% at 12 years . The progressive increasing of seropositivity related to age suggests that reinfections may be frequent . This study shows an important spread of this bacteria in the preadolescent population of an African country.

Nihon Kokyuki Gakkai Zasshi, 1999 Feb, 37(2), 125 - 9
{Pulmonary nocardiosis associated with Cushing's syndrome}; Dohchin A et al.; A 54-year-old man was admitted for further investigation of multiple nodules disclosed by a chest roentgenogram . Adrenocorticotropic hormone (ACTH)-dependent Cushing's syndrome was diagnosed because serum ACTH and serum cortisol levels were elevated with a loss of diurnal rhythm . Because several extensive examinations, including inferior petrosal sinus sampling, did not detest ACTH-producing tumors, the patient was also given a diagnosis of occult ectopic ACTH syndrome . The nodules disclosed on chest roentgenograms increased gradually in size and number, and some were cavitary . Bronchial secretion samples obtained by fiberoptic bronchoscopy contained numerous Nocardia asteroides bacteria . After treatment with sulfamethoxazole-trimethoprim, the nodules gradually disappeared, leaving only scars . Although mitotane had been continuously administered to inhibit the synthesis of intrinsic corticosteroids, pulmonary nocardiosis relapsed in the patient following the termination of sulfamethoxazole-trimethoprim therapy.

Kansenshogaku Zasshi, 1999 Feb, 73(2), 172 - 8
{Evaluation of mycobacteria growth indicator tube (MGIT), an automated culture system for detection of mycobacteria from clinical specimens}; Kobayashi I et al.; We compared the Mycobacteria Growth Indicator Tube 960 (MGIT 960) and Ogawa medium (OM) for the detection of mycobacteria (acid fast bacteria: AFB) using 882 sputum specimens . Overall, 120 strains of AFB were isolated by the MGIT 960 system and 99 strains of AFB were isolated by using OM . As far as Mycobacterium tuberculosis is concerned, 88 and 71 isolates were achieved by the MGIT 960 and OM respectively . A total of 28 isolates (18 isolates of M . tuberculosis and 10 isolates of nontuberculous mycobacteria: NTM) were detected by the MGIT 960 only whereas only 2 isolates (1 M . tuberculosis and 1 NTM) were detected by OM only . Of these sputum specimens, 72 were smear positive for AFB . The rates of smear negative but culture positive specimens were 8.0% (65 out of 809) for the MGIT 960 system and 6.2% (50 out of 809) for OM . The contamination rate for MGIT 960 was only 1.2% . The average time required for detection of M . tuberculosis was 14.1 days by the MGIT 960 system and 24.6 days by OM . For the NTM, the average detection time were 8.3 days for the MGIT 960 system and 22.8 days for OM . These results indicate that the MGIT 960 system allows detection of mycobacteria significantly faster than OM.

Cytometry, 1999 Apr 1, 35(4), 346 - 52
Cytofluorometry and fluorescence confocal laser scanning microscopy (CLSM) in the study of neutral lipid dynamics in Paramecium primaurelia mating types during cell line development; Ramoino P et al.; BACKGROUND: In Paramecium primaurelia, an exconjugant cell can produce two lines with different mating capacities . Mating type II cells can form a higher food vacuole number and digest the nutrient taken up in a shorter time; thus, mating type II cells grow at a faster rate than do mating type I cells . The present study was done to determine whether cells that ingest more nutrients also have a larger amount of storage lipids . METHODS: Quantitative and qualitative determinations of neutral lipids were obtained by means of cytofluorometry and fluorescence confocal laser scanning microscopy (CLSM), respectively, by using nile red on cells in different physiologic states . RESULTS: Lipid droplet number and neutral lipid content were higher in mating type II cells than in mating type I cells in the early logarithmic growth phase (i.e., immature well-fed cells) . These values were reversed during the middle and the late logarithmic phases and became equal in the stationary phase (i.e., mature starved cells) . In well-fed cells maintained with food excess, differences in neutral lipid content between the two mating types also were present in mature cells . CONCLUSIONS: Although differences between mating type I and mating type II lines were not correlated to cell size, a relation was found between lipid content and food ingestion capacity . A depletion of bacteria in the culture medium could be responsible for the lack of differences in mature starved cells . CLSM allowed us to gather volume information about the lipid droplet distribution within the cell.

J Basic Clin Physiol Pharmacol, 1998, 9(2-4), 139 - 51
Novel aspects of the regulation of glycogen storage; Roach PJ et al.; The storage polysaccharide glycogen is widely distributed in nature, from bacteria to mammals . Study of its regulated accumulation has resulted in the discovery or elaboration of several important biochemical principles . Many aspects of the control of glycogen storage still remain poorly understood and glycogen metabolism continues to provide interesting models of more general relevance.

Histol Histopathol, 1999 Apr, 14(2), 517 - 24
Cytological and functional aspects of telomere maintenance; Dandjinou AT et al.; The fact that eukaryotic chromosomes are linear poses a special problem for their maintenance: the natural ends of chromosomes must be distinguished from ends generated by chromosomal breakage and somehow, the chromosome ends must also be fully replicated to maintain their integrity . Telomeres, the complex structures at the ends of chromosomes are thought to be instrumental for both of these functions . However, recent insights in telomere biology suggest that these terminal structures do much more than just fulfill these two basic functions . Cytological data demonstrate that telomeres may play leading roles in chromatin organization and nuclear architecture during mitosis and meiosis . Moreover, non-functional telomeres may lead to genetic instability, a common prelude to cancer . Here, we review the basic functions of telomeres during chromosome replication and discuss the cytological aspects of telomere function during mitosis and meiosis.

Riv Biol, 1998, 91(3), 425 - 57
Environmentally responsive mutator systems: toward a unifying perspective; Lieber MM; Biology has long sought a unifying principle . The behaviour of genetically controlled mutator processes adaptively responsive to stress may reflect such a principle . Related mutators exist in diverse organisms from bacteria to mammals . Such systems have evolved from one another and have defined the very evolution of organisms . Many of these mutator systems can determine in a developmental manner a ultramutability or hypermutability throughout the genome . Though the genetic control of high levels of mutability can reflect molecular features, such mutagenic processes reflect a deeper parameter involving forces and their configurations . These configurations must generate stable or uniform configurations from unstable ones throughout the genome and organism . Directed mutation becomes a generative process attuned to non-uniform forces of local niches and to the more uniform forces of a universal niche . The manner of mutagenic, attuned response depends on the level of genomic and transgenomic organization . This is reflected in hierarchies of evolution . Directed mutation is a feature of a universal, generative ordering process, and this feature is marked by a universal dimensionless constant . It is the dynamic consequence of such directed generation which ultimately confers adaptation through dynamic completion . This suggests an underlying, unitary, and necessary dynamics connecting ultramutability systems in all organisms and would serve, in complementation with a molecular approach, to elicit new and productive research avenues . One outcome would be the illustration of a unifying principle governing biological and physical phenomena.

J Biol Chem, 1999 Apr 30, 274(18), 12222 - 8
Pre-steady-state reaction of 5-aminolevulinate synthase . Evidence for a rate-determining product release; Hunter GA et al.; 5-Aminolevulinate synthase (ALAS) is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and the alpha-subclass of purple bacteria . The pyridoxal 5'-phosphate cofactor at the active site undergoes changes in absorptive properties during substrate binding and catalysis that have allowed us to study the kinetics of these reactions spectroscopically . Rapid scanning stopped-flow experiments of murine erythroid 5-aminolevulinate synthase demonstrate that reaction with glycine plus succinyl-CoA results in a pre-steady-state burst of quinonoid intermediate formation . Thus, a step following binding of substrates and initial quinonoid intermediate formation is rate-determining . The steady-state spectrum of the enzyme is similar to that formed in the presence of 5-aminolevulinate, suggesting that release of this product limits the overall rate . Reaction of either glycine or 5-aminolevulinate with ALAS is slow (kf = 0.15 s-1) and approximates kcat . The rate constant for reaction with glycine is increased at least 90-fold in the presence of succinyl-CoA and most likely represents a slow conformational change of the enzyme that is accelerated by succinyl-CoA . The slow rate of reaction of 5-aminolevulinate with ALAS is 5-aminolevulinate-independent, suggesting that it also represents a slow isomerization of the enzyme . Reaction of succinyl-CoA with the enzyme-glycine complex to form a quinonoid intermediate is a biphasic process and may be irreversible . Taken together, the data suggest that turnover is limited by release of 5-aminolevulinate or a conformational change associated with 5-aminolevulinate release.

J Med Chem, 1999 Apr 22, 42(8), 1459 - 65
Antineoplastic agents . 410 . Asymmetric hydroxylation of trans-combretastatin A-4; Pettit GR et al.; The South African willow tree Combretum caffrum has yielded a number of potent cancer cell growth inhibitors . The present SAR studies of the antineoplastic agent combretastatin A-4 (1c) were focused mainly on the olefinic bridge to determine the effects on cancer cell growth and, potentially, to better define the combretastatin A-4 binding site on tubulin . The geometric trans-isomer 3a of combretastatin A-4 was converted to the (1S,2S)- and (1R,2R)-vicinal diols 4c and 4d, respectively, under Sharpless' asymmetric dihydroxylation conditions . Cancer cell line testing showed the (1S, 2S)-diol 4c to be more potent than its enantiomer 4d . Diol 4c weakly inhibited tubulin polymerization (IC50 = 22 microM, versus 1.2 microM for combretastatin A-4), while 4d was inactive (IC50 > 40 microM) . Esterification of either stereoisomer at the diol and/or phenolic positions resulted in elimination of inhibitory activity.

Aliment Pharmacol Ther, 1999 Mar, 13 Suppl 1, 13 - 8
Review article: the role of inflammation in the pathogenesis of gastric cancer; Ernst P; Helicobacter pylori induces infiltration of the gastric mucosa by polymorphonuclear cells and macrophages, as well as T and B lymphocytes . Paradoxically, this robust immune/inflammatory response cannot clear the infection, and thus leaves the host prone to complications resulting from chronic inflammation . One adverse consequence of this inflammatory response may be gastric cancer, as inflammation has been implicated in the development of intestinal metaplasia and mutations in oncogenes that precede the development of gastric adenocarcinoma . The gastric inflammatory response is affected somewhat, by the strain of H . pylori that infects the host . Thus, the more severe clinical manifestation associated with some strains may be attributed to the higher grade of inflammation that they induce . Both H . pylori and cytokines induced during infection can stimulate the recruitment and activation of inflammatory cells including neutrophils and macrophages . When activated, these cells produce inflammatory mediators that include reactive oxygen species (ROS) . These mediators impart an oxidative stress on the cells in the immediate vicinity, in this case, the gastric epithelium . Normally, oxidative stress is neutralized by natural antioxidants such as vitamin C, however, levels of this antioxidant in the gastric juice are decreased during infection . The increased levels of oxidants and decreased antioxidants create a stress that can change many processes in the gastric epithelium . For example, an accumulation of intracellular ROS regulates the expression of many genes and can induce DNA damage . Point mutations in the DNA that disrupt the expression and function of genes that inhibit cell growth (i.e . p53) are believed to contribute to the pathogenesis of gastric cancer . Several studies suggest that epithelial cell turnover is affected by the inflammatory response to H . pylori . This notion is supported by studies describing an increase in both epithelial cell proliferation, as well as cell death by apoptosis, in response to infection . Apoptosis is a regulated process of cell death that is triggered by H . pylori as well as various inflammatory mediators, including tumour necrosis factor and interferon-gamma . Activated T-cells also kill gastric epithelial cells directly . Moreover, the host response increases the expression of receptors for H . pylori and thus increases bacterial binding and the induction of apoptosis by the bacteria . There are several other immune/inflammatory responses that contribute to epithelial cell damage mucosa and the pathogenesis of gastric cancer . For example, gastric B cells produce autoreactive antibodies that bind to gastric epithelial cells . As a consequence of this antigen-antibody complex formation, complement becomes activated suggesting that some of the inflammation and epithelial cell damage is attributable to immune-complex formation . Epithelial cell death can then stimulate the proliferative response of epithelial cell precursors . In summary, the proposed model may explain how the gastric inflammatory response contributes to the pathogenesis of cancer . This model raises the possibility that it could be preferable to identify the patients at highest risk of developing gastric cancer and then apply an intervention that eliminates the infection and inflammatory response . Alternatively, clinical interventions should at least attenuate the oxidative stress that is directly attributed to inflammation . These mechanisms have to be examined in the paediatric population.

Int Rev Cytol, 1999, 188, 203 - 55
Roles of reactive oxygen species: signaling and regulation of cellular functions; Gamaley IA et al.; Reactive oxygen species (ROS) are the side products (H2O2, O2.-, and OH.) of general metabolism and are also produced specifically by the NADPH oxidase system in most cell types . Cells have a very efficient antioxidant defense to counteract the toxic effect of ROS . The physiological significance of ROS is that ROS at low concentrations are able to mediate cellular functions through the same steps of intracellular signaling, which are activated by natural stimuli . Moreover, a variety of natural stimuli act through the intracellular formation of ROS that change the intracellular redox state (oxidation-reduction) . Thus, the redox state is a part of intracellular signaling . As such, ROS are now considered signal molecules at nontoxic concentrations . Progress has been achieved in studying the oxidative activation of gene transcription in animal cells and bacteria . Changes in the redox state of intracellular thiols are considered to be an important mechanism that regulates cell functions.

Novartis Found Symp, 1999, 221, 152 - 63; discussion 163-6
pH homeostasis in acidophiles; Matin A; The acidophilic bacteria comprise an environmentally important group that includes pathogens . A fundamental requirement for existence in strongly acidic environments is generation of a large pH gradient (delta pH) to maintain the cytoplasmic pH near neutrality and safeguard the acid-labile cell constituents . These organisms require the capacity to extrude H+ across the thermodynamic barrier imposed by the delta pH . They also need special transport proteins that can function in strongly acidic environments . The quintessential device by which the acidophiles fulfil these requirements by generating a positive-inside membrane potential (delta psi) that mitigates both the force against which H+ must be extruded, as well as the influxing force of protons into the cells . The delta psi is generated through passive as well as active mechanisms . The former constitutes an H+ diffusion as well as a Donnan potential, and the latter involves electrogenic circulation of Cl-, and the operation of an electrogenic H+/K+ (Na+) antiporter.

J Biol Chem, 1999 Apr 23, 274(17), 11995 - 2000
Adhesion of cultured bovine aortic endothelial cells to laminin-1 mediated by dystroglycan; Shimizu H et al.; Expression of dystroglycan (DG) by cultured bovine aortic endothelial (BAE) cells was confirmed by cDNA cloning from a BAE cDNA library, Northern blotting of mRNA, Western blotting of membrane proteins, and double immunostaining with antibodies against betaDG and platelet endothelial cell adhesion molecule-1 . Immunocytochemical analysis revealed localization of DG in multiple plaques on the basal side of resting cells . This patchy distribution was obscured in migrating cells, in which the most prominent staining was observed in the trailing edge anchoring the cells to the substratum . Biotin-labeled laminin-1 overlay assay of dissociated BAE membrane proteins indicated the interaction of laminin-1 with alphaDG . The laminin alpha5 globular domain fragment expressed in bacteria and labeled with biotin could also bind alphaDG on the membrane blot, and the unlabeled fragment disrupted the binding of biotin-laminin-1 to alphaDG . The interaction of biotin-laminin-1 with alphaDG was inhibited by soluble alphaDG contained in the conditioned medium from DG cDNA-transfected BAE cells and by a series of glycosaminoglycans (heparin, dextran sulfate, and fucoidan) . Soluble alphaDG in the conditioned medium inhibited the adhesion of BAE cells to laminin-1-coated dishes, whereas it had no effect on their adhesion to fibronectin . All three glycosaminoglycans that disrupted the biotin-laminin-1 binding to alphaDG inhibited BAE cell adhesion to laminin-1, whereas they failed to inhibit the adhesion to fibronectin . These results indicate a role of DG as a non-integrin laminin receptor involved in vascular endothelial cell adhesion to the extracellular matrix.

Int J Tuberc Lung Dis, 1999 Apr, 3(4), 354 - 7
Application of a computer-directed automated microscope in mycobacteriology; Somoskovi A et al.; Microscopy is currently the fastest, cheapest and most easily performed technique in mycobacteriology; it can be used in any laboratory . However, the sensitivity of microscopy is unsatisfactory and it is time-consuming . To eliminate these drawbacks, we have constructed a computer-directed automated microscope . To evaluate the equipment, we examined a total of 132 smears of sputum and 74 smears of liquid media . Manual microscopy was positive for 53 and negative for 79 sputum smears, while automated microscopy was positive for 55 and negative for 77 sputum smears . Both methods furnished 50 positive and 24 negative smears of liquid media . We conclude that the automated microscope is able to detect acid-fast bacteria, the examination procedure with the instrument is more rapid (1.8-3.5 min/slide) and it is always possible to follow the standard recommendations of microscopy.

Vet Immunol Immunopathol, 1999 Mar 1, 67(4), 327 - 40
Production and functional characterization of recombinant bovine interleukin-8 as a specific neutrophil activator and chemoattractant; Caswell JL et al.; Interleukin-8, a member of the chemokine family of cytokines, is a potent neutrophil chemoattractant in many non-rodent species . In this study, recombinant bovine interleukin-8 (rbIL-8) was expressed in bacteria as a glutathione-S-transferase fusion protein . The fusion protein was purified by glutathione-Sepharose affinity chromatography and recombinant rbIL-8 was eluted by cleaving with thrombin . The purified rbIL-8 molecule was approximately 8 kDa and was confirmed as authentic IL-8 by Western analysis . Recombinant bovine IL-8 induced specific dose-dependent in vitro chemotaxis of neutrophils at doses as low as 1.0 ng/ml, and this activity was inhibited by pre-treatment of rbIL-8 with a monoclonal antibody to ovine IL-8 . Neutrophils exposed to rbIL-8 developed pseudopodia and became elongated as determined by microscopic analysis and flow cytometry . Injection of 3.3 ng to 3.3 microg of rbIL-8 into the skin of a normal calf induced dose-dependent recruitment of neutrophils but not eosinophils . Intravascular margination of neutrophils was obvious at the injection sites from 15 to 60 min after administration of rbIL-8, and extravascular neutrophil numbers increased steadily from 1 to 18 h after injection . Neutrophils with morphologic features of apoptosis were detected in these lesions at 18 and 30 h after injection, and this correlated with reduction in the number of dermal neutrophils . These results confirm unequivocally that bovine IL-8 functions as a neutrophil, but not an eosinophil, chemoattractant in vitro and in vivo.

Farmaco, 1998 Oct-Nov, 53(10-11), 709 - 17
Synthesis and activity of cephalosporins containing an oxyiminomethylene functionality in the ortho-position of a phenyl- or phenoxyacetic acid C-7 side chain substituent; Albanese D et al.; A new series of cephalosporins having in the C-7 side chain a phenyl- or a phenoxyacetamido group bearing an oxyiminomethyl function in the ortho-position of the aromatic ring was prepared . Their in vitro activity was tested against both Gram+ and Gram- strains.

Prof Nurse, 1999 Feb, 14(5), 329 - 33
Larval therapy in wound debridement; Rayner K; The use of larvae (maggots) in wound management was popular in the 1930s . Now the advent of multi-resistant strains of bacteria has led to its reintroduction in some hospitals, when other avenues have been exhausted.

Eur J Med Res, 1999 Apr 27, 4(4), 161 - 4
Familial Mediterranean fever . No role of Mycobacterium tuberculosis in ten patients; Akcan Y et al.; BACKGROUND: Tuberculosis (TB) and Familial mediterranean fever (FMF) are two common diseases in our region, Turkey . Both share some properties in common: Both cause AA type amyloidosis and have association with some immunological abnormalities . Upon incidentally observing Mycobacterium tuberculosis in bone marrow biopsies of three patients with FMF in a previous study, we intended to elucidate this association prospectively . MATERIAL AND METHODS: In this study, we examined prospectively 10 FMF patients, 5 male and 5 female, with a median duration of 31 years disease activity . All were under colchicine therapy . They had no sign of renal involvement . The bone marrow biopsies of these patients were examined for the presence of M . tuberculosis by Polymerase chain reaction (PCR), BACTEC culture and pathological stains . Pathological examination was performed for the existence of granuloma and amyloid deposition by hematoxylin-eosin, Crystal Violet and Congo red stains . RESULTS: The examination of all bone marrow specimens by the mentioned methods suggest that Mycobacterium tuberculosis has no role in the ethiopathogenesis of FMF . Although the patients had a positive family history of 60% for tuberculosis and in 80% of them with positive tuberculin skin test . CONCLUSIONS: We concluded that although there seemed to be a kind of association between both diseases, this relationship is not via the direct existence of bacteria itself . Considering high family history and skin test positivity, one should look for the presence of autoimmune mechanisms under this suspicious relationship between tuberculosis and FMF . Also, this is the first study examined the state of amyloidosis in the bone marrow at an earlier stage of FMF without overt renal findings.

Gut, 1999 May, 44(5), 643 - 52
Mast cells are an important cellular source of tumour necrosis factor alpha in human intestinal tissue; Bischoff SC et al.; BACKGROUND: Several inflammatory disorders of the intestine are characterised by enhanced expression of tumour necrosis factor alpha (TNF-alpha) . Monocytes and macrophages have been suggested as a major cellular source of TNF-alpha in human gut, whereas mast cells, although known to be capable of producing TNF-alpha, have been poorly examined in this respect . AIMS: To investigate whether human intestinal mast cells can produce TNF-alpha, and which factors regulate TNF-alpha production in these cells . METHODS: Mast cells were isolated from surgery tissue specimens of patients undergoing bowel resection because of cancer . Immunohistochemical studies were performed in biopsy specimens derived from 13 patients (two healthy controls, four with Crohn's disease, four with ulcerative colitis, three others) . TNF-alpha mRNA and protein expression were studied in vitro by polymerase chain reaction, RNAse protection assay, western blot, and enzyme linked immunosorbent assay in isolated purified human intestinal mast cells stimulated by IgE receptor crosslinking, intestinal bacteria, and lipopolysaccharide . Cellular localisation of TNF-alpha was examined by immunohistochemistry . RESULTS: TNF-alpha mRNA and protein were expressed constitutively in isolated human intestinal mast cells . Expression of TNF-alpha mRNA and release of TNF-alpha protein were substantially enhanced by IgE receptor crosslinking and by coculture of mast cells with intestinal bacteria; lipopolysaccharide had only marginal effects . Immunohistochemical studies revealed that approximately 60% of the lamina propria cells with immunoreactivity for TNF-alpha were mast cells . CONCLUSIONS: The data show that mast cells are an important source of TNF-alpha in the human intestinal mucosa.

EMBO J, 1999 Apr 15, 18(8), 2284 - 93
Dynamics and efficiency in vivo of UGA-directed selenocysteine insertion at the ribosome; Suppmann S et al.; The kinetics and efficiency of decoding of the UGA of a bacterial selenoprotein mRNA with selenocysteine has been studied in vivo . A gst-lacZ fusion, with the fdhF SECIS element ligated between the two fusion partners, gave an efficiency of read-through of 4-5%; overproduction of the selenocysteine insertion machinery increased it to 7-10% . This low efficiency is caused by termination at the UGA and not by translational barriers at the SECIS . When the selenocysteine UGA codon was replaced by UCA, and tRNASec with anticodon UGA was allowed to compete with seryl-tRNASer1 for this codon, selenocysteine was found in 7% of the protein produced . When a non-cognate SelB-tRNASec complex competed with EF-Tu for a sense codon, no effects were seen, whereas a non-cognate SelB-tRNASec competing with EF-Tu-mediated Su7-tRNA nonsense suppression of UGA interfered strongly with suppression . The induction kinetics of beta-galactosidase synthesis from fdhF'-'lacZ gene fusions in the absence or presence of SelB and/or the SECIS element, showed that there was a translational pause in the fusion containing the SECIS when SelB was present . The results show that decoding of UGA is an inefficient process and that using the third dimension of the mRNA to accommodate an additional amino acid is accompanied by considerable quantitative and kinetic costs.

Oral Microbiol Immunol, 1999 Feb, 14(1), 49 - 55
Adherence of Peptostreptococcus micros morphotypes to epithelial cells in vitro; Kremer BH et al.; Peptostreptococcus micros, which is associated with oral and non-oral mixed anaerobic infections, occurs in three colony morphotypes, the smooth type, the rough type and the smooth variant of the rough type . These types differ in surface structures; the rough type expresses large fibrillar surface appendages, which are absent on the surface of both the smooth and the smooth variant of the rough type . To determine the role of these surface structures in adherence we characterized the adherence of the three morphotypes of P . micros to epithelial cells in vitro . Although all three types adhered well to epithelial cells, adhering numbers of the rough type were significantly lower than those of the smooth and the smooth variant of the rough type . Protease treatment increased the adherence of the rough type of the level of the two other types . The adherence of all three types was reduced more than 85% by treatment with 10 mM sodium periodate . Furthermore, the adherence was pH independent and could not be blocked by incubation with antisera to the bacteria . In addition, we determined the capacity to invade epithelial cells by P . micros . In an acridine orange assay such invasion could not be detected . Our results suggest that the adherence of P . micros to epithelial cells is mediated by periodate-sensitive extracellular polysaccharides and that the protruding fibril-like protein surface structures of the rough type have an obstructive effect on the adherence.

J Clin Microbiol, 1999 May, 37(5), 1426 - 30
Distribution of Porphyromonas gingivalis strains with fimA genotypes in periodontitis patients; Amano A et al.; Fimbriae (FimA) of Porphyromonas gingivalis are filamentous components on the cell surface and are thought to play an important role in the colonization and invasion of periodontal tissues . We previously demonstrated that fimA can be classified into four variants (types I to IV) on the basis of the nucleotide sequences of the fimA gene . In the present study, we attempted to detect the four different fimA genes in saliva and plaque samples isolated from patients with periodontitis using the PCR method . Four sets of fimA type-specific primers were designed for the PCR assay . These primers selectively amplified 392-bp (type I), 257-bp (type II), 247-bp (type III), and 251-bp (type IV) DNA fragments of the fimA gene . Positive PCR results were observed with reference strains of P . gingivalis in a type-specific manner . All other laboratory strains of oral and nonoral bacteria gave negative results . The sensitivity of the PCR assay for fimA type-specific detection was between 5 and 50 cells of P . gingivalis . Clinical samples were obtained from saliva and subgingival plaque from deep pockets (>/=4 mm) of 93 patients with periodontitis . Bacterial genomic DNA was isolated from the samples, and the targeted fragments were amplified by PCR . The presence of P . gingivalis was demonstrated in 73 patients (78.5%), and a single fimA gene was detected in most patients . The distribution of the four fimA types among the P . gingivalis-positive patients was as follows: type I, 5.4%; type II, 58.9%; type III, 6 . 8%; type IV, 12.3%; types I and II, 6.8%; types II and IV, 2.7%; and untypeable, 6.8% . P . gingivalis with type II fimA was detected more frequently in the deeper pockets, and a significant difference of the occurrence was observed between shallow (4 mm) and deep (>/=8 mm) pockets . These results suggest that P . gingivalis strains that possess type II fimA are significantly more predominant in periodontitis patients, and we speculate that these organisms are involved in the destructive progression of periodontal diseases.

J Clin Microbiol, 1999 May, 37(5), 1254 - 9
Prevalence of Mycobacterium avium in slaughter pigs in The Netherlands and comparison of IS1245 restriction fragment length polymorphism patterns of porcine and human isolates; Komijn RE et al.; A significant increase in the incidence of caseous lesions in the lymph nodes of slaughter pigs prompted a large-scale investigation in five slaughterhouses in The Netherlands . In total, 158,763 pigs from 2,899 groups underwent gross examination . At least one pig with caseous lesions in the submaxillary and/or mesenteric lymph nodes was observed in each of 154 of the 2,899 groups examined (5%) . In total, 856 pigs (0.5%) were affected . As many as five pigs in each of 141 of the 154 positive groups (91.5%) had lymph node lesions . Greater numbers of pigs with affected lymph nodes were found in 13 groups (8.5%) . Four pigs had lesions in the kidneys, liver, or spleen . Acid-fast bacteria were detected by microscopic examination of 121 of 292 Ziehl-Neelsen-stained smears of caseous lesions (41%) . In a follow-up study, Mycobacterium avium complex (MAC) bacteria were isolated from 219 of 402 affected lymph nodes (54.2%) . Ninety-one of the isolated strains were analyzed by restriction fragment length polymorphism (RFLP) typing with insertion sequence IS1245 as a probe . All but 1 of these 91 strains contained IS1245 DNA, indicating that pigs in The Netherlands carried almost exclusively M . avium bacteria and no other bacteria of MAC . Only one pig isolate exhibited the bird-type RFLP pattern . MAC isolates from 191 human patients in The Netherlands in 1996 were also typed by RFLP analysis . Computer-assisted analysis showed that the RFLP patterns of 61% of the human isolates and 59% of the porcine isolates were at least 75% similar to the RFLP patterns of the other group of strains . This indicates that pigs may be an important vehicle for M . avium infections in humans or that pigs and humans share common sources of infection.

Can J Gastroenterol, 1999 Mar, 13(2), 147 - 51
Changing parenteral nutrition administration sets every 24 h versus every 48 h in newborn infants; Fox M et al.; OBJECTIVE: To determine whether changing total parenteral nutrition fluid administration sets (TAS) every 48 h rather than every 24 h results in a greater infusate contamination rate . PATIENTS AND METHODS: Prospectively, 166 infants were assigned at random to have TAS changed either every 24 h or every 48 h . Samples of the infusate were cultured to determine contamination rates of the infusate in the sets and were tested from 149 of these infants . TAS was replaced every 24 h in the control group, and 445 amino acid plus dextrose solutions (AADS) and 449 lipid emulsions samples were taken for bacterial culture . Fungal cultures were also performed on 449 samples . The study group had TAS replaced every 48 h, and 454 samples of AADS were cultured for bacteria . The numbers of lipid emulsion samples sent for bacterial culture and fungal culture were 449 and 440, respectively . Information on type of intravenous access device, administration of antibiotics and blood cultures was also collected . RESULTS: There was no difference in bacterial contamination rates for AADS or lipid emulsion from TAS changed every 24 or 48 h (c2, P>0.05) . Lipid emulsion sampled from the 24 h group showed a statistically significant higher rate of fungal contamination than specimens from the 48 h group (P<0.01) . CONCLUSIONS: Changing TAS every 48 h versus 24 h does not increase the contamination rate of infusate in newborns.

Vet Clin North Am Small Anim Pract, 1999 Mar, 29(2), 471 - 500, vi-vii
Gastrointestinal immunity in health and disease; Elwood CM et al.; The gastrointestinal lymphoid tissue (GALT) is under constant antigenic challenge from bacteria and food and must be able to distinguish between benign and pathogenic organisms . Recent advances in understanding the organization and function of GALT reveal how it is able to direct appropriate immune responses according to the nature of the antigen and how inappropriate immune responses can lead to local and systemic immunopathology and/or infection . The interaction of the normal bowel flora and GALT is critical to normal local and systemic immune function and plays a major role in the pathogenesis of some immune-related diseases . This review draws upon information from veterinary, human, and laboratory animal studies to provide an update of mechanisms and consequences of function and dysfunction in the gastrointestinal immune system.

Bioorg Med Chem Lett, 1999 Mar 8, 9(5), 781 - 6
Synthesis and electrochemical study of a new chiral tris-catecholamide analogue of enterobactin; Cheraiti N et al.; The comparison of siderophore complex redox potentials with those of physiological reductants may aid in the clarification of the mechanism of iron metabolism . In this paper, a new chiral tris-catecholamide compound N,N',N''-tris-(2,3-dihydroxybenzoyl)-1,1,1-tris-(L-methioninemethyl++ +)-ethane or H6L (11) has been synthesised in nine steps, and may mimic the release of iron from enterobactin to the agents which are directly involved in cell metabolism . The choice of methionine as a constituent of the siderophore incorporates divalent sulphur which leads to the increase of the reduction potential of the siderophore, and consequently facilitates the iron release {Fe(III)/Fe(II) redox potential E(1/2)=-0.749 V vs (SCE)}.

Mol Microbiol, 1999 Mar, 31(5), 1295 - 305
Transcription initiation in Archaea: facts, factors and future aspects; Soppa J; The basal apparatus for transcription initiation in Archaea is more closely related to the eukaryal than to the bacterial counterpart . The understanding of archaeal transcription initiation has been deepened by recent advances, which include genome sequencing, biochemical approaches and the structure determination of a protein DNA complex . Archaeal promoter elements, transcription factors, RNA polymerase and their interactions are discussed and compared with the eukaryal situation . It is emerging that transcription initiation is not uniform in Archaea . A minimal set of promoter elements and transcription factors is conserved, but the relative importance for transcription initiation can vary . Furthermore, additional basal transcription factors and promoter elements seem to be crucial in subgroups of Archaea . Finally, some aspects of global as well as gene-specific transcriptional regulation are discussed.

Proc Natl Acad Sci U S A, 1999 Apr 13, 96(8), 4615 - 20
A corrinoid-dependent catabolic pathway for growth of a Methylobacterium strain with chloromethane; Vannelli T et al.; Methylobacterium sp . strain CM4, an aerobic methylotrophic alpha-proteobacterium, is able to grow with chloromethane as a carbon and energy source . Mutants of this strain that still grew with methanol, methylamine, or formate, but were unable to grow with chloromethane, were previously obtained by miniTn5 mutagenesis . The transposon insertion sites in six of these mutants mapped to two distinct DNA fragments . The sequences of these fragments, which extended over more than 17 kb, were determined . Sequence analysis, mutant properties, and measurements of enzyme activity in cell-free extracts allowed the definition of a multistep pathway for the conversion of chloromethane to formate . The methyl group of chloromethane is first transferred by the protein CmuA (cmu: chloromethane utilization) to a corrinoid protein, from where it is transferred to H4folate by CmuB . Both CmuA and CmuB display sequence similarity to methyltransferases of methanogenic archaea . In its C-terminal part, CmuA is also very similar to corrinoid-binding proteins, indicating that it is a bifunctional protein consisting of two domains that are expressed as separate polypeptides in methyl transfer systems of methanogens . The methyl group derived from chloromethane is then processed by means of pterine-linked intermediates to formate by a pathway that appears to be distinct from those already described in Methylobacterium . Remarkable features of this pathway for the catabolism of chloromethane thus include the involvement of a corrinoid-dependent methyltransferase system for dehalogenation in an aerobe and a set of enzymes specifically involved in funneling the C1 moiety derived from chloromethane into central metabolism.

Proc Natl Acad Sci U S A, 1999 Apr 13, 96(8), 4348 - 53
The membrane-attached electron carrier cytochrome cy from Rhodobacter sphaeroides is functional in respiratory but not in photosynthetic electron transfer; Myllykallio H et al.; Rhodobacter species are useful model organisms for studying the structure and function of c type cytochromes (Cyt c), which are ubiquitous electron carriers with essential functions in cellular energy and signal transduction . Among these species, Rhodobacter capsulatus has a periplasmic Cyt c2Rc and a membrane-bound bipartite Cyt cyRc . These electron carriers participate in both respiratory and photosynthetic electron-transfer chains . On the other hand, until recently, Rhodobacter sphaeroides was thought to have only one of these two cytochromes, the soluble Cyt c2Rs . Recent work indicated that this species has a gene, cycYRs, that is highly homologous to cycYRc, and in the work presented here, functional properties of its gene product (Cyt cyRs) are defined . It was found that Cyt cyRs is unable to participate in photosynthetic electron transfer, although it is active in respiratory electron transfer, unlike its R . capsulatus counterpart, Cyt cyRc . Chimeric constructs have shown that the photosynthetic incapability of Cyt cyRs is caused, at least in part, by its redox active subdomain, which carries the covalently bound heme . It, therefore, seems that this domain interacts differently with distinct redox partners, like the photochemical reaction center and the Cyt c oxidase, and allows the bacteria to funnel electrons efficiently to various destinations under different growth conditions . These findings raise an intriguing evolutionary issue in regard to cellular apoptosis: why do the mitochondria of higher organisms, unlike their bacterial ancestors, use only one soluble electron carrier in their respiratory electron-transport chains?

RNA, 1999 Apr, 5(4), 562 - 73
Nonsense mutations in the alcohol dehydrogenase gene of Drosophila melanogaster correlate with an abnormal 3' end processing of the corresponding pre-mRNA; Brogna S; From bacteria to mammals, mutations that generate premature termination codons have been shown to result in the reduction in the abundance of the corresponding mRNA . In mammalian cells, more often than not, the reduction happens while the RNA is still associated with the nucleus . Here, it is reported that mutations in the alcohol dehydrogenase gene (Adh) of Drosophila melanogaster that generate premature termination codons lead to reduced levels of cytoplasmic and nuclear mRNA . Unexpectedly, it has been found that the poly(A) tails of Adh mRNAs and pre-mRNAs that carry a premature termination codon are longer than in the wild-type transcript . The more 5' terminal the mutation is, the longer is the poly(A) tail of the transcript . These findings suggest that the integrity of the coding region may be required for accurate mRNA 3' end processing.

Cell, 1999 Apr 2, 97(1), 75 - 84
Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism; Velankar SS et al.; We have determined two different structures of PcrA DNA helicase complexed with the same single strand tailed DNA duplex, providing snapshots of different steps on the catalytic pathway . One of the structures is of a complex with a nonhydrolyzable analog of ATP and is thus a "substrate" complex . The other structure contains a bound sulphate ion that sits in a position equivalent to that occupied by the phosphate ion produced after ATP hydrolysis, thereby mimicking a "product" complex . In both complexes, the protein is monomeric . Large and distinct conformational changes occur on binding DNA and the nucleotide cofactor . Taken together, these structures provide evidence against an "active rolling" model for helicase action but are instead consistent with an "inchworm" mechanism.

Probl Tuberk, 1999, (1), 30 - 1
{Results of polyresistant tuberculosis treatment: data of the Santarishkes Republican tuberculous hospital}; Mishkinis K et al.; In 1994-1996 the Santarishkes republican tuberculous hospital (Lithuania) has admitted 98 patients (82 males, 16 females) with polyresistant tuberculosis (M . tuberculosis were resistant to at least isoniazid and rifampicin) . Of these, 13 patients were untreated, 17 had recurrences, 68 had chronic tuberculosis . After polyresistance of M . tuberculosis was found, the patients received an individual treatment with sensitive drugs . 20 patients were operated . The absence of bacteria was achieved only in 24 (24.5%) patients . Tuberculous lesions in the lungs disappeared and clinical symptoms relieved in 14 of them . In 74(75.5%) nonresponders the course of the disease was unfavourable . Negative treatment outcomes were observed in 38.5%, 64.7% and 85.3% of new-onset, recurrent and chronic cases, respectively.

Probl Tuberk, 1999, (1), 12 - 3
{Analysis of tuberculosis morbidity in the southeastern district of Moscow}; Kovaleva SI et al.; The present-day poor epidemiological conditions are marked by an increase in tuberculosis detection rates (1.43%) with low coverage (28.0%) of the population with preventive fluorographic surveys and a high proportion (39.0%) of young patients (aged 18-39 years) among new cases . The shares of advanced and acute forms of tuberculosis were 7.2 and 11.4%, respectively . A decay phase was detected in 50.6%, 52.6% of patients isolated bacteria . In children, tuberculosis morbidity increased by 28.6% mainly among the unregistered extrafamilial contacts . Most patients (75.8%) detected upon their referral to the polyclinics and general hospitals had a high proportion of extrapulmonary tuberculosis . Thus, the above trends of morbidity show its high potential level.

Br J Gen Pract, 1998 Nov, 48(436), 1751 - 4
Why patients consult when they cough: a comparison of consulting and non-consulting patients; Cornford CS; BACKGROUND: Although it is the commonest symptom presented to general practitioners (GPs), little is known about why someone decides to consult with a cough . AIM: To describe the illness behaviour of patients with a cough . METHOD: Patients who had consulted a GP because of a cough, and a group of subjects who had recently had a cough but had not consulted, were interviewed in a qualitative study that investigated how they made sense of their illness . RESULTS: Consulting patients understood their cough to be abnormally severe, whereas non-consulting subjects regarded their cough as 'normal' and mild . Consulting patients thought the cough would interfere with social roles and non-consulting subjects did not . The consulting patients were much more likely to be worried about the cough than the non-consulting subjects . In particular, half of the consulting patients were worried about their hearts, whereas the non-consulting subjects were not . The two groups did not distinguish bacteria from viruses, and did not differ in beliefs about the role of antibiotics that they thought were needed for severe coughs . Both groups had concerns about pollution . CONCLUSIONS: For consulting patients, cough breached the taken for granted property' of health that the non-consulting subjects with a cough were able to maintain . Cough, for the consulting patients, was not a trivial illness.

Am J Physiol, 1999 Apr, 276(4 Pt 1), L650 - 8
Effects of endotoxin on surfactant protein A and D stimulation of NO production by alveolar macrophages; Wright JR et al.; Surfactant protein (SP) A and SP-D affect numerous functions of immune cells including enhancing phagocytosis of bacteria and production of reactive species . Previous studies have shown that SP-A and SP-D bind to a variety of bacteria and to the lipopolysaccharide (LPS) components of their cell walls . In addition, purified preparations of SPs often contain endotoxin . The goals of this study were 1) to evaluate the effects of SP-A and SP-D and complexes of SPs and LPS on the production of nitric oxide metabolites by rat alveolar macrophages and 2) to evaluate methods for the removal of endotoxin with optimal recovery of SP . Incubation of SP-A or SP-D with polymyxin, 100 mM N-octyl-beta-D-glucopyranoside, and 2 mM EDTA followed by dialysis was the most effective method of those tested for reducing endotoxin levels . Commonly used storage buffers for SP-D, but not for SP-A, inhibited the detection of endotoxin . There was a correlation between the endotoxin content of the SP-A and SP-D preparations and their ability to stimulate production of nitrite by alveolar macrophages . SP-A and SP-D treated as described above to remove endotoxin did not stimulate nitrite production . These studies suggest that the functions of SP-A and SP-D are affected by endotoxin and illustrate the importance of monitoring SP preparations for endotoxin contamination.

Genomics, 1999 Apr 15, 57(2), 306 - 9
A 1.5-Mb contig within the cat eye syndrome critical region at human chromosome 22q11.2; Johnson A et al.; We have constructed a 1.5-Mb contig spanning the distal half of the critical region for cat eye syndrome on human chromosome 22 from D22S543 to D22S181 . The contig consists of 20 P1 artificial chromosome (PAC) clones and 11 bacterial artificial chromosome (BAC) clones screened from 2 BAC and 2 PAC libraries . Continuous overlap between the clones was confirmed using vectorette PCR and riboprobes . Despite the instability of this region in a previous YAC contig, only 1 BAC showed a minor instability and then in only one isolation . This contig is now providing the basis for genomic sequencing and gene identification in the cat eye syndrome critical region .

Plant Physiol, 1999 Apr, 119(4), 1483 - 96
Oxidative turnover of soybean root glutamine synthetase . In vitro and in vivo studies
Ortega JL, Roche D, Sengupta-Gopalan C.
Glutamine synthetase (GS) is the key enzyme in ammonia assimilation and catalyzes the ATP-dependent condensation of NH3 with glutamate to produce glutamine . GS in plants is an octameric enzyme . Recent work from our laboratory suggests that GS activity in plants may be regulated at the level of protein turnover (S.J . Temple, T.J . Knight, P.J . Unkefer, C . Sengupta-Gopalan {1993} Mol Gen Genet 236: 315-325; S.J . Temple, S . Kunjibettu, D . Roche, C . Sengupta-Gopalan {1996} Plant Physiol 112: 1723-1733; S.J . Temple, C . Sengupta-Gopalan {1997} In C.H . Foyer, W.P . Quick, eds, A Molecular Approach to Primary Metabolism in Higher Plants . Taylor & Francis, London, pp 155-177) . Oxidative modification of GS has been implicated as the first step in the turnover of GS in bacteria . By incubating soybean (Glycine max) root extract enriched in GS in a metal-catalyzed oxidation system to produce the.OH radical, we have shown that GS is oxidized and that oxidized GS is inactive and more susceptible to degradation than nonoxidized GS . Histidine and cysteine protect GS from metal-catalyzed inactivation, indicating that oxidation modifies the GS active site and that cysteine and histidine residues are the site of modification . Similarly, ATP and particularly ATP/glutamate give the enzyme the greatest protection against oxidative inactivation . The roots of plants fed ammonium nitrate showed a 3-fold increase in the level of GS polypeptides and activity compared with plants not fed ammonium nitrate but without a corresponding increase in the GS transcript level . This would suggest either translational or posttranslational control of GS levels.

J Bacteriol, 1999 Apr, 181(8), 2655 - 8
Azorhizobium caulinodans PII and GlnK proteins control nitrogen fixation and ammonia assimilation; Michel-Reydellet N et al.; We herein report that Azorhizobium caulinodans PII and GlnK are not necessary for glutamine synthetase (GS) adenylylation whereas both proteins are required for complete GS deadenylylation . The disruption of both glnB and glnK resulted in a high level of GS adenylylation under the condition of nitrogen fixation, leading to ammonium excretion in the free-living state . PII and GlnK also controlled nif gene expression because NifA activated nifH transcription and nitrogenase activity was derepressed in glnB glnK double mutants, but not in wild-type bacteria, grown in the presence of ammonia.

J Bacteriol, 1999 Apr, 181(8), 2430 - 9
The CtrA response regulator mediates temporal control of gene expression during the Caulobacter cell cycle; Reisenauer A et al.; In its role as a global response regulator, CtrA controls the transcription of a diverse group of genes at different times in the Caulobacter crescentus cell cycle . To understand the differential regulation of CtrA-controlled genes, we compared the expression of two of these genes, the fliQ flagellar gene and the ccrM DNA methyltransferase gene . Despite their similar promoter architecture, these genes are transcribed at different times in the cell cycle . PfliQ is activated earlier than PccrM . Phosphorylated CtrA (CtrA approximately P) bound to the CtrA recognition sequence in both promoters but had a 10- to 20-fold greater affinity for PfliQ . This difference in affinity correlates with temporal changes in the cellular levels of CtrA . Disrupting a unique inverted repeat element in PccrM significantly reduced promoter activity but not the timing of transcription initiation, suggesting that the inverted repeat does not play a major role in the temporal control of ccrM expression . Our data indicate that differences in the affinity of CtrA approximately P for PfliQ and PccrM regulate, in part, the temporal expression of these genes . However, the timing of fliQ transcription but not of ccrM transcription was altered in cells expressing a stable CtrA derivative, indicating that changes in CtrA approximately P levels alone cannot govern the cell cycle transcription of these genes . We propose that changes in the cellular concentration of CtrA approximately P and its interaction with accessory proteins influence the temporal expression of fliQ, ccrM, and other key cell cycle genes and ultimately the regulation of the cell cycle.

Lett Appl Microbiol, 1999 Mar, 28(3), 211 - 5
Growth of moulds inoculated into commercial mineral water; Fujikawa H et al.; The growth of mould spores of Penicillium sp . and Cladosporium sp . inoculated in a commercial mineral water product was studied . The strains had been isolated as fungal foreign bodies in commercial mineral waters . In product A, which was not originally sterilized and was contaminated with psychrophilic bacteria, the inoculated mould spores of the strains did not grow; no increases in viable colony counts or beta-glucans concentration in the samples were observed during storage . In a sterilized product A, inoculated spores of the strains grew into visible foreign bodies . The viable colony counts and the beta-glucans concentration in the samples increased during storage . These results showed that in a sterilized mineral water product, mould spores could grow into visible foreign bodies.

C R Acad Sci III, 1999 Feb-Mar, 322(2-3), 167 - 75
Adaptive response and induced resistance; Joiner MC et al.; Cellular stress responses are upregulated following exposure to radiation and other DNA-damaging agents . Therefore radiation response can be dose dependent so that small acute exposures (and possibly exposures at very low dose rates?) are more lethal per unit dose than larger exposures above a threshold (typically 10-40 cGy) where induced radioprotection is triggered . We have termed these interlinked phenomena low-dose hypersensitivity (HRS) and induced radioresistance (IRR) as the dose increases . HRS/IRR has been recorded in cell-survival studies with yeast, bacteria, protozoa, algae, higher plant cells, insect cells, mammalian and human cells in vitro, and in studies on animal normal-tissue models in vivo . There is indirect evidence that cell survival-related HRS/IRR in response to single doses is a manifestation of the same underlying mechanism that determines the well-known adaptive response in the two-dose case and that it can be triggered by high- and low-LET radiations as well as a variety of other stress-inducing agents such as hydrogen peroxide and chemotherapeutic agents . Little is currently known about the precise nature of this underlying mechanism, but there is evidence that it operates by increasing the amount and rate of DNA repair, rather than by indirect mechanisms such as modulation of cell-cycle progression or apoptosis . Changed expression of some genes, only in response to low and not high doses, may occur within a few hours of irradiation and this would be rapid enough to explain the phenomenon of induced radioresistance although its specific molecular components have yet to be identified . Net cancer risk is a balance between cell transformation and cell kill . Our known low-dose cell-survival responses suggest that lethality may more than compensate for transformation at low radiation doses . However, adaptive reduction in sensitivity to radio-mutation has also been reported, which implies the existence also of enhanced mutation following very low single doses . So far this has not been confirmed, but provided the trigger dose for mutational protection was lower than the trigger dose for protection against cytotoxicity, cell killing would still dominate over at least the first 10 cGy of low-LET exposure . This would lead to a non-linear, threshold, dose-risk relationship and even provide some explanation for anecdotal reports of apparent 'health promoting' effects and lowered cancer risk from very low exposure to ionising radiation.

C R Acad Sci III, 1999 Feb-Mar, 322(2-3), 143 - 9
Mechanisms of mutagenesis in mammalian cells . Application to human thyroid tumours; Sarasin A et al.; Mutations are defined as stable and irreversible modifications of the normal genetic message due to small changes in the number or type of bases, or to large modifications of the genome such as deletions, insertions or chromosome rearrangements . These lesions are due to either polymerase errors during normal DNA replication or unrepaired DNA lesions, which will give rise to mutations through a mutagenic pathway . The molecular process leading to mutagenesis depends largely on the type of DNA lesions . Base modifications, such as 8-oxo-guanine or thymine glycol, both induced by ionizing radiations (IR), are readily replicated leading to direct mutations, usually base-pair substitutions . The 8-oxo-G gives rise predominantly to G to T transversions, the type of mutations found in ras or p53 gene from IR-induced tumors . Bulky adducts produced by chemical carcinogens or UV-irradiation are usually repaired by the nucleotide excision repair (NER) pathway which is able to detect structural distortion in the normal double-strand DNA backbone . These lesions represent a blockage to DNA and RNA polymerases as well as some signal for p53 accumulation in the damaged cell . In the absence of repair, these lesions could be eventually replicated owing to the induction of specific proteins at least in bacteria during the SOS process . The precise nature of the error-prone replication across an unexcised DNA lesion in the template is not fully understood in detailed biochemical terms, in mammalian cells . IR basically produce a very large number of DNA lesions from unique base modifications to single- or double-strand breaks and even complex DNA lesions due to the passage of very high energy particles or to a local re-emission of numerous radicals . The breakage of the double-helix is a difficult lesion to repair . Either it will result in cell death or, after an incorrect recombinational pathway, it will induce frameshifts, large deletions or chromosomal rearrangements . Most of the IR-induced mutations are recessive ones, requiring therefore a second genetic event in order to exhibit any harmful effect and a long latency period before the development of a radiation-induced tumor . The fact that IR essentially induced deletions and chromosomal translocations renders very difficult the use of the p53 gene as a marker for mutation analysis . In agreement with the type of lesions induced by IR, it is interesting to point out that the presence has been observed, in a vast majority of radiation-induced papillary thyroid carcinomas (PTC), of an activated ret proto-oncogene originated by the fusion of the tyrosine kinase 3' domain of this gene with the 5' domain of four different genes . These ret chimeric genes which are due to intra- or inter-chromosomal translocations, were called RET/PTC1 to PTC5 . The RET/PTC rearrangements were found in PTC from children contaminated by the Chernobyl fall-out as well as in tumours from patients with a history of therapeutic external radiation, with a frequency of 60-84% . This frequency was only 15% in 'spontaneous' PTC . The type of ret chimeric gene predominantly originated by the accidental or therapeutic IR was different . Indeed, PTC1 was present in 75% of the tumours linked to a therapeutic radiation and PTC3 in 75% of the Chernobyl ones . The other forms of RET/PTC were observed in only a minority of the post-Chernobyl PTC (< 20%) . The difference in the frequency of PTC1 and PTC3 in both types of PTC, is statistically significant (P < 10(-5), Fischer's exact test) . In two of the post-therapeutic radiation PTC, RET/PTC1 and PTC3 were simultaneously present . A PTC1 gene was also observed in 45% of the adenomas appearing after therapeutic radiation . The long-period of latency between exposure to IR and the appearance of thyroid tumours is probably due to the conversion of a heterozygote genotype of IR-induced mutations to a homozygote one . It will be interesting to use this time lag in accidental or therapeutic-irradiated p

J Virol, 1999 May, 73(5), 3810 - 7
A novel cellular protein, p60, interacting with both herpes simplex virus 1 regulatory proteins ICP22 and ICP0 is modified in a cell-type-specific manner and Is recruited to the nucleus after infection; Bruni R et al.; Herpes simplex virus 1 encodes two multifunctional regulatory proteins, infected-cell proteins 22 and 0 (ICP22 and ICP0) . ICP0 is a promiscuous transactivator, whereas ICP22 is required in vivo and for efficient replication and expression of a subset of late (gamma2) genes in rodent or rabbit cell lines and in primary human cell strains (restrictive cells) but not in HEp-2 or Vero (permissive) cells . We report the identification in the yeast two-hybrid system of a cellular protein designated p60 that interacts with ICP22 . This protein (apparent Mr of 60,000) has not been previously described and has no known motifs . Analyses of p60 revealed the following . (i) p60 bound fast-migrating, underprocessed wild-type ICP22 and ICP22 lacking the carboxyl-terminal 24 amino acids but not ICP22 lacking the carboxyl-terminal 40 amino acids, whereas the previously identified cellular protein p78 (R . Bruni and B . Roizman, J . Virol . 72:8525-8531, 1998) bound all forms of ICP22 . The interaction of p60 with only one isoform of ICP22 supports that hypothesis that each isoform of herpes simplex virus proteins performs a specific function that may be different from that of other isoforms . (ii) p60 also bound ICP0; the binding of ICP0 was independent of that of ICP22 . (iii) p60 localized in uninfected rabbit skin cells in both nuclei and cytoplasm . In rabbit skin cells infected with wild-type virus, p60 was posttranslationally processed to a higher apparent Mr but was not redistributed . Posttranslational processing required the presence of the genes encoding ICP22 and UL13 protein kinase . (iv) In uninfected HEp-2 cells, p60 localized primarily in nuclei . Soon after infection with wild-type virus, the p60 localized in discrete small nuclear structures with ICP0 . Late in infection, both ICP0 and p60 tended to disperse but p60 did not change in apparent Mr . The localization of p60 was independent of ICP22, but p60 tended to be more localized in small nuclear structures and less dispersed in cells infected with mutants lacking the genes encoding the UL13 or US3 protein kinases . The results suggest that posttranslational modification of p60 is mediated either by ICP0 (permissive cells) or by ICP22 and UL13 protein kinase (restrictive rabbit skin cells) and that the restrictive phenotype of rabbit skin cells may be related to the failure to process p60 by mutants lacking the genes encoding UL13 or ICP22.

Protein Eng, 1999 Feb, 12(2), 155 - 62
Mutation of a highly conserved aspartate residue in subdomain IX abolishes Fer protein-tyrosine kinase activity; Cole LA et al.; Before the structure of cAMP-dependent protein kinase had been solved, sequence alignments had already suggested that several highly conserved peptide motifs described as kinase subdomains I through XI might play some functional role in catalysis . Crystal structures of several members of the protein kinase superfamily have suggested that the nearly invariant aspartate residue within subdomain IX contributes to the conformational stability of the catalytic loop by forming hydrogen bonds with backbone amides within subdomain VI . However, substitution of this aspartate with alanine or threonine in some protein kinases have indicated that these interactions are not essential for activity . In contrast, we show here that conversion of this aspartate to arginine abolished the catalytic activity of the Fer protein-tyrosine kinase when expressed either in mammalian cells or in bacteria . Structural modeling predicted that the catalytic loop of the FerD743R mutant was disrupted by van der Waal's repulsion between the side chains of the substituted arginine residue in subdomain IX and histidine-683 in subdomain VI . The FerD743R mutant model predicted a shift in the peptide backbone of the catalytic loop, and an outward rotation of histidine-683 and arginine-684 side chains . However, the position and orientation of the presumptive catalytic base, aspartate-685, was not substantially changed . The proposed model explains how substitutions of some, but not all residues could be tolerated at this nearly invariant aspartate in kinase subdomain IX.

Math Biosci, 1999 Mar 15, 157(1-2), 217 - 36
Resistance of a food chain to invasion by a top predator; Kooi BW et al.; We study the invasion of a top predator into a food chain in a chemostat . For each trophic level, a bioenergetic model is used in which maintenance and energy reserves are taken into account . Bifurcation analysis is performed on the set of nonlinear ordinary differential equations which describe the dynamic behaviour of the food chain . In this paper, we analyse how the ability of a top predator to invade the food chain depends on the values of two control parameters: the dilution rate and the concentration of the substrate in the input . We investigate invasion by studying the long-term behaviour after introduction of a small amount of top predator . To that end we look at the stability of the boundary attractors; equilibria, limit cycles as well as chaotic attractors using bifurcation analysis . It will be shown that the invasibility criterion is the positiveness of the Lyapunov exponent associated with the change of the biomass of the top predator . It appears that the region in the control parameter space where a predator can invade increases with its growth rate . The resulting system becomes more resistant to further invasion when the top predator grows faster . This implies that short food chains with moderate growth rate of the top predator are liable to be invaded by fast growing invaders which consume the top predator . There may be, however, biological constraints on the top predator's growth rate . Predators are generally larger than prey while larger organisms commonly grow slower . As a result, the growth rate generally decreases with the trophic level . This may enable short food chains to be resistant to invaders . We will relate these results to ecological community assembly and the debate on the length of food chains in nature.

Clin Infect Dis, 1999 Mar, 28(3), 514 - 9
Differential tumor necrosis factor alpha production in simian immunodeficiency virus-infected rhesus macaques coinfected with Mycobacterium avium; Newman GW et al.; Mycobacterium avium infections are the third most common opportunistic infection in patients with AIDS . Simian immunodeficiency virus (SIV)-infected rhesus macaques naturally acquire M . avium infections from the environment, and their clinical symptoms are similar to those observed in AIDS patients . We characterized concurrent infection with SIV and M . avium in monkeys on the basis of the growth of the bacteria in macrophages (Mphis) from rhesus macaques and the ability of M . avium to induce SIV replication and tumor necrosis factor alpha (TNF-alpha) production . The simian M . avium isolate grew significantly better than did an isolate from an AIDS patient or a chicken isolate (P = .001); it induced significantly more TNF-alpha production in Mphis from SIV-positive and SIV-negative monkeys than did the isolate from an AIDS patient (P = .013) . No significant increase in SIV replication was seen in the M . avium isolates, and no correlation was found between increased SIV replication and increased TNF-alpha production . In addition, Mphis from monkeys infected with M . avium during late-stage SIV disease produced less TNF-alpha when stimulated with virulent M . avium.

Drugs, 1999 Mar, 57(3), 309 - 25
Prospects for the therapeutic use of anticancer vaccines; Chamberlain RS; Although a century has passed since initial attempts were made to stimulate the immune system to destroy tumour, the immunotherapy of cancer is still in the early stages . Historically, a variety of specific and nonspecific immunostimulatory strategies have been administered with only modest clinical success . However, recent advances in tumour immunology, most notably the identification of genes encoding for cancer regression antigens, have paved the way for the development of a variety of novel and specific vaccine approaches . These include vaccines based on tumour cells, carbohydrates, peptides and heat-shock proteins, DNA-based vaccination, and the use of recombinant bacteria and viruses to deliver antigens or the DNA coding for them . While several of these approaches have yielded exciting clinical results, a number of immunological and host obstacles to the successful application of cancer vaccines remain . Further research is needed on the optimum choice of antigen, delivery vector, adjuvant and administration regimen.

Am Fam Physician, 1999 Mar 15, 59(6), 1547 - 56, 1561-2
Vulvodynia and vulvar vestibulitis: challenges in diagnosis and management; Metts JF; Vulvodynia is a problem most family physicians can expect to encounter . It is a syndrome of unexplained vulvar pain, frequently accompanied by physical disabilities, limitation of daily activities, sexual dysfunction and psychologic distress . The patient's vulvar pain usually has an acute onset and, in most cases, becomes a chronic problem lasting months to years . The pain is often described as burning or stinging, or a feeling of rawness or irritation . Vulvodynia may have multiple causes, with several subsets, including cyclic vulvovaginitis, vulvar vestibulitis syndrome, essential (dysesthetic) vulvodynia and vulvar dermatoses . Evaluation should include a thorough history and physical examination as well as cultures for bacteria and fungus, KOH microscopic examination and biopsy of any suspicious areas . Proper treatment mandates that the correct type of vulvodynia be identified . Depending on the specific diagnosis, treatment may include fluconazole, calcium citrate, tricyclic antidepressants, topical corticosteroids, physical therapy with biofeedback, surgery or laser therapy . Since vulvodynia is often a chronic condition, regular medical follow-up and referral to a support group are helpful for most patients.

J Infect Dis, 1999 May, 179(5), 1139 - 44
Enteropathogens and other factors associated with severe disease in children with acute watery diarrhea in Lima, Peru; Cama RI et al.; To evaluate enteropathogens and other factors associated with severe disease in children with diarrhea, 381 children <5 years of age with diarrhea and moderate to severe dehydration (in-patients) and 381 age-, sex-, and date-of-visit-matched children with mild diarrhea (out-patients) presenting to a hospital in Peru, were studied . Rotavirus was detected in 52% of the in-patients and 35% of the out-patients (odds ratio {OR}=2.3, 95% confidence interval {95% CI}= 1.6-3.2); 95% of the rotaviruses among in-patients were of serotypes G1-G4 . The risk of severe diarrhea was particularly great in children who were not exclusively breast-fed in early infancy and who also lacked piped water in their homes (for children with both characteristics OR=6.8, 95% CI=3.6-12.8) . The high prevalence of rotavirus and its association with severe diarrhea underscores the need for rotavirus vaccines . Interventions to educate mothers and improve access to safe water should augment the impact of rotavirus vaccines in preventing severe diarrhea.

Nippon Eiseigaku Zasshi, 1999 Jan, 53(4), 596 - 600
{Epidemiological study on association of periodontal disease and total and differential leukocyte counts}; Ogawa Y et al.; Periodontal disease is defined as inflammation that is caused by bacteria in dental plaque . This disease is liable to be a factor contributing to the high leukocyte count over an extended period . Furthermore, a number of prospective epidemiologic studies have shown that the leukocyte count is a good predictor of ischemic heart disease (IHD) . However there have been few epidemiological studies of the relationship between periodontal disease and the leukocyte count . The purpose of this study was to investigate the relationship between the total and differential leukocyte counts and oral conditions of Japanese factory workers who were classified according to their smoking habits . The 1,167 subjects were male factory workers employed with a chemical factory in Osaka, Japan . The oral conditions recorded were periodontal status (Community Periodontal Index of Treatment Needs, CPITN) . The relationship between the total and differential leukocyte counts and the CPITN score of subjects who were classified according to their smoking habits was investigated in 1996 and 1997 . In both the current smokers and nonsmokers, the subjects with severe periodontal disease, in contrast to the normal subjects, exhibited total leukocyte, neutrophil and monocyte counts that were significantly high for 1 year during follow-up studies . The periodontal disease is shown that causes the total leukocyte and neutrophil counts related to the development of IHD to remain at a high level.

Pharmacol Ther, 1999 Feb, 81(2), 77 - 89
Structure-function relationships of acid ribonucleases: lysosomal, vacuolar, and periplasmic enzymes; Irie M; It is surprising that only relatively recently has attention been directed to the characterization of the properties of acid ribonucleases (RNases), leading to some understanding of their biochemistry and their functional roles . The present review summarizes current progress in this field under the following general topics: (1) the wide distribution of acid RNases in organisms from viruses to animals; (2) recent findings concerning their primary and three-dimensional structure; (3) the structure-function relationship of acid RNases, with a fungal RNase from Rhizopus niveus as a model enzyme; (4) the unique localization of acid RNases in the periplasm of bacteria, vacuoles in plants, and lysosomes of animals and protozoa; and (5) the diversity of physiological roles, depending on the organism, such as self-incompatibility factors and defense proteins in some plants, the surface protein of an animal virus related to pathogenicity, and possible relationship to human cancer.

Rev Sci Tech, 1999 Apr, 18(1), 104 - 21
{Prevention of and attention to emergencies in South America}; Barcos LO et al.; The authors review the policies designed to prevent and deal with animal health emergencies which have been implemented in countries of South America . They describe the evolution of the epidemiological situation of the continent, the new arrangements for international trade in animals and products of animal origin arising from the creation of the World Trade Organization (WTO), and the consequences of such developments for livestock production in South America . Veterinary systems used to prevent and deal with emergencies in the eleven OIE Member Countries on the continent are described, together with emerging problems which confront the Veterinary Services of the continent, namely: exotic diseases, abnormal occurrence of endemic diseases subject to control programmes, faults in food-safety mechanisms, diseases which have an environmental impact, and problems connected with animal welfare . The emergencies which present the greatest risk to South America are foot and mouth diseases, transmissible spongiform encephalopathies, the porcine reproductive and respiratory syndrome, food poisoning, Newcastle disease and fowl plague . Other problems are the appearance of new strains of existing agents, and the presence of resistant individuals among species of bacteria or harmful arthropods . The authors emphasise the need to co-ordinate the prevention of emergencies with development work at the international level, particularly regional and international agreements, harmonization of procedures, progress in animal health and public health, risk analysis, etc . These systems and methods of prevention have a contribution to make in enhancing the potential of animal production in South America, and the adoption of stricter health and quality standards, according to criteria established by the WTO Agreement on the Application of Sanitary and Phytosanitary Measures.

Vet Microbiol, 1999 Mar 12, 65(3), 185 - 94
Preliminary studies on the prevalence of Mycoplasma bovis mastitis in dairy in cattle in Australia; Ghadersohi A et al.; A highly sensitive and specific PCR (MB-PCR) was used in preliminary studies to detect M . bovis in milk samples to investigate its association with high somatic cell count (SCC), an indicator of subclinical mastitis and one of the factors in down grading the quality of milk . A total of 186 and 167 herds were tested with 43% and 62% of herds positive for M . bovis in Victoria and North Queensland, respectively . The quarter milks from 52 cows with persistently high SCC were tested by MB-PCR and culture to investigate the association of M . bovis with major mastitis pathogens (MMP) . M . Bovis was detected in 77% of cows of which 19% alone had M . bovis without any other bacteria, 17% had M . bovis in combination with major mastitis pathogens and 40% had M . bovis in combination with non-major mastitis pathogens . We believe that M . bovis is widespread in dairy cattle and has the potential to produce disease alone or to predispose the udder to disease caused by major mastitis and environmental pathogens . These studies have revealed a hitherto unrecognised high prevalence of M . bovis in dairy cattle in North Queensland and Victoria in Australia . These initial studies also give a clear association between M . bovis and elevated somatic cell counts.

Am J Vet Res, 1999 Mar, 60(3), 310 - 5
Granuloma development in cattle after intratonsilar inoculation with Mycobacterium bovis; Palmer MV et al.; OBJECTIVE: To examine the temporal development of tuberculous lesions in cattle inoculated with Mycobacterium bovis . ANIMALS: 15 mature crossbred cows obtained from a herd with no history of M bovis infection . PROCEDURE: Inoculation of cattle was done by intratonsilar instillation of 1.48 X 10(5) to 5.4 X 10(7) colony-forming units of M bovis strain 2045T . At 3 to 4 hours, 4 weeks, 6 weeks, and 8 weeks after inoculation, tissues were examined for gross and microscopic lesions and processed for isolation of M bovis . RESULTS: Retropharyngeal lymph nodes from cattle examined 4 weeks after inoculation contained microgranulomas consisting of aggregates of macrophages with few neutrophils . Retropharyngeal lymph nodes from all cattle examined 6 and 8 weeks after inoculation contained multiple, large, coalescing granulomas consisting of central areas of necrosis with mild fibrosis, numerous macrophages, lymphocytes, plasma cells, multinucleated giant cells, and neutrophils . Three of 8 cattle examined 6 or 8 weeks after inoculation had lesions in nonretropharyngeal sites with morphologic characteristics similar to that seen in retropharyngeal lymph node granulomas from cattle examined 4 weeks after inoculation . CONCLUSION: Granulomas can develop in draining lymph nodes of cattle in as little as 4 weeks after inoculation via intratonsilar instillation of M bovis . Intralesional morphologic changes between 4 and 6 weeks after inoculation indicate an increase in cellular chemotaxis and differentiation . Dissemination of bacteria to distant sites most likely was by lymphatic and hematogenous routes after establishment of the primary infection in retropharyngeal lymph nodes.

Syst Appl Microbiol, 1999 Feb, 22(1), 39 - 44
Phylogenetic relationships of a large marine Beggiatoa; Teske A et al.; Based upon 16S rRNA sequence and phenotypic similarities, a large, uncultured Beggiatoa sp . from the Bay of Concepcion (Chile), is very closely related to the Chilean Thioploca species Thioploca araucae., whose filaments grow as sheathed bundles . The formation of sheathed filament bundles, the key character to distinguish the genus Thioploca from Beggiatoa, places closely related filamentous sulfur-oxidizing bacteria into two different genera, incongruent with 16S rRNA-defined clades.

Syst Appl Microbiol, 1999 Feb, 22(1), 22 - 7
Characterization of the koala biovar of Chlamydia pneumoniae at four gene loci--ompAVD4, ompB, 16S rRNA, groESL spacer region; Wardrop S et al.; Koalas are infected with two species of Chlamydia, C . pecorum and C . pneumoniae . While it is known that significant genetic diversity occurs in the C . pecorum strains infecting koalas, very little is known about the C . pneumoniae strains that infect this host . In the current study, 10 isolates of koala C . pneumoniae were analysed at four gene loci and found to be different to both the human and horse C . pneumoniae strains at all loci (biovar differences ranging from 0.3% at groESL up to 9.0% at ompAVD4) . All koala biovar isolates studied were found to be 100% identical at ompAVD4 (all 10 isolates) and at ompB (all three isolates) gene . This lack of allelic polymorphisms at ompAVD4 has now been observed for koala C . pneumoniae, human C . pneumoniae, guinea pig inclusion conjuctivitis C . psittaci and feline conjuctivitis C . psittaci and may be correlated to a lack of antibody response to the chlamydial major outer membrane protein (MOMP) in these same strain/host combinations . This study also provides the first documented case of natural C . pneumoniae infection causing a severe and extended respiratory episode in a captive koala population . This captive episode is in contrast to most free-range observations in which koala C . pneumoniae is rarely documented as causing respiratory, ocular or urogenital tract disease.

Appl Environ Microbiol, 1999 Apr, 65(4), 1463 - 9
Bacterivory rate estimates and fraction of active bacterivores in natural protist assemblages from aquatic systems
Gonzalez JM.
Unlike the fraction of active bacterioplankton, the fraction of active bacterivores (i.e., those involved in grazing) during a specified time period has not been studied yet . Fractions of protists actively involved in bacterivory were estimated assuming that the distributions of bacteria and fluorescently labeled bacteria (FLB) ingested by protists follow Poisson distributions . Estimates were compared with experimental data obtained from FLB uptake experiments . The percentages of protists with ingested FLB (experimental) and the estimates obtained from Poisson distributions were similar for both flagellates and ciliates . Thus, the fraction of protists actively grazing on natural bacteria during a given time period could be estimated . The fraction of protists with ingested bacteria depends on the incubation time and reaches a saturating value . Aquatic systems with very different characteristics were analyzed; estimates of the fraction of protists actively grazing on bacteria ranged from 7 to 100% in the studied samples . Some nanoflagellates appeared to be grazing on specific bacterial sizes . Evidence indicated that there was no discrimination for or against bacterial surrogates (i.e., FLB); also, bacteria were randomly encountered by bacterivorous protists during these short-term uptake experiments . These analyses made it possible to estimate the ingestion rates from FLB uptake experiments by counting the number of flagellates containing ingested FLB . These results represent the first reported estimates of active bacterivores in natural aquatic systems; also, a proposed protocol for estimating in situ ingestion rates by protists represents a significant improvement and simplification to the current protocol and avoids the tedious work of counting the number of ingested FLB per protist.

Biopolymers, 1999 Apr 15, 49(5), 361 - 72
A light-harvesting antenna protein retains its folded conformation in the absence of protein-lipid and protein-pigment interactions; Kikuchi J et al.; The first study by nmr of the integral membrane protein, the bacterial light-harvesting (LH) antenna protein LH1 beta, is reported . The photosynthetic apparatus of purple bacteria contains two different kinds of antenna complexes (LH1 and LH2), which consist of two small integral membrane proteins alpha and beta, each of approximately 6 kDa, and bacteriochlorophyll and carotenoid pigments . We have purified the antenna polypeptide LH1 beta from Rhodobacter sphaeroides, and have recorded CD spectra and a series of two-dimensional nmr spectra . A comparison of CD spectra of LH1 beta observed in organic solvents and detergent micelles shows that the helical character of the peptide does not change appreciably between the two milieus . A significantly high-field shifted methyl signal was observed both in organic solvents and in detergent micelles, implying that a similar three-dimensional structure is present in each case . However, the 1H-nmr signals observed in organic solvents had a narrower line width and better resolution, and it is shown that in this case organic solvents provide a better medium for nmr studies than detergent micelles . A sequential assignment has been carried out on the C-terminal transmembrane region, which is the region in which the pigment is bound . The region is shown to have a helical structure by the chemical shift values of the alpha-CH protons and the presence of nuclear Overhauser effects characteristic of helices . An analysis of the amide proton chemical shifts of the residues surrounding the histidine chlorophyll ligand suggests that the local structure is well ordered even in the absence of protein-lipid and protein-pigment interactions . Its structure was determined from 348 nmr-derived constraints by using distance geometry calculations . The polypeptide contains an alpha-helix extending from Leu19 (position of cytoplasmic surface) to Trp44 (position of periplasmic surface) . The helix is bent, as expected from the amide proton chemical shifts, and it is similar to the polypeptide fold of the previously determined crystal structure of Rhodopseudomonas acidophila Ac10050 LH2 beta (S . M . Prince et al., Journal of Molecular Biology, 1997, Vol . 268, pp . 412-423) . It is concluded that the polypeptide conformation of this region may facilitate assembly of the LH complex.

J Clin Eng, 1996 Mar-Apr, 21(2), 149 - 55
Electrosurgery smoke: hazards and protection; O'Grady KF et al.; Early discussion regarding smoke produced by both surgical lasers and electrosurgical machines concluded that the smoke produced by these instruments was little more than a malodorous nuisance . Animal and human studies to date, however, have suggested that this smoke is, indeed, dangerous . This smoke has been shown to be mutagenic and can contain bacteria and viruses, the HIV virus being the most notable . Furthermore, these particles are small enough to penetrate deep within the respiratory tract . In response to the concerns raised by these hazards, commercial smoke evacuation systems have been designed to greatly reduce the number of hazardous particles, as well as the noxious odor produced by electrosurgery and laser surgery . The efficacy of these systems, however, is dependent o n usage and placement close to the surgical site . This review paper presents the potential hazards of electrosurgical smoke, along with some guidelines on how to properly protect hospital staff and patients from these hazards.

J Appl Biomater, 1994 Summer, 5(2), 151 - 7
Six bioabsorbable polymers: in vitro acute toxicity of accumulated degradation products; Taylor MS et al.; Bioabsorbable polymer implants may provide a viable alternative to metal implants for internal fracture fixation . One of the potential difficulties with absorbable implants is the possible toxicity of the polymeric degradation products especially if they accumulate and become concentrated . Accordingly, material evaluation must involve dose-response toxicity data as well as mechanical properties and degradation rates . In this study the toxicity and rates of degradation for six polymers were determined, along with the toxicity of their degradation product components . The polymers studied were poly(glycolic acid) (PGA), two samples of poly(L-lactic acid) (PLA) having different molecular weights, poly(ortho ester) (POE), poly(epsilon-caprolactone) (PCL), and poly(hydroxy butyrate valerate) (5% valerate) (PHBV) . Polymeric specimens were incubated at 37 degrees C in 0.05 M Tris buffer (pH 7.4 at 37 degrees C) and sterile deionized water . The solutions were not changed during the incubation intervals, providing a worst-case model of the effects of accumulation of degradation products . The pH and acute toxicity of the incubation solutions and the mass loss and logarithmic viscosity number of the polymer samples were measured at 10 days, 4, 8, 12, and 16 weeks . Toxicity was measured using a bioluminescent bacteria, acute toxicity assay system . The acute toxicity of pure PGA, PLA, POE, and PCL degradation product components was also determined . Degradation products for PHBV were not tested . PGA incubation solutions were toxic at 10 days and at all following intervals . The lower molecular weight PLA incubation solutions were not toxic in buffer but were toxic by 4 weeks in water.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1999 Apr, 65(4), 1570 - 7
Production of poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) and poly(4-hydroxybutyric acid) without subsequent degradation by Hydrogenophaga pseudoflava; Choi MH et al.; A Hydrogenophaga pseudoflava strain was able to synthesize poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) {P(3HB-co-4HB)} having a high level of 4-hydroxybutyric acid monomer unit (4HB) from gamma-butyrolactone . In a two-step process in which the first step involved production of cells containing a minimum amount of poly(3-hydroxybutyric acid) {P(3HB)} and the second step involved polyester accumulation from the lactone, approximately 5 to 10 mol% of the 3-hydroxybutyric acid (3HB) derived from the first-step culture was unavoidably reincorporated into the polymer in the second cultivation step . Reincorporation of the 3HB units produced from degradation of the first-step residual P(3HB) was confirmed by high-resolution 13C nuclear magnetic resonance spectroscopy . In order to synthesize 3HB-free poly(4-hydroxybutyric acid) {P(4HB)} homopolymer, a three-stage cultivation technique was developed by adding a nitrogen addition step, which completely removed the residual P(3HB) . The resulting polymer was free of 3HB . However, when the strain was grown on gamma-butyrolactone as the sole carbon source in a synthesis medium, a copolyester of P(3HB-co-4HB) containing 45 mol% 3HB was produced . One-step cultivation on gamma-butyrolactone required a rather long induction time (3 to 4 days) . On the basis of the results of an enzymatic study performed with crude extracts, we suggest that the inability of cells to produce 3HB in the multistep culture was due to a low level of 4-hydroxybutyric acid (4HBA) dehydrogenase activity, which resulted in a low level of acetyl coenzyme A . Thus, 3HB formation from gamma-butyrolactone is driven by a high level of 4HBA dehydrogenase activity induced by long exposure to gamma-butyrolactone, as is the case for a one-step culture . In addition, intracellular degradation kinetics studies showed that P(3HB) in cells was completely degraded within 30 h of cultivation after being transferred to a carbon-free mineral medium containing additional ammonium sulfate, while P(3HB-co-4HB) containing 5 mol% 3HB and 95 mol% 4HB was totally inert in interactions with the intracellular depolymerases . Intracellular inertness could be a useful factor for efficient synthesis of the P(4HB) homopolymer and of 4HB-rich P(3HB-co-4HB) by the strain used in this study.

Aliment Pharmacol Ther, 1999 Feb, 13(2), 135 - 44
Review article: the potential role of nitric oxide in chronic inflammatory bowel disorders; Perner A et al.; The aetiology of the chronic inflammatory bowel diseases-ulcerative colitis and Crohn's disease-as well as 'microscopic colitis'-both collagenous (COC) and lymphocytic colitis (LC)-remains unknown . Autoimmune mechanisms, cytokine polymorphism, commensal bacteria, infectious agents and vascular impairment have all been proposed as playing important roles in the pathogenesis of this spectrum of diseases . A variety of proinflammatory mediators, including tumour necrosis factor alpha, interleukin-1beta, interferon gamma, leukotriene B4 and platelet activating factor, promote the adherence of phagocytes to the venular endothelium and extravasation of these cells into the colonic mucosa . In addition to large amounts of nitric oxide (NO), injurious peroxynitrite may be formed in the epithelium by the inducible nitric oxide synthase (iNOS), which is considered to elicit cytotoxicity by the generation of superoxide with reduced L-arginine availability . In active ulcerative colitis, and to a lesser extent in Crohn's disease, a greatly increased production of NO has been demonstrated by indirect and direct measurements . Surprisingly, even higher rates of production have been observed in COC-a condition which is never associated with injurious inflammation . The latter observation favours the notion that NO promotes mucosal integrity . Further evidence for a protective role of NO in chronic inflammatory bowel disorders is provided by the observation of increased susceptibility to the induction of experi mental colitis in 'knock-out' mice deficient in iNOS . Selective inhibitors of iNOS activity, as well as topical L-arginine, may therefore prove beneficial in inflammatory bowel disease by reducing the production of superoxide by iNOS, while only the former option may be expected to reduce diarrhoea in chronic inflammatory bowel disorders . Clearly, further experimental work needs to be done before testing topical L-arginine in human inflammatory bowel disease.

J Periodontol, 1999 Feb, 70(2), 185 - 8
Periodontitis and anti-neutrophil cytoplasmic antibodies in systemic lupus erythematosus and rheumatoid arthritis: a comparative study; Novo E et al.; BACKGROUND: This investigation was designed to determine and compare the distribution pattern of anti-neutrophil cytoplasmic antibodies (ANCA) in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in the presence or absence of periodontal disease . METHODS: Sera of 30 patients with SLE and 30 with RA were tested for ANCA utilizing an indirect enzyme immunosorbent assay (ELISA) directed to a neutrophil granular extract and 6 neutrophil granule proteins . A control group of 20 healthy individuals showing neither evidence of periodontal disease nor systemic compromise was also included in this study . RESULTS: For RA, the number of ANCA-positive sera was very low but was evenly distributed among patients with and without periodontitis . Conversely, a high number of ANCA-positive sera in SLE was found mostly in individuals presenting periodontal compromise . A statistically significant association between ANCA and periodontitis in SLE patients was found (P <0.005, chi square test) . CONCLUSIONS: A marked difference in the number and distribution of ANCA with respect to periodontitis between RA and SLE was found . Hyperresponsiveness of B cells and polyclonal B activation to periodontopathic bacteria in SLE might be accountable for the high numbers of ANCA and the close association observed between those autoantibodies and periodontitis in SLE.

Chemosphere, 1999 Apr, 38(8), 1797 - 810
Modulation of metabolic activity prevents degradation of sorbed toluene; Roch F et al.; The factors affecting the ability of a bacterial species to degrade different amounts of toluene (8.5 to 217 mg/g) sorbed to granular activated carbon (GAC), in an aqueous solution of mineral salts, were investigated . After 144 days the amounts of toluene remaining on one type of GAC ranged from 7.5 to 9.5 mg/g, and the aqueous concentrations of toluene ranged from 2 to 7 micrograms/L . Neither bacterial death nor an inhibition by accumulating by-products could explain why the remaining toluene had not been degraded . However, at these low concentrations of toluene, and probably because of cell starvation, bacteria were observed to be more than 100-times less efficient to degrade toluene than at high concentrations . We propose that this low degradation ability is responsible for the presence of residual toluene on the GAC, and that this mechanism may contribute to the persistence of low concentrations of sorbed pollutants in the environment.

Clin Orthop, 1999 Mar, (360), 22 - 9
Pathophysiology of posttraumatic osteomyelitis; Tsukayama DT; Understanding the pathophysiology of posttraumatic osteomyelitis is crucial as researchers attempt to meet the challenge of developing more effective strategies for the management and prevention of this infection . Some aspects of pathogenesis have been well described, including the important roles of the extent of soft tissue injury, bacterial attachment to necrotic bone and fixation devices, and bacterial contamination at the time of injury . More recently, the importance of early wound coverage in preventing osteomyelitis has been emphasized . Now some of the cellular interactions that promote infection and tissue damage are beginning to be understood . Trauma can have deleterious effects on host response to infection through its activation of certain cytokines . These cytokines, mainly produced by cells of the immune system, regulate the action of polymorphonuclear leukocytes, macrophages, and lymphocytes . Bacteria have been shown to use diverse tactics to initiate and maintain infection that lead to host defense impairment, decreased efficacy of antibiotics, and direct tissue damage . New insights into the pathophysiology of osteomyelitis may lead to the innovative therapeutic approaches needed to improve the standard of care for this infection.

Biotechnol Bioeng, 1999 Feb, 62(4), 402 - 411
A method of graphically analyzing substrate-inhibition kinetics; Wang J et al.; A model of substrate inhibition for enzyme catalysis was extended to describe the kinetics of photosynthetic production of ethylene by a recombinant cyanobacterium, which exhibits light-inhibition behavior similar to the substrate-inhibition behavior in enzyme reactions . To check the validity of the model against the experimental data, the model equation, which contains three kinetic parameters, was transformed so that a linear plot of the data could be made . The plot yielded reasonable linearity, and the parameter values could be estimated from the plot . The linear-plot approach was then applied to other inhibition kinetics including substrate inhibition of enzyme reactions and inhibitory growth of bacteria, whose analyses would otherwise require nonlinear least-squares fits or data measured in constrained ranges . Plots for three totally different systems all showed reasonable linearity, which enabled visual validation of the assumed kinetics . Parameter values evaluated from the plots were compared with results of nonlinear least-squares fits . A normalized linear plot for all the results discussed in this work is also presented, where dimensionless rates as a function of dimensionless concentration lie in a straight line . The linear-plot approach is expected to be complementary to nonlinear least-squares fits and other currently used methods in analyses of substrate-inhibition kinetics .

Chem Biol, 1999 Apr, 6(4), R91 - R105
Catalytic promiscuity and the evolution of new enzymatic activities; O'Brien PJ et al.; Several contemporary enzymes catalyze alternative reactions distinct from their normal biological reactions . In some cases the alternative reaction is similar to a reaction that is efficiently catalyzed by an evolutionary related enzyme . Alternative activities could have played an important role in the diversification of enzymes by providing a duplicated gene a head start towards being captured by adaptive evolution.

Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3479 - 85
Biochemical evolution III: polymerization on organophilic silica-rich surfaces, crystal-chemical modeling, formation of first cells, and geological clues; Smith JV et al.; Catalysis at organophilic silica-rich surfaces of zeolites and feldspars might generate replicating biopolymers from simple chemicals supplied by meteorites, volcanic gases, and other geological sources . Crystal-chemical modeling yielded packings for amino acids neatly encapsulated in 10-ring channels of the molecular sieve silicalite-ZSM-5-(mutinaite) . Calculation of binding and activation energies for catalytic assembly into polymers is progressing for a chemical composition with one catalytic Al-OH site per 25 neutral Si tetrahedral sites . Internal channel intersections and external terminations provide special stereochemical features suitable for complex organic species . Polymer migration along nano/micrometer channels of ancient weathered feldspars, plus exploitation of phosphorus and various transition metals in entrapped apatite and other microminerals, might have generated complexes of replicating catalytic biomolecules, leading to primitive cellular organisms . The first cell wall might have been an internal mineral surface, from which the cell developed a protective biological cap emerging into a nutrient-rich "soup." Ultimately, the biological cap might have expanded into a complete cell wall, allowing mobility and colonization of energy-rich challenging environments . Electron microscopy of honeycomb channels inside weathered feldspars of the Shap granite (northwest England) has revealed modern bacteria, perhaps indicative of Archean ones . All known early rocks were metamorphosed too highly during geologic time to permit simple survival of large-pore zeolites, honeycombed feldspar, and encapsulated species . Possible microscopic clues to the proposed mineral adsorbents/catalysts are discussed for planning of systematic study of black cherts from weakly metamorphosed Archaean sediments.

Parasitol Res, 1999 Apr, 85(4), 331 - 6
Evaluation of the effect of peptidyl membrane-interactive molecules on avian coccidia; Martin A et al.; This study examined the lytic effect of seven different synthetic peptidyl membrane-interactive molecules (Peptidyl-MIMs) on sporozoites of five different species of Eimeria infecting chickens and merozoites of two different species that infect chickens . All Peptidyl-MIMs (pMIMs) demonstrated antiparasitic effects at concentrations of 1-50 microM during incubation periods varying from 1 to 20 min . In addition, electron microscopy showed that ultrastructural degeneration of the pellicle of sporozoite stages of the parasites occurred within 5-10 min of exposure to 5-microM concentrations of three different pMIMs . Pore-like openings were seen in the pellicle of the sporozoites at the ultrastructural level, which indicated that the pMIMs had the same mechanism of action on the parasites as that reported from studies done on bacteria . A reduction in lesion scores was seen in chickens treated orally with 10-, 50-, or 75-microM concentrations of two different proteolytic stabilized (methylated) pMIMs after challenge with three different species of avian coccidia in battery-cage trials . Collectively these data indicate that pMIMs may be useful in the control of coccidiosis in poultry.

Vet Pathol, 1999 Mar, 36(2), 161 - 3
Inflammatory pseudotumor in a cat with cutaneous mycobacteriosis; Miller MA et al.; A 5-year-old, castrated male, domestic Shorthair Cat had an ulcerated mass with fistulous tracts on the left hind paw . Homogeneous tan tissue diffusely infiltrated the dermis and subcutis of the paw and extended proximally so that, short of amputation, complete excision was not feasible . Biopsy specimens consisted of granulation tissue with marked proliferation of spindle cells . Neutrophils and histiocytic cells were scattered among the spindle cells . The histiocytic cells had abundant foamy or vacuolated cytoplasm, but features of granulomatous inflammation, such as epithelioid macrophages or granuloma formation, were not observed . The initial impression was inflammatory granulation tissue, but the degree of fibroplasia prompted inclusion of fibrosarcoma in the differential diagnosis . Cutaneous mycobacteriosis was diagnosed when numerous acid-fast bacteria were identified with Kinyoun's stain; Mycobacterium avium was subsequently cultured . The cat was euthanatized because of lack of response to enrofloxacin therapy . At necropsy, lesions were localized to the hind limb . Not only is mycobacteriosis an uncommon cause of cutaneous masses in cats, but this case was unusual because of the lack of granuloma formation and the similarity of the mass to a spindle cell tumor.

Trends Biochem Sci, 1999 Feb, 24(2), 64 - 8
Non-mitochondrial ATP transport; Winkler HH et al.; Exchange of organelle ATP with cytosolic ADP through the ADP/ATP carrier is a well-characterized feature of mitochondrial metabolism . Obligate intracellular bacteria, such as Rickettsia prowazekii, and higher-plant plastids possess another type of adenylate transporter, which exchanges bacterial or plastidic ADP for ATP from the eukaryotic (host cell) cytoplasm . The bacterial and plastidic transporters are similar but do not share significant sequence similarities with the mitochondrial carrier . Recent molecular and biochemical studies are providing deeper insight into the functional and evolutionary relationships between the bacterial and the plant transport proteins.

Trends Biochem Sci, 1999 Feb, 24(2), 47 - 53
The domains of death: evolution of the apoptosis machinery; Aravind L et al.; Recent progress in research into programmed cell death has resulted in the identification of the principal protein domains involved in this process . The evolution of many of these domains can be traced back in evolution to unicellular eukaryotes or even bacteria, where the domains appear to be involved in other regulatory functions . Cell-death systems in animals and plants share several conserved domains, in particular the family of apoptotic ATPases; this allows us to suggest a plausible, even if still incomplete, scenario for the evolution of apoptosis.

Heredity, 1999 Feb, 82 ( Pt 2), 163 - 9
Cross-order transfer of Wolbachia from Muscidifurax uniraptor (Hymenoptera: Pteromalidae) to Drosophila simulans (Diptera: Drosophilidae); Van Meer MM et al.; Bacteria of the genus Wolbachia are widespread in arthropods and can induce different effects on the host such as cytoplasmic incompatibility (CI), thelytoky (T) or feminization (F) . In some Wolbachia-infected hosts, no effect (N) has been found . Successful transfer of Wolbachia by microinjection from one host to an uninfected one has been established with CI, F, N-Wolbachia but not with T-Wolbachia . In this paper a transfer experiment of T-Wolbachia from the parasitoid Muscidifurax uniraptor to Drosophila simulans is described . The infection could be detected in the new host for several generations by polymerase chain reaction (PCR) . However, no specific effects on the host were detected, and the bacteria were not stably maintained.

Acta Cytol, 1999 Mar-Apr, 43(2), 201 - 6
Cytologic findings in vitreous fluids . Analysis of 74 specimens; Liu K et al.; OBJECTIVE: To review the cytologic findings of vitreous specimens and propose a simplified approach to them . STUDY DESIGN: Seventy-four vitreous specimens from 60 patients obtained either during a pars plana vitrectomy or by vitreous aspiration were reviewed . Clinical correlation was obtained on all patients . RESULTS: Findings suggestive of a specific disorder were present in 30 specimens (41%); cytologic examination of the remaining 44 showed nonspecific changes . A lymphoproliferative disorder, the intraocular malignancy suspected most often in this series, was identified in eight specimens (11%) . Large cell lymphomas were evident in 5 specimens, 2 specimens were suspicious for lymphoma, and 1 specimen was consistent with plasmacytoma . Twelve specimens (16%) contained hemorrhage . In rare instances, specific infectious agents, such as parasites (5%), bacteria (1%) and fungi (3%), could be identified . The diagnosis of viral infections required ancillary studies . Lens fragments were identified in four cases (5%), and a diagnosis of lens-induced endophthalmitis could be rendered in one case (1%) . Changes consistent with sarcoidosis were present in 3% of cases . CONCLUSION: Based on this experience with vitreous specimens submitted for clinical reasons, we found that they could be divided into three broad diagnostic categories: inflammation/infection (54 specimens/41 patients), hemorrhage (12 specimens/12 patients) and malignancy (8 specimens/7 patients).

J Dent Res, 1999 Mar, 78(3), 735 - 42
Extracts of Actinobacillus actinomycetemcomitans induce apoptotic cell death in human osteoblastic MG63 cells; Morimoto Y et al.; Whether an extracellular component of periodontal-disease-causing bacteria induces apoptotic cell death in bone-related cells is unknown . To study the effects on osteoblasts of extracts obtained from sonicated Actinobacillus actinomycetemcomitans and Prevotella intermedia, we cultured human osteoblastic cell lines MG63 and Saos-2 cells and mouse osteoblastic cell line MC3T3-E1 cells in the presence of such extracts . The addition of the extracts from Actinobacillus actinomycetemcomitans induced cell death in MG63 cells in a dose- and time-dependent fashion over the concentration range of 0.1 to 10 microg/mL . By contrast, the extracts from Prevotella intermedia did not induce cell death in these cells, even in the presence of 10 microg/mL protein . By using the Hoechst 33342 staining technique, we observed marked nuclear condensation and fragmentation of chromatin in MG63 cells treated with the extracts of Actinobacillus actinomycetemcomitans . DNA ladder formation, a hallmark of apoptosis, also was detected in MG63 cells treated with extracts from Actinobacillus actinomycetemcomitans . In MG63 cells, DNA ladder formation was dose-dependent, with a maximal effect at a concentration of 10 microg/mL, and time-dependent, from 12 to 48 hrs . However, the extracts from Prevotella intermedia did not induce DNA fragmentation in MG63, Saos-2, or MC3T3-E1 cells . The extracts from Actinobacillus actinomycetemcomitans did not induce cell death and DNA fragmentation in Saos-2 and MC3T3-E1 cells . Sonicated extracts of Actinobacillus actinomycetemcomitans that had been treated with heat and trypsin did not induce DNA ladder formation in MG63 cells, suggesting that the apoptosis-inducing factors are proteinaceous . Cycloheximide prevented the Actinobacillus actinomycetemcomitans-induced DNA ladder formation in MG63 cells in a dose-dependent fashion, suggesting that new gene transcription and protein synthesis are regulated for Actinobacillus actinomycetemcomitans-induced apoptosis in MG63 cells . Our results indicate that apoptosis in alveolar bone cells induced by Actinobacillus actinomycetemcomitans plays an important role in periodontal diseases.

Mult Scler, 1999 Feb, 5(1), 17 - 21
Acute/relapsing experimental autoimmune encephalomyelitis: induction of long lasting, antigen-specific tolerance by syngeneic bone marrow transplantation; Karussis D et al.; Experimental autoimmune encephalomyelitis (EAE) is an inducible autoimmune disease widely used as a model of the acute/relapsing stage of multiple sclerosis . We have previously shown that treatment of EAE-mice with high doses of cyclophosphamide (CY) (350 mg kg), followed by syngeneic bone marrow transplantation (SBMT), completely abrogates the clinical paralytic signs and even prevents the appearance of new relapses in the chronic-relapsing model of the disease . In the present study we examined whether this treatment protocol induces long term tolerance and whether this tolerance is antigen-specific . EAE was induced by immunization with spinal cord homogenate (MSCH) in complete Freund's adjuvant (CFA) . The treatment with CY and SBMT was performed on day 6 post immunization . Treated and untreated mice were rechallenged with MSCH, or a non-relevant antigen (OVA) in CFA at various stages after the first paralytic attack . In contrast to previous data showing that animals recovering from acute EAE are usually refractory to re-induction of the disease, repeated injections of MSCH at different sites from the initial immunization, followed by i.v . injection of inactivated Bordetella bacteria, 2, 4 and 6 months after the initial EAE-induction, caused a severe and usually lethal relapse in all the untreated, control animals . Mice treated with CY and SBMT were resistant to all rechallenges with the same encephalitogenic inoculum . Following the second rechallenge, peripheral lymph node cells were examined in vitro for their proliferative responses to myelin antigens or to OVA . Lymphocytes obtained from CY+SBMT treated mice did not proliferate in vitro in response to myelin basic protein (MBP), but proliferated against OVA, when immunized with this antigen, after SBMT . Adoptive transfer of lymphocytes from tolerant mice to naive recipients did not transfer resistance to EAE-induction . Our results indicate that high doses of CY, followed by SBMT, induce long term antigen-specific tolerance presumably by a mechanism of clonal deletion or anergy.

J Bacteriol, 1999 Apr, 181(7), 2267 - 72
Structure-function study of MalF protein by random mutagenesis; Tapia MI et al.; MalF is one of the two integral inner membrane proteins of the maltose-maltodextrin transport system . To identify functional regions in this protein, we characterized a collection of malF mutants obtained by random mutagenesis . We analyzed their growth on maltose and maltodextrins, the steady-state levels and subcellular localization of the mutant proteins, and the subcellular localization of MalK . Only 2 of the 21 MalF mutant proteins allowed growth on maltose and maltodextrins . Most mutations resulting in immunodetectable proteins mapped to hydrophilic domains, indicating that insertions affecting transmembrane segments gave rise to unstable or lethal proteins . All MalF mutant proteins, even those C-terminally truncated or with large N-terminal deletions, were inserted into the cytoplasmic membrane . Having identified mutations leading to reduced steady-state level, to partial mislocation, and/or to misfolding, we were able to assign to some regions of MalF a role in the assembly of the MalFGK2 complex and/or in the transport mechanism.

J Bacteriol, 1999 Apr, 181(7), 2038 - 43
Twelve pil genes are required for biogenesis of the R64 thin pilus; Yoshida T et al.; The IncI1 plasmid R64 produces two kinds of sex pili: a thin pilus and a thick pilus . The thin pilus, which belongs to the type IV family, is required only for liquid matings . Fourteen genes, pilI to -V, were found in the DNA region responsible for the biogenesis of the R64 thin pilus (S.-R . Kim and T . Komano, J . Bacteriol . 179:3594-3603, 1997) . In this study, we introduced frameshift mutations into each of the 14 pil genes to test their requirement for R64 thin pilus biogenesis . From the analyses of extracellular secretion of thin pili and transfer frequency in liquid matings, we found that 12 genes, pilK to -V, are required for the formation of the thin pilus . Complementation experiments excluded the possible polar effects of each mutation on the expression of downstream genes . Two genes, traBC, were previously shown to be required for the expression of the pil genes . In addition, the rci gene is responsible for modulating the structure and function of the R64 thin pilus via the DNA rearrangement of the shufflon . Altogether, 15 genes, traBC, pilK through pilV, and rci, are essential for R64 thin pilus formation and function.

FEBS Lett, 1999 Feb 26, 445(2-3), 333 - 7
Chemical modification of lysine side chains of cyclodextrin glycosyltransferase from Thermoanaerobacter causes a shift from cyclodextrin glycosyltransferase to alpha-amylase specificity; Alcalde M et al.; Cyclodextrin glycosyltransferases and alpha-amylases are two groups of enzymes with related secondary structures . However, cyclodextrin glycosyltransferases display transferase activities not present in alpha-amylases, probably derived from the existence of two more domains and different amino acid sequences . The hydrolytic activity of cyclodextrin glycosyltransferases is generally quite low, except for two cyclodextrin glycosyltransferases from termophiles . In this work, we have carried out the chemical modification (with acetic anhydride) of the amino groups of cyclodextrin glycosyltransferase from Thermoanaerobacter to assess their contributions to protein function . The acetylated cyclodextrin glycosyltransferase showed a significant reduction of its cyclization, coupling and disproportionation activities . Surprisingly, the hydrolytic (saccharifying) activity was slightly enhanced . These results suggest the participation of one or more lysine side chains in the interactions contributing to the transferase activity, either in any of the S11 subsites or in the acceptor binding site.

Pediatr Infect Dis J, 1999 Mar, 18(3), 271 - 5
An epidemic of a pertussis-like illness caused by Chlamydia pneumoniae; Hagiwara K et al.; BACKGROUND: Between June and July, 1994, we encountered an epidemic of a pertussis-like illness in adolescents in a junior high school located in a rural area of Japan . The purposes of this study were to record the clinical manifestations and to identify an etiology . PATIENTS AND METHODS: We interviewed patients and parents and we performed physical examinations on patients with cough during the epidemic . The chest radiographs were also reviewed by us . To identify an etiology we performed culture and serologic studies for a variety of bacteria, Mycoplasma, chlamydiae and viruses . Polymerase chain reaction (PCR) for Chlamydia pneumoniae was carried out on throat swab specimens . RESULTS: Of a total of 230 students 136 (59%) had severe cough illnesses . One developed pneumonia, 9 had bronchitis and the remaining 126 (93%) presented upper respiratory tract infections (URI) . The mean duration of cough in cases with URI was 17.4 days and that in cases with bronchitis and pneumonia was 30.4 days . Serology and/or cultures for Bordetella pertussis, Bordetella parapertussis, Mycoplasma pneumoniae, Chlamydia trachomatis, Chlamydia psittaci or viruses were negative . Detection of C . pneumoniae infection was carried out in 46 patients with pneumonia, bronchitis or URI by serology and PCR . The patient with pneumonia, 7 of 7 patients with bronchitis and 32 (84%) of 38 patients with URI were documented to be infected by C . pneumoniae either by serology, PCR or both tests . CONCLUSION: An epidemic of a pertussis-like illness in a junior high school population was caused by C . pneumoniae.

J Immunother, 1999 Mar, 22(2), 93 - 102
Quantitation of T-cell receptor frequencies by competitive PCR: generation and evaluation of novel TCR subfamily and clone specific competitors; McKee MD et al.; T cell receptor (TCR) V gene usage has been used to characterize the immune response to bacteria, viruses, allografts, self antigens, tumor antigens, and superantigens . Sensitive methods to detect changes in the frequency of TCR subfamilies or clonotypes might be useful in evaluating the efficacy of vaccines against infectious agents, immunotherapy treatments for cancer patients, or the status of autoimmune diseases . Two HLA-A2 restricted CTL clones expressing BV17 were isolated from a tumor infiltrating lymphocytes (TIL) culture of a patient with metastatic melanoma . One clone recognized the MART-1(27-35) peptide and the other clone recognized the gp100(209-217) peptide . The frequency of each of these CTL clones in an expanding TIL culture was measured using a novel competitive RT-PCR (cRT-PCR) strategy . cRT-PCR uses a single primer pair to amplify template cDNA simultaneously with a modified DNA competitor molecule . A rapid two-step PCR technique followed by a single cloning step was used to generate a TCR BV17 subfamily specific competitor or competitors specific for the MART-1(27-35) reactive CTL clone (CO-41) and the gp100(209-217) reactive CTL clone (CO-4) . Each competitor contained a segment of the TCR BC region that served as an internal reference standard . Using the BV17 competitor we were able to accurately and reproducibly measure cDNA templates at a frequency as low as 1/100,000 using cDNA samples of known TCRBV subfamily composition . This competitor was used to monitor the frequency of BV17 expressing T cells in the TIL and PMBC of a patient with metastatic melanoma . We determined that the frequency of BV17 expressing T cells increased from 4.5% of the culture on day 35 to 60.7% of the culture on day 58 . Expansion of the BV17 subfamily was due predominantly to the expansion of the CO-4 clone . This method can be used to meaningfully quantify the precursor frequency of T cell mRNA in prepared samples via TCR subfamily or TCR sequence specific primers.

Dis Aquat Organ, 1999 Jan 29, 35(2), 101 - 5
Different prevalences of Renibacterium salmoninarum detected by ELISA in Alaskan chinook salmon Oncorhynchus tshawytscha spawned from freshwater and seawater; Meyers TR et al.; Soluble antigen of Renibacterium salmoninarum (Rs) was detected by a polyclonal enzyme-linked immunosorbent assay (ELISA) at significantly higher prevalences in adult chinook salmon Oncorhynchus tshawytscha that matured in freshwater than in the same cohort of fish spawned after maturation in seawater . The cumulative results were consistent during 4 yr of comparison at the Little Port Walter Hatchery on Baranof Island, Alaska, USA . Possible causes for this difference are discussed . Maturation of chinook salmon broodstock in seawater has become a practical strategy at this hatchery to reduce the prevalence of Rs-positive parent fish and the numbers of culled eggs.

J Immunol, 1999 Mar 15, 162(6), 3481 - 90
Two constituents of the initiation complex of the mannan-binding lectin activation pathway of complement are encoded by a single structural gene; Stover CM et al.; Mannan-binding lectin (MBL) forms a multimolecular complex with at least two MBL-associated serine proteases, MASP-1 and MASP-2 . This complex initiates the MBL pathway of complement activation by binding to carbohydrate structures present on bacteria, yeast, and viruses . MASP-1 and MASP-2 are composed of modular structural motifs similar to those of the C1q-associated serine proteases C1r and C1s . Another protein of 19 kDa with the same N-terminal sequence as the 76-kDa MASP-2 protein is consistently detected as part of the MBL/MASP complex . In this study, we present the primary structure of this novel MBL-associated plasma protein of 19 kDa, MAp19, and demonstrate that MAp19 and MASP-2 are encoded by two different mRNA species generated by alternative splicing/polyadenylation from one structural gene.

J Biol Chem, 1999 Apr 2, 274(14), 9803 - 11
Cloning, expression, and substrate specificity of MeCPA, a zinc carboxypeptidase that is secreted into infected tissues by the fungal entomopathogen Metarhizium anisopliae; Joshi L et al.; To date zinc carboxypeptidases have only been found in animals and actinomycete bacteria . A cDNA clone (MeCPA) for a novel fungal (Metarhizium anisopliae) carboxypeptidase (MeCPA) was obtained by using reverse transcription differential display polymerase chain reaction to identify pathogenicity genes . MeCPA resembles pancreatic carboxypeptidases in being synthesized as a precursor species (418 amino acids) containing a large amino-terminal fragment (99 amino acids) . The mature (secreted) form of MeCPA shows closest amino acid identity to human carboxypeptidases A1 (35%) and A2 (37%) . MeCPA was expressed in an insect cell line yielding an enzyme with dual A1 + A2 specificity for branched aliphatic and aromatic COOH-terminal amino acids . However, in contrast to the very broad spectrum A + B-type bacterial enzymes, MeCPA lacks B-type activity against charged amino acids . This is predictable as key catalytic residues determining the specificity of MeCPA are conserved with those of mammalian A-type carboxypeptidases . Thus, in evolutionary terms the fungal enzyme is an intermediate between the divergence of A and B forms and the differentiation of the A form into A1 and A2 isoforms . Ultrastructural immunocytochemistry of infected host (Manduca sexta) cuticle demonstrated that MeCPA participates with the concurrently produced endoproteases in procuring nutrients; an equivalent function to digestive pancreatic enzymes.

J Biol Chem, 1999 Apr 2, 274(14), 9175 - 82
Barley BLZ2, a seed-specific bZIP protein that interacts with BLZ1 in vivo and activates transcription from the GCN4-like motif of B-hordein promoters in barley endosperm; Onate L et al.; A barley endosperm cDNA, encoding a DNA-binding protein of the bZIP class of transcription factors, BLZ2, has been characterized . The Blz2 mRNA expression is restricted to the endosperm, where it precedes that of the hordein genes . BLZ2, expressed in bacteria, binds specifically to the GCN4-like motif (GLM; 5'-GTGAGTCAT-3') in a 43-base pair oligonucleotide derived from the promoter region of a Hor-2 gene (B1-hordein) . This oligonucleotide also includes the prolamin box (PB; 5'-TGTAAAG-3') . Binding by BLZ2 is prevented when the GLM is mutated to 5'-GTGctTCtc-3' but not when mutations affect the PB . The BLZ2 protein is a potent transcriptional activator in a yeast two-hybrid system where it dimerizes with BLZ1, a barley bZIP protein encoded by the ubiquitously expressed Blz1 gene . Transient expression experiments in co-bombarded developing barley endosperms demonstrate that BLZ2 transactivates transcription from the GLM of the Hor-2 gene promoter and that this activation is also partially dependent on the presence of an intact PB . A drastic decrease in GUS activity is observed in co-bombarded barley endosperms when using as effectors equimolar mixtures of Blz2 and Blz1 in antisense constructs . These results strongly implicate the endosperm-specific BLZ2 protein from barley, either as a homodimer or as a heterodimer with BLZ1, as an important transcriptional activator of seed storage protein genes containing the GLM in their promoters.

Plant Mol Biol, 1999 Feb, 39(3), 565 - 75
A novel flower-specific Arabidopsis gene related to both pathogen-induced and developmentally regulated plant beta-1,3-glucanase genes; Delp G et al.; Beta-1,3-glucanases are usually associated with plant defense responses, although some are also developmentally or hormonally regulated . We characterized two Arabidopsis genes linked in a tandem array, BG4 and BG5, encoding putative novel isoforms of beta-1,3-glucanase . The deduced polypeptides, BG4 and BG5, were highly similar to each other (89% amino acid identity) but only moderately related (32 to 41% amino acid identity) to the different categories of previously characterized beta-1,3-glucanases, suggesting that BG4 and BG5 may represent a novel class of beta-1,3-glucanases in plants . Neither of the genes was responsive to pathogen or SA induction in contrast to the previously identified Arabidopsis beta-1,3-glucanases, nor could we detect any developmental or hormonally induced expression in the vegetative parts of the plants . Both RNA blot and in situ hybridization data demonstrated that the BG4 gene was specifically expressed in the style and septum of the ovary, suggesting that the corresponding protein is involved in the reproductive process of the plant.

Biosystems, 1999 Jan, 49(1), 71 - 8
The mechanical advantages of DNA; Norris V et al.; The elastic properties of DNA and the contractile activities of enzymes involved in transcription, translation and supercoiling may have contributed to the ability of early cells (protocells) to withstand turgor pressure . In the hypothesis proposed here, resistance to turgor resulted from (1) the elastic properties of DNA which was connected to the membrane by association with positively charged lipids and with membrane peptides, (2) the coupled transcription-translation-insertion of peptides into membrane--transertion--which connected membranes with phase-condensed DNA, and (3) the action of topoisomerases which supercoiled and shortened DNA . The existence of a negative feedback system is also proposed to explain how weakened regions of membrane were preferentially strengthened . It may prove possible to test this hypothesis by studying transertion using optical tweezers and by studying wall-less L-form bacteria.

Protein Sci, 1999 Mar, 8(3), 689 - 92
Common structural features of MAPEG -- a widespread superfamily of membrane associated proteins with highly divergent functions in eicosanoid and glutathione metabolism; Jakobsson PJ et al.; A novel superfamily designated MAPEG (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism), including members of widespread origin with diversified biological functions is defined according to enzymatic activities, sequence motifs, and structural properties . Two of the members are crucial for leukotriene biosynthesis, and three are cytoprotective exhibiting glutathione S-transferase and peroxidase activities . Expression of the most recently recognized member is strongly induced by p53, and may therefore play a role in apoptosis or cancer development . In spite of the different biological functions, all six proteins demonstrate common structural characteristics typical of membrane proteins . In addition, homologues are identified in plants, fungi, and bacteria, demonstrating this superfamily to be generally occurring.

Sports Med, 1999 Feb, 27(2), 73 - 80
Exercise and immune function . Recent developments; Nieman DC et al.; Comparison of immune function in athletes and nonathletes reveals that the adaptive immune system is largely unaffected by athletic endeavour . The innate immune system appears to respond differentially to the chronic stress of intensive exercise, with natural killer cell activity tending to be enhanced while neutrophil function is suppressed . However, even when significant changes in the level and functional activity of immune parameters have been observed in athletes, investigators have had little success in linking these to a higher incidence of infection and illness . Many components of the immune system exhibit change after prolonged heavy exertion . During this 'open window' of altered immunity (which may last between 3 and 72 hours, depending on the parameter measured), viruses and bacteria may gain a foothold, increasing the risk of subclinical and clinical infection . However, no serious attempt has been made by investigators to demonstrate that athletes showing the most extreme post-exercise immunosuppression are those that contract an infection during the ensuing 1 to 2 weeks . This link must be established before the 'open window' theory can be wholly accepted . The influence of nutritional supplements, primarily zinc, vitamin C, glutamin and carbohydrate, on the acute immune response to prolonged exercise has been measured in endurance athletes . Vitamin C and glutamine have received much attention, but the data thus far are inconclusive . The most impressive results have been reported in the carbohydrate supplementation studies . Carbohydrate beverage ingestion has been associated with higher plasma glucose levels, an attenuated cortisol and growth hormone response, fewer perturbations in blood immune cell counts, lower granulocyte and monocyte phagocytosis and oxidative burst activity, and a diminished pro- and anti-inflammatory cytokine response . It remains to be shown whether carbohydrate supplementation diminishes the frequency of infections in the recovery period after strenuous exercise . Studies on the influence of moderate exercise training on host protection and immune function have shown that near-daily brisk walking compared with inactivity reduced the number of sickness days by half over a 12- to 15-week period without change in resting immune function . Positive effects on immunosurveillance and host protection that come with moderate exercise training are probably related to a summation effect from acute positive changes that occur during each exercise bout . No convincing data exist that moderate exercise training is linked with improved T helper cell counts in patients with HIV, or enhanced immunity in elderly participants.

Microb Pathog, 1999 Feb, 26(2), 53 - 63
Downregulation of a protective Actinobacillus pleuropneumoniae antigen during the course of infection; Hennig I et al.; The persistence of Actinobacillus pleuropneumoniae in convalescent pigs significantly contributes to the distribution of disease . The downregulation of protective antigens in vivo as one possible mechanism responsible for this phenomenon was investigated using the small iron-regulated transferrin binding protein (TbpB-protein) as exemplary protective antigen . From a total of 21 pigs experimentally infected with A . pleuropneumoniae serotype 7 in three trials, bronchoalveolar lavage fluid (BALF) was obtained on day 1 or 2, day 7, day 14 and day 21 . Employing double immunofluorescence of BALF with a monoclonal anti-TbpB antibody and an A . pleuropneumoniae -specific anti-polysaccharide antiserum a statistically significant decrease of the percentage of A . pleuropneumoniae bacteria strongly expressing TbpB protein was observed during the course of infection . These results were supported by in vitro incubation of A . pleuropneumoniae in medium supplemented with BALF . In addition, it was found that TbpB-expression in BALF from day 7 after infection could not be inhibited by the substitution of iron . These results suggest (i) the downregulation of protective antigens is one possible mechanism allowing bacterial persistence, (ii) in vitro induction in the presence of BALF mimics the in vivo situation, and (iii) TbpB expression is additionally regulated by an iron-independent mechanism .

Biochemistry, 1999 Mar 23, 38(12), 3711 - 8
Lysine-313 of 5-aminolevulinate synthase acts as a general base during formation of the quinonoid reaction intermediates; Hunter GA et al.; 5-Aminolevulinate synthase catalyzes the condensation of glycine and succinyl-CoA to form CoA, carbon dioxide, and 5-aminolevulinate . This represents the first committed step of heme biosynthesis in animals and some bacteria . Lysine 313 (K313) of mature murine erythroid 5-aminolevulinate synthase forms a Schiff base linkage to the pyridoxal 5'-phosphate cofactor . In the presence of glycine and succinyl-CoA, a quinonoid intermediate absorption is transiently observed in the visible spectrum of purified murine erythroid ALAS . Mutant enzymes with K313 replaced by glycine, histidine, or arginine exhibit no spectral evidence of quinonoid intermediate formation in the presence of glycine and succinyl-CoA . The wild-type 5-aminolevulinate synthase additionally forms a stable quinonoid intermediate in the presence of the product, 5-aminolevulinate . Only conservative mutation of K313 to histidine or arginine produces a variant that forms a quinonoid intermediate with 5-aminolevulinate . The quinonoid intermediate absorption of these mutants is markedly less than that of the wild-type enzyme, however . Whereas the wild-type enzyme catalyzes loss of tritium from {2-3H2}-glycine, mutation of K313 to glycine results in loss of this activity . Titration of the quinonoid intermediate formed upon binding of 5-aminolevulinate to the wild-type enzyme indicated that the quinonoid intermediate forms by transfer of a single proton with a pK of 8.1 +/- 0.1 . Conservative mutation of K313 to histidine raises this value to 8.6 +/- 0.1 . We propose that K313 acts as a general base catalyst to effect quinonoid intermediate formation during the 5-aminolevulinate synthase catalytic cycle.

Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 31 - 45 Epub 1999 Jan 01.
Structure analysis of bovine heart cytochrome c oxidase at 2.8 A resolution; Tomizaki T et al.; The crystal structure of bovine heart cytochrome c oxidase has been determined at 2.8 A resolution by the multiple isomorphous replacement (MIR) method with three heavy-atom derivatives . An asymmetric unit of the crystal has a molecular weight of 422 kDa . Eight heavy atoms as main sites of a CH3HgCl derivative were clearly located by solving the difference Patterson function . The electron density obtained by the MIR method was refined by density modification, consisting of solvent flattening, histogram matching and non-crystallographic symmetry averaging . The enzyme exhibits a dimeric structure in the crystal . Out of 3606 amino-acid residues in 26 subunits in the dimer, 3560 residues were located in the electron-density map . The structure was refined by X-PLOR . The final R factor and the free R factor were 0.199 and 0.252 at 2.8 A resolution, respectively . One monomer in the dimeric structure with a stronger packing interaction has a lower averaged temperature factor than the other, by 16 A2 . The region +/-12 A from the centre of the transmembrane part is almost 100% alpha-helix, despite the glycine residue content being as high as 7.1% in the transmembrane region . The residues around haem a of animals have evolved away from those of bacteria in contrast with the residues of the haem a3 . The hierarchy of the structural organization of the enzyme complex has been proposed on the basis of intersubunit interactions.

Acta Crystallogr D Biol Crystallogr, 1999 Feb, 55 ( Pt 2), 369 - 78
Solution of the structure of the cofactor-binding fragment of CysB: a struggle against non-isomorphism; Verschueren KH et al.; The elucidation of the structure of CysB(88-324) by multiple isomorphous replacement (MIR) techniques was seriously delayed by problems encountered at every stage of the analysis . There was extensive non-isomorphism both between different native crystals and between native and heavy-atom-soaked crystals . The heavy-atom substitution was invariably weak and different soaking experiments frequently led to substitution at common sites . These correlated heavy-atom binding sites resulted in an overestimation of the phase information . Missing low-resolution reflections in the native data set, constituting only 2% of the total observations, reduced the power of density modification and phase refinement . Finally, the extensive dimer interface made it difficult to isolate a single molecule in the course of model building into the MIR maps . The power of maximum likelihood refinement (REFMAC) was exploited in solving the structure by means of iterative cycles of refinement of a partial model, initially comprising only 30% of the protein atoms in the final coordinate set . This technique, which uses experimental phases, can automatically discriminate the correct and incorrect parts of electron-density maps and give properly weighted combined phases which are better than the experimental or calculated ones . This allowed the model to be gradually extended by manual building into improved electron-density maps . A model generated in this way, containing just 50% of the protein atoms, proved good enough to find the transformations needed for multi-crystal averaging between different crystal forms . The averaging regime im-proved the phasing dramatically such that the complete model could be built . The problems, final solutions and some possible causes for the observed lack of isomorphism are discussed.

Acta Crystallogr D Biol Crystallogr, 1999 Apr, 55 ( Pt 4), 880 - 2
Crystallization and preliminary crystallographic analysis of the major horse allergen Equ c 1; Gregoire C et al.; The secreted protein Equ c 1 is the major component responsible for the induction of specific IgE antibodies in patients sensitized to horse allergens . Equ c 1 belongs to the lipocalin superfamily of hydrophobic ligand-binding proteins, which also includes other known allergens . Equilibrium sedimentation and gel-filtration studies demonstrate that both the glycosylated form of Equ c 1 purified from horse salivary glands and the non-glycosylated recombinant form expressed in bacteria exist predominantly as dimers in solution . As observed for other dimeric lipocalins, acidic pH and low protein concentration favour dimer dissociation . The recombinant form of Equ c 1 has been crystallized using ammonium sulfate as a precipitant . The crystals belong to the tetragonal space group P41212 with cell parameters a = b = 84.0, c = 56.1 A, and contain a single molecule in the asymmetric unit . A complete data set from native crystals was collected at the synchrotron source in Hamburg to 2.9 A resolution using a frozen crystal, and structure determination is in progress.

J Infect, 1999 Jan, 38(1), 12 - 7
Detection of Chlamydia trachomatis DNA in cervical samples with regard to infection by human papillomavirus; Lehmann M et al.; OBJECTIVE: The correlation between human papillomavirus (HPV) and Chlamydia, trachomatis infections was evaluated in 144 patients with normal cytology or with atypical squamous cells of undetermined significance (ASCUS) . METHODS: Cervical samples were analysed using polymerase chain reaction (PCR) and non-radioactive Southern blot analysis . Specificity and sensitivity of two C . trachomatis PCR systems: major outer membrane protein (MOMP)-PCR and plasmid-PCR were determined . Southern blot hybridization of the PCR amplicons was done using 5' and 3' biotinylated oligonucleotide probes . RESULTS: All cervical samples were tested by the plasmid-PCR due to a 10 times higher sensitivity compared to the MOMP-PCR . To determine the specificity of our C . trachomatis primer sets different bacteria and viruses which can cause urogenital infections were analysed . Comparison of the probes revealed an increased sensitivity of the 5' and 3' double-biotinylated probe vs . the 5' biotinylated probe . The infection rate of C . trachomatis in cervical samples of HPV-positive patients was 10.3% (three out of 29) vs . 1.7% (two out of 115; P< or =0.05) in HPV-negative patients . In patients HPV-X (unsequenced HPV-types) positive the rate was 14.3% (one out of seven) vs . 2.9% (four out of 137: P = 0.2) in HPV-X negative patients . In high risk (HR) HPV-positive cervical samples the infection rate was 9.1% (two out of 22) vs . 2.5% (three out of 122; P = 0.14) in HR HPV-negative samples . Chlamydia trachomatis frequency of patients with cytological changes (ASCUS) was 27.3% (three out of 11) vs . 1.5% (two out of 1 33) in patients with normal cytology (P = 0.003) . The highest prevalence rate of C . trachomatis-positive cervical samples (50%; one out of two) was found in HR HPV-positive patients with cytological changes (ASCUS) vs . 5% (one out of 20) in HR HPV-positive patients with normal cytology (P = 0.17) . Patients negative for HPV and positive for ASCIIS have a C . trachomatis rate of 22.2% (two out of nine) vs . HPV-negative patients with normal cytology (none out of 106; P = 0.006) and vs . HR HPV-negative patients with normal cytology (0.9%; one out of 113; P = 0.014) . CONCLUSIONS: There appears to be a correlation between cervical HPV and cervical C . trachomatis infections . The prevalence rate of C . trachomatis was significantly higher in patients with abnormal cytology (ASCUS) vs . normal cytology.

Glycobiology, 1999 Apr, 9(4), 407 - 14
Complete glycan structure of the S-layer glycoprotein of Aneurinibacillus thermoaerophilus GS4-97; Schaffer C et al.; Isolate GS4-97 was purified from an extraction juice sample of an Austrian beet sugar factory and affiliated to the newly described species Aneurinibacillus thermoaerophilus . It is closely related to the type strain of this species, A.thermoaerophilus L420-91(T), and possesses a square surface layer (S-layer) array composed of identical glycoprotein monomers as its outermost cell envelope component . By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified S-layer showed an apparent molecular mass of approximately 109,000 . After thorough proteolytic degradation of this material by pronase E and purification of the reaction mixture by gel permeation, chromatofocusing, and reversed-phase chromatography, a homogeneous glycopeptide fraction was obtained which was subjected to one- and two-dimensional nuclear magnetic resonance spectroscopy . The combined chemical and spectroscopic evidence, together with N-terminal sequencing, suggest the following structure of the O-glycosidically linked S-layer glycan chain of the glycopeptide: This is the first description of a beta-d-GalNAc-Thr linkage in glycoproteins.

Mol Med Today, 1999 Jan, 5(1), 32 - 9
Virulence factors of Helicobacter pylori: implications for vaccine development; Suerbaum S et al.; Helicobacter pylori is one of the most common infectious diseases in humans and causes gastritis, peptic ulcer disease and malignant tumours of the stomach . This review discusses how H . pylori can colonize the human stomach, an ecological niche that is protected against all other bacteria . Knowledge about the virulence factors of H . pylori has accumulated rapidly over the last decade . Together with the information contained in the complete H . pylori genome sequence, this knowledge is now being applied in the search for a vaccine against this global pathogen.

Ostomy Wound Manage, 1999 Jan, 45(1A Suppl), 80S - 85S; quiz 86S-87S
Pressure ulcer debridement and cleansing: a review of current literature; Rodeheaver GT; Optimal wound healing can only occur in a clean wound . Cleaning the wound of inflammatory stimuli such as devitalized tissue, reactive chemicals, and bacteria involves the procedures of debridement and cleansing . Debridement can be accomplished surgically, mechanically (wet-to-dry gauze), or chemically (autolysis or topical enzymes) . Several recent studies are summarized that document the benefits of each of these techniques . Cleansing is the process of using fluid to remove loosely adherent foreign material . Numerous wound cleansing solutions and novel irrigation systems have been introduced in recent years . The potential benefits and harms of these products are discussed.

Biochem J, 1999 Apr 1, 339 ( Pt 1), 127 - 34
ATPase activity associated with the magnesium-protoporphyrin IX chelatase enzyme of Synechocystis PCC6803: evidence for ATP hydrolysis during Mg2+ insertion, and the MgATP-dependent interaction of the ChlI and ChlD subunits; Jensen PE et al.; Insertion of Mg2+ into protoporphyrin IX catalysed by the three-subunit enzyme magnesium-protoporphyrin IX chelatase (Mg chelatase) is thought to be a two-step reaction, consisting of activation followed by Mg2+ chelation . The activation step requires ATP and two of the subunits, ChlI and ChlD (I and D respectively), and it has been speculated that this step results in the formation of an I-D-ATP complex . The subsequent step, in which Mg2+ is inserted into protoporphyrin, also requires ATP and the third subunit, H, in addition to ATP-activated I-D complex . In the present study, we examine the interaction of the I and D subunits of the Mg chelatase from the cyanobacterium Synechocystis PCC 6803 . We demonstrate the purification of an I-D complex, and show that ATP and Mg2+ are absolute requirements for the formation of this complex, probably as MgATP . However, ATP may be replaced by the slowly hydrolysable analogue, adenosine 5'-{gamma-thio}triphosphate, and, to a minor extent, by ADP and the non-hydrolysable ATP analogue, adenosine 5'-{beta,gamma-imido}triphosphate, all of which suggests that ATP hydrolysis is not necessary for the formation of the ChlI-ChlD complex . A sensitive continuous assay was used to detect ATPase activity during Mg2+ chelation, and it was found that the maximum rate of ATP hydrolysis coincided with the maximum rate of Mg2+ insertion . The rate of ATP hydrolysis depended on factors that determined the rate of Mg2+ chelation, such as increasing the concentration of the H subunit and the concentration of protoporphyrin . Thus ATP hydrolysis has been identified as an absolute requirement for the chelation step . The I subunit possessed strong ATPase activity when assayed on its own, whereas the D subunit had no detectable activity, and when the I and D subunits were assayed in combination, the ATPase activity of the I subunit was repressed.

Infect Immun, 1999 Apr, 67(4), 1962 - 6
Vaccination and protection of pigs against pleuropneumonia with a vaccine strain of Actinobacillus pleuropneumoniae produced by site-specific mutagenesis of the ApxII operon; Prideaux CT et al.; The production of toxin (Apx)-neutralizing antibodies during infection plays a major role in the induction of protective immunity to Actinobacillus pleuropneumoniae reinfection . In the present study, the gene encoding the ApxII-activating protein, apxIIC, was insertionally inactivated on the chromosome of a serovar 7 strain, HS93 . Expression of the structural toxin, ApxIIA, and of the two genes required for its secretion, apxIB and apxID, still occurs in this strain . The resulting mutant strain, HS93C- Ampr, was found to secrete the unactivated toxin . Pigs vaccinated with live HS93C- Ampr via the intranasal route were protected against a cross-serovar challenge with a virulent serovar 1 strain of A . pleuropneumoniae . This is the first reported vaccine strain of A . pleuropneumoniae which can be delivered live to pigs and offers cross-serovar protection against porcine pleuropneumonia.

Infect Immun, 1999 Apr, 67(4), 1917 - 21
Effect of temperature on growth, hemagglutination, and protease activity of Porphyromonas gingivalis; Percival RS et al.; Bacteria persisting in periodontal pockets are exposed to elevated temperatures during periods of inflammation . Temperature is an environmental factor that can modulate gene expression . Consequently, in the present study we examined the effect of temperature on the expression of virulence determinants by the periodontopathogen, Porphyromonas gingivalis . P . gingivalis W50 was grown in a complex medium under hemin excess at pH 7.0 and at a constant temperature of either 37, 39, or 41 degrees C; cultures were monitored for protease and hemagglutinin activity . P . gingivalis grew well at all three temperatures . An increase in growth temperature from 37 to 39 degrees C resulted in a 65% reduction in both total arginine- and lysine-specific activities (P < 0.01) . A further rise in growth temperature to 41 degrees C led to even greater reductions in arginine-specific (82%; P < 0.001) and lysine-specific (73%; P < 0 . 01) activities . These reductions were also associated with an altered distribution of individual arginine-specific enzyme isoforms . At 41 degrees C, there was a disproportionate reduction in the level of the heterodimeric RI protease, which also contains adhesin domains . The reduction also correlated with a markedly diminished hemagglutination activity of cells, especially in those grown at 41 degrees C, and a reduced immunoreactivity with a monoclonal antibody which recognizes gene products involved in hemagglutination . Thus, as the environmental temperature increased, P . gingivalis adopted a less aggressive phenotype, while retaining cell population levels . The coordinate down-regulation of virulence gene expression in response to an environmental cue linked to the intensity of the host inflammatory response is consistent with the clinically observed cyclical nature of disease progression in periodontal diseases.

Infect Immun, 1999 Apr, 67(4), 1789 - 97
Rapid local expression of interleukin-12, tumor necrosis factor alpha, and gamma interferon after cutaneous Francisella tularensis infection in tularemia-immune mice; Stenmark S et al.; Francisella tularensis LVS is an effective live vaccine strain used for cutaneous vaccination against tularemia in man . In mice, injection of LVS causes invasive disease and subsequent development of immunity that is characterized by effective control of otherwise lethal doses of the organism . In the present investigation, it is shown that LVS-immune mice controlled an intradermal infection much more effectively than did naive mice; bacterial counts in skin samples were 1.5 to 2.0 log10 lower 24 h after injection and 6 log10 lower 72 h after injection in immune mice . Moreover, in contrast to naive mice, no bacteria were demonstrated in samples from livers and spleens of immune mice . By immunohistochemistry, skin samples from immune mice showed an intense staining for interleukin-12 (IL-12) and a moderate staining for tumor necrosis factor alpha (TNF-alpha) at 24 h postinoculation, after which staining for both cytokines faded . In naive mice, the staining for IL-12 was weak at all time points and no staining for TNF-alpha was observed . No staining for gamma interferon (IFN-gamma) was observed in any group before 72 h . At that time point, skin samples from immune mice showed moderate staining and skin samples from naive mice showed weak staining . Reverse transcriptase PCR showed an induction of mRNA of the three cytokines in the skin within the first day after injection . A quantitative analysis demonstrated higher IFN-gamma and TNF-alpha mRNA levels in immune mice at 24 h postinoculation . In conclusion, immunization with F . tularensis LVS conferred a capability to respond to cutaneous reinfection, with rapid local expression of IL-12, TNF-alpha, and IFN-gamma, and this expression was paralleled by containment and mitigation of the infection . The cytokine response may be part of a local barrier function of the skin, important to host protection against tularemia.

Infect Immun, 1999 Apr, 67(4), 1723 - 8
An Arcanobacterium (Actinomyces) pyogenes mutant deficient in production of the pore-forming cytolysin pyolysin has reduced virulence; Jost BH et al.; Pyolysin (PLO), the hemolytic exotoxin expressed by Arcanobacterium (Actinomyces) pyogenes, is a member of the thiol-activated cytolysin family of bacterial toxins . Insertional inactivation of the plo gene results in loss of expression of PLO with a concomitant loss in hemolytic activity . The plo mutant, PLO-1, has an approximately 1 . 8-log10 reduction in the 50% infectious dose compared to that for wild-type A . pyogenes in a mouse intraperitoneal infection model . Studies involving cochallenge of wild-type and PLO-1 bacteria resulted in recovery of similar numbers of both strains, suggesting that PLO production is required for survival in vivo . Recombinant, His-tagged PLO (His-PLO) is cytotoxic for mouse peritoneal macrophages and J774 cells in a dose-dependent manner . Protection against challenge with A . pyogenes could be afforded by vaccination with formalin-inactivated His-PLO, suggesting that PLO is a host-protective antigen, as well as a virulence determinant.

Clin Exp Pharmacol Physiol, 1999 Mar, 26(3), 198 - 205
The cholinergic 'pitfall': acetylcholine, a universal cell molecule in biological systems, including humans; Wessler I et al.; 1 . Acetylcholine (ACh) represents one of the most exemplary neurotransmitters . In addition to its presence in neuronal tissue, there is increasing experimental evidence that ACh is widely expressed in pro- and eukaryotic non-neuronal cells . Thus, ACh has been detected in bacteria, algae, protozoa, tubellariae and primitive plants, suggesting an extremely early appearance of ACh in the evolutionary process . 2 . In humans, ACh and/or the synthesizing enzyme, choline acetyltransferase, has been demonstrated in epithelial cells (airways, alimentary tract, urogenital tract, epidermis), mesothelial (pleura, pericardium) and endothelial and muscle cells . In addition, immune cells express the non-neuronal cholinergic system (i.e . the synthesis of ACh can be detected in human leucocytes (granulocytes, lymphocytes and macrophages)), as well as in rat microglia in vitro . 3 . The widespread expression of non-neuronal ACh is accompanied by the ubiquitous expression of cholinesterase activity, which prevents ACh from acting as a classical hormone . 4 . Non-neuronal ACh mediates its cellular actions in an auto- and paracrine manner via the activation of the widely expressed nicotinic and muscarinic acetylcholine receptors, which can interfere with virtually all cellular signalling pathways (ion channels and key enzymes) . 5 . Non-neuronal ACh appears to be involved in the regulation of basic cell functions, such as mitosis, cell differentiation, organization of the cytoskeleton, cell-cell contact, secretion and absorption . Non-neuronal ACh also plays a role in the regulation of immune functions . All these qualities together may mediate the so-called 'trophic property' of ACh . 6 . Future experiments should be designed to analyse the cellular effects of ACh in greater detail . The involvement of the non-neuronal cholinergic system in the pathogenesis of chronic inflammatory diseases should be investigated to open up new therapeutic strategies.

Antonie Van Leeuwenhoek, 1998 Nov, 74(4), 245 - 51
Coproporphyrin excretion by Azorhizobium caulinodans under micro-aerobic conditions; Pronk AF et al.; Azorhizobium caulinodans ORS571 was found to excrete moderate amounts of a fluorescent pigment into the culture medium in response to dissolved oxygen tensions below 1.0 kPa . The pigment was identified as coproporphyrin, on the basis of its optical and fluorescence spectra . FixLJ and fixK mutant derivatives of ORS571 were found to excrete 25-fold higher amounts of coproporphyrin under micro-aerobic conditions than the wild type strain . These observations suggest that A . caulinodans switches from an aerobic to an anaerobic coproporphyrinogen oxidase when the dissolved oxygen tension falls below 1.0 kPa and that the fixLJ and fixK genes are involved in the regulation of expression of the anaerobic coproporphyrinogen oxidase.

J Infect Dis, 1999 Mar, 179 Suppl 2, S318 - 25
Progress in gene therapy for chronic granulomatous disease; Malech HL; Progress in development of gene therapy for chronic granulomatous disease (CGD), an inherited defect in leukocyte oxidase deficiency, is reviewed . The use of retrovirus vectors to transfer oxidase enzyme subunit cDNA sequence into hematopoietic progenitors results in correction of oxidase activity in neutrophils differentiating from transduced progenitors . In CGD mouse knockouts (X-linked gp91phox-deficient CGD and autosomal recessive p47phox-deficient CGD), gene therapy correction of the CGD defect resulted in appearance of oxidase-normal neutrophils in the peripheral blood and increased host resistance to challenge with fungi or bacteria . In a phase I clinical trial of ex vivo gene therapy of p47phox-deficient CGD, prolonged production (2-6 months) of a low number (1:5000) of oxidase-normal neutrophils was achieved . This therapy might prove beneficial in a setting of prolonged infection in CGD patients, in which even transient production of autologous gene-corrected neutrophils might augment host defense.

Rozhl Chir, 1998 Dec, 77(12), 567 - 73
{Use of genetic methods in the detection of pathogens in complications of extensive surgical procedures}; Dendis M et al.; The method of the polymerase chain reaction (PCR) is used in the detection of septic conditions and monitoring of infectious agents in asymptomatic patients after organ transplantations and extensive cardiosurgical operations . The method allows semiquantitative and quantitative detection of bacteria, micromycetes and viruses and from the systematic quantitative follow-up of pathogen levels it is possible to draw conclusions on the prognosis of the patients condition or the success of the therapeutic procedure . The author demonstrates on practical examples of examined patients the possibilities of the method as compared with hitherto used ones . As examples two patients are quoted after surgery of a valve on account of bacterial endocarditis and three patients after transplantation of life important organs where the presence if cytomegalovirus was detected.

Ophthalmology, 1999 Mar, 106(3), 490 - 6
Acute corneal necrosis after excimer laser keratectomy for hyperopia; Mietz H et al.; OBJECTIVE: To describe a new, rare clinical complication after routine excimer laser photorefractive keratectomy to correct hyperopia . DESIGN: Case report with clinicopathologic correlation . MAIN OUTCOME MEASURES: Four weeks after treatment with excimer laser, a perforating keratoplasty was performed for persistent corneal opacities . The corneal button was examined using light and electron microscopy . Special immunohistochemical stains were used to detect apoptosis . RESULTS: The patient developed corneal opacities, endothelial precipitates, and a fibrinous exudate in the anterior chamber after the laser treatment . The changes did not respond to therapy directed against bacteria, fungi, and Acanthamoeba . All examinations and special stains were negative for micro-organisms . By light microscopy, an anterior zone of corneal necrosis was present with a moderate amount of acute inflammatory cells . At the interface between necrotic and viable corneal stroma, keratocytes with typical features of apoptosis were detected by immunohistochemistry and electron microscopy . CONCLUSION: This is the first full histopathologic report of a case of acute corneal necrosis with signs of apoptosis after excimer laser therapy of the cornea . Surgeons should be aware of this rare but potentially severe complication.

Mol Biochem Parasitol, 1999 Jan 25, 98(2), 253 - 64
A signal recognition particle receptor gene from the early-diverging eukaryote, Giardia lamblia; Svard SG et al.; The molecular mechanisms for targeting and translocation of secreted proteins are highly conserved from bacteria to mammalian cells, although the machinery is more complex in higher eukaryotes . To investigate protein transport in the early-diverging eukaryote, Giardia lamblia, we cloned the gene encoding the alpha subunit (SRalpha) of the signal recognition particle (SRP) receptor . SRalpha is a small GTPase that functions in SRP-ribosome targeting to the ER . Sequence and phylogenetic analyses showed that SRalpha from G . lamblia is most homologous to SRalpha proteins from higher eukaryotes, although it lacks some conserved motifs . Specifically, giardial SRalpha has an N-terminal extension that enables SRalpha of higher eukaryotes to interact with a beta subunit that anchors it in the ER membrane . While the C-terminal regions are similar, giardial SRalpha lacks a prominent 13 amino acid regulatory loop that is characteristic of higher eukaryotic versions . Thus, giardial SRalpha resembles that of higher eukaryotes, but likely diverged before the advent of the regulatory loop . The 1.8 kb SRalpha transcript has extremely short untranslated regions (UTRs): a 1-2 nt 5'- and a 9 nt 3' UTR with the polyadenylation signal overlapping with the stop codon . RT-PCR, Northern and Western analyses showed that SRalpha is present at relatively constant levels during vegetative growth and encystation, even though there are extensive changes in endomembrane structures and secretory activity during encystation . Imnuno-EM showed that SRalpha localizes to ER-like structures, strengthening the observation of a typical ER in G . lamlia . Unexpectedly, SRalpha was also found in the lysosome-like peripheral vacuoles, suggesting unusual protein traffic in this early eukaryote . Our results indicate that the eukaryotic type of cotranslational transport appeared early in the evolution of the eukaryotic cell.

Dig Dis Sci, 1999 Mar, 44(3), 542 - 6
Bacteribilia and cholangitis after percutaneous transhepatic biliary drainage for malignant biliary obstruction; Nomura T et al.; Cholangitis often develops after percutaneous transhepatic biliary drainage (PTBD) for malignancy . The aims of this retrospective study were to clarify whether or not bacteribilia and cholangitis increase with time after PTBD and to define the pathogenesis of bacteribilia and cholangitis after PTBD . One hundred twenty-eight patients underwent PTBD for malignancy . Both the cumulative incidences of bacteribilia (77%) and cholangitis (28%) showed an increase with time after PTBD . In 78% of patients with bacteribilia, bacteria from intestinal flora were detected in bile . Catheter malfunction (N = 17) or the presence of undrained bile ducts (N = 7) induced cholangitis . Proximal obstruction, because it often accompanied undrained ducts, had higher incidences of bacteribilia (P = 0.04) and cholangitis (P < 0.0001) than did distal obstruction . In conclusion, bacteribilia and cholangitis increase in incidence with time after PTBD . The primary cause of bacteribilia is the transpapillary reflux of intestinal flora . Catheter malfunction or undrained ducts cause cholangitis, provided underlying bacteribilia is present.

Bioorg Khim, 1998 Nov, 24(11), 839 - 41
{Structure of glycerophosphate-containing O-specific polysaccharide from Pseudoalteromonas sp . KMM 639}; Gorshkova RP et al.; On the basis of acid hydrolysis, dephosphorylation, methylation, and 13C NMR spectroscopy data, the O-specific polysaccharide of Pseudoalteromonas sp . KMM 639 was shown to be a glycerophosphate-containing polymer built of repeating disaccharide units of the following structure: {formula: see text}

Anal Chem, 1999 Mar 1, 71(5), 1087 - 91
Two-layer sample preparation: a method for MALDI-MS analysis of complex peptide and protein mixtures; Dai Y et al.; The analytical performance of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for direct analysis of peptide and protein mixtures is strongly dependent on the sample and matrix preparation . A two-layer sample preparation method is demonstrated to be very effective for analyzing complex mixtures . In this method, the first layer on the MALDI probe is the densely packed matrix microcrystals formed by fast solvent evaporation of a matrix solution . A mixture solution containing both matrix and sample is then deposited onto the first layer to form uniform analyte/matrix micrococrystals . It is found that the addition of matrix to the second-layer sample solution proves to be critical in analyzing mixtures of peptides and proteins covering a broad mass range . The effect of solvent conditions for preparing the second-layer solution is discussed . The application of this method is demonstrated for the analysis of cow's milk where milk proteins as well as peptide fragments produced from proteins by indigenous proteinases are detected . Direct analyses of peptides and proteins from a bacteria extract and crude egg white are also illustrated.

J Mol Evol, 1999 Apr, 48(4), 482 - 92
Identification of new members of the GS ADP-forming family from the de novo purine biosynthesis pathway; Kanai S et al.; Most living organisms can synthesize isosinate from 5-phosphoribosyl 1-pyrophosphate in the de novo purine biosynthesis pathway, which is basically composed of 10 reaction steps . Phosphoribosylglycinamide synthetase (GARS) catalyzes the second step of the pathway . We found that the enzyme shows weak, but significant, sequence similarity to phosphoribosylglycinamide formyltransferase 2 (GART2) and the ATPase domain of phosphoribosylaminoimidazole carboxylase (AIRCA), which catalyze the third and sixth steps of the pathway, respectively . In addition, the three enzymes were similar in amino acid sequence to biotin carboxylase (BC) and carbamoylphosphate synthetase (CPS), which are the members of the GS ADP-forming family . This family has been identified through a tertiary structure comparison and includes glutathione synthetase, d-alanine:d-alanine ligase, BC, succinyl-CoA synthetase beta-chain, and phosphoribosylaminoimidazole-succinocarboxamide synthase . Molecular phylogenetic analysis based on a multiple alignment of GARS, GART2, AIRCA, BC, and CPS suggests that GART2 is more closely related to AIRCA than to GARS among the three enzymes from the pathway, though the three enzymes are relatively close to each other within the GS ADP-forming family . Moreover, the analysis showed that archaeal GARS had diverged before the speciation between bacteria and eucarya.

Biochemistry, 1999 Mar 16, 38(11), 3362 - 8
MerR cross-links to the alpha, beta, and sigma 70 subunits of RNA polymerase in the preinitiation complex at the merTPCAD promoter; Kulkarni RD et al.; MerR, the metalloregulator of the mercury resistance (mer) operon, binds the operator (merO)between -10 and -35 of the merTPCAD promoter (PT) and sequesters RNA polymerase (RNAP) in a closed complex . MerR represses PT until Hg(II) induces it to underwind merO DNA and thus facilitate open complex formation . We used cross-linking to determine if direct contacts between MerR and RNAP also occur during this process . MerR cross-linked to the alpha, beta, and sigma70 subunits of RNAP alone, indicating stable contacts which were further stabilized upon forming the preinitiation complex at PT . Hg(II) did not eliminate any of the MerR-RNAP cross-links but did increase the relative abundance of a MerR dimer conformer . Interference by MerR with self-cross-links among RNAP subunits and the formation of an electrophoretically stable association between MerR and RNAP also indicated MerR-RNAP interactions . This is the first evidence for stable physical contacts between MerR and RNAP and for a Hg(II)-induced allosteric change in MerR in the transcription-competent complex.

J Spinal Disord, 1999 Feb, 12(1), 17 - 26
Anterior debridement, fusion, and extrafocal stabilization in the treatment of osteomyelitis of the spine; Krodel A et al.; To simplify and shorten the rehabilitation after anterior debridement and fusion in pyogenic and tuberculous osteomyelitis of the spine, the role of additional extrafocal dorsal transpedicular instrumentation was studied . Thirty-three (10 female, 23 male) patients were followed up in a prospective study and controlled with an average follow-up period of 22.1 months after the operation with clinical and neurologic check-up, blood test, and serial radiographs . Solid bony fusion and healing of the infection was achieved in all patients . Preoperative deformities could be corrected, and there were no life-threatening complications . Dorsal extrafocal stabilization offered the advantage of braceless rehabilitation without adding unpredictable risks.

Kansenshogaku Zasshi, 1999 Jan, 73(1), 15 - 9
{Resuscitation from the viable but nonculturable state of Helicobacter pylori}; Kurokawa M et al.; It is well known that the change of state from the culturable to not culturable on pathogenic organisms could easily occurred . These states of bacteria were so called viable but nonculturable (VNC) . The coccoid form of H . pylori is seems to be in this state . The possibility of resuscitation of H . pylori was examined in vitro . The coccoid form bacteria was treated with Ammonium Sulfate and heat shock before culture, and cultured in Brucella broth containing, sodium pyruvate, laked human erythrocyte and serum . The growth of the rod spiral form was found in the coccoid form bacteria, and further the bacteria could grow on blood agar and Skirrow agar as well as the original strain . It was strongly suggested that the resuscitation from VNC state of cells to culturable form consists of two processes, stimulation and supplemented of appropriate nutrition.

Proc Natl Acad Sci U S A, 1999 Mar 16, 96(6), 3188 - 93
The role of an alternative sigma factor in motility and pilus formation in the cyanobacterium Synechocystis sp . strain PCC6803; Bhaya D et al.; Disruption of a gene for an alternative sigma factor, designated sigF, in the freshwater, unicellular cyanobacterium Synechocystis sp . strain PCC6803 resulted in a pleiotropic phenotype . Most notably, this mutant lost phototactic movement with a concomitant loss of pili, which are abundant on the surface of wild-type cells . The sigF mutant also secreted both high levels of yellow-brown and UV-absorbing pigments and a polypeptide that is similar to a large family of extracellular proteins that includes the hemolysins . Furthermore, the sigF mutant had a dramatically reduced level of the transcript from two tandemly arranged pilA genes (sll1694 and sll1695), which encode major structural components of type IV pili . Inactivation of these pilA genes eliminated phototactic movement, though some pili were still present in this strain . Together, these results demonstrate that SigF plays a critical role in motility via the control of pili formation and is also likely to regulate other features of the cell surface . Furthermore, the data provide evidence that type IV pili are required for phototactic movement in certain cyanobacteria and suggest that different populations of pili present on the Synechocystis cell surface may perform different functions.

Int J Biol Macromol, 1999 Jan, 24(1), 19 - 26
Prediction of a conserved, neutralizing epitope in ribosome-inactivating proteins; Lebeda FJ et al.; The secondary structures, side-chain solvent accessibilities, and superpositioned crystal structures of the A-chain of ricin and four other plant rRNA N-glycosidases (ribosome-inactivating proteins, RIPs) were examined . Previously, a 26-residue fragment from the A-chain of ricin was determined to bind to a neutralizing monoclonal antibody . The region in the native ricin A-chain, to which this peptide corresponds, is solvent-exposed and contains a negatively charged residue that has been hypothesized to participate in the toxin's function, namely, rRNA binding and/or enzymatic activity . This region appears to be conserved in all of the structurally defined plant RIPs examined . Moreover, other plant RIPs, whose tertiary structures are, as yet, unknown, were predicted to have an analogous, solvent-exposed region containing a conserved, negatively charged residue . By analogy, these conserved structural and functional features lead to the suggestion that this exposed region represents a logical starting point for experiments designed to locate neutralizing epitopes in these RIPs . In contrast, the tertiary structure of the analogous region in a bacteria-derived RIP (Shiga toxin) is a less solvent-exposed, truncated loop and is a structure that is not as likely to be a neutralizing epitope . Because most of the amino acid residues are not conserved within this exposed region, these RIPs are predicted to be antigenically distinct.

Nucleic Acids Res, 1999 Apr 1, 27(7), 1609 - 18
DNA polymerase beta-like nucleotidyltransferase superfamily: identification of three new families, classification and evolutionary history; Aravind L et al.; A detailed analysis of the polbeta superfamily of nucleotidyltransferases was performed using computer methods for iterative database search, multiple alignment, motif analysis and structural modeling . Three previously uncharacterized families of predicted nucleotidyltransferases are described . One of these new families includes small proteins found in all archaea and some bacteria that appear to consist of the minimal nucleotidyltransferase domain and may resemble the ancestral state of this superfamily . Another new family that is specifically related to eukaryotic polyA polymerases is typified by yeast Trf4p and Trf5p proteins that are involved in chromatin remodeling . The TRF family is represented by multiple members in all eukaryotes and may be involved in yet unknown nucleotide polymerization reactions required for maintenance of chromatin structure . Another new family of bacterial and archaeal nucleotidyltransferases is predicted to function in signal transduction since, in addition to the nucleotidyltransferase domain, these proteins contain ligand-binding domains . It is further shown that the catalytic domain of gamma proteobacterial adenylyl cyclases is homologous to the polbeta superfamily nucleotidyltransferases which emphasizes the general trend for the origin of signal-transducing enzymes from those involved in replication, repair and RNA processing . Classification of the polbeta superfamily into distinct families and examination of their phyletic distribution suggests that the evolution of this type of nucleotidyltransferases may have included bursts of rapid divergence linked to the emergence of new functions as well as a number of horizontal gene transfer events.

Gut, 1999 Apr, 44(4), 456 - 62
Role of apoptosis induced by Helicobacter pylori infection in the development of duodenal ulcer; Kohda K et al.; BACKGROUND: Helicobacter pylori affects gastric epithelium integrity by acceleration of apoptosis . However, it remains unclear what product of the bacteria causes apoptosis, or whether or not the apoptosis is involved in the development of ulcers . AIMS: To elucidate the factor from H pylori that causes acceleration of apoptosis and the role of apoptosis in the development of duodenal ulcer in H pylori infection . PATIENTS: Five H pylori negative healthy volunteers, 47 H pylori positive patients with duodenal ulcer, and 35 H pylori positive patients with gastric ulcer . METHODS: An endoscopic examination was carried out to diagnose ulcers and determine their clinical stage . To analyse apoptosis, a cell cycle analysis was performed using biopsy specimens . RESULTS: There was a significant correlation between the urease activity of the H pylori strain and the level of apoptosis induced by this bacterial strain . Moreover, in duodenal ulcer patients infected with H pylori, the patients with an active ulcer exhibited a significantly higher level of apoptosis than those with ulcers at both the healing and scarring stages . CONCLUSION: These findings suggest that acceleration of apoptosis in the antral mucosa caused by the urease of H pylori plays a crucial role in the development of ulcers in the duodenum.

Nat Struct Biol, 1999 Mar, 6(3), 278 - 84
Crystal structure of a phospholipase D family member; Stuckey JA et al.; The first crystal structure of a phospholipase D (PLD) family member has been determined at 2.0 A resolution . The PLD superfamily is defined by a common sequence motif, HxK(x)4D(x)6GSxN, and includes enzymes involved in signal transduction, lipid biosynthesis, endonucleases and open reading frames in pathogenic viruses and bacteria . The crystal structure suggests that residues from two sequence motifs form a single active site . A histidine residue from one motif acts as a nucleophile in the catalytic mechanism, forming a phosphoenzyme intermediate, whereas a histidine residue from the other motif appears to function as a general acid in the cleavage of the phosphodiester bond . The structure suggests that the conserved lysine residues are involved in phosphate binding . Large-scale genomic sequencing revealed that there are many PLD family members . Our results suggest that all of these proteins may possess a common structure and catalytic mechanism.

Aust N Z J Surg, 1999 Mar, 69(3), 210 - 3
Role of abdominal drains in perforated duodenal ulcer patients: a prospective controlled study; Pai D et al.; BACKGROUND: Duodenal ulcer perforation is a common emergency in south India, with about 100-120 cases being treated at Jawaharalal Institute of Post Graduate Medical Education and Research each year . The routine to date has been to leave two tube drains: one in the Morrison's pouch and one in the pelvis after omental patch closure . This study was conducted to test the efficacy and safety of drain usage routinely after duodenal ulcer perforation closure with Roscoe Graham omental patch technique . METHODS: In this prospective controlled study, 44 patients formed the test group (without drains) and 75 patients formed the control group (with abdominal drainage) . Only patients of perforated duodenal ulcer closed with Roscoe Graham omental patch technique were included in the study . The incidence of postoperative fever, wound infection, time for return of bowel function and duration of hospital stay were noted . Details of drainage noted were the mean amount of daily drainage, mean time of drain removal and occurrence of drain-related complications . Peritoneal fluid, wound discharges, drain tips and drain wounds were cultured . Abdominal ultrasound was performed in all patients in the second postoperative week or if earlier indicated to detect intra-abdominal collections . RESULTS: It was found that there was no difference in incidence or duration of postoperative pyrexia, return of bowel function or postoperative hospital stay between the two groups . Routine use of drains was not effective in preventing postoperative fluid collection nor in decreasing the incidence of intra-abdominal abscesses . The migration of bacteria from the exterior to the peritoneal cavity via the drain was also demonstrated . Drains were found to cause morbidity including intestinal obstruction . CONCLUSION: The routine use of drains was found to be neither safe nor effective in patients of perforated duodenal ulcer treated by omental patch closure.

Ann Clin Lab Sci, 1999 Jan-Mar, 29(1), 1 - 8
Using a cutoff of <10 ppm for breath hydrogen testing: a review of five years' experience; Karcher RE et al.; To assess the clinical use of the breath hydrogen test in a large community hospital using a <10 ppm cutoff, we reviewed 222 tests performed over a 5-year period to evaluate patients for disaccharidase deficiency or bacterial overgrowth of the small intestine . Of these, the vast majority (195) were for lactose malabsorption, although fructose (17), sucrose (8) and lactulose (2) were also occasionally administered . One hundred eleven tests (50 percent) were positive, with an increase of at least 10 ppm hydrogen above the fasting level and a maximum value most commonly observed (42.3 percent of the time) at 3 hours post-administration . Only 34 patients (15.3 percent) had symptoms noted during the test, as compared with 185 (83.3 percent) who had experienced persistent intestinal problems prior to the test . Recent conditions which may have caused intestinal distress, such as transient disaccharidase deficiency, infections, surgery or other disorders like Crohn's disease, ulcerative colitis or food poisoning, were recorded in only 14 cases . Patterns consistent with bacterial overgrowth of the small intestine were observed in only 3 cases . Of 111 positives, 9 cases had increases between 10 and 20 ppm hydrogen and 7 showed the increase in the 3-hour sample, possibly reflecting a delayed transit through the intestine . Final diagnoses in 6 of these where information was available were for conditions other than malabsorption . We conclude that using a rise of 10 ppm to interpret a positive test does not contribute significantly to an increased frequency of false positives, but that patients with increases between 10 and 20 ppm probably are not lactase deficient.

Int J Oral Maxillofac Implants, 1999 Jan-Feb, 14(1), 94 - 100
Microleakage at the abutment-implant interface of osseointegrated implants: a comparative study; Gross M et al.; Microleakage can occur at the abutment-implant (A-I) interface in osseointegrated implants and may cause malodor and inflammation of peri-implant tissues . The degree of microleakage at the A-I interface of 5 implant systems was comparatively assessed at varying closing torques . Using colored tracing probes driven by a 2-atm pressure system, the interface microleakage of Branemark, Sulzer Calcitek, 3i, ITI, and Steri-Oss implants was determined spectrophotometrically . Microleakage through the A-I interface occurred in all systems, with variability between systems, samples, and closing torques . As closing torque increased from 10 Ncm to 20 Ncm to manufacturers' recommended closing torques, microleakage decreased significantly (P < .005) for all systems . Analysis of variance showed significant interaction between closing torques and the time course of microleakage, and between systems and the time course of microleakage (P < .001) . The results indicate that fluids and small molecules are capable of passing through the interface of all the A-I assemblies studied . Presumably in an in situ situation, fluids containing bacterial byproducts and nutrients required for bacterial growth may pass through the interface gap, contributing in part to clinically observed malodor and peri-implantitis.

J Bacteriol, 1999 Mar, 181(6), 1875 - 82
In vivo role of catalase-peroxidase in synechocystis sp . strain PCC 6803; Tichy M et al.; The katG gene coding for the only catalase-peroxidase in the cyanobacterium Synechocystis sp . strain PCC 6803 was deleted in this organism . Although the rate of H2O2 decomposition was about 30 times lower in the DeltakatG mutant than in the wild type, the strain had a normal phenotype and its doubling time as well as its resistance to H2O2 and methyl viologen were indistinguishable from those of the wild type . The residual H2O2-scavenging capacity was more than sufficient to deal with the rate of H2O2 production by the cell, estimated to be less than 1% of the maximum rate of photosynthetic electron transport in vivo . We propose that catalase-peroxidase has a protective role against environmental H2O2 generated by algae or bacteria in the ecosystem (for example, in mats) . This protective role is most apparent at a high cell density of the cyanobacterium . The residual H2O2-scavenging activity in the DeltakatG mutant was a light-dependent peroxidase activity . However, neither glutathione peroxidase nor ascorbate peroxidase accounted for a significant part of this H2O2-scavenging activity . When a small thiol such as dithiothreitol was added to the medium, the rate of H2O2 decomposition in the DeltakatG mutant increased more than 10-fold, indicating that a thiol-specific peroxidase, for which thioredoxin may be the physiological electron donor, is present . Oxidized thioredoxin is likely to be reduced again by photosynthetic electron transport . Therefore, under laboratory conditions, there are only two enzymatic mechanisms for H2O2 decomposition present in Synechocystis sp . strain PCC 6803 . One is catalyzed by a catalase-peroxidase, and the other is catalyzed by thiol-specific peroxidase.

J Wildl Dis, 1999 Jan, 35(1), 105 - 7
Absence of tuberculosis in free-ranging deer in Nebraska; Steffen DJ et al.; Lymph nodes from 271 white-tailed deer (Odocoileus virginianus) and mule deer (Odocoileus hemionus) in Nebraska (USA) were examined microscopically for tuberculoid lesions . Lymph nodes lesions in at least one node were found in 12 deer . Lesions were examined with Zeihl-Neelson acid fast stains and by polymerase chain reactions using M . bovis specific probes . No evidence of tuberculosis was found . The small granulomatous lesions were likely caused by other bacteria.

Zhonghua Bing Li Xue Za Zhi, 1997 Apr, 26(2), 103 - 5
{Severe combined immunodefficiency disease, 4 autopsy case reports}; Yan P et al.; OBJECTIVE: To study the pathological changes of severe combined immunodefficiency disease (SCID) . METHODS: 4 cases with SCID proven by autopsy examination . RESULTS: All patients were male with an average age of 5 moths . The major clinical manifestations included severe repeated infection with bacteria, virus, fungi and pneumocystis carinii . Autopsy examination revealed hypoplasia of thymus, spleen and lymph nodes . 1 case died from pneumocystis carinii pneumonia and BCG vaccine disease, 2 cases died of severe general infection and 1 case died from large cell lymphoma . CONCLUSIONS: The main diagnosis criteria for SCID are hypoplasia of both T and B lymphocytes, the pathologic changes of SCID are very complex.

Gene, 1999 Mar 4, 228(1-2), 233 - 42
Dorsal-B, a splice variant of the Drosophila factor Dorsal, is a novel Rel/NF-kappaB transcriptional activator; Gross I et al.; The Drosophila transcription factor Dorsal, a member of the Rel/NF-kappaB family of proteins, plays a key role in the establishment of dorsoventral polarity in the early embryo and is also involved in the immune response . Here, we present evidence that the primary transcript of dorsal can be alternatively spliced, generating Dorsal-B, a new Rel/NF-kappaB family member . Dorsal and Dorsal-B are identical in the N-terminal region, which comprises both a DNA-binding domain and a dimerization domain . However, Dorsal-B lacks the nuclear localization signal located at the end of the Rel domain of Dorsal and is totally divergent in the C-terminal portion . Although Dorsal-B by itself is not able to induce the expression of a kappaB-controlled Luciferase reporter gene, we demonstrate that its C-terminal portion has transactivating properties . Analysis of the dorsal-B expression pattern indicates that the splicing is tissue-specific and excludes a putative role in early embryogenesis . However, dorsal-B synthesis is enhanced upon septic injury, and this challenge induces a nuclear accumulation of the protein in fat body cells suggesting that it may be involved in the immune response.

Clin Cardiol, 1999 Feb, 22(2), 85 - 90
Lack of association between prior infection with Chlamydia pneumoniae and acute or chronic coronary artery disease; Altman R et al.; BACKGROUND: Higher than normal serologic titers and the detection of bacteria within atheroma have suggested an association between Chlamydia pneumoniae (C . pneumoniae) infection and coronary heart disease (CHD), but the relationship has not been well established . HYPOTHESIS: The study was designed to establish a lack of relationship between chronic C . pneumoniae infection and CHD . METHODS: Chlamydia-specific IgG-antibody was assayed using an indirect immunofluorescence test in the serum of 159 patients with severe arterial disease and 203 patients with a heart valve prostheses and no demonstrable CHD . Fatal and nonfatal vascular events and systemic thromboembolism were recorded over a 2-year period . RESULTS: In the arterial group 107 patients (67.3%) and in the valvular group 120/203 (59.1%) were positive for C . pneumoniae antibody . The number of patients with fatal or nonfatal vascular events (double end point) in the arterial and valvular groups was 23 and 2, respectively (p < .0001) . Triple end points (fatal plus nonfatal vascular events plus thromboembolism) were also more frequent in the arterial group (p < 0.002) . The prevalence of chlamydia antibody positivity was the same in the arterial and valvular groups, and the occurrence of clinical events was also the same for chlamydia-positive (227 patients) as for chlamydia-negative (135 patients) . After adjustment for confounding variables, only arterial disease was a predictive factor for double (OR 17.0; 95% CI 3.94-73.3) or triple (OR 3.12; 95% CI 1.56-6.25) end points . CONCLUSION: We find C . pneumoniae chronic infection not to be an independent risk factor for acute or chronic arterial disease.

Dev Biol, 1999 Mar 15, 207(2), 445 - 56
The synthesis of a small heat shock/alpha-crystallin protein in Artemia and its relationship to stress tolerance during development; Liang P et al.; Fertilized oocytes of the brine shrimp Artemia franciscana undergo either ovoviviparous or oviparous development, yielding free-swimming larvae (nauplii) or encysted gastrulae (cysts), respectively . Encystment is followed by diapause, wherein metabolism is greatly reduced; the resulting cysts are very resistant to extreme stress, including desiccation and long-term anoxia . The synthesis of p26, a small heat shock/alpha-crystallin protein produced only in oviparously developing Artemia, is shown in this paper to be transcriptionally regulated . A p26 mRNA of about 0.7 kb was detected on Northern blots in the second day after oocyte fertilization . It peaked as embryos encysted and declined rapidly when activated cysts resumed development . The appearance of p26 protein, as indicated by immunoprobing of Western blots, followed mRNA by 1 day; it also increased as encystment occurred but remained constant during postgastrula development of cysts . However, p26 underwent a marked reduction during emergence of nauplii and could not be detected in cell-free extracts of second-instar larvae . p26 entered nuclei of encysting embryos soon after synthesis and was localized therein as late as instar II, when it was restricted to a small set of salt gland nuclei . First-instar larvae derived from cysts were more thermotolerant than larvae that had developed ovoviviparously, but synthesis of p26 was not induced by heat under the experimental conditions employed . Additionally, transformed bacteria synthesizing p26 were more thermotolerant than bacteria that lacked the protein . The results support the proposal that p26, a developmentally regulated protein synthesized during embryo encystment, has chaperone activity in vivo and protects the proteins of encysted Artemia from stress-induced denaturation .

Curr Opin Microbiol, 1998 Oct, 1(5), 524 - 9
The superbugs: evolution, dissemination and fitness; Morris A et al.; Since the introduction of antibiotics, bacteria have not only evolved elegant resistance mechanisms to thwart their effect, but have also evolved ways in which to disseminate themselves or their resistance genes to other susceptible bacteria . During the past few years, research has revealed not only how such resistance mechanisms have been able to evolve and to rapidly disseminate, but also how bacteria have, in some cases, been able to adapt to this new burden of resistance with little or no cost to their fitness . Such adaptations make the control of these superbugs all the more difficult.

Curr Opin Microbiol, 1998 Oct, 1(5), 598 - 610
Global dinucleotide signatures and analysis of genomic heterogeneity; Karlin S; Early biochemical experiments measuring nearest neighbor frequencies established that the set of dinucleotide relative abundance values (dinucleotide biases) is a remarkably stable property of the DNA of an organism . Analyses of currently available genomic sequence data have extended these earlier results, showing that the dinucleotide biases evaluated for successive 50 kb segments of a genome are significantly more similar to each other than to those of sequences from more distant organisms . From this perspective, the set of dinucleotide biases constitutes a 'genomic signature' that can discriminate sequences from different organisms . The dinucleotide biases appear to reflect species-specific properties of DNA stacking energies, modification, replication, and repair mechanisms . The genomic signature is useful for detecting pathogenicity islands in bacterial genomes.

J Chromatogr B Biomed Sci Appl, 1999 Feb 5, 722(1-2), 153 - 77
Purification of chaperonins; Quaite-Randall E et al.; The availability of protein samples of sufficient quality and in sufficient quantity is a driving force in biology and biotechnology . Protein samples that are free of critical contaminants are required for specific assays . Large amounts of highly homogeneous and reproducible material are needed for crystallography and nuclear magnetic resonance studies of protein structure . Protein-based therapeutic factors used in human medicine must not contain any contaminants that might interfere with treatment . The roles played by molecular chaperones in protein folding and in many cellular processes make these proteins very attractive candidates as biochemical reagents, and the class of chaperones called chaperonins is one of the most important candidates . Methods for successfully purifying chaperonins are needed to advance the field of chaperonin-mediated protein folding . This article outlines the strategies and methods used to obtain pure chaperonin samples from different biological sources . The objective is to help new researchers obtain better quality samples of chaperonins from many new organisms.

Endocrinology, 1999 Mar, 140(3), 1236 - 44
Recombinant growth differentiation factor-9 (GDF-9) enhances growth and differentiation of cultured early ovarian follicles; Hayashi M et al.; Transgenic mice with deletion of the GDF-9 (growth differentiation factor-9) gene are characterized by the arrest of ovarian follicle development at the primary stage . Based on the hypothesis that GDF-9 is important for early follicle development, we isolated rat GDF-9 complementary DNA (cDNA) and generated recombinant GDF-9 protein to study its physiological role . Using bacteria-derived GDF-9-glutathione S-transferase (GST) fusion protein, specific antibodies to the mature form of GDF-9 was generated . Immunohistochemical staining of ovarian sections indicated the localization of GDF-9 protein in the oocyte of primary, secondary and preantral follicles, whereas immunoblotting demonstrated the secretion of GDF-9 by mammalian cells transfected with GDF-9 cDNAs . Recombinant GDF-9 was shown to be an N-glycosylated protein capable of stimulating early follicle development . Growth of preantral follicles isolated from immature rats was enhanced by treatment with either GDF-9 or FSH whereas the combined treatment showed an additive effect . In addition, treatment with GDF-9, like forskolin, also stimulated inhibin-alpha content in explants of neonatal ovaries . In contrast, the stimulatory effects of GDF-9 were not mimicked by amino-terminal tagged GDF-9 that was apparently not bioactive . Thus, the present study demonstrates the important role of GDF-9 in early follicle growth and differentiation . The availability of recombinant bioactive GDF-9 allows future studies on the physiological role of GDF-9 in ovarian development in vivo.

J Biol Chem, 1999 Mar 12, 274(11), 7226 - 37
Transglutaminase cross-linking properties of the small proline-rich 1 family of cornified cell envelope proteins . Integration with loricrin; Candi E et al.; Small proline-rich 1 (SPR1) proteins are important for barrier function in stratified squamous epithelia . To explore their properties, we expressed in bacteria a recombinant human SPR1 protein and isolated native SPR1 proteins from cultured mouse keratinocytes . By circular dichroism, they possess no alpha or beta structure but have some organized structure associated with their central peptide repeat domain . The transglutaminase (TGase) 1 and 3 enzymes use the SPR1 proteins as complete substrates in vitro but in different ways: head domain A sequences at the amino terminus were used preferentially for cross-linking by TGase 3, whereas those in head domain B sequences were used for cross-linking by TGase 1 . The TGase 2 enzyme cross-linked SPR1 proteins poorly . Together with our data base of 141 examples of in vivo cross-links between SPRs and loricrin, this means that both TGase 1 and 3 are required for cross-linking SPR1 proteins in epithelia in vivo . Double in vitro cross-linking experiments suggest that oligomerization of SPR1 into large polymers can occur only by further TGase 1 cross-linking of an initial TGase 3 reaction . Accordingly, we propose that TGase 3 first cross-links loricrin and SPRs together to form small interchain oligomers, which are then permanently affixed to the developing CE by further cross-linking by the TGase 1 enzyme . This is consistent with the known consequences of diminished barrier function in TGase 1 deficiency models.

J Biol Chem, 1999 Mar 12, 274(11), 7082 - 8
Cloning and expression of a wheat (Triticum aestivum L.) phosphatidylserine synthase cDNA . Overexpression in plants alters the composition of phospholipids; Delhaize E et al.; We describe the cloning of a wheat cDNA (TaPSS1) that encodes a phosphatidylserine synthase (PSS) and provides the first strong evidence for the existence of this enzyme in a higher eukaryotic cell . The cDNA was isolated on its ability to confer increased resistance to aluminum toxicity when expressed in yeast . The sequence of the predicted protein encoded by TaPSS1 shows homology to PSS from both yeast and bacteria but is distinct from the animal PSS enzymes that catalyze base-exchange reactions . In wheat, Southern blot analysis identified the presence of a small family of genes that cross-hybridized to TaPSS1, and Northern blots showed that aluminum induced TaPSS1 expression in root apices . Expression of TaPSS1 complemented the yeast cho1 mutant that lacks PSS activity and altered the phospholipid composition of wild type yeast, with the most marked effect being increased abundance of phosphatidylserine (PS) . Arabidopsis thaliana leaves overexpressing TaPSS1 showed a marked enhancement in PSS activity, which was associated with increased biosynthesis of PS at the expense of both phosphatidylinositol and phosphatidylglycerol . Unlike mammalian cells where PS accumulation is tightly regulated even when the capacity for PS biosynthesis is increased, plant cells accumulated large amounts of PS when TaPSS1 was overexpressed . High levels of TaPSS1 expression in Arabidopsis and tobacco (Nicotiana tabacum) led to the appearance of necrotic lesions on leaves, which may have resulted from the excessive accumulation of PS . The cloning of TaPSS1 now provides evidence that the yeast pathway for PS synthesis exists in some plant tissues and provides a tool for understanding the pathways of phospholipid biosynthesis and their regulation in plants.

J Biol Chem, 1999 Mar 12, 274(11), 6817 - 9
Switch from an aquaporin to a glycerol channel by two amino acids substitution; Lagree V et al.; The MIP (major intrinsic protein) proteins constitute a channel family of currently 150 members that have been identified in cell membranes of organisms ranging from bacteria to man . Among these proteins, two functionally distinct subgroups are characterized: aquaporins that allow specific water transfer and glycerol channels that are involved in glycerol and small neutral solutes transport . Since the flow of small molecules across cell membranes is vital for every living organism, the study of such proteins is of particular interest . For instance, aquaporins located in kidney cell membranes are responsible for reabsorption of 150 liters of water/day in adult human . To understand the molecular mechanisms of solute transport specificity, we analyzed mutant aquaporins in which highly conserved residues have been substituted by amino acids located at the same positions in glycerol channels . Here, we show that substitution of a tyrosine and a tryptophan by a proline and a leucine, respectively, in the sixth transmembrane helix of an aquaporin leads to a switch in the selectivity of the channel, from water to glycerol.

Curr Opin Microbiol, 1998 Dec, 1(6), 698 - 706
Time at the end of the millennium: the Neurospora clock; Loros JJ; In the past two years we have entered the log phase for unraveling the molecular clockworks . Rapid progress in understanding the Neurospora clock has been complemented by a flood of information from diverse systems including cyanobacteria, insects and mice . There are broadly conserved features in transcription/translation based feedback loops . Conservation is also found at the sequence level, from fungi to mammals, in the PAS domains of the heterodimeric partners of the transcription factors that act as the positive components of the feedback cycle . Pivotal PAS proteins from Neurospora, the WCs, provide an evolutionary link connecting the clock in insects and mammals to the fungi and to light-harvesting proteins from bacteria.

Curr Opin Microbiol, 1998 Dec, 1(6), 669 - 73
The cyanobacterial circadian system: a clock apart; Golden SS et al.; Several new molecular components of the circadian clocks of animals, fungi, and bacteria have been unveiled in the past two years . Enough parts are now identified to indicate that there is more than one way to build a biological clock, although there are parallels in the cycling molecular events among disparate groups of organisms.

Curr Opin Microbiol, 1998 Feb, 1(1), 88 - 95
Oral pathogens: from dental plaque to cardiac disease; Meyer DH et al.; Oral bacteria exhibit highly specific adherence mechanisms and as a result they colonize and cause disease principally in the oral cavity . Oral pathogens, however, can produce systemic disease and are known causative agents of infective endocarditis . Recent studies have revealed that periodontal disease per se is also a statistically significant risk factor for cardiovascular disease . A link between the two diseases is the secretion and systemic appearance in periodontitis of pro-inflammatory cytokines capable of eliciting effects associated with atherosclerosis and coronary heart disease.

J Invertebr Pathol, 1999 Mar, 73(2), 173 - 81
Histopathological effects of tannic acid on the midgut epithelium of some aquatic Diptera larvae; Rey D et al.; The impact of tannins on larval Nematocera was investigated by an extensive survey of the relative toxicity of tannic acid in Diptera larvae representative of mosquito communities from alpine hydrosystems (Culicidae, Chaoboridae, Chironomidae, and Simuliidae) together with a nonindigenous vector competent Culicidae species . Bioassays indicate that exposure to tannic acid at concentrations from 0.25 to 4 mM is deleterious for Culex pipiens, Simulium variegatum, and Chironomus annularius, but not for Aedes, Anopheles, Culiseta, and Chaoborus species . Histopathological observations reveal that, among the target organs of tannic acid, mainly the midgut epithelium is affected by treatment . However, the extent of degeneration varies according to the taxon, the duration of the treatment, and the concentrations assayed . The vulnerability of epithelial cells differs among cell types, clear cells of the anterior midgut showing symptoms of intoxication before dark cells of the posterior midgut . The toxic effects of tannic acid are discussed, particularly in comparison to those of insecticidal bacteria, in order to evaluate the potential for use of tannins in the regulation of larval populations of dipteran pests .

Nihon Kokyuki Gakkai Zasshi, 1998 Dec, 36(12), 1062 - 5
{Resection of asymptomatic infected bulla associated with pulmonary tuberculosis}; Yamazaki A et al.; The patient was a 49-year-old man . A physical examination in 1995 had disclosed the presence of a bulla in his left lung . Chest x-ray films taken in February 1997 disclosed niveau formation as an abnormal shadow in contact with the bulla . The patient was given a diagnosis of an infected bulla, and underwent an operation for partial resection of the S 6 portion of the left lung . The intracystic fluid was serous and no general bacteria or mycobacteria were detected . However, histopathological examination found granulation tissue associated with caseous necrosis and containing mycobacteria . The patient is currently receiving tuberculostatic agents on an outpatient basis.

Mutat Res, 1999 Mar 8, 424(1-2), 83 - 95
Lipid peroxidation-DNA damage by malondialdehyde; Marnett LJ; Malondialdehyde is a naturally occurring product of lipid peroxidation and prostaglandin biosynthesis that is mutagenic and carcinogenic . It reacts with DNA to form adducts to deoxyguanosine and deoxyadenosine . The major adduct to DNA is a pyrimidopurinone called M1G . Site-specific mutagenesis experiments indicate that M1G is mutagenic in bacteria and is repaired by the nucleotide excision repair pathway . M1G has been detected in liver, white blood cells, pancreas, and breast from healthy human beings at levels ranging from 1-120 per 108 nucleotides . Several different assays for M1G have been described that are based on mass spectrometry, 32P-postlabeling, or immunochemical techniques . Each technique offers advantages and disadvantages based on a combination of sensitivity and specificity . Application of each of these techniques to the analysis of M1G is reviewed and future needs for improvements are identified . M1G appears to be a major endogenous DNA adduct in human beings that may contribute significantly to cancer linked to lifestyle and dietary factors . High throughput methods for its detection and quantitation will be extremely useful for screening large populations .

J Mol Biol, 1999 Mar 12, 286(5), 1379 - 87
Asymmetric recognition of psoralen interstrand crosslinks by the nucleotide excision repair and the error-prone repair pathways; Barre FX et al.; Psoralen is an asymmetric photoreactive intercalator with a furane and a pyrone side . When intercalated at 5'-TpA-3' sites and upon UVA irradiation, the psoralen can react with the thymine residues on both strands, introducing an interstrand crosslink . Using psoralen-coupled triple-helix-forming oligonucleotides, psoralen interstrand crosslinks can be site-specifically introduced in the coding sequence of URA3, a yeast auxotrophic marker carried on plasmid vectors . In addition, crosslinks introduced via a triple-helix-forming oligonuleotide are oriented with the furane side of the psoralen associated with a specific strand of the target sequence . Here, the transformation efficiency, the mutation frequency and the mutational spectra of site-specifically placed and oriented crosslinks were examined in yeast cells . We found that the nature of the targeted mutations depended on the crosslink orientation: bypass of the pyrone-adducted thymine yielded T-->A or T-->C substitutions and A insertions, while bypass of the furane-adducted thymine yielded T-->G substitutions and G insertions . Thus, the structure of the damage strongly influences the choice of the nucleotide incorporated during translesion synthesis . In addition, the observed pattern of mutagenesis suggests a coupling to transcription, similar to the one observed in mammalian cells . Finally, the substitutions affected only the coding strand when the pyrone link of the psoralen crosslink was on this strand, whereas they affected both strands when the pyrone link was on the transcribed strand, suggesting that the incision preference of psoralen crosslinks, which has been observed with purified uvrABC proteins in bacteria, is conserved in live eucaryotic cells .

Infect Control Hosp Epidemiol, 1999 Feb, 20(2), 136 - 44
Selecting respirators for control of worker exposure to infectious aerosols; McCullough NV et al.; A method for selecting respirators for protection in infectious aerosol environments was developed, building on a procedure used to choose respiratory protection for environments containing nonbiological contaminants . Modifications in the traditional respirator selection method are proposed for situations where information on occupational exposure limits, toxicity, and airborne concentrations is absent . Toxicity is determined from risk rankings proposed by a variety of organizations . The nature of the activity allows assessment of source generation, which is combined with room volume and airflow to obtain a ranking of airborne concentration . Finally, concentration and toxicity ranks determine a minimum assigned protection factor, which corresponds to a respirator class . Case studies are presented to illustrate the proposed decision logic . For each situation, the procedure yielded choices that were both protective and reasonable . These results suggest that the procedure will be applicable to a variety of settings for a range of infectious organisms.

J Anim Sci, 1999 Jan, 77(1), 180 - 6
Processing, mixing, and particle size reduction of forages for dairy cattle; Heinrichs AJ et al.; Adequate forage amounts in both physical and chemical forms are necessary for proper ruminal function in dairy cows . Under conditions in which total amounts of forage or particle size of the forage are reduced, cows spend less time ruminating and have a decreased amount of buoyant digesta in the rumen . These factors reduce saliva production and allow ruminal pH to fall, depressing activity of cellulolytic bacteria and causing a prolonged period of low ruminal pH . Insufficient particle size of the diet decreases the ruminal acetate-to-propionate ratio and reduces ruminal pH . The mean particle size of the diet, the variation in particle size, and the amount of chemical fiber (i.e., NDF or ADF) are all nutritionally important for dairy cows . Defining amounts and physical characteristics of fiber is important in balancing dairy cattle diets . Because particle size plays such an important role in digestion and animal performance, it must be an important consideration from harvest through feeding . Forages should not be reduced in particle size beyond what is necessary to achieve minimal storage losses and what can be accommodated by existing equipment . Forage and total mixed ration (TMR) particle sizes are potentially reduced in size by all phases of harvesting, storing, taking out of storage, mixing, and delivery of feed to the dairy cow . Mixing feed causes a reduction in size of all feed particles and is directly related to TMR mixing time; field studies show that the longest particles (>27 mm) may be reduced in size by 50% . Forage and TMR particle size as fed to the cows should be periodically monitored to maintain adequate nutrition for the dairy cow.

Dig Dis Sci, 1999 Feb, 44(2), 237 - 42
Helicobacter pylori water extract induces interleukin-8 production by gastric epithelial cells; Kassai K et al.; In Helicobacter pylori-associated gastric mucosal injury, interleukin (IL) -8, a potent leukocyte chemoattractant, is produced by epithelial cells infected by H . pylori and directs neutrophils to the gastric mucosa . According to previous studies, the IL-8 production requires direct contact between the bacteria and epithelial cells . The aims of the present study were to determine whether an H . pylori water extract (HPE) induces IL-8 production by gastric epithelial cells and to characterize IL-8-inducing substances in HPE . Extracts were prepared from a standard strain and from strains obtained from patients with gastric ulcers . After addition of HPE to MKN 45 cells, a gastric cancer cell line, IL-8 in supernatants and IL-8 mRNA were measured by immunoassay and reverse transcription-polymerase chain reaction, respectively . For characterization, active fractions obtained by gel filtration of standard-strain HPE were treated by heating or trypsinization . To study the signal pathway leading to IL-8 production, inhibitors for protein kinase A (PKA), protein kinase C (PKC), or protein tyrosine kinase (PTK) were incubated with MKN45 cells before HPE stimulation . HPE from the standard strain and one of these clinical strains induced IL-8 production . Lipopolysaccharide or cagA in the strains showed no correlation with IL-8 concentration . Standard-strain HPE induced IL-8 mRNA expression in MKN 45 cells . Gel filtration localized activity to a low-molecular-weight fraction of about 7 kDa, which was resistant to heat and trypsin digestion . PKC inhibitors significantly blocked HPE-induced IL-8 production by MKN 45 cells; however, the PKA inhibitor or PTK inhibitors showed a partial inhibitory effect . HPE contains a nonprotein substance of low molecular weight that is responsible for IL-8 induction in gastric epithelial cells . This induction is mainly dependent on the activation of PKC but partially also dependent on PKA or PTK.

Teratog Carcinog Mutagen, 1998, 18(6), 303 - 8
Lack of genotoxicity of silver iodide in the SCE assay in vitro, in vivo, and in the Ames/microsome test; Eliopoulos P et al.; Silver iodide was evaluated for mutagenicity in the Ames/microsome test (strains TA 1535, TA 102, TA 97, and TA 98) and for the ability to induce Sister Chromatid Exchanges (SCE) in human cultured lymphocytes and in P388 lymphocytic leukemia cells cultured in the mouse peritoneal cavity . From the cytogenetic in vitro studies, it was observed that silver iodide, either in acetone solutions or as a suspension with polyacrilamide, scarcely causes a doubling effect on SCEs at nearly toxic concentrations (1 microg/ml) . Such a doubling effect by silver iodide on SCEs in P388 leukemia cells in vivo was not achieved even after using 100 microg/g mouse body weight . In the Ames/microsome test actually a doubling effect on revertants was only isolately achieved with 30 microg/ml in TA 102 (S9-) and at 150 microg/ml in TA 97 (S9+) doses, which appear to be nearly toxic for bacteria.

Proc Natl Acad Sci U S A, 1999 Mar 2, 96(5), 2491 - 6
Cloning, expression, and genetic mapping of Sema W, a member of the semaphorin family; Encinas JA et al.; The semaphorins comprise a large family of membrane-bound and secreted proteins, some of which have been shown to function in axon guidance . We have cloned a transmembrane semaphorin, Sema W, that belongs to the class IV subgroup of the semaphorin family . The mouse and rat forms of Sema W show 97% amino acid sequence identity with each other, and each shows about 91% identity with the human form . The gene for Sema W is divided into 15 exons, up to 4 of which are absent in the human cDNAs that we sequenced . Unlike many other semaphorins, Sema W is expressed at low levels in the developing embryo but was found to be expressed at high levels in the adult central nervous system and lung . Functional studies with purified membrane fractions from COS7 cells transfected with a Sema W expression plasmid showed that Sema W has growth-cone collapse activity against retinal ganglion-cell axons, indicating that vertebrate transmembrane semaphorins, like secreted semaphorins, can collapse growth cones . Genetic mapping of human SEMAW with human/hamster radiation hybrids localized the gene to chromosome 2p13 . Genetic mapping of mouse Semaw with mouse/hamster radiation hybrids localized the gene to chromosome 6, and physical mapping placed the gene on bacteria artificial chromosomes carrying microsatellite markers D6Mit70 and D6Mit189 . This localization places Semaw within the locus for motor neuron degeneration 2, making it an attractive candidate gene for this disease.

Proc Natl Acad Sci U S A, 1999 Mar 2, 96(5), 1852 - 7
Unexpected crucial role of residue 225 in serine proteases; Guinto ER et al.; Residue 225 in serine proteases of the chymotrypsin family is Pro or Tyr in more than 95% of nearly 300 available sequences . Proteases with Y225 (like some blood coagulation and complement factors) are almost exclusively found in vertebrates, whereas proteases with P225 (like degradative enzymes) are present from bacteria to human . Saturation mutagenesis of Y225 in thrombin shows that residue 225 affects ligand recognition up to 60,000-fold . With the exception of Tyr and Phe, all residues are associated with comparable or greatly reduced catalytic activity relative to Pro . The crystal structures of three mutants that differ widely in catalytic activity (Y225F, Y225P, and Y225I) show that although residue 225 makes no contact with substrate, it drastically influences the shape of the water channel around the primary specificity site . The activity profiles obtained for thrombin also suggest that the conversion of Pro to Tyr or Phe documented in the vertebrates occurred through Ser and was driven by a significant gain (up to 50-fold) in catalytic activity . In fact, Ser and Phe are documented in 4% of serine proteases, which together with Pro and Tyr account for almost the entire distribution of residues at position 225 . The unexpected crucial role of residue 225 in serine proteases explains the evolutionary selection of residues at this position and shows that the structural determinants of protease activity and specificity are more complex than currently believed . These findings have broad implications in the rational design of enzymes with enhanced catalytic properties.

Hepatology, 1999 Mar, 29(3), 737 - 45
Glycine and uridine prevent D-galactosamine hepatotoxicity in the rat: role of Kupffer cells; Stachlewitz RF et al.; Extrahepatic factors, such as increased gut permeability and bacteria from the gut, have been shown to play a role in D-galactosamine toxicity in rats . Because bacterial endotoxin activates Kupffer cells, the purpose of this study was to clarify the role of Kupffer cells in the mechanism of D-galactosamine hepatotoxicity in rats and determine whether uridine, a compound that rescues animals from D-galactosamine toxicity, affects Kupffer cells . Rats were fed control or glycine (5%) containing diets to prevent Kupffer cell activation or treated with gadolinium chloride (GdCl3, 20 mg/kg) to destroy Kupffer cells selectively before injection of D-galactosamine (500 mg/kg, intraperitoneally) . D-galactosamine caused panlobular focal hepatocellular necrosis, polymorphonuclear cell infiltration, and increased serum transaminases significantly at 24 hours . Dietary glycine or pretreatment with GdCl3 prevented these effects . D-galactosamine caused a transient increase in circulating endotoxin that was maximal at 1 hour and was blunted significantly by dietary glycine . Additionally, antisera to tumor necrosis factor-alpha (TNF-alpha) prevented hepatotoxicity caused by D-galactosamine . Moreover, apoptosis in hepatocytes caused by D-galactosamine occurred before necrosis (6 hours) and was prevented by glycine, GdCl3, TNF-alpha antiserum, and uridine . Thus, it was hypothesized that TNF-alpha from Kupffer cells causes apoptosis after D-galactosamine administration in the rat . Indeed, increases in TNF-alpha messenger RNA (mRNA) were detected as early as 2.5 hours after D-galactosamine treatment . Previous work proposed that uridine blocks D-galactosamine toxicity by preventing inhibition of mRNA synthesis . In view of these results, the possibility that uridine might affect Kupffer cells was investigated . Uridine significantly blunted the increase in {Ca2+}i and release of TNF-alpha caused by endotoxin in isolated Kupffer cells and prevented apoptosis caused by D-galactosamine treatment in vivo . These data support the hypothesis that uridine prevents D-galactosamine hepatotoxicity not only by rescuing the hepatocyte in the late phases of the injury but also preventing TNF-alpha release from Kupffer cells thereby blocking apoptosis that occurs early after D-galactosamine treatment . Taken together, these data strongly support the role of Kupffer cell activation by endotoxin early after D-galactosamine treatment as an important event in the mechanism of hepatotoxicity in the rat.

Anesteziol Reanimatol, 1998 Nov-Dec, (6), 45 - 9
{Morphology of myorenal syndrome}; Zimina LN et al.; A total of 114 autopsy specimens and 34 specimens of surgical material from patients with the myorenal syndrome are analyzed . All patients had symptoms of acute renal (hepatorenal) failure which caused death during the first two weeks of the disease . In later periods, death was caused by septic complications . Manifest morphological changes, caused by injuries, metabolic disorders, or sepsis were detected in many organs and tissues in all cases . The sources of septic complications were wounds, decompression incisions, fasciotomies, shunts, and catheters . After combined therapy of wounds with adsorbents, antibiotics, proteolytic enzymes, and laser exposure the bacteria lost their virulence and died and the wounds were cleansed.

Appl Environ Microbiol, 1999 Mar, 65(3), 1009 - 14
High-affinity methane oxidation by a soil enrichment culture containing a type II methanotroph; Dunfield PF et al.; Methanotrophic bacteria in an organic soil were enriched on gaseous mixing ratios of <275 parts per million of volume (ppmv) of methane (CH4) . After 4 years of growth and periodic dilution (>10(20) times the initial soil inoculum), a mixed culture was obtained which displayed an apparent half-saturation constant {Km(app)} for CH4 of 56 to 186 nM (40 to 132 ppmv) . This value was the same as that measured in the soil itself and about 1 order of magnitude lower than reported values for pure cultures of methane oxidizers . However, the Km(app) increased when the culture was transferred to higher mixing ratios of CH4 (1,000 ppmv, or 1%) . Denaturing gradient gel electrophoresis of the enrichment grown on <275 ppmv of CH4 revealed a single gene product of pmoA, which codes for a subunit of particulate methane monooxygenase . This suggested that only one methanotroph species was present . This organism was isolated from a sample of the enrichment culture grown on 1% CH4 and phylogenetically positioned based on its 16S rRNA, pmoA, and mxaF gene sequences as a type II strain of the Methylocystis/Methylosinus group . A coculture of this strain with a Variovorax sp., when grown on <275 ppmv of CH4, had a Km(app) (129 to 188 nM) similar to that of the initial enrichment culture . The data suggest that the affinity of methanotrophic bacteria for CH4 varies with growth conditions and that the oxidation of atmospheric CH4 observed in this soil is carried out by type II methanotrophic bacteria which are similar to characterized species.

Biochem Biophys Res Commun, 1999 Feb 24, 255(3), 602 - 7
SRPK1 and LBR protein kinases show identical substrate specificities; Papoutsopoulou S et al.; Arginine/serine protein kinases constitute a novel class of enzymes that can modify arginine/serine (RS) dipeptide motifs . SR splicing factors that are essential for pre-mRNA splicing and the lamin B receptor (LBR), an integral protein of the inner nuclear membrane, are among the best characterized proteins that contain RS domains . Two SR Protein-specific Kinases, SRPK1 and SRPK2, have been shown to phosphorylate specifically the RS motifs of the SR family of splicing factors and play an important role in regulating both the spliceosome assembly and their intranuclear distribution, whereas an LBR-associated kinase, that specifically phosphorylates a stretch of RS repeats located at the NH2-terminal region of LBR, has been recently purified and characterized from turkey erythrocyte nuclear envelopes . Using synthetic peptides representing different regions of LBR and recombinant proteins produced in bacteria we now demonstrate that SRPK1 modifies LBR with similar kinetics and on the same sites as the LBR kinase, that are also phosphorylated in vivo . These data provide significant evidence for a new role of SRPK1 in addition to that of pre-mRNA splicing .

Protein Expr Purif, 1999 Mar, 15(2), 207 - 12
One-step affinity purification of the G protein betagamma subunits from bovine brain using a histidine-tagged G protein alpha subunit; Tanaka T et al.; An efficient one-step affinity purification of bovine brain G protein betagamma subunits (betagamma's) is described . The betagamma's, in a detergent extract of brain membranes, are first dissociated from the alpha subunits (alpha's), reassociated with decahistidine-tagged alphail produced in bacteria, and then adsorbed onto Ni2+-nitrilotriacetic acid-agarose via the histidine tag . This mild adsorption retained the high activity of the ligand alpha's, in contrast to the commonly used chemical crosslinking methods . A wash step with a buffer containing chaotropic ions (SCN-) completely removed contaminating proteins that were refractory to washes with high concentrations of detergents, after which the highly purified betagamma's were eluted with a buffer containing Al3+, Mg2+, and F- ions . The obtained betagamma's were found to be fully functional, as assessed by their ability to support pertussis toxin-catalyzed ADP-ribosylation of alphail . Since the combination of the mild adsorption via the histidine tag and the wash with chaotropic ions can be easily applied to purifying betagamma's from various animal tissues, this new chromatographic method is expected to facilitate the purification of other membrane proteins that bind to Galpha and/or Galphabetagamma .

J Bacteriol, 1999 Mar, 181(5), 1689 - 93
Functional importance and local environments of the cysteines in the tetracycline resistance protein encoded by plasmid pBR322; Jewell JE et al.; The properties of the cysteines in the pBR322-encoded tetracycline resistance protein have been examined . Cysteines are important but not essential for tetracycline transport activity . None of the cysteines reacted with biotin maleimide, suggesting that they are shielded from the aqueous phase or reside in a negatively charged local environment.

J Bacteriol, 1999 Mar, 181(5), 1585 - 602
Complete sequence of a 184-kilobase catabolic plasmid from Sphingomonas aromaticivorans F199; Romine MF et al.; The complete 184,457-bp sequence of the aromatic catabolic plasmid, pNL1, from Sphingomonas aromaticivorans F199 has been determined . A total of 186 open reading frames (ORFs) are predicted to encode proteins, of which 79 are likely directly associated with catabolism or transport of aromatic compounds . Genes that encode enzymes associated with the degradation of biphenyl, naphthalene, m-xylene, and p-cresol are predicted to be distributed among 15 gene clusters . The unusual coclustering of genes associated with different pathways appears to have evolved in response to similarities in biochemical mechanisms required for the degradation of intermediates in different pathways . A putative efflux pump and several hypothetical membrane-associated proteins were identified and predicted to be involved in the transport of aromatic compounds and/or intermediates in catabolism across the cell wall . Several genes associated with integration and recombination, including two group II intron-associated maturases, were identified in the replication region, suggesting that pNL1 is able to undergo integration and excision events with the chromosome and/or other portions of the plasmid . Conjugative transfer of pNL1 to another Sphingomonas sp . was demonstrated, and genes associated with this function were found in two large clusters . Approximately one-third of the ORFs (59 of them) have no obvious homology to known genes.

J Bacteriol, 1999 Mar, 181(5), 1444 - 50
3-Hydroxylaminophenol mutase from Ralstonia eutropha JMP134 catalyzes a Bamberger rearrangement; Schenzle A et al.; 3-Hydroxylaminophenol mutase from Ralstonia eutropha JMP134 is involved in the degradative pathway of 3-nitrophenol, in which it catalyzes the conversion of 3-hydroxylaminophenol to aminohydroquinone . To show that the reaction was really catalyzed by a single enzyme without the release of intermediates, the corresponding protein was purified to apparent homogeneity from an extract of cells grown on 3-nitrophenol as the nitrogen source and succinate as the carbon and energy source . 3-Hydroxylaminophenol mutase appears to be a relatively hydrophobic but soluble and colorless protein consisting of a single 62-kDa polypeptide . The pI was determined to be at pH 4.5 . In a database search, the NH2-terminal amino acid sequence of the undigested protein and of two internal sequences of 3-hydroxylaminophenol mutase were found to be most similar to those of glutamine synthetases from different species . Hydroxylaminobenzene, 4-hydroxylaminotoluene, and 2-chloro-5-hydroxylaminophenol, but not 4-hydroxylaminobenzoate, can also serve as substrates for the enzyme . The enzyme requires no oxygen or added cofactors for its reaction, which suggests an enzymatic mechanism analogous to the acid-catalyzed Bamberger rearrangement.

Antimicrob Agents Chemother, 1999 Mar, 43(3), 514 - 9
Effects of the Chinese traditional medicine mao-bushi-saishin-to on therapeutic efficacy of a new benzoxazinorifamycin, KRM-1648, against Mycobacterium avium infection in mice; Shimizu T et al.; The Chinese traditional medicine mao-bushi-saishin-to (MBST), which has anti-inflammatory effects and has been used to treat the common cold and nasal allergy in Japan, was examined for its effects on the therapeutic activity of a new benzoxazinorifamycin, KRM-1648 (KRM), against Mycobacterium avium complex (MAC) infection in mice . In addition, we examined the effects of MBST on the anti-MAC activity of murine peritoneal macrophages (M phi s) . First, MBST significantly increased the anti-MAC therapeutic activity of KRM when given to mice in combination with KRM, although MBST alone did not exhibit such effects . Second, MBST treatment of M phi s significantly enhanced the KRM-mediated killing of MAC bacteria residing in M phi s, although MBST alone did not potentiate the M phi anti-MAC activity . MBST-treated M phi s showed decreased levels of reactive nitrogen intermediate (RNI) release, suggesting that RNIs are not decisive in the expression of the anti-MAC activity of such M phi populations . MBST partially blocked the interleukin-10 (IL-10) production of MAC-infected M phi s without affecting their transforming growth factor beta (TGF-beta)-producing activity . Reverse transcription-PCR analysis of the lung tissues of MAC-infected mice at weeks 4 and 8 after infection revealed a marked increase in the levels of tumor necrosis factor alpha, gamma interferon (IFN-gamma), IL-10, and TGF-beta mRNAs . KRM treatment of infected mice tended to decrease the levels of the test cytokine mRNAs, except that it increased TGF-beta mRNA expression at week 4 . MBST treatment did not affect the levels of any cytokine mRNAs at week 8, while it down-regulated cytokine mRNA expression at week 4 . At week 8, treatment of mice with a combination of KRM and MBST caused a marked decrease in the levels of the test cytokines mRNAs, especially IL-10 and IFN-gamma mRNAs, although such effects were obscure at week 4 . These findings suggest that down-regulation of the expression of IL-10 and TGF-beta is related to the combined therapeutic effects of KRM and MBST against MAC infection.

Wien Klin Wochenschr, 1998 Dec 23, 110(24), 866 - 9
Surface receptors of neutrophils towards B . burgdorferi; Cinco M et al.; The spirochetal agent of Lyme borreliosis, Borrelia burgdorferi, is able to induce an infection which develops in three stages: an early, localized infection, disseminated infection and a third stage, chronic infection, which probably indicates that a protected niche has been established in one or more tissues, where the spirochetes persist even if a specific immune response has been initiated . During the first stage, immediately after their entry into the host tissue, B . burgdorferi meet the motile phagocytic cells, neutrophils and monocytes; this is followed by consequent phagocytosis and killing . Although the rate and mechanism of this killing is not entirely clear, there is evidence that phagocytosis by both neutrophils and monocytes proceeds even in the absence of specific antibodies . We have demonstrated in both neutrophils and CHO Mac-1 (CR3 integrin) transfected cells, that one phagocyte receptor which is involved in B . burgdorferi adhesion in non osponic phagocytosis is the CR3 complement receptor known as integrin alpha m beta 2 . Both recognition domains of the integrin, the iC3b site and the COOH terminal lectin site, bind to B . burgdorferi . Data presented here show that inhibition of adhesion on CR3 Mac-1 transfected cells and neutrophils is induced by mannose as well as by N-acetyl-D-glucosamine, sugars known to be specific inhibitors of the COOH terminal lectin-site of the integrin CR3 . The inhibitory effect was serum complement independent . On the contrary, monoclonal antibody VIM12 directed towards the lectin domain not only failed to inhibit but improved adhesion, suggesting that, as a consequence of the binding, the integrin becomes more receptive to B . burgdorferi attachment at the I domain . Pretreatment of the borrelias with NalO4 eliminated adhesion, suggesting that the sugar residue/s recognized by CR3 is located on the bacteria.

Wien Klin Wochenschr, 1998 Dec 23, 110(24), 850 - 5
Molecular epidemiology of the aetiological agents of Lyme borreliosis; Baranton G et al.; Ten species are up to now recognized among Borrelia burgdorferi sensu lato complex . Among those, only three (Borrelia burgdorferi sensu stricto, B . garinii and B . afzelii) are reported to be pathogenic for humans and each responsible for a predominant clinical form of Lyme borreliosis . Each species is characterized by its vectors (Ixodidae), its host spectrum, its organotropism (for the pathogenic ones) and its geographical repartition . Borrelia are strictly parasitic and essentially clonal bacteria . Our goal was to explore the diversity of this bacterial complex . We selected, by several molecular markers, atypical isolates and compared them to already known species representative strains by RFLP or sequencing . The results show an unexpected diversity at a level which could be a species one leading to the conclusion that the structure of the Borrelia burgdorferi sensu lato complex is a high number of small (by their populations) clones among which emerge some large ones usually corresponding to the pathogenic species . Our data also allow to speculate on when, where and how these species evolved and migrated.

Curr Opin Microbiol, 1999 Feb, 2(1), 35 - 9
Molecular and cellular biology of pneumococcal infection; Tuomanen E; The complete pneumococcal genome sequence was released in November, 1997 . This advance has been combined with new understanding of physiology and pathogenesis including characterization of a family of surface proteins adducted to choline, regulation of virulence, and the regulon for natural DNA transformation . These developments in our understanding of molecular and cellular biology of pneumococcal infection will allow the development of new vaccines and antibiotics against this community acquired pathogen.

J Mol Biol, 1999 Mar 5, 286(4), 1241 - 9
Conformational changes in the chaperonin GroEL: new insights into the allosteric mechanism; de Groot BL et al.; Conformational changes are known to play a crucial role in the function of the bacterial GroE chaperonin system . Here, results are presented from an essential dynamics analysis of known experimental structures and from computer simulations of GroEL using the CONCOORD method . The results indicate a possible direct form of inter-ring communication associated with internal fluctuations in the nucleotide-binding domains upon nucleotide and GroES binding that are involved in the allosteric mechanism of GroEL . At the level of conformational transitions in entire GroEL rings, nucleotide-induced structural changes were found to be distinct and in principle uncoupled from changes occurring upon GroES binding . However, a coupling is found between nucleotide-induced conformational changes and GroES-mediated transitions, but only in simulations of GroEL double rings, and not in simulations of single rings . This provides another explanation for the fact that GroEL functions a double ring system .

J Biol Chem, 1999 Mar 5, 274(10), 6183 - 9
Acid-catalyzed lactonization of alpha2,8-linked oligo/polysialic acids studied by high performance anion-exchange chromatography; Zhang Y et al.; Recent studies from many laboratories revealed remarkable structural, distributional, and functional diversities of oligo/polysialic acids (OSA/PSA) that exist in organisms ranging from bacteria to man . These diversities are further complicated by the fact that OSA/PSA spontaneously form lactones under even mildly acidic conditions . By using high performance anion-exchange chromatography (HPAEC) with nitrate eluents, we found that lactonization of alpha2,8-linked OSA/PSA (oligo/poly-Neu5Ac, oligo/poly-Neu5Gc and oligo/poly-KDN) proceeds readily, and the lactonization process displays three discrete stages . The initial stage is characterized by limited lactonization occurring between two internal sialic acid residues, reflected by a regular pattern of lactone peaks interdigitated with non-lactonized peaks on HPAEC . In the middle stage, multiple lactonized species are formed from a molecule with a given degree of polymerization (DP), in which the maximum number of lactone rings formed equals DP minus 2 . At the final stage, completely lactonized species become the major components, resulting in drastic changes in the physicochemical properties of the sample . Interestingly, the smallest lactonizable OSA are tetramer, trimer, and dimer at the initial, middle, and final stages, respectively . At any of the stages, OSA/PSA of higher DP lactonize more rapidly, but all the lactone rings rapidly open up when exposed to mild alkali . Lactonized OSA/PSA are resistant to both enzyme- and acid-catalyzed glycosidic bond cleavage . The latter fact was utilized to obtain more high DP oligo/poly(alpha2,8-Neu5Gc) chains from a polysialoglycoprotein . Our results should be useful in preparation, storage, and analysis of OSA/PSA . Possible biological significance and bioengineering potentials of lactonization are discussed.

Nippon Rinsho, 1999 Jan, 57(1), 111 - 5
{The appropriate time for the assessment of Helicobacter pylori eradication}; Ishizuka J et al.; As all of the guidelines on the management of H . pylori infection suggest, the assessment of the eradication is generally performed at least 4 weeks after the completion of eradication treatment . However, H . pylori occasionally re-appears after one month, even the successful eradication was confirmed by the guideline . In this study, we investigated the appropriate time for the assessment of H . pylori eradication, mainly by using 13C urea breath tests (UBT) as a positive standard . From July 1992 to December 1997, 386 patients with H . pylori infection received eradication treatments . The presence of H . pylori was assessed by rapid urease test, UBT, culture and histologic examination . Eradication of the bacteria was determined by the negative results in all of these four tests . At 4 weeks after completion of therapy, 312 cases (80.8%) were judged as being free of H . pylori . Mean observation period was up to 12 months, and 113 cases were followed up to more than 1 year, and 50 cases were followed up to more than 2 years . H . pylori had re-appeared in 3 cases after 3 months, 1 case after 6 months, 2 cases after 12 months, and 1 case 24 months after the treatment, respectively . For the purpose of more accurate diagnosis, the assessment of eradication of H . pylori should be performed at 1 year after the completion of therapy . Since all the recrudescence could be diagnosed with UBT earlier and be confirmed by the other tests later, UBT is recommended as a useful methods in the assessment of Helicobacter pylori eradication.

Rinsho Biseibutshu Jinsoku Shindan Kenkyukai Shi, 1998, 9(2), 59 - 65
Experience and clinical usefulness of BACTEC MGIT 960; Tazawa Y et al.; Recently, an automatic instrument for culturing acid-fast bacteria in liquid media has been introduced in Japan to accelerate and increase the detection of the bacteria as well as the work e ciency of test lab technicians . We compared the MGIT (Mycobacterium Growth Indicator Tube) method and the two conventional culture methods used in our lab by testing 176 specimens (130 cases) submitted for acid-fast testing between July 27 and September 18, 1998 . BACTEC MGIT 960 (Nippon Becton Dickinson) was used in the MGIT method while Ogawa K medium and Kudo PD medium were used for our conventional culture methods . Of the 176 specimens, 26 specimens were MGIT positive, 22 specimens were Ogawa K medium positive, and 21 specimens were Kudo PD medium positive . Among these three culture methods, M . tuberculosis was found in 8 specimens, M . avium was found in 9 specimens, M . intracellulare was found in 4 specimens, and other acid-fast bacteria were found in 5 specimens . It took an average of 13.5 days to detect M . tuberculosis by the MGIT method, 23.0 days by the Ogawa K medium, and 24.3 days by the Kudo PD medium, suggesting that the MGIT method is signi cantly faster than the conventional methods (p < 0.05) . The MGIT method took an average of 5.7 days to detect M . avium and M . intracellulare while the two conventional methods required more than 20 days, suggesting that the MGIT method is signi cantly faster than the conventional methods (p < 0.05) . The BACTEC MGIT 960 was extremely useful for clinical diagnosis in some of the cases and our results indicated that the MGIT method led to more rapid diagnosis and earlier treatment than the conventional methods.

Lett Appl Microbiol, 1999 Jan, 28(1), 17 - 22
Spacer 2 region and 5S rDNA variation of Wolbachia strains involved in cytoplasmic incompatibility or sex-ratio distortion in arthropods; van Meer MM et al.; Bacteria in the genus Wolbachia are widespread in arthropods and can induce sex-ratio distortion or cytoplasmic incompatibility in their hosts . The phylogeny of Wolbachia has been studied using 16S rDNA and the cell cycle gene ftsZ, but sequence variation of those genes is limited . The spacer 2 region (SR2) was amplified to determine whether this region would improve phylogenetic resolution . The SR2 of Wolbachia is 66 bp long, shows higher variation than ftsZ and has very low homology with closely related bacteria . Due to the small length of SR2 of Wolbachia, little phylogenetic information could be retrieved.

J Calif Dent Assoc, 1998 Nov, 26(11), 846 - 50
The effect of bioburden on in-depth disinfection of denture base acrylic resin; Saunders TR et al.; This study evaluated the effectiveness of three different disinfectant solutions against denture bioburden absorbed within the depth of acrylic resin . Specimens were taken from dentures that had been worn by the patients for 15 to 20 years and were scheduled for replacement.

J Gastroenterol Hepatol, 1999 Jan, 14(1), 54 - 60
Dextran sodium sulphate-induced colitis activity varies with mouse strain but develops in lipopolysaccharide-unresponsive mice; Stevceva L et al.; Bacteria and their products have been implicated in the pathogenesis of chronic Inflammatory Bowel disease . The aim of this study was to investigate the potential role of lipopolysaccharides (LPS) in the development of intestinal injury by comparing the effects of the dextran sodium sulphate (DSS)-induced model of colitis in LPS-sensitive and -insensitive mice . Experimental colitis was induced in LPS-sensitive mice (C3H/He) and their LPS-insensitive congenic strain (C3H/HeJ) . Colitis was assessed clinically using a disease activity index (derived from the three main clinical signs; diarrhoea, rectal bleeding and weight loss) and by histological scoring of the diseased colon . The clinical signs and disease activity index did not differ between the LPS-sensitive and -insensitive costrains . Similarly, histological scores did not differ significantly for either C3H strain at any time point during exposure to DSS . However, there were differences in the inflammatory response when different strains were compared (C3H vs CBA): the effects of DSS in C3H mice were immediate, more severe and mainly involved the caecum and ascending colon . These findings suggest that LPS from colonic bacteria do not play a primary role in the initiation of DSS-induced colitis and demonstrate clear differences in the responsiveness of different mouse strains to DSS.

Chemosphere, 1999 Feb, 38(5), 1135 - 44
The toxicity and concentrations of PAHs in creosote-contaminated lake sediment; Hyotylainen T et al.; Sediment samples, divided into three layers (0-10, 10-20 and 20-30 cm), were collected from 16 sites in Lake Jamsanvesi, Central Finland . The acute toxicity of pore waters and elutriates (sediment + water 1:4 v/v) were studied by bioluminescence inhibition test and by immobilisation of water fleas (Daphnia magna Straus) . Concentrations of polycyclic aromatic hydrocarbons (PAHs) in sediments and elutriates were measured by gas chromatography using flame ionization detection (GC/FID) . The highest total PAH concentration was 3.3 mg/g dry weight in the sediment and up to 1.7 mg/l in the elutriate of the uppermost (0-10 cm) layer, also being the most toxic to photoluminencent bacteria and water flea . When sediment and water mix, like during dredging operations, toxic compounds may spread from the sediment to the water column and can pose on environmental risk.

Inflamm Bowel Dis, 1999 Feb, 5(1), 33 - 9
Medical therapy for induction and maintenance of remission in pouchitis: a systematic review; Sandborn WJ et al.; OBJECTIVE: To determine the effectiveness of medical therapy (including metronidazole, bismuth carbomer enemas, oral probiotic bacteria, butyrate suppositories, and glutamine suppositories) for inducing a response or for maintaining remission in pouchitis . SEARCH STRATEGY: Studies were selected using the MEDLINE data base (1966-December 1997), abstracts from major gastrointestinal meetings, and references from published articles and reviews . Selection criteria: Four randomized controlled trials of medical therapy in adult patients with pouchitis were identified: two placebo controlled trials in active chronic pouchitis; one maintenance of remission trial comparing two active agents in chronic pouchitis; and one placebo-controlled maintenance of remission trial for chronic pouchitis . A single patient "n-of-1" trial for active chronic pouchitis was excluded . DATA COLLECTION AND ANALYSIS: Data were extracted by three independent observers based on the intention to treat principle . Extracted data were converted to 2 x 2 tables (response versus no response and medical therapy versus placebo or medical therapy versus medical therapy) and an odds ratio with 95% confidence intervals (CI) were determined as described by Cochrane and Mantel and Haenszel . In addition, the absolute risk reduction, relative risk reduction, and number needed to treat were determined . MAIN RESULTS: The odds ratios of inducing a response using oral metronidazole or bismuth carbomer foam enemas compared with placebo in active chronic pouchitis were 12.34 (95% CI 2.34-64.95) and 1.00 (95% CI 0.29-3.42), respectively . The odds ratio of maintaining remission in chronic pouchitis for oral probiotic bacteria (VSL-3) compared with placebo was 15.33 (95% CI 4.51-52.14) . There was no difference in the odds ratio of inducing symptomatic remission and then maintaining symptomatic remission after discontinuing suppressive medical therapy for chronic pouchitis with glutamine suppositories compared with butyrate suppositories, 2.75 (95% CI 0.48-15.94) . CONCLUSIONS: Metronidazole is an effective therapy for active chronic pouchitis . Bismuth carbomer foam enemas are not effective therapy for active chronic pouchitis . Oral probiotic therapy with VSL-3 is an effective therapy for maintaining remission in patients with chronic pouchitis in remission . There is no difference in maintenance of symptomatic remission in patients with chronic pouchitis treated with glutamine versus butyrate suppositories, and it is unknown whether glutamine and butyrate are equally effective or ineffective . Additional randomized, double-blind, placebo-controlled, dose-ranging clinical trials are needed to determine the efficacy of empiric medical therapies currently being used in patients with pouchitis.

Int J Syst Bacteriol, 1999 Jan, 49 Pt 1, 189 - 91
Phylogeny of marine and freshwater Shewanella: reclassification of Shewanella putrefaciens NCIMB 400 as Shewanella frigidimarina; Reid GA et al.; Dissimilatory Fe(III) reduction by Shewanella putrefaciens and related species has generated considerable interest in biochemical characterization of the pathways for anaerobic electron transfer in this organism . Two strains, MR-1 and NCIMB 400, have been extensively used, and several respiratory enzymes have been isolated from each . It has become apparent that significant sequence differences exist between homologous proteins from these strains . The 16S rRNA from NCIMB 400 was sequenced and compared to the sequences from MR-1 and other Shewanella strains . The results indicate that NCIMB 400 is significantly more closely related to the newly identified Shewanella frigidimarina than to the S . putrefaciens type strain . It is therefore proposed that NCIMB 400 should be reclassified as S . frigidimarina.

Recent Results Cancer Res, 1998, 154, 3 - 21
Epidemiological evidence of the effects of behaviour and the environment on the risk of human cancer; Doll R; The incidence of cancer in middle and old age can, in principle, be reduced by 80%-90% and the risks worldwide could be halved, although the methods required are not always socially acceptable . The proportions of fatal cancers attributable to different causes are examined under 17 headings: smoking, alcohol, pharmaceutical products, infection (parasites, bacteria, viruses) electromagnetic radiation (ionizing, ultraviolet, lower frequency) occupation, industrial products, pollution (air, water, food), physical inactivity, reproductive hormones, and diet . Smoking is the most important factor . It contributes to the production of seven types of cancer in addition to the eight that were recognized by the International Agency for Research on Cancer in 1986 and is estimated to have been responsible for 38% of cancers in men and 6% in women in Germany in 1985 . Firm estimates can also be made of the proportions of fatal cancers attributable to alcohol and ionizing radiation, and reasonable guesses can be made at the maximum effect of some of the other categories . Many of the factors act synergistically with one another, so that the risk of developing specific cancers can be modified in different ways . When all the avoidable causes are known, the sum of the proportions avoidable in different ways may add up to several hundred per cent.

Todays Surg Nurse, 1998 May-Jun, 20(3), 28 - 32
Consider using maggots; Krajacic A; Although once used to treat a multitude of conditions, maggot therapy was replaced almost entirely by the advent of antibiotics . During treatment, maggots liquify necrotic tissue and kill bacteria in the wound, stimulating healing . Despite its benefits, maggot therapy should not be used as an alternative to emergent surgery.

Biochemistry, 1999 Feb 16, 38(7), 2117 - 26
A cyanobacterial hemoglobin with unusual ligand binding kinetics and stability properties; Thorsteinsson MV et al.; The glbN gene of the cyanobacterium Nostoc commune UTEX 584 encodes a hemoprotein, named cyanoglobin, that has high oxygen affinity . The basis for the high oxygen affinity of cyanoglobin was investigated through kinetic studies that utilized stopped-flow spectrophotometry and flash photolysis . Association and dissociation rate constants were measured at 20 degrees C for oxygen, carbon monoxide, nitric oxide, and methyl and ethyl isocyanides . The association rate constants for the binding of these five ligands to cyanoglobin are the highest reported for any naturally occurring hemoglobin, suggesting an unhindered and apolar ligand binding pocket . Cyanoglobin also shows high rates of autoxidation and hemin loss, indicating that the prosthetic group is readily accessible to solvent . The ligand binding behavior of cyanoglobin was more similar to that of leghemoglobin a than to that of sperm whale myoglobin . Collectively, the data support the model of cyanoglobin function described by Hill et al . {(1996) J . Bacteriol . 178, 6587-6598}, in which cyanoglobin sequesters oxygen, and presents it to, or is a part of, a terminal cytochrome oxidase complex in Nostoc commune UTEX 584 under microaerobic conditions, when nitrogen fixation, and thus ATP demand, is maximal.

FEBS Lett, 1999 Jan 29, 443(3), 282 - 4
An Arabidopsis 14-3-3 protein can act as a transcriptional activator in yeast; Wang J et al.; The 14-3-3 proteins are a group of highly conserved and widely distributed eukaryotic proteins with diverse functions . One 14-3-3 protein, AFT1 from Arabidopsis thaliana, was found to be able to activate transcription in yeast . When fused to the DNA-binding domain of a bacterial protein LexA, AFT1 can activate transcription of reporter genes that contain LexA operator sequences in their promoters . Although the in vivo function of AFT1 is not completely known, its similarity to previously identified proteins found in transcription complexes of Arabidopsis and maize suggests that AFT1 and some other 14-3-3 proteins may activate gene expression in other systems as well.

Lupus, 1999, 8(1), 52 - 9
Role of HLA in congenital heart block: susceptibility alleles in mothers; Siren MK et al.; In congenital heart block (CHB), abnormal maternal immunisation leads to autoantibody production against SS-A/Ro and SS-B/La antigens . These maternal antibodies are transferred across the placenta to the unborn child and are believed to transmit irreversible immunological injury in developing foetal heart tissue, thus causing 3rd-degree atrioventricular block . The mothers may suffer from systemic lupus erythematosus (SLE) or primary Sjogren's syndrome (SS), but they may be asymptomatic . Women with primary SS show a typical autoimmune HLA antigen pattern, namely higher frequency of HLA B8 and DR3 than in the normal population . The HLA pattern may affect individual ability to resist infecting bacteria and viruses and to response in various ways to autoantigens . It is probable that other factors such as genetic regulation of immune response are involved in CHB . We compared the HLA class I and class II alleles of mothers having CHB children with those of women suffering from primary SS and having healthy children, and with those of healthy Finns . Antibodies against 52-kD and 60-kD SS-A/Ro and 48-kD SS-B/La antigens were compared between the two groups of mothers . Our results show that anti-SS-A/Ro antibody-positive mothers all show a strong association with known autoimmune-predisposing HLA alleles, however, the mothers of CHB children differ in some HLA class I alleles, and especially in HLA haplotypes, from mothers of healthy children . Mothers with HLA A1, Cw7, B8 and without B15 are at particularly high-risk of having CHB children.

Infect Immun, 1999 Mar, 67(3), 1493 - 500
Probing the function of Bordetella bronchiseptica adenylate cyclase toxin by manipulating host immunity; Harvill ET et al.; We have examined the role of adenylate cyclase-hemolysin (CyaA) by constructing an in-frame deletion in the Bordetella bronchiseptica cyaA structural gene and comparing wild-type and cyaA deletion strains in natural host infection models . Both the wild-type strain RB50 and its adenylate cyclase toxin deletion (DeltacyaA) derivative efficiently establish persistent infections in rabbits, rats, and mice following low-dose inoculation . In contrast, an inoculation protocol that seeds the lower respiratory tract revealed significant differences in bacterial numbers and in polymorphonuclear neutrophil recruitment in the lungs from days 5 to 12 postinoculation . We next explored the effects of disarming specific aspects of the immune system on the relative phenotypes of wild-type and DeltacyaA bacteria . SCID, SCID-beige, or RAG-1(-/-) mice succumbed to lethal systemic infection following high- or low-dose intranasal inoculation with the wild-type strain but not the DeltacyaA mutant . Mice rendered neutropenic by treatment with cyclophosphamide or by knockout mutation in the granulocyte colony-stimulating factor locus were highly susceptible to lethal infection by either wild-type or DeltacyaA strains . These results reveal the significant role played by neutrophils early in B . bronchiseptica infection and by acquired immunity at later time points and suggest that phagocytic cells are a primary in vivo target of the Bordetella adenylate cyclase toxin.

Infect Immun, 1999 Mar, 67(3), 1050 - 5
Role of antibodies against Bordetella pertussis virulence factors in adherence of Bordetella pertussis and Bordetella parapertussis to human bronchial epithelial cells; van den Berg BM et al.; Immunization with whole-cell pertussis vaccines (WCV) containing heat-killed Bordetella pertussis cells and with acellular vaccines containing genetically or chemically detoxified pertussis toxin (PT) in combination with filamentous hemagglutinin (FHA), pertactin (Prn), or fimbriae confers protection in humans and animals against B . pertussis infection . In an earlier study we demonstrated that FHA is involved in the adherence of these bacteria to human bronchial epithelial cells . In the present study we investigated whether mouse antibodies directed against B . pertussis FHA, PTg, Prn, and fimbriae, or against two other surface molecules, lipopolysaccharide (LPS) and the 40-kDa outer membrane porin protein (OMP), that are not involved in bacterial adherence, were able to block adherence of B . pertussis and B . parapertussis to human bronchial epithelial cells . All antibodies studied inhibited the adherence of B . pertussis to these epithelial cells and were equally effective in this respect . Only antibodies against LPS and 40-kDa OMP affected the adherence of B . parapertussis to epithelial cells . We conclude that antibodies which recognize surface structures on B . pertussis or on B . parapertussis can inhibit adherence of the bacteria to bronchial epithelial cells, irrespective whether these structures play a role in adherence of the bacteria to these cells.

J Mol Biol, 1999 Feb 26, 286(3), 883 - 98
Refined crystal structures of reaction centres from Rhodopseudomonas viridis in complexes with the herbicide atrazine and two chiral atrazine derivatives also lead to a new model of the bound carotenoid; Lancaster CR et al.; In a reaction of central importance to the energetics of photosynthetic bacteria, light-induced electron transfer in the reaction centre (RC) is coupled with the uptake of protons from the cytoplasm at the binding site of the secondary quinone (QB) . It has been established by X-ray crystallography that the triazine herbicide terbutryn binds to the QB site . However, the exact description of protein-triazine interactions has had to await the refinement of higher-resolution structures . In addition, there is also interest in the role of chirality in the activity of herbicides . Here, we report the structural characterisation of triazine binding by crystallographic refinement of complexes of the RC either with the triazine inhibitor atrazine (Protein Data Bank (PDB) entry 5PRC) or with the chiral atrazine derivatives, DG-420314 (S(-) enantiomer, PDB entry 6PRC) or DG-420315 (R(+) enantiomer, PDB entry 7PRC) . Due to the high quality of the data collected, it has been possible to describe the exact nature of triazine binding and its effect on the structure of the protein at high-resolution limits of 2.35 A (5PRC), 2.30 A (6PRC), and 2.65 A (7PRC), respectively . In addition to two previously implied hydrogen bonds, a third hydrogen bond, binding the distal side of the inhibitors to the protein, and four additional hydrogen bonds mediated by two tightly bound water molecules on the proximal side of the inhibitors, are apparent . Based on the high quality data collected on the RC complexes of the two chiral atrazine derivatives, unequivocal assignment of the structure at the chiral centres was possible, even though the differences in structures of the substituents are small . The structures provide explanations for the relative binding affinities of the two chiral compounds . Although it was not an explicit goal of this work, the new data were of sufficient quality to improve the original model also regarding the structure of the bound carotenoid 1,2-dihydroneurosporene . A carotenoid model with a cis double bond at the 15,15' position fits the electron density better than the original model with a 13,14-cis double bond .

Clin Exp Allergy, 1998 Dec, 28(12), 1581 - 90
Adenoviral infection inhibits allergic airways inflammation in mice; Stampfli MR et al.; BACKGROUND: Recent epidemiological studies have suggested that exposure to certain viruses and bacteria influences the development of allergy and allergic diseases, such as asthma . However, there is a paucity of experimental evidence examining the consequences of concurrent exposure to allergen and infectious agents, and the potential mechanisms by which allergic disease might be averted as a result . OBJECTIVE: To model this situation experimentally, we investigated whether a virally induced immune response, elicited by a replication-deficient human type 5 adenovirus (RDA) administered at a site distant from the airways, could inhibit ovalbumin (OVA)-induced airways eosinophilic inflammation . METHODS: C57BL/6 mice were infected intramuscularly with RDA 16h prior to intraperitoneal OVA sensitization . Cellular and cytokine responses in the lung/airways were examined after an OVA aerosol challenge . RESULTS: RDA infection significantly inhibited the inflammatory response in the lung tissue after antigen challenge . In the bronchoalveolar lavage (BAL), total cell number, eosinophils and lymphocytes were decreased by 70, 85 and 65%, respectively, after antigen challenge in RDA-treated, compared with untreated, mice . RDA infection had no effect on IgE synthesis . The levels of IL-5, IL-4 and IFNgamma in the BAL after antigen challenge were significantly lower in RDA-treated mice . In vitro production of cytokines by splenocytes in response to OVA restimulation revealed a shift from IL-4 in sensitized, PBS-treated mice, to IFNgamma in sensitized mice treated with RDA . Flow cytometric analysis revealed that RDA infection increased the proportion of CD8 T cells in the BAL; this change in T-cell subsets was accompanied by an increase in both CD4 and CD8 T cells positive for intracellular IFNgamma . Inhibition of antigen-induced airways inflammation was IFNgamma-dependent but did not require IL-12, as RDA-treatment inhibited airways inflammation in IL-12 but not IFNgamma knock-out mice . CONCLUSION: This study demonstrates that an immune response against a replication-deficient adenovirus during the initial exposure to OVA inhibits the development of airways inflammation after antigen aerosol challenge.

Ann Surg, 1999 Feb, 229(2), 279 - 85
Immune response capacity after human splenic autotransplantation: restoration of response to individual pneumococcal vaccine subtypes; Leemans R et al.; OBJECTIVE: To evaluate features of general immune function, in particular the restoration of the humoral immune response to pneumococcal capsular polysaccharides, in humans undergoing a spleen autotransplantation after splenectomy because of trauma . SUMMARY BACKGROUND DATA: After splenectomy, patients have an increased risk of overwhelming infection or sepsis involving encapsulated bacteria such as pneumococci . The value of human spleen autotransplantation after splenectomy because of trauma has long been questioned . Mononuclear phagocyte system function appeared to be similar to that in splenectomized persons . The presence of specific antipneumococcal antibodies would allow other parts of the mononuclear phagocyte system, such as those in the liver, to phagocytose opsonized bacteria . METHODS: Ten consecutive patients undergoing splenectomy followed by autotransplantation were compared with the next 14 consecutive patients undergoing splenectomy alone . After a minimum of 6 months, the patients were vaccinated with 23-valent pneumococcal vaccine . Blood samples were taken at the time of vaccination and after 3 and 6 weeks for antipneumococcal capsular polysaccharides IgM and IgG enzyme-linked immunosorbent assay against types 3, 4, 6, 9, 14, and 23 . Splenic regrowth was evaluated by scintigraphy . RESULTS: Surprisingly, several of the nonautotransplanted patients showed scintigraphic activity, indicating the presence of either accessory spleens or traumatic seeding (splenosis) . Significant antibody titer increases (more than twofold) were found for both IgM and IgG in the autotransplanted patients . Splenectomized-only patients showed no significant increase in Ig levels in patients without splenic regrowth and partial improvement in patients with splenosis/accessory spleens . CONCLUSIONS: Considering this significant antipneumococcal antibody increase, spleen autotransplants can be expected to permit an adequate humoral response to pneumococcal infections and presumably also to other TI-2 antigens, and to protect against overwhelming postsplenectomy infection or sepsis.

Scand J Immunol, 1999 Jan, 49(1), 45 - 50
Identification of arthritogenic adjuvants of self and foreign origin; Lorentzen JC; The lack of defined triggers for human inflammatory joint diseases warrants efforts to identify candidate molecules . For this task, it may be an important lead that nonspecific activation of the immune system can precipitate arthritis in rats . Consequently, arthritis-prone rat strains were used to search for disease-triggering factors among molecules which initially induce innate defence reactions rather than specific immune responses . A variety of immunological adjuvants were investigated by intradermal injection into DA and LEW.1AV1 rats and monitoring of clinical signs for 30 days . Several arthritogenic cell-wall structures from yeast and bacteria were identified, such as beta-glucan, lipopolysaccharide and trehalosedimycolate . The test procedures also revealed arthritogens of chemical origin, such as dioctadecyldiammoniumbromide (DDA = C38H80NBr) and heptadecane (C17H36) . Furthermore, it allowed the precise definition of arthritogenic determinants of lipids, since C16H34 induced arthritis, whereas the closely related linear hydrocarbons C16H32, C16H33Br and C15H32 did not . The observed pathogenicity of organic lipids raised the question of whether endogenous lipids can also precipitate arthritis . Indeed, this was true for the cholesterol precursor squalene (C30H50) . In conclusion, this article describes the rational use of arthritis-prone rat strains to identify arthritogenic factors of both foreign and self origin . Although structurally unrelated, the pathogenic molecules defined here share the feature of being nonspecific triggers of the immune system . This consolidates a general principle for the induction of adjuvant arthritis which may provide clues to the aetiology of human arthritides, including rheumatoid arthritis.

J Dairy Sci, 1999 Jan, 82(1), 143 - 52
Starch and stage of maturity of grass silage: site of digestion and intestinal nutrient supply in dairy cows; van Vuuren AM et al.; The interaction between the quality of grass silage and starch supplementation on ruminal digestion was studied in an experiment with a 2 x 2 factorial design using four dairy cows . Treatment factors were grass silage harvested after either 21 or 37 d of regrowth and two concentrations of steam-flaked corn starch (0 or 4 kg/d) . Ruminal volume and flow of duodenal digesta were estimated . When forage was harvested at a more mature stage, only minor effects were noted for silage composition and, consequently, ruminal and intestinal digestion . The addition of starch to the diet tended to reduce ruminal digestion of neutral detergent fiber . The reduction in ruminal digestion was not compensated by increased digestion in the large intestine . Starch increased duodenal nonammonia N flow because of an increase in bacterial N flow . The increase in bacterial N was accompanied by a reduction in the escape of feed N from the rumen . Results from this study indicate that the addition of ruminally available starch to diets based on grass silage reduced ruminally degradable neutral detergent fiber and increased the duodenal supply of protein . These effects have to be taken into account to predict production responses to extra starch.

Curr Opin Chem Biol, 1999 Feb, 3(1), 39 - 46
Extremozymes; Hough DW et al.; Extremozymes offer new opportunities for biocatalysis and biotransformations as a result of their extreme stability . From recent work, major approaches to extending the range of applications of extremozymes have emerged . Both the discovery of new extremophilic species and the determination of genome sequences provide a route to new enzymes, with the possibility that these will lead to novel applications . Of equal importance, protein engineering and directed evolution provide approaches to improve enzyme stability and modify specificity in ways that may not exist in the natural world.

Curr Opin Chem Biol, 1999 Feb, 3(1), 22 - 7
C-H activation; Holland HL; Although the biocatalytic activation of carbon-hydrogen bonds by oxidation to alcohols was one of the first biotransformations to be exploited by the chemical industry, this remarkable process continues to be the subject of intense research activity . Recent developments in this area have centred on the use of new biocatalysts and substrates for C-H activation, in particular on the use of recombinant strains of yeast and bacteria expressing useful hydroxylase enzymes, on mechanistic work using hydroxylase enzymes that relies on the tools of molecular biology to address outstanding questions, particularly those of substrate selection and product predictability, and on the application of these systems for hydroxylation and heteroatom dealkylation reactions.

Curr Biol, 1999 Feb 11, 9(3), R97 - 9
Cell-mediated immunity: dealing a direct blow to pathogens; Kaufmann SH; Cytotoxic T lymphocytes are essential for defence against viral infections . Recent data demonstrating direct killing of intracellular bacteria by granulysin, a protein released from the granules of cytotoxic T lymphocytes, emphasize the contribution of these lymphocytes to the control of tuberculosis.

Mol Med, 1998 Dec, 4(12), 795 - 806
C1 inhibitor gene sequence facilitates frameshift mutations; Bissler JJ et al.; Mutations disrupting the function or production of C1 inhibitor cause the disease hereditary angioneurotic edema . Patient mutations identified an imperfect inverted repeat sequence that was postulated to play a mechanistic role in the mutations . To test this hypothesis, the inverted repeat was cloned into the chloramphenicol acetyltransferase gene in pBR325 and its mutation rate was studied in four bacterial strains . These strains were selected to assay the effects of recombination and superhelical tension on mutation frequency . Mutations that revert bacteria to chloramphenicol resistance (Cmr) were scored . Both pairs of isogenic strains had reversion frequencies of approximately 10(-8) . These rare reversion events in bacteria were most often a frameshift that involved the imperfect inverted repeat with a deletion or a tandem duplication, an event very similar to the human mutations . Increased DNA superhelical tension, which would be expected to enhance cruciform extrusion, did not accentuate mutagenesis . This finding suggests that the imperfect inverted repeat may form a stem-loop structure in the single-stranded DNA created by the duplex DNA melting prior to replication . Models explaining the slippage can be drawn using the lagging strand of the replication fork . In this model, the formation of a stem-loop structure is responsible for bringing the end of the deletion or duplication into close proximity.

Genes Dev, 1999 Feb 1, 13(3), 357 - 70
Amino-terminal sequences of sigmaN (sigma54) inhibit RNA polymerase isomerization; Cannon W et al.; In bacteria, association of the specialized sigmaN protein with the core RNA polymerase subunits forms a holoenzyme able to bind promoter DNA, but unable to melt DNA and initiate transcription unless acted on by an activator protein . The conserved amino-terminal 50 amino acids of sigmaN (Region I) are required for the response to activators . We have used pre-melted DNA templates, in which the template strand is unpaired and accessible for transcription initiation, to mimic a naturally melted promoter and explore the function of Region I . Our results indicate that one activity of Region I sequences is to inhibit productive interaction of holoenzyme with pre-melted DNA . On pre-melted DNA targets, either activation of sigmaN-holoenzyme or removal of Region I allowed efficient formation of complexes in which melted DNA was sequestered by RNA polymerase . Like natural pre-initiation complexes formed on conventional DNA templates through the action of activator, such complexes were heparin-resistant and transcriptionally active . The inhibitory sigmaN Region I domain functioned in trans to confer heparin sensitivity to complexes between Region I-deleted holoenzyme and pre-melted promoter DNA . Evidence that Region I senses the conformation of the promoter was obtained from protein footprint experiments . We suggest that one function for Region I is to mask a single-strand DNA-binding activity of the holoenzyme . On the basis of extended DNA footprints of Region I-deleted holoenzyme, we also propose that Region I prevents RNA polymerase isomerization, a conformational change necessary for access to and the subsequent stable association of holoenzyme with melted DNA.

FEMS Microbiol Rev, 1998 Dec, 22(5), 553 - 66
Biochemistry of coenzyme B12-dependent glycerol and diol dehydratases and organization of the encoding genes; Daniel R et al.; Glycerol and diol dehydratases exhibit a subunit composition of alpha 2 beta 2 gamma 2 and contain coenzyme B12 in the base-on form . The dehydratase reaction proceeds via a radical mechanism . The dehydratases are subject to reaction inactivation by the substrate glycerol which is caused by a cessation of the catalytic cycle because coenzyme B12 is not regenerated, instead 5'-deoxyadenosine and a catalytically inactive cobalamin are formed . The genetic organization of the dehydratase genes is quite similar in all organisms . Downstream of the dehydratase genes an open reading frame encoding a polypeptide of approximately 600 amino acids was identified which is apparently involved in the reactivation of suicide-inactivated enzyme.

FEMS Microbiol Rev, 1998 Dec, 22(5), 503 - 21
A structural comparison of molybdenum cofactor-containing enzymes; Kisker C et al.; This work gives an overview of the recent achievements which have contributed to the understanding of the structure and function of molybdenum and tungsten enzymes . Known structures of molybdo-pterin cofactor-containing enzymes will be described briefly and the structural differences between representatives of the same and different families will be analyzed . This comparison will show that the molybdo-pterin cofactor-containing enzymes represent a very heterogeneous group with differences in overall enzyme structure, cofactor composition and stoichiometry, as well as differences in the immediate molybdenum environment . Two recently discovered molybdo-pterin cofactor-containing enzymes will be described with regard to molecular and EPR spectroscopic properties, pyrogallol-phloroglucinol transhydroxylase from Pelobacter acidigallici and acetylene hydratase from Pelobacter acetylenicus . On the basis of its amino acid sequence, transhydroxylase can be classified as a member of the dimethylsulfoxide reductase family, whereas classification of the tungsten/molybdenum-containing acetylene hydratase has to await the determination of its amino acid sequence.

Annu Rev Entomol, 1999, 44, 159 - 82
Pathogens and predators of ticks and their potential in biological control; Samish M et al.; This review summarizes the literature about pathogens and predators of ticks and their potential use as biocontrol agents published since the beginning of this century . In nature, many bacteria, fungi, spiders, ants, beetles, rodents, birds, and other living things contribute significantly toward limiting tick populations, as do, for instance, the grooming activities of hosts . Experiments with the most promising potential tick biocontrol agents--especially fungi of the genera Beauveria and Metarhizium and nematodes in the families Steinernematidae and Heterorhabditidae, as well as oxpeckers--are described.

Eur J Biochem, 1998 Dec 15, 258(3), 1032 - 9
DNA synthesis exhibited by the reverse transcriptase of mouse mammary tumor virus: processivity and fidelity of misinsertion and mispair extension; Taube R et al.; We have recently expressed in bacteria an enzymatically active reverse transcriptase (RT) of mouse mammary tumor virus (MMTV), a mammalian retrovirus with a typical B-type morphology {Taube, R., Loya, S., Avidan, O., Perach, M . & Hizi, A . (1998) Biochemical J . 329, 579-587} . The purified recombinant protein was shown to possess the catalytic activities characteristic of retroviral reverse transcriptases . In the present study, we have analyzed two basic parameters characteristic of the DNA polymerase activity of the novel MMTV RT, namely the processivity and the fidelity of DNA synthesis . Two features related to fidelity were studied, the capacity to misinsert wrong nucleotides at the 3' end of the nascent DNA strand and the ability to extend 3' mispairs . The studied properties of MMTV RT were compared with those of the RT purified from virions of avian myeloblastosis virus (AMV), since AMV RT shows a relatively high sequence similarity to MMTV RT . MMTV RT shows a relative processivity of DNA synthesis which is as high as the reference AMV RT . Regarding fidelity of DNA synthesis, MMTV RT shows a fidelity of misinsertion lower than that of AMV RT, whereas its capacity to elongate mispaired DNA is lower than that of AMV RT indicating a somewhat higher fidelity . These fidelity properties are discussed also in the context of the RTs of lentiviruses, especially those of HIV, which were reported to exhibit an exceptionally low fidelity of DNA synthesis . It is clear that MMTV RT has a fidelity higher than that of lentiviral RTs.

Eur J Biochem, 1998 Dec 15, 258(3), 915 - 22
Affinities of mAbs to Tet repressor complexed with operator or tetracycline suggest conformational changes associated with induction; Pook E et al.; We isolated five monoclonal antibodies (mAbs) made against tetracycline repressor (TetR), one against the TetR tetracycline complex (Tc) and two against the TetR-tet operator (tetO) complex . The epitopes of the anti-TetR mAbs are localized in the alpha-helix-turn-alpha-helix motif (HTH), at different sites near the Tc binding pocket and at the dimerization interface . The anti-TetR-Tc and one of the anti-TetR-tetO mAbs recognize epitopes near the Tc binding pocket . The other anti-TetR-tetO mAb binds to an epitope within the HTH . Quantitative immunoprecipitation and competitive ELISA employing TetR, TetR-Tc, or TetR-tetO revealed different affinities of the mAbs for TetR in these functional states . Binding of the two mAbs to epitopes in the HTH was identical for TetR and TetR-Tc indicating the same conformation in both forms . The epitope located in the dimerization interface is bound more strongly in TetR compared to TetR-Tc, supporting the idea of different conformations of that epitope in these forms of TetR . The greatest affinity differences were found for epitopes around the Tc binding pocket . Two anti-TetR mAbs have the highest affinities for free TetR, somewhat reduced affinity for TetR-tetO and the lowest affinities for TetR-Tc . The anti-TetR-Tc mAb has a discontinuous epitope, formed in TetR-Tc, which is less well bound in TetR and not bound in the TetR-tetO complex . One anti-TetR-tetO mAb does not recognize TetR-Tc . Since the epitopes do not overlap with the respective ligand binding sites on TetR, these results are interpreted as conformational differences of the epitopes in these forms of TetR.

Proc Natl Acad Sci U S A, 1999 Feb 16, 96(4), 1744 - 9
Molecular basis for semidominance of missense mutations in the XANTHA-H (42-kDa) subunit of magnesium chelatase; Hansson A et al.; During biosynthesis of bacteriochlorophyll or chlorophyll, three protein subunits of 140, 70, and 42 kDa interact to insert Mg2+ into protoporphyrin IX . The semidominant Chlorina-125, -157, and -161 mutants in barley are deficient in this step and accumulate protoporphyrin IX after feeding on 5-aminolevulinate . Chlorina-125, -157, and -161 are allelic to the recessive xantha-h mutants and contain G559A, G806A, and C271T mutations, respectively . These mutations cause single amino acid substitutions in residues that are conserved in all known primary structures of the 42-kDa subunit . In vitro complementation and reconstitution of Mg-chelatase activity show that the 42-kDa subunits are defective in the semidominant Chlorina mutants . A mutated protein is maintained in the Chlorina plastids, unlike in the xantha-h plastids . Heterozygous Chlorina seedlings have 25-50% of the Mg-chelatase activity of wild-type seedlings . Codominant expression of active and inactive 42-kDa subunits in heterozygous Chlorina seedlings is likely to produce two types of heterodimers between the strongly interacting 42-kDa and 70-kDa subunits . Reduced Mg-chelatase activity is explained by the capacity of heterodimers consisting of mutated 42-kDa and wild-type 70-kDa protein to bind to the 140-kDa subunit . The 42-kDa subunit is similar to chaperones that refold denatured polypeptides with respect to its ATP-to-ADP exchange activity and its ability to generate ATPase activity with the 70-kDa subunit . We hypothesize that the association of the 42-kDa subunit with the 70-kDa subunit allows them to form a specific complex with the 140-kDa subunit and that this complex inserts Mg2+ into protoporphyrin IX.

Cell, 1999 Jan 8, 96(1), 131 - 41
Structure of CheA, a signal-transducing histidine kinase; Bilwes AM et al.; Histidine kinases allow bacteria, plants, and fungi to sense and respond to their environment . The 2.6 A resolution crystal structure of Thermotoga maritima CheA (290-671) histidine kinase reveals a dimer where the functions of dimerization, ATP binding, and regulation are segregated into domains . The kinase domain is unlike Ser/Thr/Tyr kinases but resembles two ATPases, Gyrase B and Hsp90 . Structural analogies within this superfamily suggest that the P1 domain of CheA provides the nucleophilic histidine and activating glutamate for phosphotransfer . The regulatory domain, which binds the homologous receptor-coupling protein CheW, topologically resembles two SH3 domains and provides different protein recognition surfaces at each end . The dimerization domain forms a central four-helix bundle about which the kinase and regulatory domains pivot on conserved hinges to modulate transphosphorylation . Different subunit conformations suggest that relative domain motions link receptor response to kinase activity.

Arch Insect Biochem Physiol, 1999, 40(1), 30 - 40
Cloning and expression of a gene encoding a Campoletis sonorensis polydnavirus structural protein; Deng L et al.; Polydnaviruses are the only known group of mutualistic viruses . They are required for successful parasitization in many braconid and ichneumonid parasitoids . The intimacy of this mutualistic association is indicated by the integration and vertical transmission of polydnaviruses in wasp genomes and by their asymptomatic, developmentally regulated replication . The evolution of this mutualism raises several interesting issues that require a better understanding of the viral genome and viral replication . To develop probes for virus replication and morphogenesis, we have begun to characterize several viral structural proteins . A 699 bp cDNA encoding the p12 viral structural protein was cloned and sequenced . The p12 gene localizes to viral segment Y and encodes a predicted protein of 92 amino acids that does not encode a signal peptide and is unrelated to known peptide or nucleic acid sequences . The p12 mRNA is detected at the onset of virus replication . mRNA titers increase with increasing rates of virus replication . Polyclonal antisera raised against histidine-tagged p12 protein expressed in bacteria reacted specifically with the p12 polypeptide in Western blots of CsPDV virions . The p12 polypeptide was not detected in non-replicative wasp or lepidopteran tissues by Western blot analyses but was readily detected in protein extracts of wasp ovaries . The data indicate that the p12 gene is a viral gene encoding a virion protein and provides a specific probe for virus replication that will be useful for studying the evolution of this group of mutualistic viruses.

Zentralbl Bakteriol, 1998 Dec, 288(4), 457 - 61
Evaluation of three methods for culturing alginate throat swabs from cystic fibrosis patients; Hoppe JE et al.; Deep throat swabs are usually cultured for potential respiratory pathogens in patients with cystic fibrosis (CF) who do not produce sputum . Using alginate swabs, three methods were examined in the present study: (i) semiquantitative processing (direct plating); (ii) mere elution of swabs in Ringer's lactate followed by plating of the eluate; and (iii) complete quantitative processing (elution plus two-step dilution series) . Throat swabs from 56 CF patients yielded 122 isolates of potential respiratory pathogens . Semiquantitative processing (n = 101 isolates) was not significantly inferior to elution (n = 104) and quantitative processing (n = 113; p > 0.05) . Since semiquantitative processing is the simplest method, it is to be preferred, provided that alginate swabs are used.

Mol Microbiol, 1998 Dec, 30(5), 1029 - 40
The type III secretion determinants of the flagellar anti-transcription factor, FlgM, extend from the amino-terminus into the anti-sigma28 domain; Chilcott GS et al.; The flagellar-specific anti-sigma factor, FIgM, inhibits the expression of late flagellar genes until the hook-basal body structure is assembled and competent for export of the flagellins and hook-associated proteins (flagellar late proteins) . FIgM monitors this assembly checkpoint by being a substrate for export via the hook-basal body structure, which includes a type III protein secretion complex . Amino acid sequence alignment of late-secreted flagellar proteins identified a region of homology present in the amino-terminus of FIgM and the other late flagellar proteins, but not in flagellar proteins secreted earlier during flagellar biosynthesis . Single amino acid substitutions at specific positions within this motif decreased the export of FIgM . Deletion of this region (S3-P11) resulted in lower intracellular FIgM levels, but did not prevent recognition and export by the flagellar-specific secretion system . Mutations were isolated in a second region of FIgM spanning residues K27 to A65 that exhibited increased anti-sigma28 activity . These FIgM 'hyperinhibitor' mutants were secreted less than wild-type FIgM . Mutations that interfere with the secretion of FIgM without abolishing anti-sigma28 activity have a negative effect upon the secretion of a His-tagged FIgM mutant that lacks anti-sigma28 activity . Models are proposed to explain the dominant negative phenotype of the FIgM secretion mutants reported in this study.

J Chem Inf Comput Sci, 1999 Jan-Feb, 39(1), 21 - 7
Application of nearest-neighbor and cluster analyses in pharmaceutical lead discovery; Stanton DT et al.; High throughput screening (HTS) programs based on diverse collections of compounds can rapidly identify leads for potential drug candidates . In cases where the compound collection is truly diverse, one may only identify a few compounds of interest . However, where a large number of hits are identified, it becomes necessary to examine the structures to determine the true number of compound classes involved so that follow-up studies may be conducted as efficiently as possible . In this case, cluster analysis is applied to determine the structural relationship among HTS hits . To efficiently expand around the region of the hit (or a class of hits) in chemical space, we have applied nearest neighbors analysis to select additional compounds from collections of a large number of commercial vendors, achieving an average hit rate in excess of 15% . Applying these techniques in a number of different cases, we obtained results that are useful for subsequent investigations of hits from HTS and other relevant molecular structures from the literature.

Acta Cytol, 1999 Jan-Feb, 43(1), 47 - 57
Semiautomated monolayer preparation of bronchial secretions using AutoCyte PREP; Motherby H et al.; OBJECTIVE: Development of a method for semiautomated preparation of purified, representative and conventionally stained monolayer smears from bronchial secretions suitable for subjective and/or automated cytodiagnosis . STUDY DESIGN: Bronchial secretions from 50 patients with and 48 without carcinoma cells of different types were collected in Saccomanno's fixative . After routine pick-and-smear processing, residual material was subjected to a mucolytic agent (ammonium thioglycolate) . Separation of cells was performed by differential centrifugation through aqueous sucrose . The pellet was automatically processed by the AutoCyte PREP system . RESULTS: Slides revealed well-preserved, slightly shrunken, homogeneously distributed cells devoid of mucus, cellular debris and bacteria in monolayer arrangement nearly without overlap . Granulocytes were eliminated to a large extent . Comparison with pick-and-smear specimens showed more tumor cells per square centimeter of slide surface in 100% of AutoCyte PREP slides . The number of tumor cells per AutoCyte PREP slide was higher in 46% and lower in 54% . Selecting slides at random and requiring at least 10 abnormal cells to establish a tumor diagnosis were achieved in 82.7% if only one, in 88.0% if two and 94.0% if seven or eight AutoCyte PREP slides were investigated . CONCLUSION: The semiautomated method yielded conventionally stained, purified monolayer smears from bronchial secretions with cellular morphology suitable for evaluation by cytologists and screening machines . Representativity of AutoCyte PREP monolayers was superior to that of pick-and-smear slides.

J Clin Microbiol, 1999 Mar, 37(3), 835 - 7
Justification for use of a single trichrome stain as the sole means for routine detection of intestinal parasites in concentrated stool specimens; Kellogg JA et al.; Of 12,321 stool samples analyzed over a 6-year interval, 870 (7.1%) were positive for a total of 1,019 parasites, of which 1,011 (99.2%) were found in trichrome-stained smears of unconcentrated specimens while only 479 (47.0%) were detected in iodine-stained smears of concentrated samples . Stool specimens were next analyzed by trichrome staining of both unconcentrated and concentrated specimens preserved in either mercury-polyvinyl alcohol (PVA) or cupric PVA . Of 2,198 specimens, 171 (7.8%) were positive for a total of 208 parasites, 192 (92.3%) and 204 (98.1%) of which were found in the unconcentrated and concentrated specimens, respectively (P < 0.05) . In our patient population, examination of a single trichrome-stained smear of a concentrated stool specimen is a cost-effective alternative to routinely analyzing both concentrated and unconcentrated specimens for parasites.

J Clin Microbiol, 1999 Mar, 37(3), 575 - 80
Rapid detection of the Chlamydiaceae and other families in the order Chlamydiales: three PCR tests; Everett KD et al.; Few identification methods will rapidly or specifically detect all bacteria in the order Chlamydiales, family Chlamydiaceae . In this study, three PCR tests based on sequence data from over 48 chlamydial strains were developed for identification of these bacteria . Two tests exclusively recognized the Chlamydiaceae: a multiplex test targeting the ompA gene and the rRNA intergenic spacer and a TaqMan test targeting the 23S ribosomal DNA . The multiplex test was able to detect as few as 200 inclusion-forming units (IFU), while the TaqMan test could detect 2 IFU . The amplicons produced in these tests ranged from 132 to 320 bp in length . The third test, targeting the 23S rRNA gene, produced a 600-bp amplicon from strains belonging to several families in the order Chlamydiales . Direct sequence analysis of this amplicon has facilitated the identification of new chlamydial strains . These three tests permit ready identification of chlamydiae for diagnostic and epidemiologic study . The specificity of these tests indicates that they might also be used to identify chlamydiae without culture or isolation.

Microb Pathog, 1999 Jan, 26(1), 35 - 43
Frequency of apolipoprotein E (APOE) allele types in patients with Chlamydia-associated arthritis and other arthritides; Gerard HC et al.; Genetic background is important in determining whether certain infecting bacteria disseminate to the joint and cause arthritis . We assessed whether APOE genotype is associated with the presence of DNA from Chlamydia or other bacteria in synovial tissues of patients with various arthritides . Nucleic acids from synovial tissues of 135 patients were screened by PCR for DNA from Chlamydia trachomatis, C . pneumoniae and other bacteria (pan-bacteria) . APOE genotype was determined by a PCR-based method for all patients in each of four resulting groups comprised of about 35 individuals each, positive for C . trachomatis only, C . pneumoniae only, other bacteria, or no bacteria . RT-PCR was used to assess synovial APOE expression . The latter assays confirmed that APOE mRNA is present in synovial tissue . Determination of APOE genotype showed that patients PCR-negative in all assays, and those positive in the C . trachomatis - and pan-bacteria- (excluding Chlamydia) directed assays, had distributions of the APOE epsilon2, epsilon3 and epsilon4 alleles mirroring those of the general population (i.e . about 8%, 79% and 13%, respectively) . In contrast, 68% of patients with C . pneumoniae DNA in synovium possessed a copy of the epsilon4 allele . These results indicate that no association exists between APOE genotype and synovial presence of C . trachomatis or other bacteria . However, individuals bearing at least one copy of the APOE epsilon4 allele may be at increased risk for synovial infection by C . pneumoniae .

J Immunol, 1999 Feb 15, 162(4), 2259 - 65
IFN-gamma-mediated control of Coxiella burnetii survival in monocytes: the role of cell apoptosis and TNF; Dellacasagrande J et al.; The treatment of infectious diseases caused by intracellular bacteria, such as Q fever, may benefit from cytokines acting on macrophages . Monocytic THP-1 cells were infected with Coxiella burnetii, the etiological agent of Q fever, and then treated with IFN-gamma . While C . burnetii multiplied in untreated monocytes, IFN-gamma reduced bacterial viability after 24 h of treatment and reached maximum inhibition after 96 h . IFN-gamma also affected the viability of infected cells . Cell death resulted from apoptosis; occurring 24 h after the addition of IFN-gamma, it reached a maximum after 48 h and was followed by necrosis . Reactive oxygen intermediates were not required for C . burnetii killing, since monocytes from patients with chronic granulomatous disease were microbicidal in response to IFN-gamma . The role of cytokines was also investigated . IFN-gamma elicited a moderate release of IL-1beta in infected monocytes . Moreover, the IL-1 receptor antagonist did not affect C . burnetii survival, suggesting that IL-1beta was not involved in the bacterial killing induced by IFN-gamma . TNF was involved in IFN-gamma-induced killing of C . burnetii and cell death . IFN-gamma induced mRNA expression and sustained secretion of TNF . Neutralizing Abs to TNF as well as Abs directed against TNF receptors I and II, significantly prevented IFN-gamma-dependent killing of C . burnetii and cell death . These results suggest that IFN-gamma promotes the killing of C . burnetii in monocytes through an apoptotic mechanism mediated in part by TNF.

Comp Biochem Physiol C Pharmacol Toxicol Endocrinol, 1998 Nov, 121(1-3), 185 - 95
Occurrence of flavin-containing monooxygenases in non-mammalian eukaryotic organisms; Schlenk D; Flavin-containing monooxygenases (FMOs) have been identified in various organisms from bacteria to humans . However, because of the importance of these enzymes in the biotransformation of xenobiotics, the majority of studies have focused almost entirely upon the mammalian forms of the enzyme . Consequently, this review is an attempt to document the occurrence of FMO expression (mRNA, proteins, activities) in non-mammalian species in an attempt to provide insight about its putative physiological and toxicological roles . Activity indicative of FMO has been observed in numerous invertebrate species but corresponding proteins or transcripts have not been identified . There is a significant gap of information pertaining to insects, echinoderms, avian, reptilian and amphibian species . Significant homology of structure and function is observed in lower vertebrates . Evidence is provided primarily from studies with piscine forms of the enzyme suggesting a possible osmoregulatory role of FMOs, especially in euryhaline species of fish.

J Virol, 1999 Mar, 73(3), 2425 - 33
Mutant influenza viruses with a defective NS1 protein cannot block the activation of PKR in infected cells; Hatada E et al.; A short model genome RNA and also the genome RNA of influenza A virus bearing both 5'- and 3'-terminal common sequences activated the interferon-induced double-stranded-RNA-dependent protein kinase, PKR, by stimulating autophosphorylation in vitro . The activated PKR catalyzed phosphorylation of the alpha subunit of eucaryotic translation initiation factor 2 (eIF2alpha) . The NS1 protein efficiently eliminated the PKR-activating activity of these RNAs by binding to them . Two mutant NS1 proteins, each harboring a single amino acid substitution at different regions, exhibited temperature sensitivity in their RNA binding activity in the mutant virus-infected cell lysates as well as when they were prepared as fusion proteins expressed in bacteria . The virus strains carrying these mutant NS1 proteins exhibited temperature sensitivity in virus protein synthesis at the translational level, as reported previously, and could not repress the autophosphorylation of PKR developing during the virus growth, which is normally suppressed by a viral function(s) . As a result, the level of eIF2alpha phosphorylation was elevated 2.5- to 3-fold . The defect in virus protein synthesis was well correlated with the level of phosphorylation of PKR and eIF2alpha.

Equine Vet J, 1999 Jan, 31(1), 61 - 7
Differences in second-intention wound healing between horses and ponies: histological aspects; Wilmink JM et al.; The histological aspects of second-intention healing were studied in 5 horses and 5 ponies . Biopsies were taken weekly from standardised wounds on the metatarsus and femoral biceps muscle of one horse and one pony . Sections were stained to enable cell counting and the detection of DNA synthesis, fibrin, smooth muscle actin (SMA), collagen, and bacteria . In the ponies, the number of polymorphonuclear leucocytes (PMNs) was high during the first 3 weeks and subsequently decreased rapidly . In the horses, the initial number of PMNs was lower, but remained persistently elevated during the evaluation period . PMNs were found mainly in the superficial zones . Significantly more fibrin was present in the wounds of the horses . No significant differences were observed in the number of fibroblasts, the amounts of SMA and collagen . However, myofibroblasts were significantly less regularly organised in the wounds of the horses, particularly in the metatarsal wounds . The mitotic activity of the epithelium was temporally reduced in week 3 . The mitotic activity of the granulation tissue was initially high but declined rapidly from week 1 onwards, with the exception of the metatarsal wounds of the horses, in which mitotic activity remained significantly higher . Histology confirmed and explained the macroscopical differences in wound healing between horses and ponies by the strict organisation of the myofibroblasts and the more effective acute inflammation in the ponies . Stimulation of the organisation of myofibroblasts and improvement of the efficacy of the inflammatory response in horses may therefore result in better second-intention wound healing in horses in clinical practice.

Dig Dis Sci, 1999 Jan, 44(1), 116 - 24
Reactions from rat gastric mucosa during one year of Helicobacter pylori infection; Li H et al.; The aim of the present study was to investigate responses from the gastric mucosa of rats during long-term H . pylori infection . Twenty-four Sprague-Dawley rats were inoculated with a mouse-adapted strain of human H . pylori (vacA+, cagA+), 16 uninfected rats served as controls . Three to six rats from each group were killed two weeks or two, six, or 12 months later . At sacrifice, blood was sampled and the gastric mucosa was taken for bacterial culture, histology, immunocytochemistry and in situ hybridization . H . pylori colonized the antrum in 23/24 inoculated rats; with time the density of bacteria increased . The inflammation in the antral mucosa was mild to moderate and was dominated by infiltration of lymphocytes and macrophages . Serum H . pylori-specific IgG2a was significantly increased in the infected rats . The frequency of epithelial cell apoptosis was significantly increased in the early months of infection . The mucosal expression of trefoil peptide mRNA remained unchanged . We conclude that after one year of H . pylori infection in rats, the mucosal responses were rather mild, indicating that the animals may adapt to the infection by mechanisms which remain to be identified.

Hepatogastroenterology, 1998 Nov-Dec, 45(24), 2219 - 23
The importance of increasing the number of gastric biopsies in the diagnosis of Helicobacter pylori; Gur G et al.; BACKGROUND/AIMS: The role of Helicobacter pylori in various gastroduodenal diseases is universally accepted . In this study, we aimed to determine the proper number and sites of the gastric biopsies in order to achieve the highest diagnostic yield through the use of a urease test and histopathology . We also compared the histological findings encountered in patients who had Helicobacter pylori (H . pylori) colonization . METHODOLOGY: Fifty patients referred for upper gastrointestinal endoscopy for dyspeptic complaints were included in the study . Our mapping protocol included 2 biopsies from antrum and 2 biopsies from corpus . We obtained 2 biopsies from each biopsy site for urease test and histopathological assessment . Golden standard positivity for the presence of H . pylori colonization was defined as concomitantly positive urease test and histologically detected bacteria found at the same biopsy site . RESULTS: Forty-three patients had H . pylori colonization . Colonization rates of H . pylori, sensitivities of urease testing, and histopathology in 4 biopsy sites were not statistically different . Sensitivity of urease testing was 81.4% for 1 biopsy and 100% for 4 cumulative biopsies . Sensitivities of histological assessment were 93% and 100% for 1 and 4 biopsies, respectively . CONCLUSIONS: Results of this study suggest that 2 biopsies for urease testing and 1 biopsy for histopathology obtained from the antrum or corpus of the stomach were sufficient to obtain the highest statistically significant diagnostic sensitivity.

Planta, 1999 Jan, 207(3), 325 - 34
A unique reaction in a common pathway: mechanism and function of chorismate synthase in the shikimate pathway; Macheroux P et al.; Chorismate synthase, the seventh enzyme in the shikimate pathway, catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate to chorismate which is the last common precursor in the biosynthesis of numerous aromatic compounds in bacteria, fungi and plants . The enzyme has an absolute requirement for reduced FMN as a cofactor, although the 1,4-anti elimination of phosphate and the C(6proR)-hydrogen does not involve a net redox change . The role of the reduced FMN in catalysis has long been elusive . However, recent detailed kinetic and bioorganic approaches have fundamentally advanced our understanding of the mechanism of action, suggesting an initial electron transfer from tightly bound reduced flavin to the substrate, a process which results in C-O bond cleavage . Studies on chorismate synthases from bacteria, fungi and plants revealed that in these organisms the reduced FMN cofactor is made available in different ways to chorismate synthase: chorismate synthases in fungi--in contrast to those in bacteria and plants--carry a second enzymatic activity which enables them to reduce FMN at the expense of NADPH . Yet, as shown by the analysis of the corresponding genes, all chorismate synthases are derived from a common ancestor . However, several issues revolving around the origin of reduced FMN, as well as the possible regulation of the enzyme activity by means of the availability of reduced FMN, remain poorly understood . This review summarizes recent developments in the biochemical and genetic arena and identifies future aims in this field.

Exp Gerontol, 1998 Nov-Dec, 33(7-8), 813 - 26
The nitric oxide hypothesis of aging; McCann SM et al.; Nitric oxide (NO), generated by endothelial (e) NO synthase (NOS) and neuronal (n) NOS, plays a ubiquitous role in the body in controlling the function of almost every, if not every, organ system . Bacterial and viral products, such as bacterial lipopolysaccharide (LPS), induce inducible (i) NOS synthesis that produces massive amounts of NO toxic to the invading viruses and bacteria, but also host cells by inactivation of enzymes leading to cell death . The actions of all forms of NOS are mediated not only by the free radical oxidant properties of this soluble gas, but also by its activation of guanylate cyclase (GC), leading to the production of cyclic guanosine monophosphate (cGMP) that mediates many of its physiological actions . In addition, NO activates cyclooxygenase and lipoxygenase, leading to the production of physiologically relevant quantities of prostaglandin E2 (PGE2) and leukotrienes . In the case of iNOS, the massive release of NO, PGE2, and leukotrienes produces toxic effects . Systemic injection of LPS causes induction of interleukin (IL)-1 beta mRNA followed by IL-beta synthesis that induces iNOS mRNA with a latency of two and four hours, respectively, in the anterior pituitary and pineal glands, meninges, and choroid plexus, regions outside the blood-brain barrier, and shortly thereafter, in hypothalamic regions, such as the temperature-regulating centers, paraventricular nucleus containing releasing and inhibiting hormone neurons, and the arcuate nucleus, a region containing these neurons and axons bound for the median eminence . We are currently determining if LPS similarly activates cytokine and iNOS production in the cardiovascular system and the gonads . Our hypothesis is that recurrent infections over the life span play a significant role in producing aging changes in all systems outside the blood-brain barrier via release of toxic quantities of NO . NO may be a major factor in the development of coronary heart disease (CHD) . Considerable evidence has accrued indicating a role for infections in the induction of CHD and, indeed, patients treated with a tetracycline derivative had 10 times less complications of CHD than their controls . Stress, inflammation, and infection have all been shown to cause induction of iNOS in rats, and it is likely that this triad of events is very important in progression of coronary arteriosclerosis leading to coronary occlusion . Aging of the anterior pituitary and pineal with resultant decreased secretion of pituitary hormones and the pineal hormone, melatonin, respectively, may be caused by NO . The induction of iNOS in the temperature-regulating centers by infections may cause the decreased febrile response in the aged by loss of thermosensitive neurons . iNOS induction in the paraventricular nucleus may cause the decreased nocturnal secretion of growth hormone (GH) and prolactin that occurs with age, and its induction in the arcuate nucleus may destroy luteinizing hormone-releasing hormone (LHRH) neurons, thereby leading to decreased release of gonadotropins . Recurrent infections may play a role in aging of other parts of the brain, because there are increased numbers of astrocytes expressing IL-1 beta throughout the brain in aged patients . IL-1 and products of NO activity accumulate around the plaques of Alzheimer's, and may play a role in the progression of the disease . Early onset Parkinsonism following flu encephalitis during World War I was possibly due to induction of iNOS in cells adjacent to substantia nigra dopaminergic neurons leading to death of these cells, which, coupled with ordinary aging fall out, led to Parkinsonism . The central nervous system (CNS) pathology in AIDS patients bears striking resemblance to aging changes, and may also be largely caused by the action of iNOS . Antioxidants, such as melatonin, vitamin C, and vitamin E, probably play an important acute and chronic role in reducing or eliminating the oxidant damage produced by NO.

Am J Physiol, 1999 Feb, 276(2 Pt 1), G479 - 84
Role of endotoxin in intestinal reperfusion-induced expression of E-selectin; Bauer P et al.; Products of enteric bacteria, including endotoxin {lipopolysaccharide (LPS)}, have been implicated in the acute inflammatory responses elicited by ischemia and reperfusion (I/R) of the small intestine . The objective of this study was to assess the contribution of LPS to the increased E-selectin expression observed in the intestinal vasculature after I/R . The dual radiolabeled monoclonal antibody technique was used in LPS-sensitive (C3HeB/FeJ) and LPS-insensitive (C3H/HeJ) mice that were exposed to either exogenous LPS or to gut I/R (45 min ischemia, 5 h reperfusion) . LPS elicited a dose-dependent (0.5-50 microgram LPS/animal) increase in E-selectin expression (at 3 h) in LPS-sensitive mice, whereas LPS-insensitive mice were largely unresponsive . E-selectin expression was increased fivefold by I/R in the small bowel of both LPS-sensitive and -insensitive mice . These results indicate that, although exogenous LPS is capable of eliciting profound dose-dependent increases in E-selectin expression, endogenous LPS does not contribute significantly to I/R-induced expression of this endothelial cell adhesion molecule.

Am J Physiol, 1999 Feb, 276(2 Pt 1), C291 - 9
Preservation of murine embryos in a state of dormancy at 4 degreesC; Wiggins PM et al.; With the aim of improving preservation of blood products and organs for transplantation, we designed solutions to induce a state of dormancy in cells and tissues at 4 degreesC . The solutions were devoid of combinations of ions (e.g., K+, Rb+, Cs+, and NH+4 with HCO-3, H2PO-4, and Cl-) that are believed to break down low-density water in the entrance compartments of ion channels, resulting in cyclical open states (normal water) and closed states (low-density water) . The total osmolality was always 0.29-0.3 osmol/kgH2O, made up of combinations of a di- or trisaccharide, a compatible solute, sodium sulfate, citrate, or chloride, and 1.75 mM CaCl2 . The end point was the ability of murine embryos to progress to hatching in culture after preservation in such a solution at 4 degreesC . Embryos hatched after 5 or 6 days in some preservative solutions compared with 1-3 days in most saline solutions; survival was improved by pretreatment with sodium butyrate.

Mol Biol Cell, 1999 Feb, 10(2), 393 - 406
The small Mr Ras-like GTPase Rap1 and the phospholipase C pathway act to regulate phagocytosis in Dictyostelium discoideum; Seastone DJ et al.; The function of the small-Mr Ras-like GTPase Rap1 remains largely unknown, but this protein has been demonstrated to regulate cortical actin-based morphologic changes in Dictyostelium and the oxidative burst in mammalian neutrophils . To test whether Rap1 regulates phagocytosis, we biochemically analyzed cell lines that conditionally and modestly overexpressed wild-type {Rap1 WT(+)}, constitutively active {Rap1 G12T(+)}, and dominant negative {Rap1 S17N(+)} forms of D . discoideum Rap1 . The rates of phagocytosis of bacteria and latex beads were significantly higher in Rap1 WT(+) and Rap1 G12T(+) cells and were reduced in Rap1 S17N(+) cells . The addition of inhibitors of protein kinase A, protein kinase G, protein tyrosine kinase, or phosphatidylinositide 3-kinase did not affect phagocytosis rates in wild-type cells . In contrast, the addition of U73122 (a phospholipase C inhibitor), calphostin C (a protein kinase C inhibitor), and BAPTA-AM (an intracellular Ca2+ chelator) reduced phagocytosis rates by 90, 50, and 65%, respectively, suggesting both arms of the phospholipase C signaling pathways played a role in this process . Other protein kinase C-specific inhibitors, such as chelerythrine and bisindolylmaleimide I, did not reduce phagocytosis rates in control cells, suggesting calphostin C was affecting phagocytosis by interfering with a protein containing a diacylglycerol-binding domain . The addition of calphostin C did not reduce phagocytosis rates in Rap1 G12T(+) cells, suggesting that the putative diacylglycerol-binding protein acted upstream in a signaling pathway with Rap1 . Surprisingly, macropinocytosis was significantly reduced in Rap1 WT(+) and Rap1 G12T(+) cells compared with control cells . Together our results suggest that Rap1 and Ca2+ may act together to coordinate important early events regulating phagocytosis.

Mod Pathol, 1999 Jan, 12(1), 82 - 7
Congenital syphilis in a newborn: an immunopathologic study; Guarner J et al.; A 3-week-old girl presented to the emergency room with respiratory distress and generalized maculopapular rash . The newborn was hospitalized with a presumptive diagnosis of congenital syphilis, but she died after 2 days of therapy . Tissue from the gastrointestinal tract, brain, liver, spleen, and lung was studied by using direct fluorescent antibody and immunohistochemical analysis (IHC) for Treponema pallidum . The inflammatory infiltrate was characterized by using IHC against CD3, CD20, CD68, and smooth muscle actin . The diagnosis of congenital syphilis was confirmed by demonstrating spirochetes in tissues with IHC and direct fluorescent antibody examination . IHC showed abundant treponemes in the small intestine and liver and occasional spirochetes in the meninges . Bacteria were seen as intact spirochetes, granular staining, or large extracellular collections of antigen . A constant pathologic feature throughout the tissues was concentric macrophage (CD68-positive) infiltrate around vessels, giving an onion-skin appearance . IHC identified the macrophages as the prime immune response in congenital syphilis.

Novartis Found Symp, 1998, 217, 160 - 72; discussion 172-7
Analysis of the genome of Mycobacterium tuberculosis H37Rv; Cole ST et al.; The powerful combination of genomics and bioinformatics is providing a wealth of information about Mycobacterium tuberculosis, the aetiological agent of human tuberculosis, that will facilitate the conception and development of new therapies . The starting point for genome sequencing was the integrated map of the 4.4 Mb circular chromosome of the widely used, virulent reference strain, M . tuberculosis H37Rv . Cosmids and bacterial artificial chromosomes were selected from ordered libraries and subjected to systematic shotgun sequence analysis . This approach simplified sequence assembly as the genome is rich in repetitive DNA . In common with most bacteria, > 90% of the potential coding capacity is used, and probable or tentative functions could be attributed to > 70% of the genes . The potential biological roles of two of the principal driving forces in genome dynamics, insertion sequence elements and polymorphic multigene families are discussed.

Am J Gastroenterol, 1999 Jan, 94(1), 252 - 6
Fatal invasive gastric mucormycosis occurring with emphysematous gastritis: case report and literature review; Cherney CL et al.; Emphysematous gastritis is an often lethal, rare clinical entity referring to air bubbles in the wall of the stomach produced by gas-forming bacteria . Invasive gastrointestinal mucormycosis is an unusual clinical presentation of this invasive fungal disease . We report the first case of invasive gastric mucormycosis occurring with emphysematous gastritis, and review the literature regarding both of these clinical entities.

Adv Biochem Eng Biotechnol, 1999, 64, 101 - 54
Bioactive agents from natural sources: trends in discovery and application; Grabley S et al.; About 30% of the worldwide sales of drugs are based on natural products . Though recombinant proteins and peptides account for increasing sales rates, the superiority of low-molecular mass compounds in human diseases therapy remains undisputed mainly due to more favorable compliance and bioavailability properties . In the past, new therapeutic approaches often derived from natural products . Numerous examples from medicine impressively demonstrate the innovative potential of natural compounds and their impact on progress in drug discovery and development . However, natural products are currently undergoing a phase of reduced attention in drug discovery because of the enormous effort which is necessary to isolate the active principles and to elucidate their structures . To meet the demand of several hundred thousands of test samples that have to be submitted to high-throughput screening (HTS) new strategies in natural product chemistry are necessary in order to compete successfully with combinatorial chemistry . Today, pharmaceutical companies have to spend approximately US $350 million to develop a new drug . Currently, approaches to improve and accelerate the joint drug discovery and development process are expected to arise mainly from innovation in drug target elucidation and lead finding . Breakthroughs in molecular biology, cell biology, and genetic engineering in the 1980 s gave access to understanding diseases on the molecular or on the gene level . Subsequently, constructing novel target directed screening assay systems of promising therapeutic significance, automation, and miniaturization resulted in HTS approaches changing the industrial drug discovery process drastically . Furthermore, elucidation of the human genome will provide access to a dramatically increased number of new potential drug targets that have to be evaluated for drug discovery . HTS enables the testing of an increasing number of samples . Therefore, new concepts to generate large compound collections with improved structural diversity are desirablePublication Types:

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