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J Natl Cancer Inst, 1997 Jun 18, 89(12), 848 - 56 Carcinogenicity of the drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone in the rat; Komulainen H et al.; BACKGROUND: Several epidemiologic studies have suggested that the consumption of chlorinated drinking water may be associated with the development of certain cancers in humans . 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a byproduct of the chemical reactions that occur in chlorinated drinking water, has been found to be mutagenic in bacteria and mammalian cells; however, its potential to cause tumors in animals has not been tested previously . PURPOSE: The objective of this study was to evaluate the carcinogenicity of MX in rats given MX in their drinking water . METHODS: MX was administered to male and female Wistar rats (50 rats per dose group) in drinking water for 104 weeks at concentrations yielding the average daily doses of MX of 0.4 mg/kg of animal weight (low dose), 1.3 mg/kg (mid dose), and 5.0 mg/kg (high dose) for males and 0.6 mg/kg, 1.9 mg/kg, and 6.6 mg/kg for females, respectively . Control rats received water from the same source used for preparation of the MX dose formulations (after its adjustment to the same pH range) . Body weight, clinical signs, and food and water consumption were recorded regularly . At the end of the treatment period, the animals were killed and full histopathologic analysis was performed on 47 tissues and all lesions . RESULTS: Dose-dependent increases in tumor incidence were observed in rats given MX-containing drinking water; the same MX doses had no obvious toxic effects on animals . MX consumption increased most drastically the prevalence of follicular adenoma (up to 43% and 72% in high-dose males and females, a test {one-sided} for positive trend in all dose groups P = .0045 and P = .0000, respectively) and carcinoma (55% {P = .0000} and 44% {P = .0000}, respectively) in thyroid glands and cholangioma in the liver (8% {P = .0009} and 66% {P = .0000} in the high-dose males and females, respectively) . Among rats given the higher doses of MX in their drinking water, cortical adenomas of the adrenal glands were increased in both sexes, alveolar and bronchiolar adenomas of the lungs and Langerhans' cell adenomas of the pancreas were increased in males, and lymphomas, leukemias, and adenocarcinomas and fibroadenomas of the mammary glands were increased in females . Even the lowest MX dose studied was carcinogenic . CONCLUSION: MX is a potent carcinogen in both male and female rats, and it causes tumors at doses that are not overtly toxic to rats . IMPLICATIONS: Although these findings cannot be extrapolated to humans, MX should be studied as a candidate risk factor in the possible association between consumption of chlorinated drinking water and cancer in humans. FEBS Lett, 1997 Jun 16, 409(3), 421 - 5 Redox chemistry of cobalamin and iron-sulfur cofactors in the tetrachloroethene reductase of Dehalobacter restrictus; Schumacher W et al.; Respiration of Dehalobacter restrictus is based on reductive dechlorination of tetrachloroethene . The terminal component of the respiratory chain is the membrane-bound tetrachloroethene reductase . The metal prosthetic groups of the purified enzyme have been studied by optical and EPR spectroscopy . The 60-kDa monomer contains one cobalamin with Em(Co{1+/2+}) = -350 mV and Em(Co{2+/3+}) > 150 mV and two electron-transferring {4Fe-4S}(2+;1+) clusters with rather low redox potentials of Em approximately -480 mV . The cob(II)alamin is present in the base-off configuration . A completely reduced enzyme sample reacted very rapidly with tetrachloroethene yielding base-off cob(II)alamin rather than trichlorovinyl-cob(III)alamin. Structure, 1997 Jun 15, 5(6), 735 - 9 Out of the blue: the photocycle of the photoactive yellow protein; Schlichting I et al.; A first real glance at the structural, spectral and temporal interplay that constitutes the photocycle of the photoactive yellow protein (PYP) has been obtained from a combination of time-resolved crystallography with mutational analysis and spectroscopic studies. J Immunol, 1997 Jun 15, 158(12), 5868 - 73 Four conserved promoter motifs regulate transcription of the gene encoding human complement component C2; Sullivan KE; The early complement components of the classical activation pathway of the complement cascade include the components C1, C4, C2, and C3 . These components act in concert to opsonize bacteria, clear immune complexes, and produce inflammatory mediators . They are not structurally homologous nor are they coordinately regulated . The expression of the early complement components is divergent in terms of cytokine responsiveness and tissue specificity . The only pattern of expression shared by the early complement components is inducibility by gamma-IFN and expression in cells of hepatic or monocytic lineage . Nevertheless, four novel conserved promoter motifs were identified in the 5' flanking region of multiple early complement component promoters . Mutation of these four motifs in the C2 promoter decreased transcription in hepatoma cells in transient transfection analyses, and a synthetic promoter consisting of just the four motifs supported transcription in hepatoma cells . Electrophoretic mobility shift assays demonstrate that three of the four conserved elements bind DNA-binding proteins in a tissue-specific manner . One DNA-binding protein is expressed ubiquitously, but the other three are restricted to cells of monocytic or hepatic lineage . Two of the DNA-binding proteins appear to be members of the zinc-finger family of transcription factors . Therefore, these four motifs appear to bind DNA-binding proteins that may function in the tissue-specific expression of C2 . Conservation of these four motifs in multiple early complement component genes suggests that these may represent a conserved transcriptional strategy. J Am Vet Med Assoc, 1997 Jun 15, 210(12), 1784 - 7 Ocular diseases of llamas: 194 cases (1980-1993) Gionfriddo JR, Gionfriddo JP, Krohne SG. OBJECTIVE: To identify ocular and adnexal diseases to which llamas in North America are susceptible, to determine prevalence of these diseases in llamas, and to compare prevalences of the major ocular diseases of llamas, cattle, and horses . DESIGN: Retrospective study . ANIMALS: 194 llamas, 4,937 cows, and 11,950 horses with ocular disease . PROCEDURE: Medical records of all llamas entered into the Veterinary Medical Database between 1980 and 1993 were reviewed . Data on ocular structures affected and types of ocular disease were compiled . Prevalences of uveitis, corneal ulcers, and ocular squamous cell carcinoma in llamas were compared with prevalences in cattle and horses . RESULTS: 194 of 3,243 (6%) llamas had at least 1 ocular disease . The proportion of llamas that had ocular disease was significantly higher than the proportions of cattle or horses . The most frequently affected ocular structure in llamas was the cornea, and ulcerative keratitis was the most common corneal disease . The second most commonly affected structure was the uveal tract . Cataracts were reported in 20 (10%) of the llamas with ocular problems . Eyelid disorders, retinal diseases, glaucoma, and ocular or adnexal neoplasia were reported infrequently in llamas . CLINICAL IMPLICATIONS: Results suggest that corneal disease is common in llamas and is usually secondary to trauma . Uveitis may also be common in llamas, but llamas do not appear to be highly susceptible to glaucoma, ocular neoplasia, or to direct corneal invasion by bacteria such as Moraxella sp. J Mol Biol, 1997 Jun 13, 269(3), 440 - 55 Cytochrome cd1 structure: unusual haem environments in a nitrite reductase and analysis of factors contributing to beta-propeller folds; Baker SC et al.; The central tunnel of the eight-bladed beta-propeller domain of cytochrome cd1 (nitrite reductase) is seen, from a 1.28 A resolution structure, to contain hydrogen donors and acceptors that are satisfied by interaction either with water or the d1 haem . The d1 haem, although bound by an extensive network of hydrogen bonds, is not distorted in its binding pocket and is confirmed to have exactly the dioxoisobacteriochlorin structure proposed from chemical studies . A biological rationale is advanced for the undistorted structure of the d1 haem and the large number of hydrogen bonds it makes . The beta-propeller domain can be closely superimposed on that of methanol dehydrogenase despite the enzymes sharing no common sequence motifs and using a different set of interactions to "Velcro" close the propeller . The sequence and likely structural relationships between cytochrome cd1 or methanol dehydrogenase and other predicted eight-bladed beta-propeller domains in proteins, such as the pyrolloquinoline quinone-dependent alcohol dehydrogenase, are discussed and compared with other propeller proteins . From sequencing the nirS gene of Thiosphaera pantotropha, it is established that the amino acid sequence deduced previously in part from X-ray diffraction data at lower resolution was largely correct, as was the proposal that eight N-terminal amino acid residues were not seen in the structure . The unusual haem iron environments in both the c-type cytochrome domain, with His/His coordination, and the d1-type cytochrome domain with Tyr/His coordination are related to the functions of the redox centres. J Biol Chem, 1997 Jun 13, 272(24), 15521 - 6 In vitro recognition of specific DNA targets by AlcR, a zinc binuclear cluster activator different from the other proteins of this class; Lenouvel F et al.; AlcR is the transactivator mediating transcriptional induction of the alc gene cluster in Aspergillus nidulans . The AlcR DNA-binding domain consists of a zinc binuclear cluster different from the other members of the Zn2Cys6 family by several features . In particular, it is able to bind to symmetric and asymmetric sites with the same affinity, with both sites being functional in A . nidulans . Here, we show that unlike the other proteins of the Zn2Cys6 binuclear cluster family, AlcR binds most probably as a monomer to its cognate targets . Two molecules of the AlcR protein can simultaneously bind in a noncooperative manner to inverted repeats . The consensus core has been determined precisely (5'-CCGCN-3'), and the AlcR-binding site in the aldA promoter has been localized . The sequence downstream of the zinc cluster is necessary for high affinity binding . Furthermore, our data show that the use of the carrier protein glutathione S-transferase in AlcR binding experiments introduces an important bias in the recognition of DNA sites due to its tertiary dimeric structure. J Biol Chem, 1997 Jun 13, 272(24), 15452 - 8 Effects of mutating specific residues present near the amino terminus of 2'-5'-oligoadenylate synthetase; Ghosh A et al.; In this study, we investigated the role of specific amino acid residues present near the amino terminus of the 9-2 isozyme of 2'-5'-oligoadenylate synthetase . In vitro expression of deletion mutants showed that residues 1-9 are required for enzyme activity . Within this region, residues 3, 7, and 8 were found to be conserved among all known isozymes of 2'-5'-oligoadenylate synthetase . Mutation of these residues singly or in combination resulted in partial or total loss of enzyme activity . Substitution of the proline residue at position 7 by different residues caused a partial or complete loss of activity . The properties of the inactive P7Q mutant were further explored by expressing the protein in bacteria . The bacterially expressed protein was also enzymatically inactive . The mutant protein could bind the substrate ATP and the activator double-stranded RNA normally . Oligomerization properties of the protein were examined by an affinity-based interaction assay and by glycerol gradient centrifugation; there was no detectable difference between the wild type and the P7Q mutant . These results demonstrated the importance of the proline residue at position 7 in conferring enzyme activity to the protein without affecting its other properties. J Biol Chem, 1997 Jun 13, 272(24), 15167 - 73 MST/MLK2, a member of the mixed lineage kinase family, directly phosphorylates and activates SEK1, an activator of c-Jun N-terminal kinase/stress-activated protein kinase; Hirai S et al.; c-Jun N-terminal kinases/stress-activated protein kinases (JNKs/SAPKs) are mitogen-activated protein kinase (MAPK)-related protein kinases that are involved in several cellular events, including growth, differentiation, and apoptosis . Mixed lineage kinases (MLKs) form a family of protein kinases sharing two leucine zipper-like motifs and a kinase domain whose primary structure is similar to both the tyrosine-specific and the serine/threonine-specific kinase classes . We have reported that a member of the MLK family, MUK/DLK/ZPK, can activate JNK/SAPK in vivo, and here we show that another member of the MLK family, MST/MLK2, activates JNK/SAPK . Both MUK/DLK/ZPK and MST/MLK2 cause a slight activation of p38/Mpk2 when overexpressed in COS-1 cells, whereas MST/MLK2, but not MUK/DLK/ZPK, activates extracellular response kinase (ERK) to a certain degree . The activity of SEK1/MKK4/JNKK, a MAPK kinase class protein kinase designated as a direct activator of JNK/SAPK, is also induced by MUK/DLK/ZPK or MST/MLK2 overexpression . Furthermore, recombinant MST/MLK2 produced in bacteria directly phosphorylates and activates SEK1/MKK4/JNKK in vitro, showing that MST/MLK2 acts like a MAPK kinase kinase . Taken together, these results suggest that MLK family members are MAPK kinase kinases preferentially acting on the JNK/SAPK pathway. J Biol Chem, 1997 Jun 13, 272(24), 15128 - 34 A single mutation in the heme 4 environment of Desulfovibrio desulfuricans Norway cytochrome c3 (Mr 26,000) greatly affects the molecule reactivity; Aubert C et al.; The gene encoding Desulfovibrio desulfuricans Norway cytochrome c3 (Mr 26,000), a dimeric octaheme cytochrome belonging to the polyheme cytochrome c3 superfamily, has been cloned and successfully expressed in another sulfate reducing bacteria, D . desulfuricans G201 . The gene, named cycD, is monocistronic and encodes a cytochrome precursor of 135 amino acids with an extension at the NH2 terminus of 24 amino acids . This extension acts as a signal sequence which allows export across the cytoplasmic membrane into the periplasmic space . Tyrosine 73, which is in a close contact with the histidine sixth axial ligand to the heme 4 iron atom, has been replaced by a glutamate residue using site-directed mutagenesis . The cytochrome mutant when expressed in D . desulfuricans G201, is correctly folded and matured . A global increase of the oxidoreduction potentials of about 50 mV is measured for the Y73E cytochrome . The mutation also has a strong influence on the interaction of the cytochrome with its redox partner, the hydrogenase . This suggests, like the tetraheme cytochrome c3 (Mr 13, 000), heme 4 is the interactive heme in the cytochrome-hydrogenase complex and that alteration of the heme 4 environment can greatly affect the electron transfer reaction with its redox partner. Science, 1997 Jun 13, 276(5319), 1684 - 7 Differential effects of cytolytic T cell subsets on intracellular infection; Stenger S et al.; In analyzing mechanisms of protection against intracellular infections, a series of human CD1-restricted T cell lines of two distinct phenotypes were derived . Both CD4(-)CD8(-) (double-negative) T cells and CD8(+) T cells efficiently lysed macrophages infected with Mycobacterium tuberculosis . The cytotoxicity of CD4(-)CD8(-) T cells was mediated by Fas-FasL interaction and had no effect on the viability of the mycobacteria . The CD8(+) T cells lysed infected macrophages by a Fas-independent, granule-dependent mechanism that resulted in killing of bacteria . These data indicate that two phenotypically distinct subsets of human cytolytic T lymphocytes use different mechanisms to kill infected cells and contribute in different ways to host defense against intracellular infection. Hum Gene Ther, 1997 Jun 10, 8(9), 1087 - 93 Alveolar macrophages inhibit retrovirus-mediated gene transfer to airway epithelia; McCray PB Jr et al.; Gene transfer with integrating vectors such as recombinant retrovirus has the potential to correct inherited lung diseases permanently . As a gene therapy target, the pulmonary epithelium presents several challenges to vector delivery in vivo . Many of the host defenses that have evolved to prevent infection from inhaled bacteria or viruses represent potential barriers to gene transfer to the lung . We performed in vitro studies to determine whether two components of the innate immune system of the lung, airway surface fluid and alveolar macrophages, inhibit retroviral gene transfer to airway epithelia . Human alveolar macrophages obtained by bronchoalveolar lavage from normal subjects were left untreated or activated with lipopolysaccharide (LPS) for 3 hr in the presence of subconfluent human bronchial epithelial cells (HBE); than 4 x 10(5) cfu DA-luciferase retrovirus was added . Three days after infection, luciferase activity was measured in cell lysates . When the epithelial cells were co-cultured with LPS-activated macrophages, retroviral gene transfer to HBE cells was reduced by approximately 60% . Nonactivated macrophages decreased the transfection to approximately 55% of control values . In control experiments with either activated or inactivated macrophages but without epithelia, no luciferase activity was detected, suggesting that terminally differentiated alveolar macrophages are not infected by the recombinant retrovirus . Pretreatment of alveolar macrophages with dexamethasone restored gene transfer to approximately 60% of control values . In contrast, incubation of retrovirus with airway surface fluid had no inhibitory effect on gene transfer . These experiments document that AM inhibit retrovirus-mediated gene transfer to airway epithelia in vitro, and may represent a barrier to retroviral gene transfer in vivo . These barriers may be overcome, at least partially, with pharmacological agents. Med Device Technol, 1997 Jul-Aug, 8(6), 6 - 9 The "sterile" debate: the effects of radiation sterilization on polymers; Williams D; All medical devices have to be sterilized, but the process of killing bacteria and other organisms can also have some deleterious effects on the devices themselves . This article discusses the mechanisms by which gamma irradiation can cause polymer damage and the potential consequences of this. Cytokines Cell Mol Ther, 1997 Jun, 3(2), 127 - 35 DNA for genetic vaccination and therapy; Moelling K; DNA coding for an antigen can be directly injected into muscle or skin and stimulate an immune response against the expressed antigen . The genes expressed can be derived from pathogens (e.g . viruses or bacteria), and can either code for surface molecules, which are often the basis for conventional peptide vaccines, or from the more genetically stable internal proteins . The DNA mimics a real infection in that the antigens are produced intracellularly where they are correctly folded and where they can be presented to the immune system so that cytotoxic T cells are stimulated as a defense mechanism . The DNA is expressed at low, but long-lasting, levels which is presumably the mechanism of its efficacy . Details of the mode of action and improvements for efficacy need to be worked out . Preclinical animal studies looked very promising, but need to be verified in humans . The method is safe and simple; DNA can be easily produced and transported, and can be composed of various genes . Recently also tumor-associated antigens have been tested in preclinical animal models, for example against colon cancer and malignant melanoma . Combinations with immune modulators are being worked out for improved efficacy . Successful therapies with this kind of gene medicine would be much cheaper and therefore superior to viral vectors . However, improvements are still required to prove that hopes are justified. Genes Cells, 1997 Jun, 2(6), 359 - 67 Protein splicing: its chemistry and biology; Anraku Y; Protein splicing is a chemical reaction in which a spliced intervening polypeptide is excised from a precursor protein and the flanking N- and C-terminal regions are ligated with the peptide bond to produce two mature proteins . This unique autocatalytic reaction was first discovered in the yeast VMA1 protein, a 120kDa spliced polypeptide encoded by the VMA1 gene of Saccharomyces cerevisiae . The VMA1 protein catalyses a self protein splicing post-translationally to yield the 70 kDa catalytic subunit of the vacuolar H+-ATPase and the 50 kDa DNA endonuclease . Accumulating evidence has indicated that splicing precursors distribute widely in many organisms covering eukarya, bacteria and archaea . This article argues and summarizes current chemical and biological views on protein splicing. FEMS Microbiol Rev, 1997 Jun, 20(1-2), 151 - 75 Applications of S-layers; Sleytr UB et al.; The wealth of information existing on the general principle of S-layers has revealed a broad application potential . The most relevant features exploited in applied S-layer research are: (i) pores passing through S-layers show identical size and morphology and are in the range of ultrafiltration membranes; (ii) functional groups on the surface and in the pores are aligned in well-defined positions and orientations and accessible for binding functional molecules in very precise fashion; (iii) isolated S-layer subunits from many organisms are capable of recrystallizing as closed monolayers onto solid supports at the air-water interface, on lipid monolayers or onto the surface of liposomes . Particularly their repetitive physicochemical properties down to the subnanometer scale make S-layers unique structures for functionalization of surfaces and interfaces down to the ultimate resolution limit . The following review focuses on selected applications in biotechnology, diagnostics, vaccine development, biomimetic membranes, supramolecular engineering and nanotechnology . Despite progress in the characterization of S-layers and the exploitation of S-layers for the applications described in this chapter, it is clear that the field lags behind others (e.g . enzyme engineering) in applying recent advances in protein engineering . Genetic modification and targeted chemical modification would allow several possibilities including the manipulation of pore permeation properties, the introduction of switches to open and close the pores, and the covalent attachment to surfaces or other macromolecules through defined sites on the S-layer protein . The application of protein engineering to S-layers will require the development of straightforward expression systems, the development of simple assays for assembly and function that are suitable for the rapid screening of numerous mutants and the acquisition of structural information at atomic resolution . Attention should be given to these areas in the coming years. J Anim Sci, 1997 Jun, 75(6), 1659 - 65 Dietary vitamin E and selenium affect mastitis and milk quality; Smith KL et al.; Vitamin E and selenium (SE) are essential nutrients that are integral components of the antioxidant defense of tissues and cells . Soils in many of the important dairy regions of the world are Se-deficient, and feedstuffs grown on these soils will not provide adequate dietary Se . Cattle consuming stored forages are likely to be low in vitamin E unless supplemented, and vitamin E deficiencies are frequently observed in peripartum dairy cows . Many new intramammary infections (IMI) occur in the 2 wk before and after calving . Deficiencies of either vitamin E or Se have been associated with increased incidence and severity of IMI, increased clinical mastitis cases, and higher somatic cell counts (SCC) in individual cows and bulk tank milk . Somatic cell counts are a primary indicator of mastitis and milk quality in dairy herds . The polymorphonuclear neutrophil (PMN) is a major defensive mechanism against infection in the bovine mammary gland . A know consequence of vitamin E and Se deficiency is impaired PMN activity and postpartum vitamin E deficiencies are frequently observed in dairy cows . Dietary supplementation of cows with Se and vitamin E results in a more rapid PMN influx into milk following intramammary bacterial challenge and increased intracellular kill of ingested bacteria by PMN . Subcutaneous injections of vitamin E approximately 10 and 5 d before calving successfully elevated PMN alpha-tocopherol concentrations during the periparturient period and negated the suppressed intracellular kill of bacteria by PMN that commonly is observed around calving. Eur J Oral Sci, 1997 Jun, 105(3), 189 - 95 Trends in dental health among Icelandic urban children; Bjarnason S et al.; Caries experience, oral hygiene and caries-related salivary parameters were recorded in a 20% representative sample of 12-year-old schoolchildren in Reykjavik, Iceland in 1991 . The majority of the children was re-examined 3 years later in 1994 . Trends in prevalence of caries and salivary bacteria were assessed by comparison with an analogous earlier longitudinal study (1984-87) . Mean DFS values for 12-year-olds were 12.1 and 4.1, for 15-year-olds 23.3 and 11.3 in the earlier and later study, respectively . Reduction in DFS was 66% and 52% for the respective age groups . The decline was most pronounced in the group with low caries prevalence . Trends in caries experience were paralleled by salivary bacteria . The mean caries scores and frequency distributions of 15-year-olds in 1994 closely resembled those of 12-year-olds a decade earlier, suggesting a delay rather than a true fall in caries prevalence. Glycoconj J, 1997 Jun, 14(4), 467 - 71 Recognition of glycoconjugates by Helicobacter pylori . Comparison of two sialic acid-dependent specificities based on haemagglutination and binding to human erythrocyte glycoconjugates; Miller-Podraza H et al.; Helicobacter pylori expresses separate binding characteristics depending on growth conditions, as documented by binding to human erythrocyte glycoconjugates . Cells grown in Ham's F12 liquid medium exhibited a selective sialic acid-dependent binding to polyglycosylceramides, PGCs (Miller-Podraza et al.(1996) Glycoconjugate J13:453-60) . There was no binding to traditional sialylated glycoconjugates like shorter-chain gangliosides, glycophorin or fetuin . However, cells grown on Brucella agar bound both to PGCs and other sialylated glycoconjugates . Fetuin was an effective inhibitor of haemagglutination caused by agar-grown cells, but had no or a very weak inhibitory effect on haemagglutination by F12-grown bacteria . PGCs were strong inhibitors in both cases, while asialofetuin was completely ineffective . The results indicate that H . pylori is able to express two separate sialic acid-dependent specificities, one represented by binding to fetuin, as described before, and another represented by a selective binding to PGCs. Gut, 1997 Jun, 40(6), 782 - 9 Histochemistry of the surface mucous gel layer of the human colon; Matsuo K et al.; BACKGROUND AND AIMS: Histochemical analysis of the surface mucous gel layer of the human colon is difficult, as it dissolves in fixatives . This study was undertaken to explore the surface mucous gel layer on the normal mucosa and neoplastic tissues of the large intestine . In addition, the distribution of different mucins secreted from goblet cells was studied with a series of histochemical stains for mucins . METHODS: Twenty four surgically resected specimens were fixed in Carnoy's solution and embedded in paraffin . In four cases, the surface mucous gel layer was also studied in frozen sections . Serial sections were stained by a battery of histochemical techniques characterising mucins . RESULTS AND CONCLUSION: The surface mucous gel layer consisted of the inner and outer layers . The first covered the luminal surface of the mucosa, consisted of mucins, and showed a vertical striped pattern . The second overlaid the first, showed a lateral striped pattern, and was contaminated with bacteria and other substances . Their thickness in paraffin sections varied considerably among the sites in the large intestine, but was the thickest in the rectum and measured 12.7 (SEM 6.0) microns and 88.8 (SEM 80.1) microns respectively . Mucins forming the inner layer were obviously derived from goblet cells underlying it. Lab Anim Sci, 1997 Jun, 47(3), 222 - 55 The role of Helicobacter species in newly recognized gastrointestinal tract diseases of animals; Fox JG et al.; Because Helicobacter pylori is now known to be a significant human pathogen, experimental animal models are increasingly being used to study the pathogenesis of this organism . Unfortunately, early studies failed to establish H . pylori in animal models, and surprisingly, Koch's postulates were initially fulfilled in two human volunteers . Germfree experiments performed in pigs and pups however established that H . pylori would colonize in these animals, and gastritis was induced . Certain macaque species and cats are now known to be naturally infected with H . pylori, and these animals are susceptible to experimentally induced infection with the organism as well . Interestingly, as the ability to manipulate and grow H . pylori in vitro increased, so did the ability to colonize it in animal models . Helicobacter pylori has now experimentally induced gastritis in germfree euthymic and nude mice and in conventionally housed mice . Six additional Helicobacter species have been isolated and identified from the stomachs of various mammals, including dogs, cats, ferrets, pigs, monkeys, and cheetahs; these organisms, similar to H . pylori, are associated with variable degrees of gastritis in their hosts . In addition to the discovery of gastric helicobacters, an increasing number of Helicobacter spp . have been isolated from the distal part of the gastrointestinal tracts of mammals and birds . Importantly, in one inbred strain of mice, A/JCr, persistent infection with H . hepaticus is linked to development of hepatic adenomas and adenocarcinomas . To date, the genus Helicobacter includes 17 named species as well as other formally unnamed closely related organisms . An overview of gastric helicobacters and naturally acquired Helicobacter spp.-induced disease in laboratory animals and, where appropriate, use of animal models to study H . pylori-associated gastric disease is presented . Similarities between Helicobacter infections and the epidemiology of the diseases induced by these bacteria in humans and animals also are highlighted. Regul Toxicol Pharmacol, 1997 Jun, 25(3), 220 - 5 Environmental hazard assessment of pharmaceuticals; Henschel KP et al.; The pharmaceuticals and pharmaceutical metabolites salicylic acid, paracetamol, clofibrinic acid, and methotrexate were examined with regard to their biological degradability and toxicity toward algae, Daphnia, fish embryos, luminescent bacteria, ciliates, and the fish cell line BF-2 . The EC50 values calculated for the most sensitive organismic test (all except cell cultures) in each case were for salicylic acid, 37 mg/L (fish embryos); for paracetamol, 50 mg/L (Daphnia); for clofibrinic acid, 86 mg/L (fish embryos); and for methotrexate, 45 mg/L (ciliates) . However, in the case of paracetamol, clofibrinic acid, and methotrexate, the fish cell line BF-2 reacted even more sensitively with EC50 values of 19 mg/L (paracetamol), 14 mg/L (clofibrinic acid), and 3 mg/L (methotrexate) . Salicylic acid and paracetamol proved to be easily degradable . The predicted exposure concentration calculated according to the procedure of the EU Draft Phase I for new pharmaceuticals (CEC III/5504/94, draft 4) was based on the total estimated quantity of these substances consumed and indicated that their entry into the environment is theoretically possible . These results show that (1) the four tested pharmaceuticals may be present in the environment, (2) the substances led to effects in at least one ecotoxicological test, and (3) the most sensitive reactions were observed for a nonstandard test which incorporates relevant end points for the respective pharmaceuticals . This demonstrates that a limitation to the standard tests (algae, Daphnia, and fish) would have underestimated the toxicity of paracetamol, clofibrinic acid, and methotrexate . In addition to improved exposure estimates, the EU guideline should therefore contain a test strategy adapted to their modes of action, which permits the definite identification of pharmaceuticals with high ecotoxic potential, and consequently the appropriate provisions. Mol Gen Genet, 1997 Jun, 255(2), 172 - 9 Ammonium repression of the nitrite-nitrate (nasAB) assimilatory operon of Azotobacter vinelandii is enhanced in mutants expressing the nifO gene at high levels; Gutierrez JC et al.; A number of Tn5 mutants were isolated which were unable to fix nitrogen and showed enhanced ammonium repression of the nitrate/nitrite assimilation genes . They also had reduced nitrate reductase activity under fully inducing conditions . Insertions were localized within the nifB gene, and inability to fix nitrogen was shown to be due to disruption of the nifB gene . However, enhanced ammonium repression proved to be the result of constitutive expression of the downstream nifO gene from an 'out' promoter present in Tn5 . Our results suggest that molybdenum metabolism might function as a regulatory factor that acts through the nitrate reductase. Vaccine, 1997 Jun, 15(8), 808 - 9 Genetic to genomic vaccination; Johnston SA et al.; Our development of genetic immunization (unfortunately named 'DNA'-immunization at times) seems to have great promise as a vaccine delivery system . However, one is still left with the often formidable problem of discovering what gene of the pathogen to include in the genetic immunization vector . The is particularly a problem with pathogens with large genomes such as bacteria and parasites . We have developed a potential solution to this problem termed 'expression library immunization' . It involves using expression libraries in genetic immunization vectors to basically strip down the genome into protective genes . It may offer a systematic, unbiased approach to discover vaccine candidates. Vaccine, 1997 Jun, 15(8), 798 - 800 Potential advantages and risks of nucleic acid vaccines for infant immunization; Siegrist CA; Infant antibody responses are impaired due to defective B cell activation by bacterial polysaccharides, slow B cell responses to protein antigens and reduced T cell helper activities . These features result in a 'greater susceptibility to severe infections by encapsulated bacteria and the requirement for repeated doses of vaccines during the first years of life . Nucleic acid vaccines could optimize infant antibody responses by allowing the identification of novel protective antigens, by supplying missing cytokines, by the achievement of sustained immune responses or by the design of novel combination vaccines allowing a reduction of required injections . The deficient infant cellular immune responses, responsible for their susceptibility to infections with intracellular pathogens, could possibly benefit from the potential of nucleic acid vaccines to trigger strong TH1-like responses . Last, a major contribution to infant immunization would be achieved if nucleic acid vaccines proved able to circumvent maternal antibody-mediated inhibition of infant responses to vaccine antigens. Dermatol Nurs, 1997 Jun, 9(3), 163 - 70; quiz 171-2 Cutaneous hazards of the coast; Burke WA; Through recreational and commercial pursuits, more people than ever before are coming in contact with coastal waters containing a variety of bacteria, aquatic flora, and sea creatures potentially harmful to the skin . It is important for dermatology nurses to be aware of some of the more common cutaneous hazards related to the coastal environment as well as the basic treatment of these problems. Int J Parasitol, 1997 Jun, 27(6), 715 - 38 Origin of the epidermis in parasitic platyhelminths; Tyler S et al.; The epidermis of members of the major parasitic taxon Neodermata is distinctive among flatworms, being a syncytial, insunk, non-ciliated epidermis that develops through a wholesale replacement of larval epidermis at metamorphosis when the larva attacks a host . How it arose in evolution from what must have been a turbellarian-like ancestor is not immediately evident . While many turbellarian flatworms have also adopted a symbiotic way of life, the literature on ultrastructure of epidermis in these symbionts shows quite a variety of morphologies, many not so different from that of their free-living relatives . Various turbellarians do have syncytial or insunk epidermises or reduction of epidermal ciliation as is characteristic of the Neodermata, but co-occurrence in a single turbellarian of all features common to neodermatans has not been reported . Urastoma cyprinae, for example, which is ectosymbiotic on bivalves, has a ciliated cellular epidermis that is little different from what is known of epidermises of its free-living relatives . The endoparasitic Anoplodium hymanae, from the coelom of sea cucumbers, also bears a ciliated cellular epidermis, as is typical of many other rhabdocoels, but it shows marked phagocytic activity as well as incorporation of endosymbiotic bacteria . The closest similarity to neodermatan epidermis is that of the turbellarian Genostoma kozloffi, an ectosymbiont of the crustacean Nebalia: covering the bulk of the body is a non-ciliated syncytium with multiple branching connections to insunk nucleated portions, much as in epidermis of adult neodermatans and, on its ventral surface, is a field of ciliated cellular insunk epidermis resembling the epidermis of some larval neodermatans . Developmental clues to the origin of the neodermatan epidermis can be seen in turbellarian embryos . Before hatching, embryos of proseriate and triclad embryos go through 3 generations of epidermis, each replacing the next; 2 generations of epidermis are reported in the literature on rhabdocoel embryos . This process of replacement parallels the epidermal replacement that larval neodermatans undergo at metamorphosis . Ultrastructural study of developing acoel, polyclad and macrostomid embryos shows that they, too, have epidermal replacement and growth through immigration of deeper-lying cells, comparable to the processes seen in higher flatworms . Succession of distinct generations of epidermis in such animals as the proseriates, triclads and rhabdocoels is probably an adaptation to development of ectolecithal eggs, providing the means for the embryo to use yolk that resides in vitellocytes, outside its blastomeres . We propose that the Neodermata has taken advantage of this developmental mechanism, producing successive generations of epidermal cells even in its larval stages, to counter the defenses of hosts. Immunopharmacology, 1997 Jun, 36(2-3), 263 - 9 The evaluation of tissue kallikrein in Helicobacter pylori-associated gastric ulcer disease; Naidoo S et al.; Helicobacter pylori (Hp) associated ulcer disease is a common form of gastric disorder involving mucosal damage and invasion of the mucosa by polymorphic inflammatory cells with concomitant changes in the epithelial cell structure . The bacteria are thought to adhere by specific junction zones to the epithelial cell surface resulting in the degeneration of the mucosal layer . Our study was undertaken to examine the relative status of tissue kallikrein (TK) in antral and fundic biopsies, endoscopically obtained from 10 patients suspected of having gastric disorders . For histological evidence of inflammation the tissue was stained with hematoxylin and eosin and classified as mild, active, chronic and chronic active gastritis . Hp infection was determined by Giemsa staining . For localisation of TK, slide-mounted tissue sections were subjected to PAP and immunofluorescent staining using a goat anti-human TK IgG antibody . The results revealed that in the antral control tissue, removed during partial antractomy, TK was immunovisualised along the luminal border of the deep pyloric glands . The surface epithelia and superficial glands showed no labelling . The fundic control tissue revealed an absence of TK in the superficial and surface epithelial glands, but was positive in the parietal cells . The fundic biopsy specimens showed similar immunoreactivity in these areas . By contrast, in the inflammed pyloric mucosa, there was a shift of TK localisation to the basal part of the glandular cells and there was also expression of TK in the superficial glands that showed histological evidence of regeneration . In the fundic biopsies there was no change observed in the sites of TK localisation (similar to control tissue) . It was observed, that even though 8 of the 10 subjects exhibited Hp infection, the inflamed mucosa showed no discernable difference in the staining patterns between the infected and non-infected tissue sections . Our findings suggest an important role for a B1/B2 kinin antagonist in patients with gastritis. Am J Physiol, 1997 Jun, 272(6 Pt 1), G1408 - 15 Evidence for the existence of a carrier-mediated folate uptake mechanism in human colonic luminal membranes; Dudeja PK et al.; Colonic bacteria synthesize significant amounts of folate . The current studies were undertaken to examine the presence of a possible folate transporter and its characterization in apical membranes of the human colon . Apical membrane vesicles were purified from mucosal scrapings of proximal organ donor colons using a differential centrifugation and divalent cation precipitation technique . {3H}folate (PteGlu) uptake was measured by a rapid filtration technique . Our results demonstrate that {3H}PteGlu uptake into these vesicles 1) was significantly increased with decreasing pH of the incubation buffer, 2) was markedly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid but not by acetazolamide, amiloride, bumetanide, furosemide, and diphenyl 2-carboxylic acid, 3) was sensitive to temperature and osmolarity of the incubation medium, 4) was inhibited by structural analogs methotrexate and 5-methyltetrahydrofolate, 5) exhibited trans-stimulation phenomenon, 6) was potential insensitive, and 7) exhibited saturation kinetics with an apparent Michaelis constant for PteGlu of 8.2 +/- 2.4 microM and a maximal enzyme reaction rate of 19.8 +/- 2.9 pmol.mg protein-1.10 s-1 . Studies on distal colonic membranes also demonstrated the presence of a folate transporter with similar kinetic characteristics . These results demonstrate the existence of a pH-dependent, DIDS-sensitive, electroneutral carrier-mediated mechanism for folate absorption in the human colon. Food Chem Toxicol, 1997 Jun, 35(6), 573 - 81 Preliminary safety assessment of an arachidonic acid-enriched oil derived from Mortierella alpina: summary of toxicological data; Hempenius RA et al.; An arachidonic acid-enriched oil (AA-oil), derived from Mortierella alpina was subjected to a programme of studies to establish its preliminary safety for use in infant nutrition . This was addressed at two levels: (1) HPLC analysis of metabolites produced by the production strains at various conditions, and (2) an evaluation of the toxicity of the final product . The following studies were carried out on the AA-oil: gene mutation assays in bacteria and mammalian cells in vitro; chromosome aberration assays both in vitro and in vivo and acute and subacute (4-wk) oral toxicity in the rat . No known mycotoxins were produced by the production strains under the conditions tested . Further, the oil did not show mutagenic or clastogenic activity and the acute oral toxicity, expressed as the LD50 value, exceeded 20 ml/kg body weight, that is, 18.2 g/kg body weight . In the subacute oral toxicity study the AA-oil was tested as such and in combination with a docosahexaenoic-enriched oil (DHA-oil) derived from fish oil at a ratio of 2:1 (AA:DHA) . This was done because high dose levels of AA may result in adverse effects; DHA can compensate for these effects . Furthermore, human milk contains both AA and DHA at a ratio of AA:DHA of 2 to 3:1 . No obvious signs of toxicity were observed . Levels of phospholipids and triglycerides tended to be decreased in the highest dose groups . The no-observed-adverse-effect level of the AA-oil in the subacute 4-wk toxicity study was placed at the highest levels tested, namely 3000 mg AA-oil/kg body weight/day as such and in the combination of 3000 mg AA-oil and 1500 mg DHA-oil/kg body weight/day . This corresponds to an intake of 1000 mg AA/kg body weight/day, which represents approximately 37 times the infant intake of AA in human milk. Biol Chem, 1997 Jun, 378(6), 489 - 93 Role of cellular kinases in the gene expression of nonsegmented negative strand RNA viruses; De BP et al.; Nonsegmented negative strand RNA viruses package an RNA-dependent RNA polymerase composed of two subunits, a large protein L and a phosphoprotein P, for transcription and replication of their genome RNAs . The RNA polymerase activity resides within the L protein, while the P protein acts as a transcription factor or transactivator of the polymerase . Since P protein is heavily phosphorylated and phosphorylation is known to regulate function of many viral as well as cellular proteins, the role of phosphorylation of P protein in the gene expression of this group of RNA viruses has recently been investigated . Through expression in bacteria the P protein was produced in large quantity in the nonphosphorylated form and involvement of cellular kinase(s) in its phosphorylation was studied . Casein kinase II and/or protein kinase C have been shown to play a critical role in the activation of P protein in transcription . These findings have opened up a new avenue for studying an important regulatory step in virus gene expression that may lead to the development of an effective antiviral agent. Mol Biotechnol, 1997 Jun, 7(3), 207 - 16 Decontamination of polymerase chain reaction reagents for detection of low concentrations of 16S rRNA genes; Hilali F et al.; We describe a polymerase chain reaction (PCR) that allowed detection of rRNA consensus sequences from the DNA extracted from a wide range of bacterial species in amounts as low as 10 fg . To avoid false positive results with universal primers for 16S rRNA PCR, contaminating DNA had to be eliminated from the polymerase preparations . Decontamination was undertaken before PCR to optimize treatment with DNaseI, and was followed by DNase inactivation at 94 degrees C for 50 min, which eliminated contaminating DNA at concentrations of up to 100 pg . After optimization of PCR conditions for each polymerase . Deep Vent Exo-polymerase (New England Biolabs, Beverly, MA), and super-Taq polymerase (HT Biotechnology, Cambridge, UK) were more effective than Ampli-Taq polymerase (Perkin-Elmer Cetus, Norwalk CT), Ampli-Taq LD polymerase (Perkin-Elmer Cetus) or Deep-vent polymerase (New England Biolabs) . The technique described in this article might prove to be a universal method for PCR detection of small numbers of unidentified bacteria in usually sterile clinical sites, such as blood and cerebrospinal fluids, in which a broad spectrum of pathogens can be expected. Biol Pharm Bull, 1997 Jun, 20(6), 613 - 20 Effect of transcriptional regulatory sequences on autonomous replication of plasmids in transient mammalian systems; Yokoyama M et al.; Transcriptional regulatory sequences often influence the efficiency of DNA replication, directly or indirectly, in bacteria, yeast, and animal virus systems . We have tested several transcriptional regulatory sequences for affecting DNA replication, based on pUC vector, in transient systems . Autonomous replication of transfected plasmids was assayed by PCR amplification of the fragments derived from the plasmids, which had replicated in mammalian cells . By this highly efficient method of detecting replicated molecules, pUC19, but not pUC18, showed a week replication activity in transfected cells . Nucleotide sequences around the HindIII site in pUC19 were required for replication . Monomers or dimers of the octamer transcription motif of the mouse immunoglobulin heavy chain gene, inserted in multicloning sites of pUC19, could stimulate replication, while the 4- or 6-mers did not, in contrast to the results on its transcription activity . Other transcriptional elements including AP1, HSE, and E2F also stimulated replication, but neither CRE nor Sp1 binding motif did . These results suggest that at least some of the transcriptional regulatory sequences function as-modulators of DNA replication as well as of transcription. Antisense Nucleic Acid Drug Dev, 1997 Jun, 7(3), 159 - 65 In vivo metabolic profile of a phosphorothioate oligodeoxyribonucleotide; Temsamani J et al.; Antisense phosphorothioate oligodeoxyribonucleotides (PS oligonucleotides) have the ability to inhibit individual gene expression in the potential treatment of cancer and viral diseases . Following administration in vivo, PS oligonucleotides are rapidly cleared from the plasma and distributed to various organs . However, the manner in which administered oligonucleotides are metabolized in plasma and tissues is poorly understood . In this study, a 25-mer PS oligonucleotide (GEM91) complementary to the gag gene mRNA of the human immunodeficiency virus (HIV-1) was administered to mice through intravenous injections to investigate its metabolism . The PS oligonucleotide was extracted from plasma at 1 hour postadministration and from kidney and liver at 24 hours postadministration . After extraction, the PS oligonucleotide and its metabolites were tailed with dA and annealed to a dT-tailed plasmid . The recombinant plasmid was ligated and used to transform competent bacteria . The region of interest containing the PS oligonucleotide was then sequenced . Our results show that degradation of the PS oligonucleotide in plasma was primarily from the 3'-end . However, in kidney and liver, degradation was primarily from the 3'-end, but a large proportion of the PS oligonucleotide was degraded from the 5'-end as well . We also studied the metabolism of PS oligonucleotide in plasma after 2-hour intravenous infusion in HIV-infected patients . The degradation of the PS oligonucleotide in plasma was primarily from the 3'-end . This study is important in understanding the metabolism of antisense PS oligonucleotide in vivo in general but also provides guidance for designing second-generation antisense oligonucleotides with improved stability and safety profile. Plant Cell, 1997 Jun, 9(6), 957 - 65 Light-stimulated degradation of an unassembled Rieske FeS protein by a thylakoid-bound protease: the possible role of the FtsH protease; Ostersetzer O et al.; Unassembled subunits of the cytochrome b6f complex as well as components of other unassembled chloroplastic complexes are rapidly degraded within the organelle . However, the mechanisms involved in these proteolytic processes are obscure . When the Rieske FeS protein (RISP) is imported into isolated chloroplasts in vitro, some of the protein does not property assemble with the cytochrome complex, as determined by its sensitivity to exogenous protease . When assayed in intact, lysed, or fractionated chloroplasts, the imported RISP was found to be sensitive to endogenous proteases as well . The activity responsible for degradation of the unassembled protein was localized to the thylakoid membrane and characterized as a metalloprotease requiring zinc ions for its activity . The degradation rate was stimulated by light, but no involvement of ATP or redox control was observed . Instead, when the RISP that was attached to thylakoid membranes was first illuminated on ice, degradation proceeded in either light or darkness at equal rates suggesting a light-induced conformational change making the protein prone to degradation . Antibodies raised against native FtsH, a bacterial, membrane-bound, ATP-dependent, zinc-stimulated protease, effectively inhibited degradation of the unassembled RISP, suggesting a role for the chloroplastic FtsH in this process. Clin Perinatol, 1997 Jun, 24(2), 497 - 521 The great balancing acts . The pregnant woman, placenta, fetus, and infectious agents; Nahmias AJ et al.; This article conceptualizes the various balancing acts between the pregnant woman, the placenta, the fetus, and the infectious agents . Despite the very large number of infectious insults during pregnancy, the outcome of most interactions usually is a normal newborn . We have identified only one general immune defect of the fetus and neonate: The inability to respond to polysaccharide antigens; yet, a similar defect also is found with certain polysaccharides in older children and adults . The capacity of the neonate to control severe life-threatening diseases with most infectious agents and the ability of the fetus, when infected in utero, to mount sophisticated, immune responses make it conceptually advantageous to consider the older fetus and the newborn infant as "immunodelayed" rather than as immunodeficient or immature. Eur J Immunol, 1997 Jun, 27(6), 1557 - 63 The repertoire of serum IgM in normal mice is largely independent of external antigenic contact; Haury M et al.; Antigen-free (AGF) and germ-free (GF) mice, although essentially free of serum IgG, maintain normal levels of circulating IgM . Using a quantitative immunoblot assay, we have now analyzed the repertoire of serum IgM from AGF, GF, and specific pathogen-free (SPF) BALB/c mice, on large panels of natural antigens from homologous tissues and bacteria . The reactivity profiles were very similar in the three groups of mice . Multiparametric statistic evaluation of the data showed that BALB/c animals, SPF, GF, and AGF mice constitute an homogeneous group with similar immunoreactivity profiles when compared to C57BL/6 . Differences between immunoreactivity profiles of GF and AGF mice were observed, but were not statistically significant . These results suggest that the serum IgM repertoire of normal mice is strictly regulated and selected by endogenous ligands. Eur J Biochem, 1997 Jun 1, 246(2), 574 - 9 Thermodynamic significance of the lactate gradient; Hockings PD et al.; The theory that some bacteria can save energy by an energy-recycling process, in which protons are excreted with metabolic end-products with variable stoichiometry, has been examined by 1H-NMR . A method has been developed that utilises observed differences in the Hahn T2 relaxation of metabolites in the intracellular and extracellular compartments to distinguish and quantify metabolite signals originating from both compartments . It was found that the lactate electrochemical-potential gradient calculated from the fraction of lactate that is sufficiently mobile to contribute to the NMR signal was in exact balance with the proton electrochemical-potential gradient over a wide range of pH values . The conclusion was reached that previous reports of variable stoichiometry were due to 'bound' lactate at high intracellular pH that could neither contribute neither to the NMR signal nor to the lactate electrochemical-potential gradient. Front Biosci, 1997 Jun 1, 2, d232 - 41 The comparative biology of pulmonary intravascular macrophages; Longworth KE; Pulmonary intravascular macrophages are an important part of the mononuclear phagocyte system in some species of mammals, mainly sheep and other ruminants, pigs, and horses . These cells phagocytize foreign particles, cell debris and pathogens that pass through the pulmonary circulation . Species with intravascular macrophages localize intravenously injected tracer particles and bacteria predominantly in the lung rather than the liver, and exhibit pulmonary hypertension when these cells are activated . Both in vivo and in vitro studies show that pulmonary intravascular macrophages have distinct secretory and immune capabilities . Consequently, the pulmonary intravascular macrophages play an important role in pulmonary inflammation in species that have them Trends Biochem Sci, 1997 Jun, 22(6), 207 - 10 Redox signal transduction via iron-sulfur clusters in the SoxR transcription activator; Hidalgo E et al.; Protein iron-sulfur (FeS) centers have recently been implicated in the regulation of gene expression . In the redox-sensing SoxR protein, the oxidation state of {2Fe-2S} centers controls its activity as a transcription activator independent of DNA-binding ability . Thus, FeS centers allosterically link cellular oxidative stress to the expression of defense genes. Microbiology, 1997 Jun, 143 ( Pt 6), 1925 - 31 Xanthobacter flavus employs a single triosephosphate isomerase for heterotrophic and autotrophic metabolism; Meijer WG et al.; The expression of the cbb and gap-pgk operons of Xanthobacter flavus encoding enzymes of the Calvin cycle is regulated by the transcriptional regulator CbbR . In order to identify other genes involved in the regulation of these operons, a mutant was isolated with a lowered activity of a fusion between the promoter of the cbb operon and the reporter gene lacZ . This mutant was unable to grow autotrophically and had a reduced growth rate on medium supplemented with gluconate or succinate . The regulation of the gap-pgk operon in the mutant was indistinguishable from the wild-type strain, but induction of the cbb operon upon transition to autotrophic growth conditions was delayed . Complementation of the mutant with a genomic library of X . flavus resulted in the isolation of a 1.1 kb ApaI fragment which restored autotrophic growth of the mutant . One open reading frame (ORF) was present on the ApaI fragment, which could encode a protein highly similar to triosephosphate isomerase proteins from other bacteria . Cell extracts of the mutant grown under glycolytic or gluconeogenic conditions had severely reduced triosephosphate isomerase activities . The ORF was therefore identified as tpi, encoding triosephosphate isomerase . The tpi gene is not linked to the previously identified operons encoding Calvin cycle enzymes and therefore represents a third transcriptional unit required for autotrophic metabolism. J Appl Microbiol, 1997 Jun, 82(6), 763 - 8 Efficacy of three prevention strategies against legionella in cooling water systems; Kusnetsov JM et al.; The efficacy of three different prevention strategies on legionella in cooling systems was studied . The strategies were as follows: (1) water temperature was lowered; (2) water quality was improved; or (3) the system as disinfected with polyhexamethylene biguanidechloride (PHMB) biocide or with 2-bromo-2-nitropropane-1,3-diol (BNPD) biocide . Lowering of water temperature was the most effective method to reduce the concentration of legionella in cooling systems . Improving of water quality resulted in a transitory disinfection effect . The additions of PHMB or BNPD decreased the concentrations of both legionella and heterotrophic bacteria in cooling water . The effect of biocides, however, lasted at the most only a few months . If possible, lowering water temperature and improving the water quality should be the primary practices for controlling bacterial growth in cooling systems . Regular biocide treatments should be incorporated into the maintenance procedures if technical improvements cannot be done or if their efficiency is too low. Arch Dermatol, 1997 Jun, 133(6), 743 - 5 The histopathology of closed and open comedones of Favre-Racouchot disease; Sanchez-Yus E et al.; OBJECTIVE: To determine the distinction between a comedo and an infundibular cyst of Favre-Racouchot disease . SETTING: A university hospital . PATIENTS: From the 8 patients included in the study, 19 cysts and comedones were evaluated . MAIN OUTCOME MEASURE: The distinguishing features between the cysts and comedones of Favre-Racouchot disease . RESULTS: All lesions were histologically indistinguishable from the primary comedones of acne vulgaris, except for the presence of a marked actinic elastosis in the surrounding dermis . The presence of a variable number of hair shafts and an abundant amount of bacteria, which was positive in the results of Gram staining and periodic acid-Schiff reaction and intermingled with sebum and eosinophilic laminated horny material within the dilated infundibulum, characterizes a comedo and differentiates it from an infundibular cyst . CONCLUSIONS: The cysts and comedones of Favre-Racouchot disease are closed and open comedones . They can be easily differentiated from an infundibular cyst by the histopathologic features rather than by the connection to the surface. Curr Biol, 1997 Jun 1, 7(6), 397 - 407 A mammalian DNA repair enzyme that excises oxidatively damaged guanines maps to a locus frequently lost in lung cancer; Lu R et al.; BACKGROUND: Guanine residues in the genome are vulnerable to attack by free radicals and reactive oxygen species . A major lesion thus produced, 8-oxoguanine (OG), causes mutations by mis-pairing with adenine during replication . In bacteria and budding yeast, OG is removed from the genome through the action of base-excision DNA repair (BER) enzymes, which catalyze expulsion of the aberrant base and excision of its sugar moiety from the DNA backbone . Although OG is known to be produced in and cleansed from mammalian genomes, the enzymes responsible for OG repair in these cells have remained elusive . RESULTS: Here, we report the cloning and biochemical characterization of mammalian BER enzymes that specifically target OG residues in DNA . These 8-oxoguanine DNA glycosylases, hOgg1 (human) and mOgg1 (murine), are homologous to each other and to yeast Ogg1 . They also contain an active site motif - the Helix-hairpin-Helix, Gly/Pro-rich-Asp motif - characteristic of a superfamily of BER proteins with a similar core fold and active site geometry . Both hOgg1 and mOgg1 exhibit exquisite selectivity for the base opposite OG in DNA, operating with high efficiency only on OG base-paired to cytosine . Furthermore, hOgg1 and mOgg1 are unable to process a panel of alternative lesions, including 8-oxoadenine, yet bind with high affinity to synthetic abasic site analogs . The proteins operate through a classical glycosylase/lyase catalytic mechanism; mutation of a catalytically essential lysine residue results in loss of catalytic potency but retention of binding to OG-containing oligonucleotides . The hOGG1 gene is localized on the short arm of chromosome 3 (3p25/26) in a region commonly deleted in cancers . CONCLUSIONS: These results conclusively establish the existence and identity of an 8-oxoguanine DNA glycosylase/lyase in human and murine cells, completing the triad of proteins that together protect mammals from the genotoxic effects of guanine oxidation . The observation that at least one allele of hOGG1 is commonly deleted in cancer cells suggests that such cells may possess a reduced capacity to counter the mutagenic effects of reactive oxygen species, a deficiency that could increase their overall genomic instability . This speculation is fueled by recent observations that cells constitutively active for the Ras/Raf pathway constitutively produce high levels of superoxide, a known generator of OG. Curr Biol, 1997 Jun 1, 7(6), R352 - 4 Laue crystallography: Lights! Camera! action! Farber GK. Recent advances in the Laue method of X-ray data collection from protein crystals have allowed very short-lived reaction intermediates to be observed successfully. Front Biosci, 1997 Jun 01, 2, d232 - 41 The comparative biology of pulmonary intravascular macrophages; Longworth KE; Pulmonary intravascular macrophages are an important part of the mononuclear phagocyte system in some species of mammals, mainly sheep and other ruminants, pigs, and horses . These cells phagocytize foreign particles, cell debris and pathogens that pass through the pulmonary circulation . Species with intravascular macrophages localize intravenously injected tracer particles and bacteria predominantly in the lung rather than the liver, and exhibit pulmonary hypertension when these cells are activated . Both in vivo and in vitro studies show that pulmonary intravascular macrophages have distinct secretory and immune capabilities . Consequently, the pulmonary intravascular macrophages play an important role in pulmonary inflammation in species that have them J Arthroplasty, 1997 Jun, 12(4), 426 - 33 Multiple irrigation, debridement, and retention of components in infected total knee arthroplasty; Mont MA et al.; The results of 24 infected total knee arthroplasties (22 patients) that were treated by irrigation, debridement, and retention of the prosthetic components were prospectively studied . Strict criteria were used for the selection of this method of treatment . Patients had to be less than 30 days after index arthroplasty (postsurgical group) or had to have less than 30 days of knee symptoms (hematogenous group) . In addition, there had to be no radiographic signs of osteitis or evidence of a loose prosthetic component . Patients had one to three irrigation and debridement procedures depending on systemic signs, knee symptoms, or the results of knee aspirations . All of the immediate postsurgical infections (10 knees) and 10 of the 14 (71%) late hematogenously infected knees retained the prosthesis without further evidence of infection at the final follow-up visit at 48 months (range, 24-140 months) . This study shows that in selected circumstances, irrigation, debridement, and retention of the components can result in low morbidity with high success rates. Dis Colon Rectum, 1997 Jun, 40(6), 726 - 30 Neoplasia in ileal pouch mucosa after total proctocolectomy for juvenile polyposis: report of a case; Stoltenberg RL et al.; PURPOSE: Patients treated with restorative proctocolectomy for familial adenomatous polyposis or ulcerative colitis occasionally develop disease in the ileal pouch similar to that originally present in the colon . We investigated the possibility of analogous involvement in the ileal pouch of juvenile polyposis patients . METHODS: Endoscopic surveillance for neoplasia throughout the gastrointestinal tract was performed, with retrieval of all polypectomy specimens for histologic classification using the criteria of Morson . RESULTS: Multiple large juvenile polyps were found in the ileal pouch of one patient less than 10 years after restorative proctocolectomy for hereditary juvenile polyposis . The pouch was much more severely affected than the proximal ileum, small intestine, or stomach . Although most polyps had a completely benign histologic appearance, three had moderate to severe dysplasia . DISCUSSION: Mucosal changes induced by bacteria or stasis of luminal contents may promote manifestation in the ileal pouch of the disease phenotype usually more evident in the colon . Patients with severe or generalized juvenile polyposis should be considered for periodic endoscopic surveillance of the ileal pouch beginning several years after restorative proctocolectomy. J Bacteriol, 1997 Jun, 179(12), 4066 - 70 Structural studies of malate dehydrogenases (MDHs): MDHs in Brevundimonas species are the first reported MDHs in Proteobacteria which resemble lactate dehydrogenases in primary structure; Charnock C; The N-terminal sequences of malate dehydrogenases from 10 bacterial strains, representing seven genera of Proteobacteria, were determined . Of these, the enzyme sequences of species classified in the genus Brevundimonas clearly resembled those malate dehydrogenases with greatest similarity to lactate dehydrogenases . Additional evidence from subunit molecular weights, peptide mapping, and enzyme mobilities suggested that malate dehydrogenases from species of the genus Brevundimonas were structurally distinct from others in the study. J Bacteriol, 1997 Jun, 179(12), 3823 - 7 Interactions of the RepA1 protein with its replicon targets: two opposing roles in control of plasmid replication; Maas R et al.; By studying the interaction of derivatives of RepFIC miniplasmids, we were able to demonstrate that under certain conditions the RepA1 initiator protein inhibits plasmid replication . An analysis of cloned derivatives whose replication is inhibited by the RepA1 protein revealed the existence of two areas of the RepFIC genome that interact with RepA1 in the inhibition reaction . One of these areas, which occurs in the origin region, was explored by in vivo methylation protection footprinting studies . The protected area was 200 bp long and showed a definite periodicity of protected and hypersensitive sites, suggesting that RepA1 promotes a topological change in the RepFIC genome . The significance of our results is discussed in the context of plasmid replication control. Ann Plast Surg, 1997 Jun, 38(6), 553 - 62 Vacuum-assisted closure: a new method for wound control and treatment: animal studies and basic foundation; Morykwas MJ et al.; A series of basic animal studies using a new subatmospheric pressure technique (The V.A.C.) to expedite wound healing are presented . The technique entails placing an open-cell foam into the wound, sealing the site with an adhesive drape, and applying subatmospheric pressure (125 mmHg below ambient) that is transmitted to the wound in a controlled manner . Utilizing a pig model, four studies were undertaken to determine the effect of subatmospheric pressure on laser Doppler-measured blood flow in the wound and adjacent tissue (N = 5), rate of granulation tissue formation (N = 10), clearance of bacteria from infected wounds (N = 5), and measurement of nutrient flow by random-pattern flap survival (N = 5) . Blood flow levels increased fourfold when 125 mmHg subatmospheric pressure was applied . Significantly increased rates of granulation tissue formation (p < or = 0.05) occurred with both continuous (63.3 +/- 26.1%) and intermittent (103% +/- 35.3%) application . Tissue bacterial counts significantly decreased (p < or = 0.05) after 4 days of application . Random-pattern flap survival significantly increased (p < or = 0.05) by 21% compared to controls . We determined that the application of controlled subatmospheric pressure creates an environment that promotes would healing. Am J Gastroenterol, 1997 Jun, 92(6), 1044 - 5 Orthotopic liver transplantation improves small bowel motility disorders in cirrhotic patients; Madrid AM et al.; Altered small intestinal motility has been observed in patients with liver cirrhosis . Its pathophysiology remains to be defined . Our aim was to investigate the effect of orthotopic liver transplantation on small intestinal dysmotility in patients with liver disease . Two patients were studied both before and after orthotopic liver transplantation . Abnormal migrating motor complex activity and prominent clustered contractions present preoperatively normalized within 6 months after the surgical procedure . This finding might represent an additional benefit of liver transplantation considering that altered motility may be involved in bacterial overgrowth and infections observed in these patients. J Bacteriol, 1997 Jun, 179(11), 3790 - 2 Characterization of DNA binding sites for the BvgA protein of Bordetella pertussis; Karimova G et al.; Expression of virulence-associated genes in Bordetella pertussis is under the control of the pleiotropic regulator BvgA . Although previous studies have identified recognition sequences for BvgA in several promoter regions, their structures have not been clearly characterized . We show that the BvgA binding sites within the bvgp(1) and cyaA promoters consist of inverted repeats and suggest that inverted-repeat motifs may represent the recognition elements for DNA-BvgA interaction. J Bacteriol, 1997 Jun, 179(11), 3580 - 7 Characterization of Azorhizobium caulinodans glnB and glnA genes: involvement of the P(II) protein in symbiotic nitrogen fixation; Michel-Reydellet N et al.; The nucleotide sequence and transcriptional organization of Azorhizobium caulinodans ORS571 glnA, the structural gene for glutamine synthetase (GS), and glnB, the structural gene for the P(II) protein, have been determined . glnB and glnA are organized as a single operon transcribed from the same start site, under conditions of both nitrogen limitation and nitrogen excess . This start site may be used by two different promoters since the expression of a glnB-lacZ fusion was high in the presence of ammonia and enhanced under conditions of nitrogen limitation in the wild-type strain . The increase was not observed in rpoN or ntrC mutants . In addition, this fusion was overexpressed under both growth conditions, in the glnB mutant strain, suggesting that P(II) negatively regulates its own expression . A DNA motif, similar to a sigma54-dependent promoter consensus, was found in the 5' nontranscribed region . Thus, the glnBA operon seems to be transcribed from a sigma54-dependent promoter that operates under conditions of nitrogen limitation and from another uncharacterized promoter in the presence of ammonia . Both glnB and glnBA mutant strains derepress their nitrogenase in the free-living state, but only the glnBA mutant, auxotrophic for glutamine, does not utilize molecular nitrogen for growth . The level of GS adenylylation is not affected in the glnB mutant as compared to that in the wild type . Under symbiotic conditions, the glnB and glnBA mutant strains induced Fix- nodules on Sesbania rostrata roots . P(II) is the first example in A . caulinodans of a protein required for symbiotic nitrogen fixation but dispensable in bacteria growing in the free-living state. Infect Immun, 1997 Jun, 65(6), 2497 - 501 Analysis of the Legionella pneumophila fliI gene: intracellular growth of a defined mutant defective for flagellum biosynthesis; Merriam JJ et al.; Using a PCR-based strategy and degenerate oligonucleotides, we isolated a Legionella pneumophila gene that showed high sequence similarity to members of the fliI gene family . An insertion mutation that disrupted the fliI open reading frame was recombined onto the L . pneumophila chromosome and analyzed for its effects on production of flagella and intracellular growth . The mutation resulted in loss of surface-localized flagellin protein but had no effect on the ability of the bacteria to grow within cultured cells . Therefore, in spite of the fact that some aflagellar mutations render L . pneumophila unable to grow within macrophages, the isolation of this defined mutant confirms that production of flagella is not required for intracellular growth. Infect Immun, 1997 Jun, 65(6), 2100 - 6 Temporal effect of tumor necrosis factor alpha on murine macrophages infected with Mycobacterium avium; Eriks IS et al.; Members of the Mycobacterium avium complex are a family of bacteria that persist within macrophages in the face of an immune response . Elimination of these organisms is likely due to cytokine-induced macrophage activation . Because macrophage activation by tumor necrosis factor alpha (TNF-alpha) appears critical for killing of intracellular M . avium, early downregulation of TNF-alpha levels in infected macrophages has been suggested as a survival mechanism for virulent strains of M . avium . We examined the relationship between TNF-alpha and growth of M . avium strains of differing virulence, as measured by their ability to grow in murine bone marrow-derived macrophages . When exogenous TNF-alpha was added immediately following macrophage infection, significant growth inhibition of virulent M . avium strains was observed . If TNF-alpha addition was delayed by 24 h or more, growth inhibition was abrogated . To determine if early downregulation of TNF-alpha levels could explain the differential growth of virulent and avirulent strains, levels of TNF-alpha and prostaglandin E2 (PGE2), which has been shown to suppress TNF-alpha production in uninfected macrophages, were quantified over time . Upregulation of both TNF-alpha and PGE2, as measured by enzyme-linked immunosorbent assay, was evident by 6 h postinfection, indicating that the ability of M . avium to replicate in macrophages was not directly correlated with early downregulation of TNF-alpha production . However, TNF-alpha bioactivity, as measured by cytotoxicity, was significantly decreased in virulent M . avium strains at all time periods examined . Treatment of infected macrophages with gamma interferon immediately after infection resulted in significantly increased levels of nitric oxide but did not affect the growth of virulent M . avium strains . These results suggest that while significant levels of TNF-alpha are present in supernatants from all M . avium strains, levels of biologically active TNF-alpha are significantly reduced in supernatants from virulent M . avium strains . Preliminary results suggest that upregulation of the soluble p75 TNF receptor may be one mechanism by which TNF-alpha bioactivity reduction occurs. J Immunol, 1997 Jun 1, 158(11), 5418 - 23 N-acetylcysteine and alpha-tocopherol reverse the inflammatory response in activated rat Kupffer cells; Fox ES et al.; Activation of the resident macrophage populations of the reticuloendothelial system is a key component of the complex pathophysiology of sepsis . Macrophage activation leads to production and secretion of inflammatory mediators such as cytokines, vasoactive substances, free radicals, and chemokines, which have been associated with high morbidity and mortality in the septic patient . The goal of the present study was to determine whether antioxidants could suppress Kupffer cell activation at points beyond the initiation of activation . Kupffer cells were studied since they are central to the clearance of bacteria and endotoxins, and have been associated with hepatocellular dysfunction in sepsis . Cells were activated with 10 ng/ml LPS for various times whereupon N-acetylcysteine (30 mM) and alpha-tocopherol (50 microM) were added . Steady state levels of cytokine mRNA, activation of nuclear factor-kappaB, and TNF-alpha secretion were determined when expression was maximal in control cells . The results of this study show that antioxidants can be used to suppress Kupffer cell activation at points beyond the initiation of activation . Furthermore, we show that N-acetylcysteine-mediated inhibition of activation requires secondary protein synthesis, but does not modulate IkappaB-alpha mRNA expression . The inhibitory effect of these drugs occurs at the very earliest steps of the LPS signal transduction cascade as it is currently understood . The results of the present study suggest that the inflammatory response to sepsis may be controlled through appropriate antioxidant therapy. J Clin Microbiol, 1997 Jun, 35(6), 1609 - 11 Use of heteroduplex analysis to classify legionellae on the basis of 5S rRNA gene sequences; Pinar A et al.; Seventeen different species of Legionella, 12 serogroups of Legionella pneumophila, and 2 Legionella-like amoebal pathogens (LLAP1 and Sarcobium lyticum) were examined by heteroduplex analysis of PCR products of the 5S rRNA gene . Eight different banding patterns were identified, indicating that heteroduplex analysis of this gene can be used to classify these bacteria according to base substitutions between species . This classification may have future applications in clinical and epidemiological studies. J Clin Microbiol, 1997 Jun, 35(6), 1592 - 4 Rapid detection of Mycoplasma pneumoniae by an assay based on PCR and probe hybridization in a nonradioactive microwell plate format; Kessler HH et al.; A new molecular assay, based on a rapid DNA extraction protocol, PCR, and hybridization to a specific probe in a nonradioactive microwell plate format was used to detect Mycoplasma pneumoniae in bronchoalveolar fluid specimens . The sensitivity of the assay was determined to be 10 to 100 organisms with M . pneumoniae reference strains . Specificity testing with different bacteria capable of producing pneumonia showed no cross-reactivity . In a prospective study, bronchoalveolar lavage fluids obtained from patients with pneumonia were investigated with the PCR assay and compared to culture . Twelve positive samples were detected with the PCR assay . Seven of them were subsequently confirmed by culture . All patients with positive PCR results seroconverted . Application of the PCR assay described may lead to safe and early diagnosis of M . pneumoniae in patients with pneumonia. Mol Cell Biol, 1997 Jun, 17(6), 3272 - 83 Evidence that complex formation by Bas1p and Bas2p (Pho2p) unmasks the activation function of Bas1p in an adenine-repressible step of ADE gene transcription; Zhang F et al.; Bas1p and Bas2p (Pho2p) are Myb-related and homeodomain DNA binding proteins, respectively, required for transcription of adenine biosynthetic genes in Saccharomyces cerevisiae . The repression of ADE genes in adenine-replete cells involves down-regulation of the functions of one or both of these activator proteins . A LexA-Bas2p fusion protein was found to activate transcription from a lexAop-lacZ reporter independently of both BAS1 function and the adenine levels in the medium . In contrast, a LexA-Bas1p fusion activated the lexAop reporter in a BAS2-dependent and adenine-regulated fashion . The DNA binding activity of Bas2p was not needed for its ability to support activation of the lexAop reporter by LexA-Bas1p, indicating that LexA-Bas1p recruits Bas2p to this promoter . The activation functions of both authentic Bas1p and LexA-Bas1p were stimulated under adenine-repressing conditions by overexpression of Bas2p, suggesting that complex formation by these proteins is inhibited in adenine-replete cells . Replacement of Asp-617 with Asn in Bas1p or LexA-Bas1p allowed either protein to activate transcription under repressing conditions in a manner fully dependent on Bas2p, suggesting that this mutation reduces the negative effect of adenine on complex formation by Bas1p and Bas2p . Deletions of N-terminal and C-terminal segments from the Bas1p moiety of LexA-Bas1p allowed high-level activation by the truncated proteins independently of Bas2p and adenine levels in the medium . From these results we propose that complex formation between Bas1p and Bas2p unmasks a latent activation function in Bas1p as a critical adenine-regulated step in transcription of the ADE genes. Arch Microbiol, 1997 Jun, 167(6), 384 - 91 Enzymology of the degradation of (di)chlorobenzenes by Xanthobacter flavus 14p1; Sommer C et al.; Xanthobacter flavus 14p1 used 1,4-dichlorobenzene as the sole source of carbon and energy but did not grow on other (chloro)aromatic compounds . 1,4-Dichlorobenzene was attacked by a chlorobenzene dioxygenase, and the intermediate chlorocatechol was metabolized by the modified ortho pathway . All enzymes necessary to convert 1, 4-dichlorobenzene to 3-oxoadipate showed a low substrate specificity and also accepted the respective intermediates of chlorobenzene or 1, 3-dichlorobenzene degradation . Of the three compounds chlorobenzene, 1,4-dichlorobenzene, and 1,3-dichlorobenzene, the latter was the most toxic for X . flavus 14p1 . Furthermore, 1,3-dichlorobenzene did not induce chlorocatechol 1,2-dioxygenase activity of the organism . Chlorobenzene, however, induced chlorocatechol 1,2-dioxygenase, dienelactone hydrolase, and maleylacetate reductase activities . As demonstrated by chloride release, also chlorobenzene dioxygenase, chlorobenzene cis-dihydrodiol dehydrogenase, and chloromuconate cycloisomerase activities were present in chlorobenzene-induced cells, but chlorobenzene failed to support growth . Presumably a toxic compound was formed from one of the intermediates. Curr Microbiol, 1997 Jun, 34(6), 337 - 9 Genome sizes of Desulfovibrio desulfuricans, Desulfovibrio vulgaris, and Desulfobulbus propionicus estimated by pulsed-field gel electrophoresis of linearized chromosomal DNA; Devereux R et al.; Pulsed-field gel electrophoresis (PFGE) of linearized, full-length chromosomal DNA was used to estimate the genome sizes of three species of sulfate-reducing bacteria . Genome sizes of Desulfovibrio desulfuricans, Desulfovibrio vulgaris, and Desulfobulbus propionicus were estimated to be 3.1, 3.6, and 3.7 Mb, respectively.These values are double the genome sizes previously determined for two Desulfovibrio species by two-dimensional agarose gel electrophoresis of DNA cut with restriction enzymes . PFGE of full-length chromosomal DNA could provide a generally applicable method to rapidly determine bacterial genome size and organization. Am J Ind Med, 1997 Jun, 31(6), 756 - 66 A field investigation of the acute respiratory effects of metal working fluids . I . Effects of aerosol exposures; Kriebel D et al.; A study of cross-shift change in pulmonary function was conducted among workers exposed to metal working fluids (MWF) in an automobile parts manufacturing company . Three hundred eighty-six workers (216 machinists exposed to straight or soluble MWFs, and 170 nonmachinists) were studied for 1 day, performing spirometry at the beginning and end of their shift . Airborne concentrations of inhalable particulate, culturable bacteria, and endotoxin were measured . We observed an approximately threefold increase in the incidence of 5% or greater cross-shift decrement in forced expiratory volume during the first second among those with exposures above about 0.15 mg/m3, compared to those with exposures below about 0.08 mg/m3 . There was some evidence that chronic respiratory symptoms were more prevalent among machinists than among nonmachinists, notably for chronic cough . Baseline FEV1 was about 3% lower on average among those with soluble MWF exposure compared to nonmachinists . These findings are consistent with earlier studies showing respiratory effects of MWFs. J Mol Biol, 1997 May 30, 269(1), 52 - 66 The structure of an RNA "kissing" hairpin complex of the HIV TAR hairpin loop and its complement; Chang KY et al.; We have used nuclear magnetic resonance (NMR) to obtain the structure of an RNA "kissing" hairpin complex formed between the HIV-2 TAR hairpin loop and a hairpin with a complementary loop sequence . Kissing hairpins are important in natural antisense reactions; their complex is a specific target for protein binding . The complex has all six nucleotides of each loop paired to form a bent quasicontinuous helix of three coaxially stacked helices: two stems plus a loop-loop interaction helix . Experimental constraints derived from heteronuclear and homonuclear NMR data on 13C and 15N-labeled RNA led to a structure for the loop-loop helix with an average root-mean-square deviation of 0.83 (+/-0.10) A for 33 converged structures relative to the average structure . The loop-loop helix of the kissing complex is distorted compared to A-form RNA . Its major groove is blocked by the phosphodiester bonds that connect the first loop residue of each hairpin with its own stem, and it is flanked by two negatively charged phosphate clusters . The loop-loop helix has alternating helical twists between adjacent base-pairs . The base-pairs at the helix junctions are overwound and three base-pairs near the helix junctions adopt high propeller twists . All these changes reduce the distance needed for the bridging phosphodiester bonds connecting each stem and loop to cross the major groove of the loop-loop helix, and result in a deformed RNA helix with localized perturbations in the minor groove surface . The alternating helical twist pattern, plus other distortions in the loop-loop helix may be important for Rom protein recognition of the kissing hairpin complex. Proc Natl Acad Sci U S A, 1997 May 27, 94(11), 5831 - 6 Positionally cloned human disease genes: patterns of evolutionary conservation and functional motifs; Mushegian AR et al.; Positional cloning has already produced the sequences of more than 70 human genes associated with specific diseases . In addition to their medical importance, these genes are of interest as a set of human genes isolated solely on the basis of the phenotypic effect of the respective mutations . We analyzed the protein sequences encoded by the positionally cloned disease genes using an iterative strategy combining several sensitive computer methods . Comparisons to complete sequence databases and to separate databases of nematode, yeast, and bacterial proteins showed that for most of the disease gene products, statistically significant sequence similarities are detectable in each of the model organisms . Only the nematode genome encodes apparent orthologs with conserved domain architecture for the majority of the disease genes . In yeast and bacterial homologs, domain organization is typically not conserved, and sequence similarity is limited to individual domains . Generally, human genes complement mutations only in orthologous yeast genes . Most of the positionally cloned genes encode large proteins with several globular and nonglobular domains, the functions of some or all of which are not known . We detected conserved domains and motifs not described previously in a number of proteins encoded by disease genes and predicted functions for some of them . These predictions include an ATP-binding domain in the product of hereditary nonpolyposis colon cancer gene (a MutL homolog), which is conserved in the HS90 family of chaperone proteins, type II DNA topoisomerases, and histidine kinases, and a nuclease domain homologous to bacterial RNase D and the 3'-5' exonuclease domain of DNA polymerase I in the Werner syndrome gene product. J Mol Biol, 1997 May 23, 268(5), 840 - 56 Initiation of replication of plasmid pMV158: mechanisms of DNA strand-transfer reactions mediated by the initiator RepB protein; Moscoso M et al.; The initiator RepB protein of the rolling circle-replicating plasmid pMV158 has nicking-closing (topoisomerase I-like) activities on supercoiled DNA . RepB is also able to perform a strand-transfer reaction on a single-stranded DNA substrate that contains its target . Several attempts at capturing covalent protein-DNA intermediates were made to identify the mechanism of RepB-mediated activity . Whereas RepB did not generate stable complexes with its target DNA, employment of single-stranded oligonucleotides containing a chiral phosphorothioate in the target DNA allowed us to follow the process of RepB-mediated strand-transfer reaction . This reaction occurred through a number of even steps because the chirality of the phosphorothioate at the reaction site was retained after RepB-mediated strand transfer . This finding suggests the existence of a covalent intermediate during the strand-transfer reaction between the protein and its target DNA . By site-directed mutagenesis at the codon for Tyr99 of RepB, and purification and assay of activity of the mutant protein variants, we showed that the Tyr99 residue is involved in the nucleophilic attack of RepB to its cognate DNA. Proc R Soc Lond B Biol Sci, 1997 May 22, 264(1382), 725 - 30 Genotypic variation among different phenotypes within aphid clones; Lushai G et al.; Most aphid species Hemiptera: Aphididae are parthenogenetic between periods of sexual reproduction . They are also highly polyphenic, with different adult morphs occurring in the life cycle, piz . winged, wingless, asexual and sexual . It is assumed that aphids born in a parthenogenetic clonal lineage are genetically identical regardless of the final adult form with the exception of sexual forms) . Using the randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) we have found that different asexual adult phenotypes winged and wingless of some clones of two cereal aphid species (the grain aphid, Sitobion avenae (F.) and the bird-cherry aphid . Rhopalosiphum padi (L.) may be distinguished by the presence or absence of one or more RAPD-PCR bands . In three of nine clones examined, such differences were found, and Southern blotting and hybridization of the discriminating bands confirmed these to be of aphid origin, rather than due to endosymbiotic bacteria or contaminating fungi . The main 248 and 296 bp bands, in the two species respectively, were sequenced and found to be A/T rich . The smaller band showed 57% homology with white striated muscle over a stretch of 90 bp . Genomic DNA treated with dimethyl sulphoxide to remove secondary structures still showed differences in RAPD-PCR profiles between winged and wingless morphs within the unusual clones . This discovery may be widespread and therefore it is important to understand the phenomenon in relation to clonal organisms. J Exp Med, 1997 May 19, 185(10), 1785 - 92 Role of repetitive antigen patterns for induction of antibodies against antibodies; Fehr T et al.; Antibody responses against antibodies, such as rheumatoid factors, are found in several immunopathological diseases and may play a role in disease pathogenesis . Experience shows that they are usually difficult to induce experimentally . Antibodies specific for immunoglobulin constant regions (anti-allotypic) or for variable regions (anti-idiotypic) have been investigated in animal models; the latter have even been postulated to regulate antibody and T cell responses via network-like interactions . Why and how such anti-antibodies are induced during autoimmune diseases, has remained largely unclear . Because repetitively arranged epitopes in a paracrystalline structure of a viral envelope cross-link B cell receptors efficiently to induce a prompt T-independent IgM response, this study used immune complexes containing viruses or bacteria to evaluate the role of antigen pattern for induction of anti-antibody responses . We present evidence that antibodies bound to strictly ordered, but not to irregularly arranged, antigens dramatically enhance induction of anti-antibodies, already after a single immunization and without using adjuvants . The results indicate a novel link between anti-antibody responses and infectious agents, and suggest a similar role for repetitive self-antigens such as DNA or collagen involved in chronic immunopathological diseases. FEMS Microbiol Lett, 1997 May 15, 150(2), 255 - 62 Characterization of monoclonal antibodies for rapid identification of Actinomyces naeslundii in clinical samples; Thurnheer T et al.; The purpose of this study was to generate highly specific serological reagents for the quantitative identification of Actinomyces naeslundii in clinical samples, in particular dental plaque . Balb/c mice were immunized with pasteurized human A . naeslundii strains representing different genospecies and serotypes . Ten hybrid cell lines secreting monoclonal antibodies reactive with A . naeslundii were isolated and characterized . Antibody specificity was determined by indirect immunofluo-rescence and enzyme-linked immunosorbent assay using strains from 59 species and by immunofluorescence analyses of supragingival plaque from 10 gingivitis patients . Nine monoclonal antibodies reacted selectively with A . naeslundii, whereas one additionally bound to Actinomyces israelii . They recognized at least nine different epitopes with characteristic expression patterns among the test strains . Six clusters of antigenically unique or closely related strains could be distinguished . Clusters 1, 4, and 5 represented by 12, 18, and 5 strains, respectively, comprised over 80% of the A . naeslundii strains tested . All reference strains for genospecies 1 grouped with cluster 1 . Strains associated with genospecies 2 fell into clusters 4 and 5 . Tests with mutant strains indicated that three monoclonal antibodies recognize type 2 and one type 1 fimbriae of genospecies 2 . Only four isolates grouped with clusters 2 and 3 characterized by the expression of cluster-specific antigens . Interestingly, cluster 2 and 3 bacteria were markedly more abundant in vivo than indicated by their sparse representation in our strain collection . Overall, all but one of the new monoclonal antibodies should prove of value for the serological classification and rapid quantitative determination of A . naeslundii in clinical samples. Biochem J, 1997 May 15, 324 ( Pt 1), 243 - 8 Glutathione S-transferases from the white-rot fungus, Phanerochaete chrysosporium; Dowd CA et al.; A glutathione S-transferase (GST) was purified to homogeneity from the white-rot fungus, Phanerochaete chrysosporium, by affinity chromatography on glutathione-agarose followed by Mono-Q ion-exchange FPLC . This protein immunoblotted with antisera to rat Theta class GST 5-5 and also showed N-terminal sequence similarity to the Theta class, including the presence of a conserved serine residue that has been specifically implicated in catalysis in this class {Wilce, Board, Feil and Parker (1995) EMBO J . 14, 2133-2143} and other residues conserved in plant sequences . Catalytic activity was found to be highly labile in the purified protein, although preliminary evidence for activity (approx . 120 m-units/mg) with 1,2-epoxy-3-(p-nitrophenoxy)propane was obtained in some preparations . The enzyme seems to be a dimer with a subunit molecular mass of 25 kDa by SDS/PAGE . The native molecular masses estimated by non-denaturing electrophoresis and by Superose-12 gel filtration were 58 and 45 kDa respectively . A second protein purified in this study also gave low level of activity with 1,2-epoxy-3-(p-nitrophenoxy)propane and had a subunit molecular mass of 28 kDa (native size 62-63 kDa), but did not immunoblot with any GST class and seemed to be N-terminally blocked. Transplantation, 1997 May 15, 63(9), 1339 - 45 Split tolerance induced by immunotoxin in a rhesus kidney allograft model; Fechner JH Jr et al.; BACKGROUND: Renal allografts were performed in rhesus monkeys using FN18-CRM9, a potent immunotoxin capable of depleting T cells to less than 1% of baseline levels in blood and lymph nodes, as a preparative agent . We have recently reported that animals pretreated with FN18-CRM9 1 week before transplantation without further immunosuppression had prolonged graft survival time compared with control animals, and frequently became tolerant . METHODS: This report examines the alloimmune responses of recipient monkeys to the donor, including cytotoxic T lymphocyte precursor (CTLp) frequency, mixed lymphocyte response, and antidonor IgG response . RESULTS: CTLp frequencies declined significantly (P<0.01) after FN18-CRM9 treatment and renal transplantation . This decline in CTLp was initially nonspecific, as CTLp frequencies against third-party animals also declined (P<0.01) . The decrease in CTLp was maintained in five of five animals tested 6 months after transplant . However, unresponsiveness was limited to the CTL arm of the immune response as antidonor IgG was detected in four of four animals tested, and the 5-day mixed lymphocyte response stimulation index and relative response were not significantly different before and after transplant . In long-term survivors (>150 days), an increase in anti-third-party CTLp was detected 1 month after grafting with third-party skin . No change was seen in the antidonor CTLp frequency after donor skin grafting, indicating that a specific defect in the antidonor CTL response had developed . CONCLUSIONS: These data suggest that FN18-CRM9 treatment of rhesus monkeys allows the development of specific down-regulation of antidonor CTL activity in renal allograft recipients. Nucleic Acids Res, 1997 May 15, 25(10), 2035 - 6 Getting more from the two-hybrid system: N-terminal fusions to LexA are efficient and sensitive baits for two-hybrid studies; Beranger F et al.; Two-hybrid methods detect interactions between two proteins fused at the C-termini of, respectively, a DNA-binding domain and the activation domain of a transcriptional activator . Thus the N-terminus of none of these proteins is available for interaction . We have tested whether a bait protein with a reverted polarity (i.e . N-bait-LexA-C) is suitable for two-hybrid interaction . We show that such constructs give a specific interaction signal, and document two cases where the sensitivity is dramatically increased . Such constructs might lead to the identification of partners missed during classical two-hybrid screens. Proc Natl Acad Sci U S A, 1997 May 13, 94(10), 4853 - 60 Optical trapping and manipulation of neutral particles using lasers; Ashkin A; The techniques of optical trapping and manipulation of neutral particles by lasers provide unique means to control the dynamics of small particles . These new experimental methods have played a revolutionary role in areas of the physical and biological sciences . This paper reviews the early developments in the field leading to the demonstration of cooling and trapping of neutral atoms in atomic physics and to the first use of optical tweezers traps in biology . Some further major achievements of these rapidly developing methods also are considered. J Mol Biol, 1997 May 2, 268(2), 412 - 23 Apoprotein structure in the LH2 complex from Rhodopseudomonas acidophila strain 10050: modular assembly and protein pigment interactions; Prince SM et al.; The refined structure of the peripheral light-harvesting complex from Rhodopseudomonas acidophila strain 10050 reveals a membrane protein with protein-protein interactions in the trans-membrane region exclusively of a van der Waals nature . The dominant factors in the formation of the complex appear to be extramembranous hydrogen bonds (suggesting that each apoprotein must achieve a fold close to its final structure in order to oligomerize), protein-pigment and pigment-pigment interactions within the membrane-spanning region . The pigment molecules are known to play an important role in the formation of bacterial light-harvesters, and their extensive mediation of structural contacts within the membrane bears this out . Amino acid residues determining the secondary structure of the apoproteins influence the oligomeric state of the complex . The assembly of the pigment array is governed by the apoproteins of LH2 . The particular environment of each of the pigment molecules is, however, influenced directly by few protein contacts . These contacts produce functional effects that are not attributable to a single cause, e.g . the arrangement of an overlapping cycle of chromophores not only provides energy delocalisation and storage properties, but also has consequences for oligomer size, pigment distortion modes and pigment chemical environment, all of which modify the precise function of the complex . The evaluation of site energies for the pigment array requires the consideration of a number of effects, including heterogeneous pigment distortions, charge distributions in the local environment and mechanical interactions. J Biol Chem, 1997 May 2, 272(18), 11916 - 23 A novel Drosophila receptor tyrosine kinase expressed specifically in the nervous system . Unique structural features and implication in developmental signaling; Oishi I et al.; We report the identification and characterization of Dnrk (Drosophila neurospecific receptor kinase), a Drosophila gene encoding a putative receptor tyrosine kinase (RTK) highly related to the Trk and Ror families of RTKs . During Drosophila embryogenesis, the Dnrk gene is expressed specifically in the developing nervous system . The Dnrk protein possesses two conserved cysteine-containing domains and a kringle domain within its extracellular domain, resembling those observed in Ror family RTKs (Ror1, Ror2, and a Drosophila Ror, Dror) . This protein contains the catalytic tyrosine kinase (TK) domain with two putative ATP-binding motifs, resembling those observed in another Drosophila RTK (Dtrk) that mediates homophilic cell adhesion . The TK domain of Dnrk, expressed in bacteria or mammalian cells, exhibits apparent autophosphorylation activities in vitro . The TK domain lacking the distal ATP-binding motif also exhibits autophosphorylation activity, yet to a lesser extent . In addition to its TK activity, there are several putative tyrosine-containing motifs that upon phosphorylation may interact with Src homology 2 regions of other signaling molecules . Collectively, these results suggest that Dnrk may play an important role in neural development during Drosophila embryogenesis. J Biol Chem, 1997 May 2, 272(18), 11812 - 5 Identification of a protein kinase from Dictyostelium with homology to the novel catalytic domain of myosin heavy chain kinase A; Clancy CE et al.; Myosin II assembly and localization into the cytoskeleton is regulated by heavy chain phosphorylation in Dictyostelium . The enzyme myosin heavy chain kinase A (MHCK A) has been shown previously to drive myosin filament disassembly in vitro and in vivo . MHCK A is noteworthy in that its catalytic domain is unrelated to the conventional families of eukaryotic protein kinases . We report here the cloning and initial biochemical characterization of another kinase from Dictyostelium that is related to MHCK A . When the segment of this protein that is similar to the MHCK A catalytic domain was expressed in bacteria, the resultant protein displayed efficient autophosphorylation, phosphorylated Dictyostelium myosin II, and also phosphorylated a peptide substrate corresponding to a portion of the myosin II tail . We have therefore named this gene myosin heavy chain kinase B . These results provide the first confirmation that sequences in other proteins that are related to the MHCK A catalytic domain can also encode protein kinase activity . It is likely that the related segments of homology present in rat eukaryotic elongation factor-2 kinase and a putative nematode eukaryotic elongation factor-2 kinase also encode the catalytic domains of those enzymes. Clin Exp Rheumatol, 1997 May-Jun, 15 Suppl 17, S3 - 14 Joint destruction in rheumatoid arthritis: biological bases; Kingsley G et al.; The pathogenesis of rheumatoid arthritis (RA) can be explained through two main hypotheses: macrophage-fibroblast and macrophage-T cell interactions . The interplay between the various populations is influenced by a strong genetic component, which determines the severity of the disease in some cohorts of patients attending referral centers . The key question of the nature of the antigen(s) driving joint inflammation still remains unsolved . Exogenous antigens such as viruses or bacteria have long been searched for in the synovial fluids as well as in tissues, but convincing evidence of their pathogenic role are lacking . Data have been accumulated on the possible role of autoantigens, such as the spliceosomes, filaggrin, calpastatin, type II collagens, or other endogenous peptides, but no definite role regarding their potential contribution to the activation of T cells has been established . Once the process starts, a progressive recruitment of inflammatory T cells and macrophages into the joints occurs through a complex series of adhesion and migratory events . The key driving steps leading to synovial inflammation and cartilage destruction, along with the potential contribution of some key molecules, have been described, thus opening possible perspectives for a therapeutic approach. Dev Comp Immunol, 1997 May-Jun, 21(3), 277 - 85 Localization of a putative inositol 1,4,5-triphosphate receptor in the Limulus granulocyte; Solon E et al.; The horseshoe crab (Limulus polyphemus) granulocyte (GR) degranulates upon contact with bacteria and release factors that mediate an immune response . Stimulated cells produce IP3, which binds to receptors (IP3R, M.W.240-300 kD) that function to release stored Ca2+ into the cytoplasm that mediates degranulation . This mechanism is believed to mediate exocytosis in the Limulus GR but IP3R in the GR has not been shown . The present study utilized monoclonal antibody 4C11 and a commercially available anti-IP3R antibody, both of which label amino acids of the N-terminal of all known isoforms . Electron microscopy, immunohistochemistry, SDS-PAGE, and Western blot analysis, which employed the use of the two antibodies, demonstrates that a putative IP3R exists in the: plasma membrane, smooth surfaced vesicles, nucleus and nuclear membrane . We hypothesize that this putative IP3R is involved in mediating the immune response of the Limulus GR. Nihon Kyobu Shikkan Gakkai Zasshi, 1997 May, 35(5), 583 - 7 {Esophagobronchial fistula and empyema resulting from esophageal carcinoma}; Hippo Y et al.; A 59-year-old woman was admitted to the hospital with a one-month history of hemoptysis, generalized fatigue, and a high fever . A chest X-ray film obtained on admission showed a massive right-sided pleural effusion . Examination of an aspirate showed a high level of amylase, and bacteria that were the same as oral bacteria . Closed drainage yielded ichorous pus and food residues, which led us to the diagnosis of empyema caused by esophageal perforation . Esophagography and fiberoptic esophagoscopy revealed that an esophagobronchial fistula related to an advanced esophageal carcinoma had caused the empyema . Surgical resection was done, and the patient was alive at the time of this writing, 7 months after she was first treated . Esophageal carcinoma is sometimes accompanied by esophagobronchial fistula . Patients with this condition usually have severe respiratory symptoms; those presenting with empyema are rare . Esophageal carcinoma must be carefully ruled out as the cause of empyema. Z Naturforsch {C}, 1997 May-Jun, 52(5-6), 329 - 32 Benzoic acid accumulation in the Pinus thunbergii callus inoculated with the pine wood nematode, Bursaphelenchus xylophilus; Zhang H et al.; Phenylacetic acid (PA), a phytotoxic product of the bacteria accompanying the virulent nematode isolate OKD-3 was detected in the callus of Pinus thunbergii after inoculation with the nematode . The amount of PA detected was large enough to induced the formation and accumulation of benzoic acid (BA) and its conjugates in the callus . These results further support the hypotheses that PA is the pathogenic toxin and that the PA-producing bacterial strains accompanying the pathogenic nematode are the genuine pathogens of the pine wilt disease. Vet Microbiol, 1997 May, 56(1-2), 87 - 98 Development of a specific DNA probe and PCR for the detection of Mycoplasma bovis; Ghadersohi A et al.; Mycoplasma bovis is responsible for several production diseases in cattle, including mastitis, arthritis, pneumonia, abortion and infertility . Current methodologies for detecting and identifying M . bovis are time consuming and difficult . Tests which rely on antigen or antibody detection have poor sensitivity and specificity . In this paper associated protocols for the development of a hybridization probe and PCR are described . A genomic library (SauIIIA digested) was prepared from M . bovis DNA (Colindale Reference Strain: NC10131:02) and cloned into pUC19 . Colony hybridization, using a probe preparation made from purified M . bovis DNA, was used to identify colonies of interest . M . bovis DNA fragments were retrieved from recombinant plasmids by digestion with EcoRI and HindIII . This DNA was used to prepare randomly primed probes for dot blot hybridization analysis with immobilized DNA from M . bovis (two strains), M . dispar, M . agalactiae, M . bovigenitalium (two strains), M . ovipneumoniae, a Group 7 strain, M . arginini and bacteria belonging to different genera . Four probes were found to hybridize only with M . bovis and M . ovipneumoniae DNA, whereas one probe reacted with genomic DNA from only one of the two M . bovis strains . The level of sensitivity of the dot blot hybridization assay was 200 CFU (colony forming units)/mL . To enhance the sensitivity further, an M . bovis-specific PCR assay was developed . The primers were designed using sequences obtained from the probe DNA which discriminated M . bovis from all other Mycoplasma DNA tested . The minimum amount of target DNA that could be detected by the PCR assay was that isolated from 10-20 CFU/mL . The PCR assay was therefore 10 times more sensitive than dot blot hybridization. FEMS Immunol Med Microbiol, 1997 May, 18(1), 61 - 6 Genotypic differentiation of Gardnerella vaginalis by amplified ribosomal DNA restriction analysis (ARDRA); Ingianni A et al.; In total 34 strains of Gardnerella vaginalis were analyzed with various molecular techniques in order to find the possibility of dividing this single species into different genotypes . Classical ribotyping, PCR-ribotyping and restriction analysis of 16S-23S rRNA intergenic spacer sequences were all unsuccessful in genotype differentiation of these bacteria . Only amplified ribosomal DNA restriction analysis (ARDRA) was suitable in recognizing different G . vaginalis genotypes . At least 3-4 genotypes were identified with different restriction enzymes, some of which showed a prevalent distribution in certain of the centers from which they were collected . Although in this study no correlation was found between bacterial vaginosis and any of the genotypes identified, the ARDRA method could prove to be a useful tool for studying the etiopathology and epidemiology of G . vaginalis. Jpn J Antibiot, 1997 May, 50(5), 479 - 84 {Measurement of antibody titers to chlamydial infection and effects of levofloxacin in cystic cervicitis and chlamydial infection}; Chimura T; Generally, clinical symptoms such as abnormal leukorrhea are caused by C . trachomatis, an ordinary bacteria in cervical infection . The effects of levofloxacin administration at a dose of 300 mg/day for 5-14 days were investigated in the subjects (n = 66) after the discussion of cystic cervicitis . The treatment was made in combination with chloramphenicol-solcoseryl tablet for vaginal use . And it was demonstrated that treatment was effective in all subjects . Then, Sero IPALISA, an examination of IgA/IgG antibody was conducted for the screening of Chlamydia infection (n = 258) . The rate of antibody-positive case was 48/160 (30.0%) for the non-pregnant women and 26/98 (26.5%) for the pregnant . The occurrence rates for the women singing in 15-24 and 35-39 years of age were 50 and 41%, respectively . The results from the measurement of the antibody titer were as follows: the rate of IgA/IgG positive care was 61/87 for IgA and 56/87 for IgG when the cut-off index was 1.11 or more . The rates for both antibody were 11/87 (12.6%) and 24/87 (27.6%) for the indexes of 1.11-3.00 and 3.01 or more, respectively . Next, one to three courses levofloxacin at 300 mg/day for 14 days were given to 48 non-pregnant subjects infected with Chlamydia and one to two courses of clarithromycin at 400 mg/day for 14 days were given to 26 pregnant subjects . Side effects have not been noted in any care and there was no changes in the IgA/IgG antibody titer depending on these treatments. J Periodontal Res, 1997 May, 32(4), 345 - 50 Intra-familial transmission and distribution of Prevotella intermedia and Prevotella nigrescens; van Steenbergen TJ et al.; The periodontal bacteria Prevotella intermedia and Prevotella nigrescens have been recently separated from each other . The purpose of this study was to investigate the distribution and routes of transmission of these bacteria among family members . Seven patients with moderate to severe periodontitis were selected . These probands, their spouses and 14 of their children were investigated . The presence of Pr . intermedia and Pr . nigrescens was determined by culture techniques in pooled subgingival plaque samples, in the saliva, on the tongue, tonsils and buccal mucosa . Differentiation of Pr . intermedia and Pr . nigrescens was performed by enzyme electrophoretic mobility . From all 7 patients, as well as 4 spouses and 3 of the children, Pr . intermedia could be isolated . Pr . nigrescens was found in 2 of the 7 patients, in 5 of the spouses and in 5 of the 6 children aged 5-10 yr . In the 8 children aged 0-4 yr both species were seldom isolated . These data are in accordance with earlier findings that Pr . intermedia is associated with periodontitis and Pr . nigrescens with a relatively healthy periodontal condition . Ribotyping of bacteria was performed by hybridization of HindIII restriction endonuclease digests of chromosomal DNA with ribosomal DNA . Isolates from unrelated individuals always had distinct ribotypes . Indistinguishable ribotypes of Pr . intermedia and Pr . nigrescens were found both among married couples and among parents and children . This indicates that intrafamilial transmission of Pr . intermedia and Pr . nigrescens is possible both between adults and between parents and children. Plant Mol Biol, 1997 May, 34(2), 209 - 21 Characterization of nuclease activities and DNA fragmentation induced upon hypersensitive response cell death and mechanical stress; Mittler R et al.; Programmed cell death (PCD) is activated during the response of multicellular organisms to some invading pathogens . One of the key aspects of this process is the degradation of nuclear DNA which is thought to facilitate the recycling of DNA from dead cells . The PCD of tobacco plants (genotype NN) infected with tobacco mosaic virus (TMV) is accompanied by the induction of nuclease activities and the cleavage of nuclear DNA to fragments of about 50 kb . We examined the correlation between the increase in nuclease activities and the fragmentation of nuclear DNA during TMV- and bacteria-induced PCD in tobacco . We found that the increase in nuclease activities did not always correlate with fragmentation of nuclear DNA . Thus, in addition to pathogens that induce PCD, mechanical injury and infiltration of leaves with 1 M sucrose or bacteria that did not induce PCD also resulted in an increase in nuclease activities . Analysis of nuclease activities in total leaf extracts, nuclear extracts, and intercellular fluid (i.e., apoplast) revealed that at least four different nuclease activities are induced during PCD in tobacco; of these at least three appear to be secreted into the intercellular fluid . Although the latter were also induced in response to treatments that did not result in DNA fragmentation, they may function in the recycling of plant DNA during late stages of PCD when the integrity of the plasma membrane is compromised . This suggestion is supported by the finding that DNA degradation occurred late during TMV-induced PCD in tobacco . In addition, the finding of induced nuclease activities in the intercellular fluid raises the possibility that they may serve a protective function by degrading the DNA of invading pathogens. Zentralbl Veterinarmed B, 1997 May, 44(3), 129 - 32 Further characterization of alpha 2-macroglobulin binding properties of Actinomyces pyogenes; Ding H et al.; Binding of 125I-labelled alpha 2-macroglobulin (alpha 2M) to Actinomyces pyogenes was investigated . The binding of alpha 2M proved to be time dependent, saturable, and could be inhibited by unlabelled alpha 2M, but not by fibrinogen, haptoglobin, fibronectin, or albumin . The alpha 2M binding site was sensitive to treatment with proteolytic enzymes and heat, indicating its protein nature . The dissociation constant (Kd) was 4.447 x 10(-9) M, and the number of binding sites per bacterial cell was calculated to be 5200 . The kinetic analysis indicated an homogeneous population of binding sites . Binding of alpha 2M to the surface of A . pyogenes had no significant influence on the phagocytosis of the bacteria by polymorphonuclear leucocytes. J Gastroenterol Hepatol, 1997 May, 12(5), S8 - 10 Current topics on digestive disease in Korea; Choi KW; This is a summary of the research topics, of current interest, relating to digestive disease in Korea . This review is based on the subjects of the papers that were accepted for presentation at the 34th Annual Meeting of the Korean Society of Gastroenterology and the 39th Semiannual Meeting of the Korean Society of Gastrointestinal Endoscopy held November 22-24, 1995, in Seoul . The most popular topics were on Helicobacter pylori infection . These included experimental papers on the pathogenesis of bacteria-associated gastritis and the duodenal ulcer . Recently, the increase in the number of papers published on the motility of the gastrointestinal and pancreaticobiliary tracts is quite remarkable . Molecular genetic works on oncogenesis using cultured tumour cell lines, on the enzyme expression in the biopsied mucosal cells of the small intestine, and on the various expressions of viral genomes in the hepatocytes are among these recent topics of research . Clinical works, such as therapeutic endoscopy in malignant diseases and therapeutic trials in the hepatitis virus-associated chronic liver diseases are also popular topics. Plant J, 1997 May, 11(5), 967 - 82 Intracellular localization of GBF proteins and blue light-induced import of GBF2 fusion proteins into the nucleus of cultured Arabidopsis and soybean cells; Terzaghi WB et al.; The G-box is an important regulatory element found in the promoters of many different genes . Four members of an Arabidopsis gene family encoding basic leucine zipper proteins (GBFs) which bind the G-box have previously been cloned . To study GBFs, a polyclonal antibody was raised against GBF1 expressed in bacteria . This antibody also recognized GBF2 and GBF3 . Immunoblot analysis of nuclear and cytoplasmic fractions from Arabidopsis and soybean (SB-M) cell cultures indicated that over 90% of proteins detected with anti-GBF1 were cytoplasmic . Electrophoretic mobility shift assays indicated that over 90% of G-box binding activity was cytoplasmic . DNA affinity chromatography demonstrated that each protein detected with anti-GBF1 specifically bound the G-box . To study individual GBFs, DNA constructs fusing GBF1, GBF2 and GBF4 to GUS were made and assayed by transient expression in SB-M protoplasts . Of GUS:GBF1 proteins, 50-62% were localized in the cytoplasm under all conditions tested, while 97% of GUS:GBF4 was localized in the nucleus . By contrast, whereas about 50% of GUS:GBF2 was found in the cytoplasm of dark-grown cells, over 80% of this protein was found in the nucleus in cells cultured under blue light . Deletion analysis of GBF1 identified a region between amino acids 112 and 164 apparently required for cytoplasmic retention . These results suggest the intriguing possibility that limitation of nuclear access may be an important control on GBF activity . In particular, GBF2 is apparently specifically imported into the nucleus in response to light. Int J Radiat Biol, 1997 May, 71(5), 515 - 7 Whole library-amplification and labelling of human chromosome-specific composite probes for fluorescence in situ hybridization (FISH) using PCR; Oberheitmann B; Chromosome painting with human-specific, whole-plasmid library probes has become a widespread technique in cytogenetic research and radiation biology . Fast and convenient methods for probe amplification and labelling are required that conserve the complexity of the libraries and stain the entire length of selected chromosomes . The present study uses PCR for amplification and labelling of the whole bluescribe plasmid libraries pBS1, pBS4 and pBS12 using M13 forward and reverse sequencing primer . Amplification and labelling can be accomplished within a few hours with a minimum of laboratory equipment and costs . It is therefore suitable for all laboratories that do not have the facilities for growing transformed bacteria containing plasmids with inserts of human origin. Biol Chem, 1997 May, 378(5), 381 - 91 Functional analysis of the yeast 40 kDa cyclophilin Cyp40 and its role for viability and steroid receptor regulation; Warth R et al.; We have identified and characterized a homolog of the 40 kDa cyclophilins in the budding yeast Saccharomyces cerevisiae . At the amino acid level, this novel yeast cyclophilin, termed Cyp40, is 47% identical to human cyclophilin-40 . Recombinant Cyp40 produced in bacteria has a peptidyl-prolyl cis-trans isomerase activity with a catalytic efficiency (k{cat}/K{m}) of 0.5 x 10(6)M(-1)s(-1), which can be inhibited by cyclosporin A with an IC50 value of 60nM . Using a polyclonal antibody against Cyp40 we have found that Cyp40 is predominantly cytoplasmic, and that its expression is induced 3-4-fold by heat shock . Moreover, Cyp40 can be coprecipitated from yeast extracts with the cytosolic molecular chaperone Hsp90 . Surprisingly, a Cyp40-deficient yeast strain is fully viable at normal and elevated temperatures . Cyp40 is also dispensable for normal regulation of vertebrate steroid receptors in yeast . While other immunophilins could conceivably compensate a Cyp40 defect, our results are compatible with the notion that immunophilins may be fortuitous partners in the biochemically established steroid receptor-Hsp90 complex. Eur J Gastroenterol Hepatol, 1997 May, 9(5), 457 - 61 Helicobacter pylori-human polymorphonuclear leucocyte interaction in the presence of ammonia; Mayo K et al.; OBJECTIVE: To determine if the ammonia produced by Helicobacter pylori affects the phagocytic ability of human polymorphonuclear leucocytes as measured by the oxidative burst . METHODS: Interactions between opsonized urease-positive and -negative strains of H . pylori with polymorphonuclear leucocytes were studied in two series of experiments . In the first series of experiments, concentrations from 0 to 50 mM of NH4Cl were added to polymorphonuclear leucocytes . In the second series of experiments, bacteria were pre-incubated for 1 h with urea (0 to 50 mM) before addition of phagocytes . Luminol-dependent chemiluminescence was measured every 5 min over a 50-min period . The pH was verified in each treatment . RESULTS: Inhibition of chemiluminescence, increasing with concentration, was noted in all treatments when NH4Cl was added . When urea was added to urease-positive strains, chemiluminescence was significantly reduced when compared to the urease-negative strain and the zymosan control . This effect could not be attributed to a change in pH in the experiments using NH4Cl or urea at a concentration of 5 mM and 10 mM . CONCLUSION: Ammonia generated by H . pylori may contribute to the decreased activity of polymorphonuclear leucocytes in vivo. Comp Biochem Physiol B Biochem Mol Biol, 1997 May, 117(1), 61 - 74 Nongenetic variation, genetic-environmental interactions and altered gene expression . II . Disease, parasite and pollution effects; Poly WJ; The use of protein electrophoretic data for determining the relationships among species or populations is widespread and generally accepted . However, there are many confounding factors that may alter the results of an electrophoretic study and may possibly allow erroneous conclusions to be drawn in taxonomic, systematic or population studies . Measured enzyme activities can also be affected significantly . Parasites, disease and pollution can affect levels of enzyme activity, and electrophoretic results can be affected both quantitatively and qualitatively . Blood serum is particularly vulnerable to variation to variation due to disease, pollution or parasites because damaged tissues may release tissue-specific enzymes into the bloodstream . Capture, handling, chemical treatments, bacteria, natural toxins and consumed food may also contribute to variation . Potential pollution impacts at specimen collection sites should be investigated, and study organisms should be inspected and/or treated for detection and elimination of parasites and disease. Mol Microbiol, 1997 May, 24(3), 653 - 64 Analysis of the architecture of the transcription factor sigma N (sigma 54) and its domains by circular dichroism; Missailidis S et al.; Enhancer-dependent transcription in bacteria requires the alternative transcription factor sigma N (sigma 54), which forms an RNA polymerase holoenzyme that binds promoters as a transcriptionally inactive complex . We have examined the structure of sigma N by circular dichroism (CD) analysis . The sigma N protein and its domains are well structured in the absence of the core RNA polymerase subunits or promoter DNA . Denaturation of sigma N by temperature as followed by changes in CD shows a concomitant loss of secondary and tertiary structures with a melting temperature of 36 degrees C . The secondary structure displays a two-state melting curve with a second Tm of 85 degrees C . The amino-terminal Region I activation domain together with the acidic Region II does not contribute to the two-state melting . In marked contrast, the integrity of the C-terminal DNA-binding domain is required for the two-state melting . Measurements of pKb also demonstrated that a C-terminal part of sigma N, but not regions I or I + II, is required for the structural integrity of sigma N at high pH . Measurements of pKa suggested that alpha-helical structures are important in sigma N for the establishment of tertiary structural elements . The tertiary structure near ultraviolet CD signals of sigma N do not require regions I or I + II but were strongly diminished by C-terminal truncation of sigma N . Promoter DNA binding resulted in a conformational change in sigma N, permitting the determination of a binding constant . A typical B-DNA conformation was adopted by the promoter DNA . Implications for the modular domain organization of sigma N, the function of C-terminal sequences, and domain communication and its role in activation of transcription are discussed. Mol Microbiol, 1997 May, 24(3), 629 - 42 Transcriptional regulation of the macrophage-induced gene (gspA) of Legionella pneumophila and phenotypic characterization of a null mutant; Abu Kwaik Y et al.; Expression of the global stress protein gene (gspA) is induced during the intracellular infection of macrophages and upon exposure of Legionella pneumophila to in vitro stress stimuli . Transcription of gspA is regulated by two promoters, one of which is regulated by the sigma 32 heat-shock transcription factor . We utilized a gspA promoter fusion to a promoter less lacZ to probe the phagososmal 'microenvironment' for the kinetics of exposure of intracellular L . pneumophila to stress stimuli . Expression through the gspA promoter was constitutively induced by approx . 16-fold throughout the intracellular infection, and occurred predominantly through the sigma 32-regulated promoter . Expression of the gspA promoter was induced approx . 4.5-fold, 5-, 11- and 9-fold upon exposure of L . pneumophila to heat shock, oxidative stress, acid shock, and osmotic shock, respectively . An isogenic insertion mutant of L . pneumophila in gspA (strain AA224) was constructed by allelic exchange in the wild-type strain AA200 . Compared to in vitro-grown wild-type strain AA200, AA224 was more susceptible to all four in vitro stress stimuli . The wild-type phenotypes were restored to strain AA224 by complementation with a plasmid containing wild-type gspA . There was no difference between the wild-type strain and the gspA mutant in cytopathogenicity to U937 cells or in their kinetics of intracellular replication within macrophages and amoebae . However, compared to in vitro-grown bacteria, macrophage-grown and amoebae-grown AA200 and AA224 showed an equal and dramatic increase in resistance to in vitro stress stimuli . Our data showed that regardless of the capacity of L . pneumophila to subvert the microbicidal mechanisms of the macrophage, intracellular L . pneumophila is exposed to a high level of stress stimuli throughout the intracellular infection . Although the GspA protein is required for protection of the bacteria against in vitro stress stimuli, and is induced during intracellular multiplication, the loss of its function is probably compensated for by other macrophage-induced and stress-induced proteins within the intracellular environment. Biol Pharm Bull, 1997 May, 20(5), 517 - 21 Pharmacokinetic study of paeonimetabolin I, a major metabolite of paeoniflorin from paeony roots; Heikal OA et al.; Plasma concentrations of paeoniflorin (PF) and its major metabolite, paeonimetabolin I (PM-I), were estimated after oral administration of PF to rats at doses of 0.5 and 5 mg/kg . The maximal plasma concentrations (Cmax) of PF were 9.9 and 20.3, and those of PM-I were 16.5 and 101.7 ng/ml at each dose, respectively . The times to Cmax (tmax) of PF were 11.6 and 13.3, and those of PM-I were 60 and 80 min, respectively . The AUC(0-180) of PM-I were 1873 and 12358, and those of PF were 300 and 1174 ng min/ml, respectively . On the other hand, after intravenous administration of PM-I to rats at doses of 0.2 and 2 mg/kg (equal in molar ratio to 0.5 and 5 mg/kg PF), the plasma concentration of PM-I decreased rapidly and the plasma concentration-time curve profile of it fitted well with the two-compartment model at each dose, with terminal half lives (t1/2) of 90.9 and 90.6 min . The Vdss values were 0.91 and 3.79 l/kg, the CLtot values were 8.7 and 39.9 ml/min kg, and the AUC(0-180) values were 5614.1 and 13176.0 ng min/ml, at each dose, respectively . The significant increase in Vdss and CLtot with increasing doses suggested dose-dependent pharmacokinetics . When PM-I was given orally at the same doses, the following parameters were shown: Cmax of 102.2 and 285 ng/ml at tmax 6.2 and 7.5 min and AUCs of 4145.6 and 14182.1 ng min/ml, at each dose . The bioavailability (F) values were 0.8 and 1.07, respectively . These findings indicated that the high percentage of PM-I transformed by intestinal bacteria was rapidly absorbed from the gastrointestinal tract, and a significantly high concentration of PM-I, rather than PF, was present in the plasma after oral administration of PF. Mol Biochem Parasitol, 1997 May, 86(1), 37 - 47 Isolation and molecular characterization of the bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase gene from Toxoplasma gondii; Pashley TV et al.; Toxoplasma gondii is an important cause of AIDS-related opportunistic infection, manifest as toxoplasmic encephalitis . The clinical treatment of choice is the synergistic combination of antifolate agents, pyrimethamine and sulphadiazine, of which the latter targets the parasite's dihydropteroate synthase (DHPS) activity . Here, we describe the isolation of the gene encoding this activity in T . gondii . The nucleotide sequence contains an open reading frame interrupted by five introns, which encodes a protein of 664 amino acids with an M(r) of 72991 . Sequence analysis revealed that, in addition to DHPS, the predicted protein contains a second enzyme function, hydroxymethyldihydropterin pyrophosphokinase (PPPK) . This enzyme immediately precedes DHPS in the folate biosynthetic pathway . The bifunctional arrangement of the T . gondii pppk-dhps gene is the same as that observed in the related protozoan parasite, Plasmodium falciparum, and confirms previous biochemical data that these activities were inseparable . Recently, specific mutations within conserved motifs of the DHPS gene of P . falciparum have been identified which give rise to sulphonamide drug resistance . Analysis of seven clinical isolates of T . gondii did not reveal any similar mutations in this limited sample of organisms that had been subjected to drug pressure. J Dairy Sci, 1997 May, 80(5), 1005 - 28 Bovine acidosis: implications on laminitis; Nocek JE; Bovine lactic acidosis syndrome is associated with large increases of lactic acid in the rumen, which result from diets that are high in ruminally available carbohydrates, or forage that is low in effective fiber, or both . The syndrome involves two separate anatomical areas, the gastrointestinal tract and body fluids, and is related to the rate and extent of lactic acid production, utilization, and absorption . Clinical manifestations range from loss of appetite to death . Lactic acid accumulates in the rumen when the bacteria that synthesize lactic acid outnumber those that utilize lactic acid . The systemic impact of acidosis may have several physiological implications, including laminitis, a diffuse aseptic inflammation of the laminae (corium) . Although a nutritional basis for the disease exists, etiology includes a multitude of interactive factors, such as metabolic and digestive disorders, postpartum stress, and localized trauma, which lead to the release of vasoactive substances that trigger mechanisms that cause degenerative changes in the foot . The severity of laminitis is related to the frequency, intensity, and duration of systemic acidotic insults on the mechanisms responsible for the release of vasoactive substance . The critical link between acidosis and laminitis appears to be associated with a persistent hypoperfusion, which results in ischemia in the digit . Management of acidosis is critical in preventing laminitis . High producing dairy herds attempting to maximize energy intake are continually confronted with subclinical acidosis and laminitis . Management of feeding and husbandry practices can be implemented to reduce incidence of disease. J Dairy Sci, 1997 May, 80(5), 984 - 94 Uterine health and disorders; Lewis GS; Nonspecific uterine infections reduce the reproductive efficiency of cows and the profit potential of dairy farms . Fortunately, most cows do not develop severe uterine infections . The term uterine infection indicates that the uterus is contaminated with pathogenic organisms . Actinomyces pyogenes, either alone or with other bacteria, is often associated with uterine infections . When A . pyogenes was isolated from uterine fluids after d 21 postpartum, cows developed severe endometritis and were infertile at first service . However, the exact causes of uterine infections are unknown but are associated with several factors . Cows with dystocia, retained placenta, twins or still-births, and various metabolic disorders are more likely to develop metritis than are other cows . Aberrant immune function before and after calving seems to predispose cows to severe uterine infections . Few cows die from uterine infections, but cows with uterine infections are more likely to be culled for poor reproductive performance . Also, uterine infections can reduce milk production, and some treatments contaminate milk . Because they are nonspecific, uterine infections are difficult to prevent; attention to sanitation and periparturient hygiene, especially during assisted calving, may be the best defense . Evidence that aberrant immuno function predisposes cows to uterine infections indicates that methods for regulating immune function in periparturient cows have the potential for preventing or treating uterine infections. Am J Physiol, 1997 May, 272(5 Pt 1), G1249 - 57 Taurochenodeoxycholic acid ameliorates and ursodeoxycholic acid exacerbates small intestinal inflammation; Uchida A et al.; Intraluminal bacteria, food intake, and bile play important roles in indomethacin-induced small intestinal inflammation in rats . Tauroursodeoxycholic acid (TUDCA) and ursodeoxycholic acid (UDCA) inhibit hydrophobic bile acid-induced damage in various types of cells . We investigated the effects of these bile acids along with the possible influence of other bile acids on this model of inflammation . Clinical and intestinal inflammatory parameters and bile secretion were assessed after 7-day dietary bile acid pretreatments and subsequent indomethacin injections . UDCA significantly enhanced indomethacin-associated reductions in food intake and body weight, increases in gross inflammatory scores and myeloperoxidase activity, and the shortening of small intestinal length . Taurochenodeoxycholic acid (TCDCA) significantly normalized the clinical inflammatory parameters, prevented indomethacin-induced increases in the biliary contents of secondary bile acids and hydrophobicity index, and tended to attenuate the intestinal inflammation . Although elevated biliary levels of muricholic acids and a decreased hydrophobicity index were evident before indomethacin injection in the TCDCA case, these alterations could not explain the TCDCA-mediated protection . Dietary TCDCA attenuates whereas UDCA exacerbates intestinal inflammation in this model . Alterations in the bile composition (increases in UDCA and chenodeoxycholic acid) may explain the observed modification effects. Am J Physiol, 1997 May, 272(5 Pt 1), G1028 - 33 Insights into human colonic physiology obtained from the study of flatus composition; Suarez F et al.; To better understand the physiology of colonic gas production, each flatus passage of 16 subjects over a 4-h period was analyzed by gas chromatography for N2, O2, H2, CO2, CH4, and for odoriferous sulfur-containing gases . Appreciable intraindividual and enormous interindividual variability was observed, indicating that each gas passage reflected the interaction of highly variable liberation and/or removal mechanisms . The predominant flatus gas was CO2, H2, and N2 in seven, six, and three subjects, respectively . Gases produced intraluminally (H2, CO2, and CH4) comprised approximately 74% of flatus, and rapid CO2 and H2 productions were responsible for high passage rates . A positive correlation between flatus H2 and CO2 suggested that CO2, like H2, mainly was a bacterial product . Whereas methanogens and H2S-producing bacteria usually are mutually exclusive in feces, CH4 and H2S did not negatively correlate, indicating coexistence of both organisms in the colon . We conclude that analysis of flatus composition provides a novel means of assessing colonic physiology, particularly ongoing bacterial metabolism throughout the unperturbed colon. Scand J Gastroenterol, 1997 May, 32(5), 445 - 54 Colony variation of Helicobacter pylori: pathogenic potential is correlated to cell wall lipid composition; Bukholm G et al.; BACKGROUND: Differences in expression of disease after infection with Helicobacter pylori have so far been connected with host factors and bacterial interstrain variation . In this study, spontaneous and ecology-mediated intrastrain variation was examined . METHODS: Four clinical isolates of H . pylori were shown to give rise to two colony forms . Bacterial morphology was examined by electron microscopy . Bacterial fractions were examined for proteins using ion exchange chromatography and SDS-PAGE; for lipids using thin-layer chromatography, lipid anion-exchange chromatography, column chromatography on silica gel, 31P-NMR, gas chromatography and mass spectrometry . Bacterial in vitro invasiveness and adhesiveness were examined in two different systems, and urease and VacA toxin were assayed by Western blot analysis . RESULTS: H . pylori was shown to give rise to two colony forms: at normal pH the population was dominated by L colonies . One strain was chosen for further studies . Bacteria from L colonies retained VacA toxin and urease, did not invade or adhere to epithelial cells, and contained normal quantities of phosphatidylethanolamine . In a small frequency, spontaneous S colonies were formed . Bacteria from these colonies released VacA and urease, adhered to and invaded epithelial cells and contained increased amounts of lysophosphatidyl ethanolamine and phosphatidyl serine . After addition of HCl to the culture medium (pH6), almost only S colonies were formed . The results demonstrate that environmental factors, such as HCl, can change the bacterial cell wall, and thereby enhance expression of virulence factors of H . pylori in vitro . A similar in vivo variation would have implications for our understanding of the interaction between HCl secretion in the gastric mucosa and H . pylori in the development of peptic ulcer disease. J Invertebr Pathol, 1997 May, 69(3), 218 - 22 Encapsulation of the entomopathogenic nematode Steinernema feltiae in Tipula oleracea; Peters A et al.; The encapsulation response of Tipula oleracea to the entomopathogenic nematode Steinernema feltiae was investigated by exposing the insects to nematode dauer juveniles (DJs) and by injecting DJs with and without the symbiotic bacteria Xenorhabdus bovienii . The encapsulation response varied considerably between individual insect larvae . The variation could not be attributed to a more or less scattered nematode invasion over time since it was also recorded after simultaneous injection of a fixed DJ dose . The proportion of encapsulated nematodes declined with increasing dose (injected DJs/larva) from approx 80% for 1 DJ/larva to 33-34% for 20 DJ/larva . Tipula oleracea larvae were capable of encapsulating nematodes with and without symbionts inside the hemocoel; however, at doses of 10 and 20 DJ/larva, axenic nematodes were encapsulated less frequently than monoxenic nematodes . Injected axenic nematodes that were not encapsulated did not develop in T . oleracea larvae but disappeared from the insect's hemocoel . Coinjection of symbiotic bacteria increased encapsulation of axenic nematodes, showing that X . bovienii is triggering the encapsulation response of T . oleracea against S . feltiae. Arch Environ Health, 1997 May-Jun, 52(3), 200 - 7 Indoor pollution and sick building syndrome symptoms among workers in day-care centers; Li CS et al.; In this study, we investigated indoor air quality and symptoms of respiratory illness in 264 nursing workers at 28 day-care centers in Taipei . Geometric mean concentrations of indoor and outdoor bacteria were 735 colony-forming units in air (CFU/m3) and 384 CFU/m3, respectively . In addition, geometric mean concentrations of indoor and outdoor fungi were 1,212 CFU/m3 and 1,032 CFU/m3, respectively . Aspergillus, Cladosporium, and Penicillium-microfungi that occurred most commonly-were found indoors and outdoors . Geometric mean concentrations of house dust mite allergens, Der p I and Der p V, were 58 ng/g dust and 14 ng/g dust, respectively . In addition, the observed high prevalence of dampness or mold problems in the day-care centers indicated that dampness was very common in this subtropical region . We found a significant relationship between dampness and work-related sick building syndrome in the day-care-center workers . Furthermore, concentrations of fungi were lower in the day-care centers equipped with air conditioners/air cleaners than in centers that lacked such equipment . Also, Aspergillus was associated strongly with work-related sick building syndrome in the day-care-center workers. Microbiology, 1997 May, 143 ( Pt 5), 1737 - 44 Molecular analysis of mxbD and mxbM, a putative sensor-regulator pair required for oxidation of methanol in Methylobacterium extorquens AM1; Springer AL et al.; Five genes are thought to be required for transcription of methanol oxidation genes in Methylobacterium strains . These putative regulatory genes include mxcQE, which encode a putative sensor-regulator pair, and mxbDM and mxaB, whose functions are less well-understood . In this study, mxbDM in Methylobacterium extorquens AM1 were shown to be required for expression of a xylE transcriptional fusion to the structural gene for the large subunit of methanol dehydrogenase (mxaF), confirming the role of these genes in transcriptional regulation of mxaF . The nucleotide sequence suggests that mxbD encodes a histidine protein kinase with two transmembrane domains and that mxbM encodes a DNA-binding response regulator . A xylE transcriptional fusion to the putative mxbD promoter showed low-level expression in wild-type cells grown on one-carbon (C1) compounds and no detectable expression in cells grown on succinate . Deletion analysis of this promoter construct showed that the region 229-129 bp upstream of the start of mxbD is required for expression . The expression of the mxbD-xylE fusion was examined in each of the five known regulatory mutant classes . xylE expression was reduced to non-detectable levels in MxcQ and MxcE mutants, but was not affected in the other regulatory mutants or in non-regulatory mutants defective in methanol oxidation . These results suggest a regulatory hierarchy in which the sensor-regulator pair MxcQE control expression of the sensor-regulator pair MxbDM, and MxbDM in turn control expression of a number of genes involved in methanol oxidation. Microbiology, 1997 May, 143 ( Pt 5), 1729 - 36 Molecular and mutational analysis of a DNA region separating two methylotrophy gene clusters in Methylobacterium extorquens AM1; Chistoserdova L et al.; A region of 14.2 kb has been analysed that is a part of a locus on the Methylobacterium extorquens AM1 chromosome containing a number of genes involved in one-carbon (C1) metabolism, including serine cycle genes, pqq genes, regulatory methanol oxidation genes and the gene for N5,N10-methylene tetrahydrofolate dehydrogenase (mtdA) . Fifteen new ORFs have been identified within the new region, and their sequences suggest that they encode the following polypeptides: the C-terminal part of phosphoenolpyruvate carboxylase, malyl-CoA lyase, polypeptides of 9.4 and 31 kDa of unknown function, three putative subunits of an ABC-type transporter, two polypeptides similar to the products of mxaF and mxaJ from M . extorquens AM1 and other methylotrophs, a cytochrome c, three enzymes of folate metabolism, and polypeptides of 13 and 20.5 kDa with no homologues in the protein database . Ten insertion mutations have been generated in the region to determine if the newly identified genes are associated with C1 metabolism . A mutation in mclA, encoding malyl-CoA lyase, resulted in a C1-minus phenotype, while mutations in the other genes all showed a C1-plus phenotype . It was not possible to obtain null mutants in a putative folate metabolism gene, folC, implying the necessity of these folate synthesis genes for metabolism of C1 and multicarbon compounds . Mutations in the putative ABC transporter genes, the genes similar to mxaG and mxaJ, and other unidentified ORFs produced double-crossover recombinants with a C1-positive phenotype . Promoter regions have been investigated upstream of orf3 and orf4 using the promoter probe vector pHX200 . Transcription from these promoters was weak in wild-type M . extorquens AM1 but increased in regulatory mox mutants. Microbiology, 1997 May, 143 ( Pt 5), 1717 - 27 Fluorescent oligonucleotide rDNA probes that specifically bind to a common nanoflagellate, Paraphysomonas vestita; Rice J et al.; Nanoflagellates are ecologically important, but morphological identification requires techniques which are not practicable for use in quantitative studies of populations; alternative methods of accurate recognition of nanoflagellate species in mixed populations are therefore desirable . Fluorescent oligonucleotide probes which hybridize with unique sequences of the small subunit (SSU) rRNA have been exploited as 'phylogenetic stains' in the identification of bacteria . In this paper we describe the preparation and application of probes which specifically hybridize with a common nanoflagellate species, Paraphysomonas vestita . The sequence of nucleotides in the SSU rRNA gene of this flagellate was determined and compared with those of related species to select unique P . vestita sequences 18-21 nucleotides in length . Five sequences in different parts of the SSU rRNA gene were used to design 5'-fluorescently labelled oligonucleotide probes . Published sequences were used to make probes that hybridized with all eukaryotes (EUK) or any cellular organism (UNI), and probes were designed not to hybridize with rRNA (CON) . Optimum conditions for hybridization were determined . In all cases, UNI probes hybridized with the cells, but CON probes were only bound to a limited extent . All five probes targeted to P . vestita proved to be species-specific; they hybridized well with this species, but not with three other species of the same genus, nor with three more distantly related flagellate species, nor with a ciliate, nor with bacteria . These probes provide a means of quantitatively measuring the proportion of P . vestita cells in samples of mixed protists. Pediatr Pulmonol, 1997 May, 23(5), 382 - 5 Non-infectious interstitial alveolitis and foreign body pulmonary vasculitis in a child treated for acute lymphoblastic leukemia; Loriette Y et al.; We report a case of a 14-month-old girl who was treated for acute lymphoblastic leukemia but died from interstitial alveolitis associated with foreign body vasculitis . This respiratory complication arose 3 months after an allogenic bone marrow transplant . No infectious agents (bacteria, virus, or parasite) were isolated from bronchial or lung tissue samples . Respiratory complications after chemotherapy are reviewed as well as the potential origin of the intravascular foreign body. J Nutr, 1997 May, 127(5 Suppl), 903S - 906S Role of Kupffer cells, endotoxin and free radicals in hepatotoxicity due to prolonged alcohol consumption: studies in female and male rats; Thurman RG et al.; Alcohol ingestion results in increases in the release of endotoxin from gut bacteria or membrane permeability of the gut to endotoxin, or both . Female rats are more sensitive to these changes . Elevated levels of endotoxin activate Kupffer cells to release substances such as eicosanoids, tumor necrosis factor-alpha and free radicals . Prostaglandins increase oxygen uptake and most likely are responsible for the hypermetabolic state in the liver . The increase in oxygen demand leads to hypoxia in the liver, and on reperfusion, alpha-hydroxyethyl free radicals are formed that lead to tissue damage in oxygen-poor pericentral regions of the liver lobule. FEMS Microbiol Lett, 1997 May 1, 150(1), 61 - 4 Identification of a lactoferrin-binding protein in Prevotella nigrescens; de Lillo A et al.; A 40-kDa lactoferrin-binding protein was identified in a strain of Prevotella nigrescens isolated from a patient with periodontitis . The protein was purified by affinity column chromatography using a Sepharose-lactoferrin column and detergent-solubilized membranes . The N-terminal sequence revealed no apparent similarities with any other sequenced bacterial protein . The native conformation of the 40-kDa protein was a condition to bind either iron-free or iron-saturated lactoferrin . A possible function of this Lf-binding protein could be related with an iron acquisition mechanism in P . nigrescens. Vet Pathol, 1997 May, 34(3), 189 - 98 Encephalitozoon hellem in budgerigars (Melopsittacus undulatus); Black SS et al.; Microsporidiosis with concurrent megabacteriosis in budgerigar (Melopsittacus undulatus) chicks contributed to significant economic floss in a commercial pet bird aviary in Mississippi . Three budgerigar chicks, 1-2 weeks old, from the aviary were necropsied . Microscopic lesions in the chicks consisted of heavy infection of enterocytes with microsporidia (2/3; autolysis precluded critical evaluation of the intestine of chick No . 2), multifocal hepatic necrosis and inflammation with intralesional microsporidia (1/3), spherical clusters of microsporidia in the hepatic sinusoids in the absence of inflammation (1/3), and gastric megabacteriosis (3/3) . The ultrastructure of the microsporidian spores was consistent with an Encephalitozoon species . The polymerase chain reaction and Southern blot analysis were used to identify the microsporidian as Encephalitozoon hellem, an organism that has only been identified in humans . Encephalitozoon hellem causes keratoconjunctivitis and respiratory infections in humans with acquired immunodeficiency syndrome . This report presents the first confirmed case of microsporidiosis in budgerigars . The finding of E . hellem in pet birds may be important in elucidating the epidemiology of human infections with this organism. Curr Genet, 1997 May, 31(5), 396 - 400 The yeast ORF YDL202w codes for the mitochondrial ribosomal protein YmL11; Bui DM et al.; The Saccharomyces cerevisiae open reading frame YDL202w has been characterised in the course of the EUROFAN yeast genome analysis program . Disruption of YDL202w causes a respiratory deficient phenotype accompanied by a loss of mitochondrial DNA . This phenotype is usually found in mutants defective in mitochondrial replication or gene expression . YDL202w has the potential to encode a soluble protein of 249 amino acids . It shows significant similarities to the ribosomal protein L10 from various bacteria and to a previously determined amino-terminal peptide sequence of the yeast mitochondrial ribosomal protein L11 . The predicted amino-acid sequence of YDL202w starts with a stretch which has neither any correspondence in the bacterial sequences nor in the protein isolated from mitochondrial ribosomes . Furthermore, this stretch matches the requirements for a signal sequence for mitochondrial protein import . A mitochondrial location of the YDL202w gene product was proven by use of a carboxy terminally HA-tagged version . These findings clearly indicate that YDL202w encodes this mitochondrial ribosomal protein (YmL11). Arch Surg, 1997 May, 132(5), 522 - 5; discussion 525-6 Sartorius myoplasty for infected vascular grafts in the groin . Safe, durable, and effective; Maser B et al.; OBJECTIVE: To review the safety, durability, and efficacy of sartorius myoplasty in the treatment of localized vascular prosthetic graft infection in the groin . DESIGN: Case series . SETTING: University teaching hospitals . PATIENTS: Fourteen patients with 15 exposed, eroded, or overtly infected prosthetic vascular grafts in the groin, treated during 7 years . INTERVENTIONS: Groin exploration for delineation of the extent of vascular graft infection, followed by extensive perigraft debridement, then dissection and rotation of the ipsilateral sartorius muscle to cover the involved graft . MAIN OUTCOME MEASURES: Healing of groin wound with preservation of vascular graft function, limb salvage, length of hospital stay, impact of specific wound bacteria, and evidence of long-term hip dysfunction . RESULTS: During a mean hospital stay of 8.7 days, sartorius myoplasty was accomplished with 20% morbidity . Hip flexor function was initially impaired in all 14 patients, but functional deficit was negligible at late assessment . During mean follow-up of 36 months, all wounds were healed, and all limbs were salvaged . Two late deaths occurred, and 2 limbs were ultimately amputated due to progressive loss of vascular outflow . CONCLUSION: Sartorius myoplasty is a simple, safe, durable, and effective technique for preservation of locally infected or exposed vascular grafts in the groin. Microb Pathog, 1997 May, 22(5), 305 - 13 Local transient induction of inflammatory cytokines after intranasal administration of recombinant Bordetella pertussis; Remoue F et al.; Inflammatory cytokines have been described to play a critical role in the orientation and amplification of the IgA immune response . In this study, we show that the intranasal administration of a Bordetella pertussis strain expressing the protective antigen glutathione-S-transferase of Schistosoma mansoni (Sm28GST) induced an inflammatory response in the lungs of mice, characterized by the production of inflammatory cytokines, such as Tumor Necrosis Factor alpha, Interleukin-6 and Transforming-Growth Factor beta . The production and the secretion of these cytokines in lung tissues were early and transient . Their presence was observed only during the first week after administration despite the persistence of the bacteria for 1 month . Two weeks after inoculation, Interleukin-10 secretion was detected in the lungs, which could explain the decrease in the production of inflammatory cytokines . These inflammation-regulating cytokines, induced in the lungs by the presence of the bacterial vector, could be part of the process generating the local immune response, in particular the anti-Sm28GST IgA response. Am J Surg Pathol, 1997 May, 21(5), 505 - 9 Synchronous mucosa-associated lymphoid tissue lymphoma and adenocarcinoma of the stomach; Goteri G et al.; The development of simultaneous primary gastric lymphoma and carcinoma is a rare event for which a possible etiopathogenetic role for Helicobacter pylori (HP) recently has been postulated . We report a series of eight such cases diagnosed from 1980 to 1995 . In two cases, both tumors arose in a gastric stump, at 26 and 34 years, respectively, after gastric resection for a duodenal ulcer . Grossly, the lymphoma and carcinoma formed a single lesion in four cases (collision tumor); they were separated in the other four cases . Histologically, all the lymphomas fit into the category of B-cell mucosa-associated lymphoid tissue lymphoma; six of them were low-grade lymphomas and two were low-grade lymphomas with a high-grade component . The adenocarcinomas were intestinal-type in four cases, diffuse in three, and mixed in one . Regarding the depth of infiltration, four carcinomas were early gastric cancers and four were advanced . All the collision tumors contained an early gastric cancer . Our observations confirmed the association of HP with gastric lymphoma and carcinoma in 4 cases . Spiral bacteria with the features of Helicobacter heilmannii were found in one case . The occurrence of two different tumors in a gastric stump, which has not been reported previously, suggests that postgastrectomy gastritis might contribute to the development of both gastric lymphoma and carcinoma. Arthritis Rheum, 1997 May, 40(5), 945 - 54 Recognition of chlamydial antigen by HLA-B27-restricted cytotoxic T cells in HLA-B*2705 transgenic CBA (H-2k) mice; Kuon W et al.; OBJECTIVE: The association of reactive arthritis (ReA) with HLA-B27 and the presence of bacterial antigen in joints with ReA suggest that bacterial peptides might be presented by the HLA-B27 molecule and thus stimulate CD8 T cells . This study was performed to investigate the B27-restricted cytotoxic T lymphocyte (CTL) response to Chlamydia trachomatis, using the model of HLA-B27 transgenic mice . METHODS: CBA (H-2k) mice homozygous for HLA-B*2705 and human beta2-microglobulin expression were immunized with C trachomatis or with the chlamydial 57-kd heat-shock protein (hsp57) coupled to latex beads . Cytotoxicity of lymphocytes from in vivo-primed transgenic mice was tested against C trachomatis-infected targets . Blocking experiments were performed with monoclonal antibodies (MAb) against class I major histocompatibility complex molecules . RESULTS: A Chlamydia-specific lysis of both B27-transfected and nontransfected target cells was observed . This response could be inhibited by anti-B27 and anti-H2 MAb . CTL from mice immunized with hsp57 were not able to lyse Chlamydia-infected target cells, and Chlamydia-specific CTL could not destroy targets loaded with hsp57 . CONCLUSION: These results suggest the existence of at least 2 CTL populations in this mouse model: one recognizing peptide of bacteria-infected cells restricted by HLA-B*2705 and the other recognizing peptide of bacteria-infected cells restricted by the murine H-2Kk molecule . It does not appear that hsp57 is a major target for the CD8 T cell response directed against Chlamydia . This animal model opens the way for identifying bacterial epitopes presented by HLA-B27, and might thus help to clarify the pathogenesis of B27-associated diseases. J Bacteriol, 1997 May, 179(10), 3110 - 5 Characterization of three protein components required for functional reconstitution of the epoxide carboxylase multienzyme complex from Xanthobacter strain Py2; Allen JR et al.; Epoxide carboxylase from Xanthobacter strain Py2 catalyzes the reductant- and NAD+-dependent carboxylation of aliphatic epoxides to beta-keto acids . Epoxide carboxylase from Xanthobacter strain Py2 has been resolved from cell extracts by anion-exchange chromatography into three protein components, designated I, II, and III, that are obligately required for functional reconstitution of epoxide carboxylase activity . Component II has been purified to homogeneity on the basis of its ability to complement components I and III in restoring epoxide carboxylase activity . Purified component II had a specific activity for epoxide carboxylation of 41.8 mU x min(-1) x mg(-1) when components I and III were present at saturating levels . The biochemical properties of component II reveal that it is the flavin-containing NADPH:disulfide oxidoreductase that was recently shown by other means to be associated with epoxide degradation activity in Xanthobacter strain Py2 (J . Swaving, J . A . M . de Bont, A . Westphal, and A . Dekok, J . Bacteriol . 178:6644-6646, 1996) . The rate of epoxide carboxylation was dependent on the relative concentrations of the three carboxylase components . At fixed concentrations of two of the components, epoxide carboxylation rates were saturated in a hyperbolic fashion by increasing the concentration of the third variable component . Methylepoxypropane has been characterized as a time-dependent, irreversible inactivator of epoxide carboxylase activity that is proposed to be a mechanism-based inactivator of the enzyme . The addition of component I, but not that of component II or III, to methylepoxypropane-inactivated cell extracts restored epoxide carboxylase activity, suggesting that component I contains the epoxide binding and activation sites. Genome Res, 1997 May, 7(5), 522 - 31 The organization of the gamma-glutamyl transferase genes and other low copy repeats in human chromosome 22q11; Collins JE et al.; A clone map consisting of YACs, cosmids, and fosmids has been constructed covering low copy repeat regions of human chromosome 22q11 . A combination of clone restriction digest analysis, single-copy landmark content analysis, HindIII-Sau3AI fingerprinting, and sequencing of PCR products derived from clones was required to resolve the map in this region . Seven repeat-containing contigs were placed in 22q11, five containing gamma-glutamyl transferase (GGT) sequences described previously . In one case, a single interval at the resolution of the YAC map was shown to contain at least three GGT sequences after higher resolution mapping . The sequence information was used to design a rapid PCR/restriction digest technique that distinguishes the GGT loci placed in the YAC map . This approach has allowed us to resolve the previous cDNA and mapping information relating to GGT and link it to the physical map of 22q11. Genome Res, 1997 May, 7(5), 457 - 70 Clone-contig and STS maps of the hereditary hemochromatosis region on human chromosome 6p21.3-p22; Lauer P et al.; YAC-based and bacterial-clone based STS-content maps were constructed that served as the framework physical maps for the positional cloning of a candidate gene for hereditary hemochromatosis . The YAC-based map comprises 43 YACs and 86 STS and spans approximately 8 Mb of DNA between the class I region of the major histocompatibility complex on human chromosome 6p21.3 and D6S276 in 6p22 . Comparison with published maps revealed a hole in the MIT/Whitehead and CEPH YAC maps that includes the immediate region around the hemochromatosis gene itself . Approximately 3 Mb of DNA was covered by a bacterial clone contig that consists of 38 BACs, 45 PACs, 26 PI clones and one lambda phage . The bacterial clone-based STS map comprises 153 STSs . A contiguous block of 8 STSs could be amplified from both human chromosome 6 and 5 . Further characterization of selected STSs and bacterial clones by radiation hybrid mapping and fluorescence in situ hybridization, respectively, revealed the presence of a multicopy DNA segment, more than one bacterial clone length in size, which is duplicated near the chromosome-6 centromere and part of which is present in multiple copies on chromosome 5 . Possible implications of the incomplete public YAC-contig map and of the multicopy segment for physical mapping and linkage disequilibrium studies of the hemochromatosis candidate region are discussed. Genome Res, 1997 May, 7(5), 441 - 56 A 1.1-Mb transcript map of the hereditary hemochromatosis locus; Ruddy DA et al.; In the process of positionally cloning a candidate gene responsible for hereditary hemochromatosis (HH), we constructed a 1.1-Mb transcript map of the region of human chromosome 6p that lies 4.5 Mb telomeric to HLA-A . A combination of three gene-finding techniques, direct cDNA selection, exon trapping, and sample sequencing, were used initially for a saturation screening of the 1.1-Mb region for expressed sequence fragments . As genetic analysis further narrowed the HH candidate locus, we sequenced completely 0.25 Mb of genomic DNA as a final measure to identify all genes . Besides the novel MHC class 1-like HH candidate gene HLA-H, we identified a family of five butyrophilin-related sequences, two genes with structural similarity to a type 1 sodium phosphate transporter, 12 novel histone genes, and a gene we named RoRet based on its strong similarity to the 52-kD Ro/SSA lupus and Sjogren's syndrome auto-antigen and the RET finger protein . Several members of the butyrophilin family and the RoRet gene share an exon of common evolutionary origin called B30-2 . The B30-2 exon was originally isolated from the HLA class 1 region, yet has apparently "shuffled" into several genes along the chromosome telomeric to the MHC . The conservation of the B30-2 exon in several novel genes and the previously described amino acid homology of HLA-H to MHC class 1 molecules provide further support that this gene-rich region of 6p21.3 is related to the MHC . Finally, we performed an analysis of the four approaches for gene finding and conclude that direct selection provides the most effective probes for cDNA screening, and that as much as 30% of ESTs in this 1.1-Mb region may be derived from noncoding genomic DNA. Biotechniques, 1997 May, 22(5), 916 - 8, 920-2 Making hybrids of two-hybrid systems; Dagher MC et al.; Two-hybrid systems are powerful tools to find new partners for a protein of interest . However, exchange of material between two-hybrid users has been handicapped by the various versions of two-hybrid systems available and by the widely accepted idea that they are not compatible . In the present paper we show that, contrary to the dogma, the most often used two-hybrid systems may be combined by either transformation or mating assays . The protocol to be followed in each case is provided . This will greatly increase the prospects of the growing network of interacting proteins, by reconciling the "two-hybrid systems" and the "interaction trap". Proteins, 1997 May, 28(1), 1 - 9 Prediction of the structure of the replication initiator protein DnaA; Schaper S et al.; The secondary structure of DnaA protein and its interaction with DNA and ribonucleotides has been predicted using biochemical, biophysical techniques, and prediction methods based on multiple-sequence alignment and neural networks . The core of all proteins from the DnaA family consists of an "open twisted alpha/beta structure," containing five alpha-helices alternating with five beta-strands . In our proposed structural model the interior of the core is formed by a parallel beta-sheet, whereas the alpha-helices are arranged on the surface of the core . The ATP-binding motif is located within the core, in a loop region following the first beta-strand . The N-terminal domain (80 aa) is composed of two alpha-helices, the first of which contains a potential leucine zipper motif for mediating protein-protein interaction, followed by a beta-strand and an additional alpha-helix . The N-terminal domain and the alpha/beta core region of DnaA are connected by a variable loop (45-70 aa); major parts of the loop region can be deleted without loss of protein activity . The C-terminal DNA-binding domain (94 aa) is mostly alpha-helical and contains a potential helix-loop-helix motif . DnaA protein does not dimerize in solution; instead, the two longest C-terminal alpha-helices could interact with each other, forming an internal "coiled coil" and exposing highly basic residues of a small loop region on the surface, probably responsible for DNA backbone contacts. Clin Diagn Lab Immunol, 1997 May, 4(3), 279 - 84 Antibody and delayed-type hypersensitivity responses to Ochrobactrum anthropi cytosolic and outer membrane antigens in infections by smooth and rough Brucella spp; Velasco J et al.; Immunological cross-reactions between Brucella spp . and Ochrobactrum anthropi were investigated in animals and humans naturally infected by Brucella spp . and in experimentally infected rams (Brucella ovis infected), rabbits (Brucella melitensis infected), and mice (B . melitensis and Brucella abortus infected) . In the animals tested, O . anthropi cytosolic proteins evoked a delayed-type hypersensitivity reaction of a frequency and intensity similar to that observed with B . melitensis brucellin . O . anthropi cytosolic proteins also reacted in gel precipitation tests with antibodies in sera from Brucella natural hosts with a frequency similar to that observed with B . melitensis proteins, and absorption experiments and immunoblotting showed antibodies to both Brucella-specific proteins and proteins common to Brucella and O . anthropi . No antibodies to O . anthropi cytosolic proteins were detected in the sera of Brucella-free hosts . Immunoblotting with sera of Brucella-infected sheep and goats showed immunoglobulin G (IgG) to Brucella group 3 outer membrane proteins and to O . anthropi proteins of similar molecular weight . No IgG to the O-specific polysaccharide of O . anthropi lipopolysaccharide was detected in the sera of Brucella-infected hosts . The sera of sheep, goats, and rabbits infected with B . melitensis contained IgG to O . anthropi rough lipopolysaccharide and lipid A, and B . ovis and O . anthropi rough lipopolysaccharides showed equal reactivities with IgG in the sera of B . ovis-infected rams . The findings show that the immunoresponse of Brucella-infected hosts to protein antigens is not necessarily specific for brucellae and suggest that the presence of O . anthropi or some related bacteria explains the previously described reactivities to Brucella rough lipopolysaccharide and outer membrane proteins in healthy animals. Clin Immunol Immunopathol, 1997 May, 83(2), 93 - 102 Autoimmunity in myocarditis: relevance of animal models; Huber SA; Infections (viruses, bacteria, protozoa, fungi) are major etiological factors causing clinical myocarditis and dilated cardiomyopathy . In many patients and symptom-free relatives antibodies and T cells reactive to heart antigens are detected, which implies that autoimmunity is probably a major pathogenic mechanism of cardiac injury . Animal models have been established to elucidate how infections initiate autoimmunity and how autoimmune mediators cause death or transient dysfunction of myocytes . Two major types of experimental models are discussed: adjuvant-induced myocarditis, in which animals are given multiple immunizations of heart proteins (myosin, adenine nucleotide translocator); and virus-induced myocarditis, in which animals are infected with the viruses predominantly associated with the human disease. Appl Environ Microbiol, 1997 May, 63(5), 1898 - 904 The soluble methane monooxygenase gene cluster of the trichloroethylene-degrading methanotroph Methylocystis sp . strain M; McDonald IR et al.; In methanotrophic bacteria, methane is oxidized to methanol by the enzyme methane monooxygenase (MMO) . The soluble MMO enzyme complex from Methylocystis sp . strain M also oxidizes a wide range of aliphatic and aromatic compounds, including trichloroethylene . In this study, heterologous DNA probes from the type II methanotroph Methylosinus trichosporium OB3b were used to isolate souble MMO (sMMO) genes from the type II methanotroph Methylocystis sp . strain M . sMMO genes from strain M are clustered on the chromosome and show a high degree of identity with the corresponding genes from Methylosinus trichosporium OB3b . Sequencing and phylogenetic analysis of the 16S rRNA gene from Methylocystis sp . strain M have confirmed that it is most closely related to the type II methanotroph Methylocystis parvus OBBP, which, unlike Methylocystis sp . strain M, does not possess an sMMO . A similar phylogenetic analysis using the pmoA gene, which encodes the 27-kDa polypeptide of the particulate MMO, also places Methylocystis sp . strain M firmly in the genus Methylocystis . This is the first report of isolation and characterization of methane oxidation genes from methanotrophs of the genus Methylocystis. J Bacteriol, 1997 May, 179(9), 2863 - 71 Identification and characterization of Myxococcus xanthus mutants deficient in calcofluor white binding; Ramaswamy S et al.; Calcofluor white is a fluorescent dye that binds to glycans and can be used to detect extracellular polysaccharide in Myxococcus xanthus and many other bacteria . We observed that an esg mutant showed less binding to calcofluor white than wild-type cells . Unlike S-motility mutants that share this phenotypic characteristic, the esg mutant exhibited S motility . This led us to identify a collection of nine new transposon insertion mutants, designated Cds (for calcofluor white binding deficient and S motile), which exhibited a phenotype similar to that of the esg strain . The Cds phenotype was found in 0.6% of the random insertion mutants that were screened . The Cds mutants were also found to be defective in cell-cell agglutination and developmental aggregation . Extracellular matrix fibrils composed of roughly equal amounts of polysaccharide and protein have been shown to be involved in agglutination, and electron microscopic examination showed that esg and the other Cds mutants lack the wild-type level of fibrils . Analysis of total M . xanthus carbohydrate demonstrated that polysaccharide content increased by about 50% when wild-type cells entered stationary phase . This induction was reduced or eliminated in all of the Cds mutants . The degree of polysaccharide deficiency in the Cds mutants correlated with the degree of loss of agglutination and dye binding as well as with the severity of the developmental aggregation defect . Preliminary genetic characterization demonstrated that the transposon insertion mutations in three of the Cds mutants (SR53, SR171, and SR200) were loosely linked . The results of this study suggest that many genes are involved in the production of calcofluor white binding polysaccharide material found in the extracellular matrix and that the polysaccharide is fibrillar . These results are also consistent with the findings of earlier studies which indicated that fibrils function to join agglutinating cells and to form multicellular fruiting aggregates. J Bacteriol, 1997 May, 179(9), 2852 - 6 High-level genetic diversity in the vapD chromosomal region of Helicobacter pylori; Cao P et al.; Helicobacter pylori isolates from different patients are characterized by diversity in the nucleotide sequences of individual genes, variation in genome size, and variation in gene order . Genetic diversity is particularly striking in vacuolating cytotoxin (vacA) alleles . In this study, five open reading frames (ORFs) were identified within a 4.2-kb region downstream from vacA in H . pylori 60190 . One of these ORFs was closely related to the virulence-associated protein D (vapD) gene of Dichelobacter nodosus (64.9% nucleotide identity) . A probe derived from vapD of H . pylori 60190 hybridized with only 19 (61.3%) of 31 H . pylori strains tested . Sequence analysis of the vapD region in vapD-negative H . pylori strains revealed that there were two different families of approximately 0.5-kb DNA segments, which were both unrelated to vapD . The presence of vapD was not associated with any specific family of vacA alleles . These findings are consistent with a recombinational population structure for H . pylori. Am J Crit Care, 1997 May, 6(3), 204 - 9 Gut dysfunction in critically ill patients: a review of the literature; Stechmiller JK et al.; BACKGROUND: Critically ill patients are susceptible to injury of the intestinal mucosa, changes in gut permeability, and failure of intestinal defense mechanisms . These conditions put the patients at risk for infection and multiple organ dysfunction syndrome . Specific therapies are needed to prevent gut failure during critical illness . OBJECTIVE: The purpose of this literature review is to provide a better understanding of the normal defense mechanisms of the gut and alterations associated with ischemia-reperfusion injury, risk of infection, and the link to multiple organ dysfunction syndrome in critically ill patients . Implications for early enteral stimulation and nutrition are included . METHODS: Medical and nursing studies on the intestinal response to critical illness and on the implications for early enteral nutrition in critically ill patients were reviewed . RESULTS: Significant advances have been made in understanding the normal defense mechanisms of the gut, including barrier and immune functions . Translocation of bacteria, mediators of the inflammatory response, and the microcirculation play a role in the response to critical illness . Enteral nutrition that includes glutamine and arginine enhances gut function and improves patients' outcomes in some clinical states . DISCUSSION: Further research should focus on specific strategies to enhance gut function, prevent loss of gut integrity, and improve patients' outcomes . These strategies include maintaining mesenteric blood flow, using gastric tonometry to assess oxygenation, inhibiting inflammatory mediators, and using growth factors to modify the metabolic state in patients who are critically ill. Infect Immun, 1997 May, 65(5), 1980 - 4 A role for fimbriae in Porphyromonas gingivalis invasion of oral epithelial cells; Njoroge T et al.; Isogenic mutants of Porphyromonas gingivalis which differ in the expression of fimbriae were used to examine the contribution of fimbriae in invasion of a human oral epithelial cell line (KB) . At a multiplicity of infection of 100, the wild-type P . gingivalis strains 33277, 381, and A7436 exhibited adherence efficiencies of 5.5, 0.11, and 5.0%, respectively, and invasion efficiencies of 0.15, 0.03, and 0.10%, respectively . However, adherence to and invasion of KB cells was not detected with the P . gingivalis fimA mutants, DPG3 and MPG1 . Adherence of P . gingivalis wild-type strains to KB cells was completely inhibited by the addition of hyperimmune sera raised to the major fimbriae . Examination by electron microscopy of invasion of epithelial cells by the P . gingivalis wild-type strain 381 revealed microvillus-like extensions around adherent bacteria; this was not observed with P . gingivalis fim mutants . Taken together, these results indicate that the P . gingivalis major fimbriae are required for adherence to and invasion of oral epithelial cells. Infect Immun, 1997 May, 65(5), 1916 - 25 Growth within macrophages increases the efficiency of Mycobacterium avium in invading other macrophages by a complement receptor-independent pathway; Bermudez LE et al.; Infections caused by organisms of the Mycobacterium avium complex occur in approximately 50 to 60% of patients with AIDS . M . avium is an intracellular pathogen that survives and multiplies within mononuclear phagocytes . In this study, we investigated the uptake of M . avium grown within macrophages (intracellular growth M . avium {IG}) by a second macrophage compared with M . avium cultured in broth (extracellular growth M . avium {EG}) . The results showed that IG was six- to eightfold more efficient than EG in entering macrophages . In addition, while an anti-CR3 antibody was able to inhibit approximately 60% of EG uptake by macrophages, it failed to inhibit the entry of IG . In contrast to EG, IG uptake into macrophages was significantly inhibited in the presence of anti-beta1-integrin and anti-transferrin receptor antibodies . Entry into macrophages by alternate receptors was associated with resistance to tumor necrosis factor alpha (TNF-alpha) stimulation . While stimulation with TNF-alpha resulted in inhibition of the growth of EG, it was not associated with inhibition of intracellular growth of IG . Investigation of the reason why M . avium is able to sense the changes in the intracellular environment triggering a change to the invasive phenotype suggests a direct relationship with macrophage apoptosis . These results suggest that intracellular growth is associated with novel mechanisms of M . avium uptake of macrophages and that those mechanisms appear to offer advantages to the bacteria in escaping the host defense. Infect Immun, 1997 May, 65(5), 1793 - 9 In vivo induction of apoptosis in the thymus by administration of mycobacterial cord factor (trehalose 6,6'-dimycolate); Ozeki Y et al.; It is reported that some bacteria or bacterial components cause thymic atrophy via the apoptotic process . The present study demonstrated for the first time in vivo induction of apoptosis in the mouse thymus by mycobacterial cord factor (CF) (trehalose 6,6'-dimycolate) . When 300 microg of purified CF from Mycobacterium tuberculosis was intravenously administered to BALB/c mice in the form of water-in-oil-in-water (w/o/w) emulsion, thymic atrophy and pulmonary granulomas were induced with a peak on day 7, whereas, in the form of liposomes, CF induced thymic atrophy on days 14 to 21 in parallel with the development of hepatic granulomas . Thymic atrophy resulted from the depletion of cortical lymphocytes via apoptosis as revealed by DNA fragmentation and karyorrhectic changes . In contrast, mycobacterial sulfatide (2,3,6,6'-tetraacyl trehalose 2'-sulfate) caused neither thymic atrophy nor granuloma formation . Compared to lipopolysaccharide-induced thymocyte apoptosis, CF (w/o/w)-induced thymocyte apoptosis developed more slowly, reached a maximum later, and lasted longer but was less intense . Although serum tumor necrosis factor alpha (TNF-alpha) levels in CF-treated mice were not significantly elevated, administration of anti-TNF-alpha antibody almost completely inhibited thymic atrophy and granuloma formation . Serum corticosterone levels were only slightly elevated by CF administration . The present results indicate that mycobacterial CF induces thymic atrophy via apoptosis, which is closely linked with granuloma formation. J Clin Microbiol, 1997 May, 35(5), 1290 - 2 Four-day incubation period for blood culture bottles processed with the Difco ESP blood culture system; Doern GV et al.; Blood culture records from 1994 to 1995 from five U.S . medical centers all using the Difco ESP continuous monitoring blood culture system were reviewed retrospectively . Among a total of 7,362 isolates of bacteria and yeasts, only 0.1% of possibly significant isolates would have been missed had blood cultures been routinely incubated for 4 days instead of the 5 days recommended by the manufacturer . Conversely, numerous contaminants, detected only on day 5, would have been eliminated by a 4-day incubation period. J Clin Microbiol, 1997 May, 35(5), 1131 - 7 Actinobacillus suis strains isolated from healthy and diseased swine are clonal and carry apxICABDvar . suis and apxIICAvar . suis toxin genes; Van Ostaaijen J et al.; Actinobacillus suis isolates recovered from both healthy and diseased pigs were characterized by biochemical testing, serotyping, restriction endonuclease fingerprinting, and apx toxin gene typing . The clinical isolates analyzed were collected over a 10-year period from approximately 40 different locations in southwestern Ontario, Canada . Little variation in the biochemical profiles of these isolates was seen, and all isolates reacted strongly with rabbit antisera prepared against one of the strains . Similarly, by using BamHI and BglII for restriction endonuclease fingerprinting (REF) analysis, all isolates were found to belong to a single REF group . Minor variations could be detected, especially in the BglII fingerprints, but overall the patterns were remarkably similar . Sequences that could be amplified by PCR with primers to the apxICA and apxIICA genes of Actinobacillus pleuropneumoniae were detected in all strains . Although no amplification was obtained with primers to the A . pleuropneumoniae apxIBD genes, sequences with homology to apxIBD were detected by hybridization . There was no evidence of apxIII homologs . Taken together, these data suggest that A . suis isolates are genotypically and phenotypically very similar, regardless of their source, and that they contain genes similar to, but not identical to, the apxICABD and apxIICA genes of A . pleuropneumoniae. Curr Microbiol, 1997 May, 34(5), 318 - 25 The presence of complete but masked freezing nuclei in various artificially constructed ice nucleation-active proteobacteria; Yankofsky SA et al.; Disparate gamma-subdivision proteobacteria artificially endowed with the same ice gene of enteric origin acquired water-freezing potential at -12 degrees C, but expressed it to varying extents under identical conditions of culture as well as after being subjected to certain post-culture treatments . Varying rates of cell-bound ice nucleus synthesis were probably not the root cause of these observed interspecies differences in nucleation-active cell frequency because potentially functional but masked ice-forming templates were found in the outer cell envelope of even initially inactive individuals taken from physiologically uniform populations of virtually all tested species . We therefore propose that the extent of bacterial ice nucleation generally reflects species-specified extent of ice nucleus sequestration. Am J Ind Med, 1997 May, 31(5), 510 - 24 Acute respiratory effects on workers exposed to metalworking fluid aerosols in an automotive transmission plant; Robins T et al.; Exposure to metalworking fluids has been linked to modest cross-shift reductions in FEV1 and occupational asthma . To identify responsible agents, we measured personal exposures to thoracic particulate (TP), viable plus nonviable thoracic bacteria (BAC), and vapor phase nicotine (VPN) (as a surrogate for tobacco particulate) among 83 machinists exposed to soluble oils and 46 dry assemblers working in an automotive transmission machining plant using biocides infrequently . The participants completed interviews and performed pre- and postshift spirometry on Monday and Thursday of the same week in each of three rounds of data collection (June 1992, January 1993, June 1993) . Generalized estimating equations were used to combine information across rounds in multiple regression models of cross-shift and cross-week changes in forced expiratory volume, I second (FEV1) and forced vital capacity (FVC) . Mean seniority was 19 years among machinists . Mean personal TP levels were 0.41 mg/m3 in machinists and 0.13 mg/m3 in assemblers . Six of the 83 machinists and none of the 46 assemblers experienced a greater than 19% cross-shift decrement in FEV1 or FVC at least once (p = .07) . In regression models using either TP or BAC, among subjects with lower baseline (Monday preshift) FEV1/FVC ratios, increasing exposure was significantly associated with increasing cross-shift decrements in FEV1 and FVC in linear models, and with increased likelihood of a 10% or greater cross-shift decrement in FEV1 or FVC in logistic models . Adjustment of TP for VPN did not affect models significantly . We conclude that clinically important cross-shift decrements in pulmonary function are associated with exposure to metalworking fluid aerosols within a high-seniority population. J Neurosci, 1997 May 1, 17(9), 3024 - 37 A hierarchy of Hu RNA binding proteins in developing and adult neurons; Okano HJ et al.; The Hu proteins are a group of antigens targeted in an immune-mediated neurodegenerative disorder associated with cancer . We have cloned and characterized four members of the Hu gene family from mouse . We find that the Hu genes encode a large number of alternatively spliced transcripts to produce a series of related neuron-specific RNA binding proteins . Despite this complexity, we have discerned several ordered features of Hu expression . In the embryo, specific Hu genes are expressed in a hierarchy during early neurogenesis . In the E16 developing cortex, mHuB is induced in very early postmitotic neurons exiting the ventricular zone, mHuD is expressed in migrating neurons of the intermediate zone, and mHuC is expressed in mature cortical plate neurons . Such a hierarchy suggests distinct functional roles for each gene in developing neurons . In the adult, all neurons express some set of Hu mRNA and protein . However, specific patterns are evident such that individual neuronal types in the hippocampus, cerebellum, olfactory cortex, neocortex, and elsewhere express from one to several Hu genes . The complexity of potential protein variants within a gene family and of different Hu family members within a neuron suggests a diverse array of function . Given the strong homologies among the Hu proteins, the Drosophila neurogenic gene elav, and the Drosophila splicing factor sxl, we predict that different combinations of Hu proteins determine different neuron-specific aspects of post-transcriptional RNA regulation . Our findings of specific developmental patterns of expression and the correlation between immune targeting of the Hu proteins and adult neurodegenerative disease suggest that the Hu proteins are critical in both the proper development and function of mature neurons. Virology, 1997 Apr 28, 231(1), 141 - 7 A specific interaction between coat protein and helper component correlates with aphid transmission of a potyvirus; Blanc S et al.; Specific binding between the coat protein (CP) and the helper component (HC) of the tobacco vein mottling potyvirus (TVMV) was characterized using a protein blotting-overlay protocol . In this in vitro assay, HC interacted with either virions or CP monomers originating from the aphid-transmissible TVMV-AT but not from the non-aphid-transmissible TVMV-NAT . There was a strong correlation between the aphid transmissibility of a series of TVMV variants having mutations in the DAG motif of the CP and their ability to bind HC . Expression of TVMV CP derivatives in bacteria allowed a precise determination of the minimum domain mediating HC binding . This domain is composed of seven amino acids, including the DAG motif (DTVDAGK), located in the N-terminus of the TVMV CP at amino acid positions 2 to 8. J Biol Chem, 1997 Apr 18, 272(16), 10756 - 60 Human pyridoxal kinase . cDNA cloning, expression, and modulation by ligands of the benzodiazepine receptor; Hanna MC et al.; Peptide fragments of a porcine benzodiazepine-binding protein were used to isolate the cDNA of a related human protein . The cDNA encodes a polypeptide of 312 amino acid residues that is homologous to a bacterial pyridoxal kinase . Transient expression of the cDNA in human embryonic kidney cells confirmed that it encodes human pyridoxal kinase . The recombinant enzyme displayed a Km value of 3.3 microM for pyridoxal and was inhibited competitively by 4-deoxypyridoxine (Ki = 2.8 microM) . Benzodiazepine receptor ligands that bound to the purified porcine protein also exerted a potent inhibitory effect on human pyridoxal kinase activity . Transcripts of the pyridoxal kinase gene were detectable in all human tissues examined, and were particularly abundant in the testes . The gene is localized on chromosome 21q22.3 and represents a candidate gene for at least one genetic disorder that has been mapped to this region (autoimmune polyglandular disease type 1). J Biol Chem, 1997 Apr 18, 272(16), 10616 - 23 Multifunctional tryptophan-synthesizing enzyme . The molecular weight of the Euglena gracilis protein is unexpectedly low; Schwarz T et al.; After developing a suitable procedure to produce large amounts of Euglena gracilis as well as a reliable protocol to purify the multifunctional tryptophan-synthesizing enzyme derived from it (Schwarz, T., Bartholmes, P., and Kaufmann, M . (1995) Biotechnol . Appl . Biochem . 22, 179-190), we here describe structural and catalytic properties of the multifunctional tryptophan-synthesizing enzyme . The kinetic parameters kcat of all five activities and Km for the main substrates were determined . The relative molecular weight under denaturing conditions as judged by SDS-polyacrylamide gel electrophoresis is 136,000 . Cross-linking as well as gel filtration experiments revealed that the enzyme exists as a homodimer . Neither intersubunit disulfide linkages nor glycosylations were detected . On the other hand, the polypeptide chains are blocked N-terminally . Complete tryptic digestion of the protomer, high pressure liquid chromatography separation of the resulting peptides, and N-terminal sequence analysis of homogenous peaks as judged by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry was performed . Depending on the sequenced peptides, alignments to all entries of the SwissProt data base resulted in both strong sequence homologies to known Trp sequences and no similarities at all . Proteolytic digestion under native conditions using endoproteinase Glu-C uncovered one major cleavage site yielding a semistable, N-terminally blocked fragment with a molecular weight of 119,000 . In addition, an increase in beta-elimination accompanied by a decrease in beta-replacement activity of the beta-reaction during proteolysis was observed. J Biol Chem, 1997 Apr 18, 272(16), 10514 - 21 Identification of the autophosphorylation sites of the Xenopus laevis Pim-1 proto-oncogene-encoded protein kinase; Palaty CK et al.; Pim-1 is an oncogene-encoded serine/threonine kinase expressed primarily in cells of the hematopoietic and germ line lineages . Previously identified only in mammals, pim-1 cDNA was cloned and sequenced from the African clawed frog Xenopus laevis . The coding region of Xenopus pim-1 encoded a protein of 324 residues, which exhibited 64% amino acid identity with the full-length human cognate . Xenopus Pim-1 was expressed in bacteria as a glutathione S-transferase (GST) fusion protein and in COS cells . Phosphoamino acid analysis revealed that recombinant Pim-1 autophosphorylated on serine and threonine and to a more limited extent on tyrosine . Electrospray ionization mass spectroscopy was undertaken to locate these phosphorylation sites, and the primary autophosphorylation site of GST-Pim-1 was identified as Ser-190 with Thr-205 and Ser-4 being minor sites . Ser-190, which immediately follows the high conserved Asp-Phe-Gly motif in catalytic subdomain VII, is also featured in more than 20 other protein kinases . To evaluate the importance of the Ser-190 site on the phosphotransferase activity of Pim-1, Ser-190 was mutated to either alanine or glutamic acid, and the constructs were expressed in bacteria as GST fusion proteins and in COS cells . These mutants confirmed that Ser-190 is a major autophosphorylation site of Pim-1 and indicated that phosphorylation of Pim-1 on the Ser-190 residue may serve to activate this kinase. Mol Gen Genet, 1997 Apr 16, 254(3), 250 - 7 A novel cytoplasmic structure containing DNA networks in Entamoeba histolytica trophozoites; Orozco E et al.; We report here the presence of cytoplasmic DNA arranged in networks in the trophozoites of the human parasite Entamoeba histolytica . Cytoplasmic DNA was detected in live trophozoites in a structure that we called EhkO, using the fluorescent dye acridine orange, and by in situ hybridization to trophozoites with a rDNA probe . The EhkO was found in the axenically grown clones A, L6 (strain HMI:IMSS) and MAVax (strain MAV) and in the polyxenically grown clone MAVpx (strain MAV) . Bacteria present in MAVpx did not cross hybridize with the DNA probe neither in in situ hybridization or in Southern blot experiments . Autoradiography of metabolically {3H}thymidine-labeled trophozoites showed the presence of EhkO, and an EhkO-enriched fraction, purified from a nuclei-free extract and examined by light microscopy, exhibited {3H}thymidine incorporation into this structure . DNA was purified from the EhkO and enriched nuclear fractions and analyzed by transmission electron microscopy . The EhkO fraction contained DNA networks resembling those of trypanosome kDNA, whereas nuclear DNA was present mainly as linear molecules and some circles . Our findings imply that E . histolytica may be taxonomically more closely related to the Trypanosomatidae than previously suspected. Virology, 1997 Apr 14, 230(2), 197 - 206 Characterization of a DNA topoisomerase encoded by Amsacta moore entomopoxvirus; Petersen BO et al.; We have identified an Amsacta moorei entomopoxvirus (AmEPV) gene encoding a DNA topoisomerase . The 333-amino acid AmEPV topoisomerase displays instructive sequence similarities to the previously identified topoisomerases encoded by five genera of vertebrate poxviruses . One hundred nine amino acids are identical or conserved among the six proteins . The gene encoding AmEPV topoisomerase was expressed in bacteria and the recombinant enzyme was partially purified . AmEPV topoisomerase is a monomeric enzyme that catalyzes the relaxation of supercoiled DNA . Like the vaccinia, Shope fibroma virus, and Orf virus enzymes, the AmEPV topoisomerase forms a covalent adduct with duplex DNA at the target sequence CCCTT decreases . The kinetic and equilibrium parameters of the DNA cleavage reaction of AmEPV topoisomerase (Kobs = 0.08 sec-1; Kcl = 0.22) are similar to those of the vaccinia virus enzyme. Biochim Biophys Acta, 1997 Apr 10, 1351(3), 341 - 58 Gene cloning and regulation of gene expression of the puc operon from Rhodovulum sulfidophilum; Hagemann GE et al.; Rhodovulum (Rhv.) sulfidophilum, unlike other nonsulfur purple bacteria, is able to synthesize the peripheral antenna complex even under fully aerobic conditions in the dark . We have obtained strong evidence that Rhv . sulfidophilum encodes only one copy of the puc operon, comprising pucB, pucA and pucC . pucB and pucA encode the beta- and alpha-polypeptides . The third ORF (pucC), downstream of pucA, has a strong homology to pucC of Rhodobacter (Rb.) capsulatus . Deletion mutation analysis indicated that the requirement for the pucC gene product for LH II expression was less strict than in Rb . capsulatus . Comparison of the deduced alpha and beta polypeptide sequences with the directly determined primary structure revealed a C-terminal processing of the alpha-subunit . Primer extension analysis showed that the pucBAC is transcribed from a sigma70-type promoter 130 bases upstream of the translational start of pucB . Transcriptional expression of the pucBAC operon in Rhv . sulfidophilum is higher, the lower the light intensity is, and is not reduced to a ground-level by the presence of oxygen . Based on lacZ fusions the relative promoter activities were, for dark aerobic:dark semiaerobic:low light anaerobic:medium light anaerobic:high light anaerobic, 5.5:7.0:2.0:1.0:0.78 . Still unidentified cis-regulatory elements or binding sites of trans-regulatory elements are apparently localized in two distinct upstream regions . Furthermore, comparison of the promoter region of the Rhv . sulfidophilum pucBAC with the promoter regions of puc operons in related species showed distinct differences in the regulatory elements . The significance of these results with respect to the regulation of transcription and the oxygen-independent synthesis of LH II from Rhv . sulfidophilum is discussed. Nature, 1997 Apr 10, 386(6625), 569 - 77 Transcriptional activation by recruitment; Ptashne M et al.; The recruitment model for gene activation stipulates that an activator works by bringing the transcriptional machinery to the DNA . Recent experiments in bacteria and yeast indicate that many genes can be activated by this mechanism . These findings have implications for our understanding of the nature of activating regions and their targets, and for the role of histones in gene regulation. Biochemistry, 1997 Apr 8, 36(14), 4203 - 11 Cyclic electron transfer in Heliobacillus mobilis involving a menaquinol-oxidizing cytochrome bc complex and an RCI-type reaction center; Kramer DM et al.; Flash-induced absorption changes arising from b-type hemes were studied on whole cells of Heliobacillus mobilis under physiological and redox-controlled conditions . The sensitivity of the monitored redox changes to inhibitors of cytochrome bc complexes and the redox potential dependence of reduction and oxidation reactions of cytochrome b-hemes demonstrate that the respective b-hemes are part of a cytochrome bc complex . Both the half-time and the extent of flash-induced reduction of cytochrome b titrated with apparent potentials of about -60 and -50 mV (both n = 2), respectively, i.e., close to the Em,7 value of the menaquinone (MK) pool, indicating a collisional interaction between menaquinol and the Qo site of the cytochrome bc complex . At strongly reducing ambient potentials (< -150 mV), a net flash-induced oxidation of b-hemes was observed in agreement with the Em,7 values of the individual hemes of -90 mV (b(h)) and -190 mV (b(l)) determined in equilibrium redox titrations on membrane fragments . From the extent of photooxidized b- and c-type hemes as well as P798+, a stoichiometry of 0.6-0.75 cytochrome bc complexes per photosynthetic reaction center was estimated . The kinetic behavior and also the energy profiles for Q-cycle turnover of the heliobacterial complex are compared to those of cytochrome bc1 complexes from purple bacteria and of cytochrome b6f complexes from chloroplasts. Anal Biochem, 1997 Apr 5, 247(1), 138 - 42 Analysis of the binding of ligands to large numbers of sites: the binding of tryptophan to the 11 sites of the trp RNA-binding attenuation protein; Saroff HA et al.; Analysis of the cooperative binding of ligands may provide insight into the underlying mechanisms of biological control . Binding of L-tryptophan at each juncture in the ring of 11 subunits of the trp RNA-binding attenuation protein exhibits cooperativity . To analyze binding of this kind we have developed both the matrix and combinatorial procedures for binding to large numbers of sites . A new, exact, and fast combinatorial procedure is presented . In this procedure the sites may be single or overlapping in a linear lattice or in the form of a ring . The dimensions of the ring of 11 binding sites for L-tryptophan on the trp RNA-binding attenuation protein are such as to prevent direct interactions between the L-tryptophan molecules . Reasonable and explicit assumptions on analyzing the binding data lead to the conclusion that binding of L-tryptophan raises the energy level of (destabilizes) the 11 membered structure. Cell, 1997 Apr 4, 89(1), 25 - 31 A novel suppressor of cell death in plants encoded by the Lls1 gene of maize; Gray J et al.; The Lls1 (lethal leaf spot1) locus of maize is defined by a recessive mutation characterized by the initiation, in a developmentally programmed manner, of necrotic lesions that expand to kill leaves cell autonomously . The loss-of-function nature of all Lls1 mutants implies that the Lls1 gene is required to limit the spread of cell death in mature leaves . We have cloned the Lls1 gene by tagging with Mutator, a transposable element system in maize, and we show that it encodes a novel protein highly conserved in plants . Two consensus binding motifs of aromatic ring-hydroxylating dioxygenases are present in the predicted LLS1 protein, suggesting that it may function to degrade a phenolic mediator of cell death. Eur J Epidemiol, 1997 Apr, 13(3), 329 - 34 Specific detection of Coxiella burnetii through partial amplification of 23S rDNA; Ibrahim A et al.; A previously published sequence of the 23S rRNA gene of Coxiella burnetii has been reported to contain an intervening sequence of 444 base pairs (bp) . The sequence information on the intervening sequence and the 23S rRNA gene was exploited to develop a specific PCR-based assay for C . burnetii . A primer set was designed that amplified a 477-bp fragment encompassing part of the intervening sequence and part of the 23S rDNA . From all of nine C . burnetii strains tested, a fragment of the expected size was amplified . As predicted from the published sequence, restriction endonuclease digestion of the PCR product from the Coxiella strains with RsaI produced two distinct fragments approximately 210- and 270-bp in size . The PCR-based method showed a detection limit of 10(2) bacteria as determined by visualization of the amplicon on an agarose gel . When experimentally infected blood was analyzed, the detection limit was 10(3) bacteria . No visible amplicons were observed when 41 bacterial strains, representing 29 species other than C . burnetii, were tested . The presence of the DNA in all bacterial samples was confirmed by amplification of a 350-bp fragment of the 16S rDNA using two universal primers . The described method proved to be specific for C . burnetii and may become a rapid and sensitive diagnostic assay for C . burnetii . The results also demonstrate that the intervening sequence within the 23S rRNA gene is generally found among isolates of C . burnetii. Pneumologie, 1997 Apr, 51 Suppl 2, 403 - 5 {PO2-affinity of oxygen-sensitive intracellular signal cascades}; Acker H; The oxygen partial pressure field in different organs ranging from 0 to 100 Torr is likely to be a mirror of oxygen sensitive intracellular signal cascades determining ion channel open probability, metabolic pathway activities and gene expression . High or low PO2 affinities of the particular signal cascade optimise the oxygen sensitive cellular response for adapting organ functions to variations of the oxygen supply conditions . The signal cascades are triggered by an oxygen sensor which is believed to be a heme protein . In some cases oxygen radicals are acting as second messengers revealing these signal cascades as an evolutionary highly conserved principle first described in bacteria. Oral Microbiol Immunol, 1997 Apr, 12(2), 121 - 5 Visualization of an extracellular mucoid layer of Treponema denticola ATCC 35405 and surface sugar lectin analysis of some Treponema species; Scott D et al.; Slime layers and capsules are common amongst medically relevant bacteria . We herein report that Treponema denticola, which has been associated with periodontitis, synthesizes or acquires an extracellular polysaccharide layer that we have observed through electron microscopy using the polysaccharide-specific dye Alcian blue and phosphotungstate . We have also visualized this extracellular layer by dark-field microscopy of Alcian blue-stained spirochete cells . A representative strain of each of the oral spirochete species T . denticola, Treponema vincentii and Treponema socranskii were differentiated by concanavalin A, phaseolus, lotus A and arachis lectins in a microtiter plate immunoassay for the detection of surface sugars. Biosci Rep, 1997 Apr, 17(2), 115 - 46 Genes of human ATP synthase: their roles in physiology and aging; Kagawa Y et al.; The reaction of ATP synthase (F0F1) is the final step in oxidative phosphorylation (OXPHOS) . Although OXPHOS has been studied extensively in bacteria, no tissue-specific functions nor bioenergetic disease, such as mitochondrial encephalomyopathy and aging occur in these organisms . Recent developments of the Human Genome Project will become an important factor in the study of mammalian bioenergetics . To elucidate the physiological roles of human F0F1, genes encoding the subunits of F0F1 were sequenced, and their expression in human cells was analyzed . The following results were obtained: A . The roles of the residues in F0F1 are not only to transform the energy of the electrochemical potential (delta mu H+) across the membrane, but also to respond rapidly to the changes in the energy demand by regulating the intramolecular rotation of F0F1 with the delta mu H+ and the inhibitors of the ATPase . B . The roles of the control regions of the F0F1 genes, are to coordinate both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) depending on the energy demand of the cells, especially in muscle, C . The cause of the age-dependent decline of ATP synthesis has been attributed to the accumulation of mutations in mtDNA . However, the involvement of nDNA in the decline is also important because of telomere shortening in somatic cells, and age-dependent mtDNA expression analyzed with rho degree cells (cells without mtDNA). Arzneimittelforschung, 1997 Apr, 47(4A), 578 - 89 Studies on the cytoprotective and antisecretory activity of ebrotidine . A review; Konturek PC et al.; Gastric mucosa is exposed to various aggressive factors such as stress, ulcerogenic drugs including acetyl-salicylic acid(ASA)-like agents, ethanol, bacteria, particularly Helicobacter pylori (Hp), and various endogenous irritants such as acid-pepsin secretion and bile salts . The maintenance of the mucosal barrier depends upon the activation of the pre-epithelial (mucus-alkali secretion), epithelial (surface-active phospholipids and rapid mucosal restitution) and post-epithelial (mucosal microcirculation, sensory nerves and mast cells) components of mucosal defense . Ebrotidine (N-{(E)-{{2-{{{2-{(diaminomethylene)amino}- 4-thiazolyl}methyl}thio}ethyl}amino}methylene}-4-bromo-benzenesulfonamid e, CAS 100981-43-9, FI-3542) is the first of a new generation of H2-receptor antagonists with both antisecretory and cytoprotective activities . Its inhibitory action is similar to that of ranitidine and approximately tenfold greater than cimetidine, and is accompanied by a small and transient increase in plasma gastrin levels . In contrast to ranitidine and other H2-receptor antagonists, ebrotidine exerts a unique cytoprotection against injury by various ulcerogens such as ethanol, ammonia, lipopolysaccharides (LPS), stress and ASA or acidified taurocholate . The mechanism of this protection by ebrotidine is not clear, but it has been shown to stimulate mucus secretion, to increase the quality of adherent mucus gel and to increase gastric mucosal blood flow (GBF), possibly due to enhanced mucosal formation of prostaglandin E2 (PGE2) and nitric oxide (NO) . The cytoprotective effects of ebrotidine were observed in rats and confirmed also in humans with gastric lesions induced by ethanol or ASA . Ebrotidine also exerts anti-Helicobacter pylori (Hp) effects by interfering with surface receptors of epithelial cells and inhibiting urease, protease and lipase activity, and by counteracting the noxious effects of Hp-related substances such as ammonia and lipopoly-saccharides (LPS). Ostomy Wound Manage, 1997 Apr, 43(3), 20 - 2, 24, 26 passim A comparison of two culturing methods for chronic wounds; Neil JA et al.; Bacterial infection has always been a potential complication of any wound . Controversy exists regarding the significance of bacteria in chronic wounds . It is important to accurately diagnose wound infection by bacterial identification and quantification in order to prevent unnecessary and/or inappropriate treatments and to minimize patient complications . The primary function of culturing is to identify infection in a wound . The tissue culture is an accepted standard for measuring infection, although swab cultures are commonplace in the clinical setting . The purpose of this study was to test the differences in bacterial counts and identification in swab and tissue cultures taken from the same wound site of 10 chronic wounds . It was hypothesized that if swab and tissue cultures are equally effective in identifying and quantifying the organisms in a chronic wound, they are equally effective methods in determining infection in the chronic wound . The reliability, validity and limitations of the study are discussed, as well as the statistical analyses and results. Appl Biochem Biotechnol, 1997 Apr, 66(1), 83 - 93 Prediction of folding stability and degradability of the de novo designed protein MB-1 in cow rumen; MacCallum JD et al.; The authors have recently reported on the design of a protein (MB-1) enriched in methionine, threonine, lysine, and leucine . The protein is intended to be produced by rumen bacteria, in a way that would provide high producing lactating cows with limiting amino acids . In this report, MB-1 stability in the rumen is assessed, i.e., where the protein might be found after cell lysis or after being secreted by rumen bacteria . Current in vitro methods used to predict proteolytic degradability in the rumen were used for MB-1, as well as other natural proteins for comparison . MB-1 was found to be more susceptible to degradation than cytochrome c and ribonuclease A . Data indicate that MB-1 will be rapidly degraded if exposed to the rumen environment without protection . The contribution of folding stability to proteolytic stability was also examined . Rumen liquor components were selected to formulate a solution compatible with constraints of thermal denaturation studies . Denaturation curves show that the natural proteins were folded at rumen temperature . The MB-1 denaturation curves indicated that MB-1 does not unfold in a cooperative transition when heated from 20 to 70 degrees C . This suggests that MB-1 structure may be progressively modified as temperature increases, and that a continuum of conformations are available to MB-1 . At 39 degrees C, a significant (50%) portion of MB-1 molecules had their tertiary structure unfolded, contributing to proteolytic degradability . Despite the unusual constraints used in MB-1 design (i.e., a maximized content in selected essential amino acids), results show that MB-1 has structural properties similar to previously reported de novo designed proteins. Ann Med, 1997 Apr, 29(2), 95 - 120 Phyto-oestrogens and Western diseases; Adlercreutz H et al.; Incidences of breast, colorectal and prostate cancer are high in the Western world compared to countries in Asia . We have postulated that the Western diet compared to the semivegetarian diet in some Asian countries may alter hormone production, metabolism or action at the cellular level by some biochemical mechanisms . Our interest has been focused on two groups of hormone-like diphenolic phyto-oestrogens of dietary origin, the lignans and isoflavonoids abundant in plasma of subjects living in areas with low cancer incidence . The precursors of the biologically active compounds detected in man are found in soybean products, whole-grain cereal food, seeds, and berries . The plant lignan and isoflavonoid glycosides are converted by intestinal bacteria to hormone-like compounds . The weakly oestrogenic diphenols formed influence sex-hormone production, metabolism and biological activity, intracellular enzymes, protein synthesis, growth factor action, malignant cell proliferation, differentiation, cell adhesion and angiogenesis in such a way as to make them strong candidates for a role as natural cancer-protective compounds . Their effect on some of the most important steroid biosynthetic enzymes may result in beneficial modulation of hormone concentrations and action in the cells preventing development of cancer . Owing to their oestrogenic activity they reduce hot flushes and vaginal dryness in postmenopausal women and may to some degree inhibit osteoporosis, but alone they may be insufficient for complete protection . Soy intake prevents oxidation of the low-density lipoproteins in vitro when isolated from soy-treated individuals and affect favourably plasma lipid concentrations . Animal experiments provide evidence suggesting that both lignans and isoflavonoids may prevent the development of cancer as well as atherosclerosis . However, in some of these experiments it has not been possible to separate the phyto-oestrogen effect from the effect of other components in the food . The isoflavonoids and lignans may play a significant inhibitory role in cancer development particularly in the promotional phase of the disease, but recent evidence points also to a role in the initiation stage of carcinogenesis . At present, however, no definite recommendations can be made as to the dietary amounts needed for prevention of disease . This review deals with all the above-mentioned aspects of phyto-oestrogens. Immunol Rev, 1997 Apr, 156, 145 - 66 The anatomical basis of intestinal immunity; Mowat AM et al.; The lymphoid tissues associated with the intestine are exposed continuously to antigen and are the largest part of the immune system . Many lymphocytes are found in organised tissues such as the Peyer's patches and mesenteric lymph nodes, as well as scattered throughout the lamina propria and epithelium of the mucosa itself . These lymphocyte populations have several unusual characteristics and the intestinal immune system is functionally and anatomically distinct from other, peripheral compartments of the immune system . This review explores the anatomical and molecular basis of these differences, with particular emphasis on the factors which determine how the intestinal lymphoid tissues discriminate between harmful pathogens and antigens which are beneficial, such as food proteins or commensal bacteria . These latter antigens normally provoke immunological tolerance, and inappropriate responses to them are responsible for immunopathologies such as food hypersensitivity and inflammatory bowel disease . We describe how interactions between local immune cells, epithelial tissues and antigen-presenting cells may be critical for the induction of tolerance and the expression of active mucosal immunity . In addition, the possibility that the intestine may act as an extrathymic site for T-cell differentiation is discussed . Finally, we propose that, under physiological conditions, immune responses to food antigens and commensal bacteria are prevented by common regulatory mechanisms, in which transforming growth factor beta plays a critical role. Acta Paediatr, 1997 Apr, 86(4), 356 - 60 Helicobacter pylori duodenal colonization in children; Bonamico M et al.; To investigate the prevalence and the significance of Helicobacter pylori duodenal colonization, endoscopic duodenal biopsies were performed in 168 children with chronic abdominal pain, gastroesophageal reflux, gastrointestinal bleeding, and malabsorption syndrome . Helicobacter pylori infection was detected in 68 children (40.4%): in 31 of them H . pylori was present in the gastric antrum, and in 37 in the duodenum also . Duodenitis was observed in 25 children with duodenal H . pylori; gastric metaplasia in 3 . Scanning electron microscopy revealed the presence of the micro-organism in 3/13 cases; the bacteria were located in the intercellular spaces and alterations of the epithelial surface were found . In conclusion, H . pylori gastritis in children is often associated with duodenal colonization which can cause duodenitis, and also without gastric metaplasia, which indicates a possible role of the micro-organism in the pathogenesis of the lesions. J Cataract Refract Surg, 1997 Apr, 23(3), 447 - 9 Keratitis from corneal anesthetic abuse after photorefractive keratectomy; Kim JY et al.; After having photorefractive keratectomy (PRK), a 29-year-old man suffered from delayed epithelial healing and corneal stromal ring infiltrates . All laboratory results including smear, culture, and biopsy for bacteria, herpes simplex virus, and Acanthamoeba were negative . The suspected cause was patient abuse of anesthetics . Subsequently, it was discovered that for 6 months, since just after the PRK, the patient had intermittently used topical proparacaine drops . After all medication was discontinued and the eye pressure patched, the corneal epithelium healed completely . Practitioners should consider the possibility of topical anesthetic abuse in cases of keratitis after PRK. Eur J Oral Sci, 1997 Apr, 105(2), 178 - 82 Are sodium lauryl sulfate-containing toothpastes suitable vehicles for xylitol? Assev S, Waler SM, Rolla G. The hypothesis to be tested in this study was that toothpastes containing sodium lauryl sulfate (SLS) is unsuitable vehicles for xylitol . The bacteriostatic (and cariostatic) effect of xylitol is assumed to be caused by intracellular accumulation of xylitol-5-P in plaque bacteria . Experiments were designed to investigate whether presence of SLS would affect the uptake of xylitol by interacting with the bacterial membranes and thus inhibit xylitol-5-P formation . It was shown in an in vitro study that even very low concentrations of the strong anionic detergent SLS inhibited uptake of xylitol and xylitol-5-phosphate formation by dental plaque totally . The mild nonionic detergent ethoxylated stearyl alcohol (30x EO) had no such effect . In vivo experiments with toothpastes containing xylitol and either the strong or the mild detergent, showed that xylitol in toothpaste with SLS was not available for the plaque bacteria and gave no adaptation to xylitol, whereas in the presence of 30x EO it was available, and a xylitol adaptation was observed . Glucose metabolism, which was also studied for the plaque samples, was not significantly affected by presence of any of the 2 detergents, indicating that the amounts of xylitol in toothpastes were presumably too low to give clinical significant effects, even when mild detergents are used. Trends Biochem Sci, 1997 Apr, 22(4), 138 - 9 Methods and reagents . Degraded DNA and gel tornados; Hengen PN; Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet . This month's column discusses a case of inexplicable DNA degradation and tornados seen in agarose gels . For details on how to partake in the newsgroup, see the accompanying box. Trends Biochem Sci, 1997 Apr, 22(4), 118 - 23 ATP-dependent proteases that also chaperone protein biogenesis; Suzuki CK et al.; The ATP-dependent proteases Clp and FtsH from bacteria, as well as mitochondrial homologs of FtsH and Lon from yeast, may act as chaperones; they mediate not only proteolysis, but also the insertion of proteins into membranes and the disassembly or oligomerization of protein complexes . The coordination of such processes with selective proteolysis may function in the quality control of protein biogenesis. Gen Pharmacol, 1997 Apr, 28(4), 485 - 7 Reductive activation of nitroheterocyclic compounds; Tocher JH; 1 . Nitroaromatic compounds are important chemotherapy agents . 2 . Their selective toxicity is determined by reduction to the biologically active form in the absence of oxygen . 3 . Nitroaromatics are extensively used in the treatment of anaerobic infections and to target hypoxic tumor cells in cancer therapy . 4 . Possible mutagenic action is related to the relative ease of nitro group reduction . 5 . The mode of action and clinical application of nitroaromatic compounds is summarized. Aliment Pharmacol Ther, 1997 Apr, 11 Suppl 1, 71 - 88 Helicobacter pylori and the risk and management of associated diseases: gastritis, ulcer disease, atrophic gastritis and gastric cancer; Kuipers EJ; This review addresses the role of H . pylori and the effect of H . pylori eradication on gastritis, peptic ulcer disease, atrophic gastritis and gastric cancer . Specific emphasis is given to various factors that influence the clinical course of this infection . H . pylori induces chronic gastritis in virtually all infected subjects . This inflammation can lead to peptic ulceration and atrophic gastritis in a considerable number of infected subjects . A minority eventually develops gastric cancer . The risk of such complications depends upon the severity of gastritis, which is determined by various host- and bacteria-related factors . Among bacterial factors, most of the evidence addresses the cagA pathogenicity island, the presence of which has been associated with more severe gastritis, peptic ulceration, atrophic gastritis and gastric cancer . Among host factors, most of the evidence focuses on acid production in response to H . pylori infection . An increase in acid secretion limits H . pylori gastritis to the antrum at the risk of duodenal ulcer disease; a reduction allows more proximal inflammation at the risk of atrophic gastritis, gastric ulcer disease, and gastric cancer . Gastritis and atrophy negatively influence acid secretion . H . pylori eradication is required in peptic ulcer disease and may be advocated in patients on profound acid suppressive therapy; it has been shown to cure gastritis and prevent ulcer recurrence . Further study is required to determine the efficacy of H . pylori eradication in the primary and secondary prevention of atrophic gastritis and gastric cancer. Aliment Pharmacol Ther, 1997 Apr, 11 Suppl 1, 11 - 20 Non-endoscopic tests in the diagnosis of Helicobacter pylori infection; Atherton JC; The urea breath test (UBT) and serological antibody detection are simpler and less expensive than endoscopic tests for the diagnosis of Helicobacter pylori . For the UBT, either non-radioactive 13C or radioactive 14C is used as an isotopic marker . 14C-UBTs are cheaper and are safe, but licensing regulations may make them inconvenient . Some UBTs have been simplified by omitting the normal test meal and encapsulating the urea to avoid metabolism by oral bacteria . These modified test need further validation, especially when used for assessing H . pylori status after treatment . Serological tests detect circulating IgG or IgA . They are of variable accuracy, the best performing as well as UBTs . Paired serum samples pre-treatment and 6 months post-treatment accurately assess treatment success . Rapid in-office tests appear less accurate and cannot be used for post-treatment assessment . In practice, for primary diagnosis of H . pylori infection, endoscopic tests are best because endoscopy allows assessment of treatment indications . Where indications already exist or taking biopsies is dangerous . UBTs or serology are suitable, but serology is cheaper and more convenient . After treatment, endoscopy is usually unnecessary and UBTs accurately assess H . pylori status at 4 weeks . Serology is an alternative only if results are not required before 6 months. Kekkaku, 1997 Apr, 72(4), 181 - 6 {Reliability of Amplicor Mycobacteria test for detection of Mycobacterium tuberculosis complex . M . avium and M . intracellulare: a cooperative study among 9 laboratories}; Abe C et al.; The Amplicor Mycobacteria, a PCR-based assay, is a rapid test for the detection of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium intracellulare in clinical samples . To estimate the reliability and reproducibility of the method, a cooperative blind study was conducted among 9 laboratories . Materials used for testing consisted of 105 sputum and 30 water samples containing known numbers of M . bovis BCG, M . avium, M . intracellulare, and samples without bacteria . Only 2 out of the 9 laboratories correctly identified the presence or absence of mycobacterial DNA in all 135 samples . In sputum samples, 6 out of the 9 laboratories detected mycobacterial DNA in all positive samples, and 4 out of the 9 laboratories correctly reported the absence of DNA in the negative samples, indicating the need for good laboratory practice and development of reference reagents to monitor the performance of the whole study, including pretreatment of clinical samples . The main problem was lack of specificity rather than lack of sensitivity . From about half of the laboratories, false-positive results were reported, however, the ratio was below 6%; 1% (1/106 sputum samples) in 3 laboratories, 1.9% (2/105) in 2 laboratories, and 5.7% (6/105) in one laboratory, respectively . These results indicate that the Amplicor Mycobacteria is quite useful for a rapid diagnosis of tuberculosis. Mol Microbiol, 1997 Apr, 24(1), 217 - 28 Chlamydia psittaci IncA is phosphorylated by the host cell and is exposed on the cytoplasmic face of the developing inclusion; Rockey DD et al.; Chlamydiae are obligate intracellular bacteria that replicate within a non-acidified vacuole called an inclusion . Chlamydia psittaci (strain GPIC) produces a 39 kDa protein (IncA) that is localized to the inclusion membrane . While IncA is present as a single 39 kDa species in purified reticulate bodies, two additional higher M(r) forms are found in C . psittaci-infected cells . This finding suggested that IncA may be post-translationally modified in the host cell . Here we present evidence that IncA is a serine/threonine phosphoprotein that is phosphorylated by host cell enzymes . This conclusion is supported by the following experimental findings: (i) treatment of infected cells with inhibitors of host cell phosphatases or kinases altered the electrophoretic migration pattern of IncA; (ii) treatment with calf intestinal alkaline phosphatase eliminated the multiple-banding pattern of IncA, leaving only the protein band with the lowest relative molecular weight; and (iii) radioimmunoprecipitation of lysates of {32P}-orthophosphate-labelled infected HeLa cells with anti-IncA antisera demonstrated that the two highest M(r) IncA bands were phosphorylated . A vaccinia-virus recombinant expressing incA was used to determine if HeLa cells can phosphorylate IncA in the absence of a chlamydial background . IncA in lysates of these cells migrated identically to that seen in C . psittaci-infected cells, indicating the host cell was responsible for the phosphorylation of the protein . Microinjection of fluorescently labelled anti-IncA antibodies into C . psittaci-infected HeLa cells resulted in immunostaining of the outer face of the inclusion membrane . Collectively, these results demonstrate that IncA is phosphorylated by the host cell, and regions of IncA are exposed at the cytoplasmic face of the inclusion. Mol Microbiol, 1997 Apr, 24(1), 141 - 53 Mycobacterial recA is cotranscribed with a potential regulatory gene called recX; Papavinasasundaram KG et al.; The recA gene of Mycobacterium smegmatis has been cloned and sequenced . The amino acid sequence of the RecA protein is highly homologous to other RecA proteins . Three other potential open reading frames were identified . One of these showed extensive homology to a protein, HypB, involved in the incorporation of nickel into hydrogenases . Another, found downstream of and overlapping recA, was similar to a gene, recX, which has been proposed to play a regulatory role related to recA function . The homology between the M . smegmatis sequence and that of Mycobacterium tuberculosis extended upstream of the recA coding region for 140 bp including a motif identical to the Cheo-box consensus sequence which has been shown to bind LexA . In addition, the transcriptional start sites were found to be identical to those identified previously for M . tuberculosis . Transcriptional fusions to the reporter gene chloramphenicol acetyltransferase (CAT) revealed that recA was DNA-damage inducible and that expression required sequences at some distance from the mapped transcriptional start sites . Although a motif with only one mismatch to the Cheo box was found in the intergenic region between orf1 and orf2 these open reading frames were not DNA-damage inducible, nor was this motif required for regulation of recA expression . Gel retardation assays revealed that the reason for this was that LexA did not bind to this sequence containing a mismatch . Reverse transcription/polymerase chain reaction analysis of M . smegmatis RNA demonstrated that recA and orf3 (recX) are within the same transcriptional unit. Mol Microbiol, 1997 Apr, 24(1), 119 - 28 The HypB protein from Bradyrhizobium japonicum can store nickel and is required for the nickel-dependent transcriptional regulation of hydrogenase; Olson JW et al.; The HypB protein from Bradyrhizobium japonicum is a metal-binding GTPase required for hydrogenase expression . In-frame mutagenesis of hypB resulted in strains that were partially or completely deficient in hydrogenase expression, depending on the degree of disruption of the gene . Complete deletion of the gene yielded a strain (JH delta Eg) which lacked hydrogenase activity under all conditions tested, including the situation as bacteroids from soybean nodules . Mutant strain JH delta 23H lacking only the N-terminal histidine-rich region (38 amino acids deleted, 23 of which are His residues) expressed partial hydrogenase activity . The activity of strain JH delta 23H was low in comparison to the wild type in 10-50 nM nickel levels, but could be cured to nearly wild-type levels by including 50 microM nickel during the derepression incubation . Studies on strains harbouring the hup promoter-lacZ fusion plasmid showed that the complete deletion of hypB nearly abolished hup promoter activity, whereas the histidine deletion mutant had 60% of the wild-type promoter activity in 50 microM NiCl2 . Further evidence that HypB is required for hup promoter-binding activity was obtained from gel-shift assays . HypB could not be detected by immunoblotting when the cells were cultured heterotrophically, but when there was a switch to microaerobic conditions (1% partial pressure O2, 10% partial pressure H2) HypB was detected, and its expression preceded hydrogenase synthesis by 3-6 h . 63Ni accumulation by whole cells showed that both of the mutant strains accumulate less nickel than the wild-type strain at all time points tested during the derepression incubation . Wild-type cultures that received nickel during the HypB expression-specific period and were then washed and derepressed for hydrogenase without nickel had activities comparable to those cells that were derepressed for hydrogenase with nickel for the entire time period . In contrast to the wild type, strain JH delta 23H cultures supplied with nickel only during the HypB expression period achieved hydrogenase activities that were 30% of those cultures supplied with nickel for the entire hydrogenase derepression period . These results indicate that the loss of the metal-binding area of HypB causes a decrease in the ability of the cells to sequester and store nickel for later use in one or more hydrogenase expression steps. Bioessays, 1997 Apr, 19(4), 347 - 52 DNA turnover and mutation in resting cells; Bridges BA; There is growing evidence that mutations can arise in non-dividing cells (both bacterial and mammalian) in the absence of chromosomal replication . The processes that are involved are still largely unknown but may include two separate mechanisms . In the first, DNA lesions resulting from the action of endogenous mutagens may give rise to RNA transcripts with miscoded bases . If these confer the ability to initiate DNA replication, the DNA lesions may have an opportunity to miscode during replication and thus could give rise to apparently 'adaptive' mutations . A second mechanism is suggested by recent work in starved bacteria, showing that there is much more turnover of chromosomal DNA than has been previously thought . This could permit polymerase errors to lead to mutations in non-dividing cells . Such cryptic DNA synthesis, which may essentially replace existing DNA rather than duplicating it, could, in principle, act as an additional source of variability on which selection may act, initially in the absence of cell division . In mammals such processes would undoubtedly have implications for germ cell mutagenesis and carcinogenesis. J La State Med Soc, 1997 Apr, 149(4), 105 - 8 Orbital complications of sinusitis; Jabor MA et al.; The ethmoid and maxillary sinuses are the sinuses most frequently involved with orbital infections . Routes of spread are by direct extension through bone and indirectly through the valveless venous plexuses of the orbit, nose, and sinus . Classification of orbital infections can be thought of as either preseptal or postseptal and then further subdivided from there . The diagnosis has been greatly improved by the use of CT scans; however this should never be the only entity used for diagnosis . The determination of visual acuity (VA) is the single most important finding and will ultimately determine treatment modality . Orbital infections show different and more virulent bacteria than does non-complicated sinusitis and this should be reflected in the choice of medical and surgical therapy . Medical management is well accepted for infections without abscesses, and surgery is generally needed for infections with an abscess. Eur J Cell Biol, 1997 Apr, 72(4), 362 - 9 Isolation and morphofunctional characterization of mussel digestive gland cells in vitro; Robledo Y et al.; The aim of the present study was to isolate and characterize digestive gland cells of the bivalve mollusc Mytilus galloprovincialis Lmk . The digestive gland of bivalves is a complex organ composed of digestive and connective tissues but it is also invaded by the reproductive tissue as gametogenesis proceeds . The digestive tissue is comprised of stomach and intestinal epithelial cells, ciliated and non-ciliated duct cells, digestive cells and basophilic cells . the last two cell types are found lining the epithelium of the blind-ending digestive tubules and are the main focus of this study . Two different approaches were assayed for cell isolation, i.e., explant culture techniques and mechanical plus enzymatical digestion techniques . Cell viability was tested by trypan blue exclusion, neutral red uptake and ultrastructural analysis . The explant cultures were often contaminated with bacteria and spermatozoa and, moreover, cells migrating out of the explants possibly corresponded to hemocytes and not to digestive tissue cells . Mechanical plus enzymatical digestion with collagenase was concluded to be the method of choice for digestive cell isolation, with a percentage of about 30% of isolated cells corresponding to digestive cells . The ultrastructure of digestive cells isolated with the latter procedure closely resembled that of mussel digestive cells in vivo. Arch Biochem Biophys, 1997 Apr 1, 340(1), 83 - 9 Preparation and properties of a 3,4-dihydroxycinnamic acid chromophore variant of the photoactive yellow protein; Devanathan S et al.; Native photoactive yellow protein (PYP) is reversibly bleached by laser excitation at the 446-nm wavelength maximum, during which the trans-4-hydroxycinnamic acid chromophore (covalently bound via a thioester to Cys 69) is isomerized, causing the protein to undergo a conformational change . We have reconstituted the holoprotein from recombinant apoprotein plus thiophenol thioester-activated chromophore and have also successfully attached a synthetic 3,4-dihydroxycinnamic acid chromophore and purified the resultant variant . The reconstituted recombinant protein has the same spectral and photochemical properties as the native protein . However, the absorption maximum of the protein with the dihydroxy chromophore variant is red-shifted to 458 nm, with an additional shoulder at about 342 nm . Following a laser flash, the rate constants for the first phase of bleaching in both the native and the variant proteins are too large to measure with the present apparatus . The second bleaching phase is only marginally accessible in the variant and has a rate constant (k approximately 2.3 x 10(4) s-1) at least an order of magnitude larger than that of the native PYP . In contrast, the rate constant for recovery of absorbance in the variant (k approximately 0.15 s-1) is about 40-fold smaller than for native PYP and is insensitive to pH (the native protein has a biphasic 16-fold variation in rate constant with pH) . We previously observed similar changes in kinetic rate constants for protein denatured by urea or alcohols, which suggests that the dihydroxy protein is less stable than the native PYP . This was confirmed by measurement of protein unfolding in guanidine hydrochloride . We conclude from these results that the binding site is too small to accommodate the dihydroxybenzene ring of the variant chromophore without introducing strain into the protein, which is then reflected in the kinetic properties of the photocycle. Arch Biochem Biophys, 1997 Apr 1, 340(1), 1 - 9 Serine/threonine phosphorylation of orphan receptor hepatocyte nuclear factor 4; Jiang G et al.; We showed previously that hepatocyte nuclear factor 4 (HNF-4) defines a new subclass, Group IV, of nuclear receptors . In order to determine whether members of this subclass are phosphorylated, HNF-4 was overexpressed to high levels in insect cells using a baculovirus expression system . The baculovirus-expressed HNF-4 (HNF4.BV) was characterized and compared to HNF-4 overexpressed in transiently transfected mammalian (COS-7) cells (HNF4.COS) . The results indicate that both HNF4.BV and HNF4.COS are phosphorylated although HNF4.BV was hypophosphorylated relative to HNF4.COS . Phosphoamino acid analysis showed that HNF-4 is phosphorylated mainly on serine and to a lesser extent on threonine residues . Phosphopeptide mapping revealed 13 phosphopeptides for HNF4.COS, only 9 of which were present in the HNF4.BV sample . DNA-binding studies also showed that HNF4.BV binds DNA with a lower specificity and affinity, as measured by the equilibrium dissociation constant (Kd), than does HNF4.COS . Partial proteolytic digestion experiments also revealed that HNF4.BV and HNF4.COS adopt somewhat different three-dimensional conformations . Since glycosylation of HNF4.BV was ruled out by a number of methods and since HNF-4 expressed in bacteria exhibited an even lower DNA-binding affinity than HNF4.BV, we propose that serine/theronine phosphorylation may play a role in the DNA-binding activity of HNF-4 and, therefore, possibly of other Group IV receptors as well. J Immunol, 1997 Apr 1, 158(7), 3464 - 73 Analysis of intestinal lymphocytes in mouse colitis mediated by transfer of CD4+, CD45RBhigh T cells to SCID recipients; Aranda R et al.; Transfer of specific T lymphocyte subsets isolated from the spleens of healthy donor mice into immunodeficient SCID mice leads to chronic intestinal inflammation with characteristics similar to those of human inflammatory bowel disease (IBD) . CD4+, CD45RBhigh cells cause disease, whereas CD4+, CD45RBlow and CD8+, CD45RBhigh cells do not . Despite this difference, we demonstrate that all three T cell populations reconstitute the intraepithelial and lamina propria compartments of both small and large intestines of SCID recipients . Therefore, infiltration of lymphocytes alone is not sufficient for disease development . CD4+ lymphocytes that have trafficked to the SCID intestine exhibit a phenotype characteristic of normal mucosal lymphocytes . This includes high expression of alpha E integrin and CD69, expression of CD8 alpha alpha homodimers in some of the intraepithelial lymphocytes, as well as low expression of CD62L and CD45RB . The phenotype of the infiltrating mucosal cells is indistinguishable, with respect to the cell surface markers tested, regardless of whether the starting donor population is CD45RBhigh or CD45RBlow . Severe inflammation is restricted primarily to the colon despite lymphocyte infiltration throughout the length of the intestine . This suggests that some property of the colon microenvironment contributes to inflammation . Consistent with this, transfer of CD4+, CD45RBhigh cells to SCID mice that have significantly reduced numbers of enteric flora results in attenuation of the wasting and colitis . Fewer numbers of donor lymphocytes are recovered from the intraepithelial and lamina propria compartments of reduced flora SCID mice . We hypothesize that the ability of pathogenic cells to traffic to the intestine and mediate colitis may be driven by T cell reactivity to bacteria or bacterial products. J Immunol, 1997 Apr 1, 158(7), 3344 - 52 Immunity to Chlamydia trachomatis is mediated by T helper 1 cells through IFN-gamma-dependent and -independent pathways; Perry LL et al.; Mucosal immunity to Chlamydia trachomatis in a mouse model of female genital tract infection is mediated predominantly by Th1-type cells, as shown by in vivo neutralization of cytokines involved in the Th1 vs Th2 pathways . Neutralization of IL-12 was associated with an apparent decrease in the infiltration of CD4+ T cells into infected tissues, systemic reductions in the production of IFN-gamma, and prolonged shedding of high levels of bacteria . Neutralization of IL-4 had no detectable effect on host immunity or on bacterial clearance . To dissociate the protective role of IL-12 from that of IL-12-induced IFN-gamma, resistance to C . trachomatis was compared in IL-12-depleted and IFN-gamma-deficient animals . IL-12-depleted mice displayed minimal bacterial clearance for 1 mo post-infection but eventually resolved genital tract infections completely . IFN-gamma-deficient mice, on the other hand, cleared 99.9% of genital Chlamydia within the first 3 wk but then developed systemic disease associated with dissemination of bacteria to multiple organs . Animals surviving this stage often maintained low level persistent infections within the urogenital tract . These results indicate that the bulk of chlamydial clearance from the genital mucosa is mediated by an IL-12-dependent, IFN-gamma-independent mechanism, while prevention of disseminated disease requires the action of IFN-gamma. Infect Immun, 1997 Apr, 65(4), 1497 - 504 Differential trafficking of live and dead Mycobacterium marinum organisms in macrophages; Barker LP et al.; We characterized the Mycobacterium marinum phagosome by using a variety of endocytic markers to follow the path of the bacteria through a mouse macrophage cell line . Using a laser confocal microscope, we found that the majority of viable M . marinum cells were in nonacidic vacuoles that did not colocalize with the vacuolar proton ATPase (V-ATPase), the calcium-independent mannose-6-phosphate receptor (CI-M6PR), or cathepsin D . In contrast, heat-killed organisms and latex beads were in acidic vacuoles which contained the V-ATPase, the CI-M6PR, and cathepsin D . A population of vesicles that contained live M . marinum labeled with the lysosomal glycoprotein LAMP-1, but the percentage of vacuoles that labeled was lower than for heat-killed organisms or latex beads . When testing live and heat-killed Mycobacterium tuberculosis, we found levels of colocalization with LAMP- and cathepsin D comparable to those for the M . marinum isolate . We conclude that M . marinum, like M . tuberculosis, can circumvent the host endocytic pathway and reside in an intracellular compartment which is not acidic and does not fuse with lysosomes . In addition, we describe a system for sampling a large population of intracellular organisms by using a laser confocal microscope. Proc Soc Exp Biol Med, 1997 Apr, 214(4), 302 - 17 Iodine metabolism and thyroid-related functions in organisms lacking thyroid follicles: are thyroid hormones also vitamins? Eales JG. Thyroid-related functions in organisms devoid of follicular thyroid tissue have been reviewed . In the lamprey, a primitive vertebrate, the larva concentrates iodide and synthesizes thyroid hormones (TH) by iodoperoxidase (IP)-mediated iodination of a thyroglobulin (TG)-like molecule in a subpharyngeal afollicular endostyle . The endostyle is the thyroid homolog, and it reorganizes into a follicular thyroid at metamorphosis to the adult . Ascidians and amphloxus, invertebrate protochordate relatives of vertebrates, also concentrate iodide and synthesize TH in a subpharyngeal afollicular endostyle, but the endostyle never transforms to follicles . Ascidian plasma contains L-thyroxine and its more biologically active derivative 3,5,3'-triiodo-L-thyronine, and TH receptors exist, but TH effects are poorly understood . No other invertebrates possess an endostyle . Several invertebrates concentrate iodide at other sites and form protein-incorporated iodohistidines and iodotyrosines; however, de novo iodothyronine biosynthesis through IP-mediated TG iodination has not been established . Nevertheless, TH occur in invertebrates, and exogenous iodothyrosines or iodothyronines have effects on jellyfish, insects, and sea urchins . Furthermore, gut bacteria metabolize TH, and plants may synthesize TH by nonenzymatic oxidative iodination . Thus, TH occur in many organisms and, after ingestion and enteric absorption, can enter the food chain . Indeed, sea urchin larvae obtain TH required to induce metamorphosis from plant diatoms . Thyroid hormones can therefore have vitamin-like effects and, in conjunction with vitamin D, and possibly with other steroids, may be more aptly termed vitamones . Availability of exogenous TH has implications for models of invertebrate and vertebrate TH metabolism and iodine salvaging, and it may explain the prominent and probable ancestral role of peripheral mechanisms in regulating thyroidal status. Int J Syst Bacteriol, 1997 Apr, 47(2), 535 - 40 Mycobacterium mageritense sp . nov; Domenech P et al.; Strains of a new species of rapidly growing, nonphotochromogenic mycobacteria, Mycobacterium mageritense, were isolated from human sputum . The growth characteristics, acid fastness, and mycolic acids of the isolates were consistent with those of Mycobacterium species . The isolates were identified as members of a new species by performing a biochemical analysis and DNA-DNA hybridization experiments, and by comparing the sequences of several conserved genes, such as the 16S rRNA, hsp65, and sodA genes . A phylogenetic analysis in which 16S rRNA and sodA sequences were used identified M . mageritense as a novel distinct species and placed M . mageritense between members of the Mycobacterium fortuitum complex and the thermotolerant rapidly growing group . Our results demonstrate that the taxonomic value of sodA sequence analysis in the genus Mycobacterium is similar to the well-established value of 16S rRNA sequence analysis. Int J Syst Bacteriol, 1997 Apr, 47(2), 377 - 80 Phylogenetic relationships and uncertain taxonomy of Pedomicrobium species; Cox TL et al.; The phylogenetic relationships among the species of the genus Pedomicrobium were studied by comparing their 16S rRNA sequences . The Pedomicrobium species form a coherent phylogenetic cluster within the genera of the hyphal budding bacteria in the alpha-Proteobacteria . The sequences of two strains of Pedomicrobium australicum were obtained from DNAs extracted from nonviable freeze-dried cells, which are the only source of material available, and were found to be almost identical (level of similarity, 99.9%) . Overall, the Pedomicrobium species are closely related, with sequence similarities ranging from 96.2 to 99.9% . Pedomicrobium manganicum is phylogenetically the most distantly related species and exhibits the lowest similarity (96.2%) with Pedomicrobium americanum . Australian isolate Pedomicrobium sp . strain ACM 3067, P . americanum, and P . australicum are all very highly related, with similarities greater than 99% . Pedomicrobium sp . strain ACM 3067 is most closely related to P . australicum (level of similarity, 99.6%) and P . americanum (99.4%) . These manganese-oxidizing species are more closely related to the iron-oxidizing species Pedomicrobium ferrugineum than to the other manganese-oxidizing species, P . manganicum . Taxonomic uncertainties resulting from the loss of the type culture of P . australicum are discussed. J Leukoc Biol, 1997 Apr, 61(4), 459 - 68 Macrophage-parasite relationship in theileriosis . Reversible phenotypic and functional dedifferentiation of macrophages infected with Theileria annulata; Sager H et al.; Theileria annulata is a tick-transmitted protozoan parasite of cattle, which transforms cells of macrophage (Mphi) or B cell lineage . Bone marrow cells, bone marrow cell-derived, and monocyte-derived Mphi were infected with T . annulata sporozoites, and the resulting cell lines were assessed for surface marker expression and function . Transformed lines expressed histocompatibility complex (MHC) class-I and II, CD44, CD45, and the myeloid marker DH598-surface markers CD14, CD11b, M-M7, TH57A, and to a lesser extent CD11a/CD18, CD11c, and ACT(B), were down-regulated . Likewise, transformed cells failed to express Mphi functions (Fc-receptor-mediated phagocytosis, phorbol myristate acetate-induced oxidative burst, lipopolysaccharide-induced tumor necrosis factor alpha, and nitric oxide generation and procoagulant activity up-regulation) . Mphi origin was assured by homogeneity of the starting population, cloning of cells by limiting dilution, and repeated microscopic and flow cytometric monitoring of the cell lines . Elimination of the parasite by treatment with BW720c resulted in the re-acquisition of monocyte lineage properties, as evidenced by up-regulation of CD14, and by re-acquisition of the capacity to ingest opsonized sheep red blood cells and bacteria . Thus, Mphi transformed by T . annulata appear to undergo a process of parasite-induced dedifferentiation but reassume the differentiated phenotype upon elimination of the parasite. J Bacteriol, 1997 Apr, 179(8), 2777 - 82 Cloning of the dapB gene, encoding dihydrodipicolinate reductase, from Mycobacterium tuberculosis; Pavelka MS Jr et al.; Diaminopimelate (DAP) is used by bacteria for the synthesis of lysine . In many species of bacteria, including mycobacteria, DAP is also used for peptidoglycan biosynthesis . In this report we describe the cloning of the dapB gene encoding dihydrodipicolinate reductase (DHPR), which catalyzes a key branch point reaction in the bacterial DAP biosynthetic pathway, from Mycobacterium tuberculosis . Analyses of the DapB proteins from different bacterial species suggest that two different classes of DHPR enzymes may exist in bacteria. J Egypt Soc Parasitol, 1997 Apr, 27(1), 183 - 95 A study on Demodex folliculorum in rosacea; Abd-El-Al AM et al.; A random sample of 16 female patients suffering from papulopustular rosacea (PPR) as well as (16) normal female healthy subjects as control group were adopted in this study to assess of Demodex folliculorum pathogenesis . It was done through determination of mite density using a standard skin surface biopsy 10.5 cm2 from different designated 6 areas on the face, and scanning electron microscopic study (SEM) as well as total IgE estimation . A trial of treatment using Crotamiton 10% cream with special program was also attempted . All subjects ranged between 35-55 years old . All patients with rosacea and 15 of the control group i.e . 75.93% were found to harbour mites . The mean mite counts by site distribution were 28.6 & 6.9 on the cheeks, followed by 14.5 & 3.0 on the forehead and lastly 6.8 & 0.8 on the chin in PPR and control groups respectively . The total mean mite count in patients was 49.9 initially and 7.9 after treatment . In the control group it was 10.7 & 10.6 respectively . The mean total IgE was 169.4 & 168.4 and 96.3 & 98.4 in PPR and control groups respectively Light and scanning electron microscopy revealed that all mites were pointing in one direction . Some of them were containing bacteria inside their gut and on their skin . After treatment 3 cases (18.75%) were completely cured, 10 cases (62.5%) gave moderate response while 3 cases (18.75) have no response . In conclusion, this study supports the pathogenic role of D . folliculorum in rosacea. Appl Environ Microbiol, 1997 Apr, 63(4), 1610 - 6 The nodular microsymbionts of Gymnostoma spp . are Elaeagnus-infective Frankia strains; Navarro E et al.; The phylogenetic relationships of Frankia strains infective on Gymnostoma with other Frankia strains was analyzed . Partial sequencing of the 16S rDNA and use of specific primers showed that the Frankia strains present in Gymnostoma are phylogenetically close to Elaeagnus-infective strains . This finding was confirmed by using the sequences of the hypervariable nifDK intergenic spacer . The strains present in Gymnostoma nodules were close to one another . Clustered with Elaeagnus-infective strains, and distantly related to Casuarina and Alnus-infective strains . Morphological observations of strains and cross-inoculation trials showed that Gymnostoma-infective strains are indistinguishable from Elaeagnus-infective strains . Results of both phenotypic and genotypic approaches indicate that Gymnostoma-infective strains are Elaeagnus infective and not Casuarina infective. Appl Environ Microbiol, 1997 Apr, 63(4), 1594 - 7 Detection of Methylobacterium species by 16S rRNA gene-targeted PCR; Nishio T et al.; We designed PCR primers for specific amplification of the 16S rRNA genes of seven species of the genus Methylobacterium . All of the pairwise species tested were successfully differentiated by PCR detection with a combination of five primer sets, with the exception of M . extorquens and M . rhodesianum . These primers did not cross-react with closely related bacteria in the alpha subclass tested. Appl Environ Microbiol, 1997 Apr, 63(4), 1256 - 60 Group-specific 16S rRNA hybridization probes for determinative and community structure studies of Butyrivibrio fibrisolvens in the rumen; Forster RJ et al.; Oligonucleotide probes covering three phylogenetically defined groups of Butyrivibrio spp . were successfully designed and tested . The specificity of each probe was examined by hybridization to rRNAs from an assortment of B . fibrisolvens isolates as well as additional ruminal and nonruminal bacteria . The sensitivity of the hybridization method was determined by using one of the probes (probe 156) . When RNA was extracted from a culture of OB156, the probe was able to detect target cells at densities as low as 10(4) cells/ml . When 10(4) or more target cells/ml were added to cattle rumen samples, detectable hybridization signals were obtained with 1,000 ng of total RNA loaded onto the nylon membrane . In contrast, the sensitivity was reduced to 10(6) target cells/ml at 100 ng of RNA per slot . The probes were used to type 19 novel Butyrivibrio isolates . The phylogenetic placement was confirmed by partial 16S rRNA gene sequencing . The use of the probes in community-based studies indicated that the Butyrivibrio groups examined in this paper did not represent a significant portion of the bacterial 16S rRNA pool in the rumen of the cattle, sheep, and deer examined. Arch Environ Contam Toxicol, 1997 Apr, 32(3), 260 - 7 Assessment of sediment toxicity using different trophic organisms; Cheung YH et al.; The main aim of the present project is to study the feasibility of using different trophic organisms for evaluating the toxicity of dredged sediments arising in Hong Kong . A total of eight sediment samples (duplicate samples collected from four selected sites: Kowloon Bay, Tsing Yi,Chek Lap Kok, and Double Haven) of Hong Kong coastal waters were analyzed for the total concentrations of As, Cd, Cr, Cu, Hg, Ni, Pb, and Zn, total organic carbon, acid volatile sulfides, simultaneously extracted metals, redox potential, and 12 organic micropollutants . The sediment elutriates were also analysed for the various metal concentrations, as well as contents of ammonia-N, nitrate, total sulfide, sulfate, and total organic carbon . Elutriate Sediment Toxicity Tests (ESTT) were also conducted, using two microalgae (Skeletonema costatum, a diatom and Dunaliella tertiolecta, a flagellate), juvenile shrimp (Metapenaeus ensis) and juvenile fish (Trachinotus obtaus) . Two commercially available tests using bacteria (Microtox Test and Toxi-Chromotest) also were employed to test both the solid phase and elutriates of the sediments . The results of Microtox test on the solid phase, and bioassay tests using diatom on the sediment elutriate, especially the former, were correlated significantly (p < 0.05) with a number of physico-chemical properties of sediments and elutriates . It is recommended that a combination of a liquid-phase bioassay using diatom and a solid-phase bioassay using Microtox test should be used for screening a large number of sediment samples . However, the presence of ammonia in the sediments containing a high content of organic matter seemed to interfere the detection of contamination impacts. Nat Struct Biol, 1997 Apr, 4(4), 285 - 91 The C-terminal half of the anti-sigma factor, FlgM, becomes structured when bound to its target, sigma 28; Daughdrill GW et al.; The interaction between the flagellum specific sigma factor, sigma 28, and its inhibitor, FlgM, was examined using multidimensional heteronuclear NMR . Here we observe that free FlgM is mostly unfolded, but about 50% of the residues become structured when bound to sigma 28 . Our analysis suggests that the sigma 28 binding domain of FlgM is contained within the last 57 amino acids of the protein while the first 40 amino acids are unstructured in both the free and bound states . Genetic analysis of flgM mutants that fail to inhibit sigma 28 activity reveal amino acid changes that are also isolated to the C-terminal 57 residues of FlgM . We postulate that the lack of structure in free and bound FlgM is important to its role as an exported protein. Nat Biotechnol, 1997 Apr, 15(4), 369 - 72 Genetically engineered plants producing opines alter their biological environment; Oger P et al.; Little is known about the consequences of releasing genetically engineered plants (GEP) into the environment . Using opine-producing GEP, we show that transgenic plants alter their biological environment, more precisely the root-associated bacterial populations . The alterations were both transgene-specific and target population-specific . Therefore, assessment studies on the introduction of a given transgene into a GEP will be valid on the given transgene . Evidence of any transgene-associated biological effect will depend on the determination of the pertinent target populations, the identification of which is a key step of such studies. Am J Ind Med, 1997 Apr, 31(4), 403 - 13 Respiratory symptoms and lung function abnormalities among machine operators in automobile production; Sprince NL et al.; This cross-sectional study was designed to assess differences in prevalence of respiratory symptoms and lung function between machine operators exposed to semisynthetic or soluble metal-working fluids (MWFs) and unexposed assemblers and to assess exposure-response relationships with MWF type, total aerosol, endotoxin, culturable bacteria and fungi . We evaluated 183 machine operators and 66 assemblers from one large automobile transmission plant using questionnaires, spirometry data, and cross-shift assessment of both lung function and respiratory symptoms . We found that airborne exposures to total aerosol, endotoxin, culturable bacteria and fungi were higher in machine operations than in the assembly area . There was a correlation between bulk and airborne culturable bacteria, but not between bulk and airborne culturable fungi . Machine operators had significantly more usual cough, usual phlegm, work-related chest tightness and post-shift symptoms of chest tightness, throat irritation, and cough compared with assemblers . We found exposure-response relationships between respiratory symptoms and total aerosol, as well as culturable fungi and bacteria . Associations with endotoxin were not strong or consistent, possibly because airborne levels were generally low . Cross-shift lung function decrements did not differ between machine operators and assemblers and there were no associations with MWF or specific exposures . The finding of respiratory symptoms at low levels of exposure in this study suggests the need to re-assess total aerosol thresholds . Associations between airborne fungal exposures and respiratory symptoms need further study to characterize sources of exposure other than MWF in machining operations. J Bacteriol, 1997 Apr, 179(7), 2401 - 9 Interactions of OxyR with the promoter region of the oxyR and ahpC genes from Mycobacterium leprae and Mycobacterium tuberculosis; Dhandayuthapani S et al.; In contrast to the intact oxyR gene (a homolog of the central regulator of peroxide stress response in enteric bacteria) in Mycobacterium leprae, this gene is inactive in all strains of M . tuberculosis . In both species, oxyR is divergently transcribed from ahpC, which encodes a homolog of alkyl hydroperoxide reductase . To initiate investigations of the regulation of oxidative stress in mycobacteria and consequences of the elimination of oxyR in M . tuberculosis, in this work we tested the hypothesis that mycobacterial OxyR acts as a DNA binding protein and analyzed its interactions with the oxyR and ahpC promoters . M . leprae OxyR was overproduced and purified, and its binding to the oxyR-ahpC intergenic region of M . leprae was demonstrated . By using a sequential series of overlapping DNA fragments, the minimal OxyR binding site was delimited to a 30-bp DNA segment which included a palindromic sequence conforming with the established rules for the LysR family of regulators . A consensus sequence for the mycobacterial OxyR recognition site (cTTATCggc-N3-gccGATAAg) was deduced based on its conservation in different mycobacteria . A variance in two potentially critical nucleotides within this site was observed in M . tuberculosis, in keeping with its reduced affinity for OxyR . Transcription of plasmid-borne M . leprae oxyR and ahpC was investigated in M . smegmatis and M . bovis BCG by S1 nuclease protection and transcriptional fusion analyses . Two mRNA 5' ends were detected in each direction: (i) P1oxyR and P2oxyR and (ii) P1ahpC and P2ahpC . The binding site for OxyR overlapped P1oxyR, reminiscent of the autoregulatory loops controlling expression of oxyR in enteric bacteria and characteristic of the LysR superfamily in general . This site was also centered 65 bp upstream of P1ahpC, matching the usual position of LysR-type recognition sequences in relationship to positively controlled promoters . Superimposed on these features was the less orthodox presence of multiple transcripts and their unique arrangement, including a region of complementarity at the 5' ends of the P2ahpC and P2oxyR mRNAs, suggesting the existence of complex regulatory relationships controlling oxyR and ahpC expression in mycobacteria. J Bacteriol, 1997 Apr, 179(7), 2281 - 8 Posttranscriptional regulation of Caulobacter flagellin genes by a late flagellum assembly checkpoint; Anderson DK et al.; Flagellum formation in Caulobacter crescentus requires ca . 50 flagellar genes, most of which belong to one of three classes (II, III, or IV) . Epistasis experiments suggest that flagellar gene expression is coordinated with flagellum biosynthesis by two assembly checkpoints . Completion of the M/S ring-switch complex is required for the transition from class II to class III gene expression, and completion of the basal body-hook structure is required for the transition from class III to class IV gene expression . In studies focused on regulation of the class IV flagellin genes, we have examined fljK and fljL expression in a large number of flagellar mutants by using transcription and translation fusions to lacZ, nuclease S1 assays, and measurements of protein stability . The fljK-lacZ and fljL-lacZ transcription fusions were expressed in all class III flagellar mutants, although these strains do not make detectable 25- or 27-kDa flagellins . The finding that the fljK-lacZ translation fusion was not expressed in the same collection of class III mutants confirmed that fljK is regulated posttranscriptionally . The requirement of multiple class III genes for expression of the fljK-lacZ fusion suggests that completion of the basal body-hook is an assembly checkpoint for the posttranscriptional regulation of this flagellin gene . Deletion analysis within the 5' untranslated region of fljK identified a sequence between +24 and +38 required for regulation of the fljK-lacZ fusion by class III genes, which implicates an imperfect 14-bp direct repeat in the posttranscriptional regulation of fljK . Our results show that fljL is also regulated posttranscriptionally by class III and unclassified flagellar genes, apparently by a mechanism different from the one regulating fljK. J Invest Dermatol, 1997 Apr, 108(4), 476 - 81 All-trans-retinoic acid inhibition of Pro alpha1(I) collagen gene expression in fetal rat skin fibroblasts: identification of a retinoic acid response element in the Pro alpha1(I) collagen gene; Meisler NT et al.; The current study was undertaken to determine the mechanism by which the retinoid all-trans-retinoic acid regulates pro alpha1(I) collagen gene expression in fetal rat skin fibroblasts . FRS fibroblasts were stably transfected with the ColCat3.6 plasmid, which contains a portion of the 5' flanking region of the rat pro alpha1(I) collagen gene linked to a reporter gene, chloramphenicol acetyltransferase . The effect of t-RA on CAT activity was determined as a function of concentration and incubation time . Maximal inhibition of CAT activity by t-RA occurred at 10(-8) M after 48 h of treatment . Transforming growth factor-beta1 did not block the inhibitory effect of t-RA on CAT activity . Computer sequence analysis of the 3.6-kb DNA fragment that contains the promoter for the rat pro alpha1(I) collagen gene identified a direct repeat RARE sequence composed of one diverse (5'-AGTAGA-3') and one idealized (5'-GGGTCA-3') half site located at positions -1345 and -1335, respectively . Two nuclear retinoid receptors that were expressed in bacteria, retinoic acid receptor-gamma and retinoid X receptor-alpha, were found to bind specifically to a double-stranded oligonucleotide containing the RARE in gel mobility shift assays . Mutation of the idealized half-site eliminated the binding of receptor proteins to the oligonucleotide . Gel mobility shift assays using nuclear protein extracts prepared from t-RA-treated FRS fibroblasts showed that binding to the oligonucleotide containing the RARE was decreased from control values . The same assays performed with the mutated oligonucleotide resulted in only slight binding . These studies indicate that t-RA downregulates the promoter activity of the rat pro alpha1(I) collagen gene by decreasing the binding of nuclear protein to the RARE sequence in the 5' flanking region of the gene. Brain Res, 1997 Mar 28, 752(1-2), 99 - 106 Application of silicon microphysiometry to tissue slices: detection of metabolic correlates of selective vulnerability; Ajilore OA et al.; The silicon microphysiometer is a recently developed instrument which measures rates of proton efflux in real time from small numbers of cultured cells . Since the main products of cellular metabolism are lactic acid and carbon dioxide, this instrument affords an indirect but sensitive measure of cellular metabolism . We previously described the use of the instrument with primary neuronal cultures (Raley-Susman, K.M., Miller, K.R., Owicki, J.C . and Sapolsky, R.M., Effects of excitotoxin exposure on metabolic rate of primary hippocampal cultures: application of silicon microphysiometer to neurobiology, J . Neurosci., 12 (1992) 773-780) . In the present report, we adapt the instrument for the indirect measurement of metabolism in tissue slices . In initial studies, we demonstrate stable measures of metabolism with low background noise in hippocampal slices . In addition, measures were relatively insensitive to slice thickness, preparation time or the possible contribution of contaminating bacteria . We then demonstrate the ability to detect metabolic correlates of selective vulnerability in individual hippocampal cell fields . Specifically, we observe a metabolic response to kainic acid that was selective for CA3-derived tissue, and a response to cyanide that was selective for CA1-derived tissue . This corresponds to the well-known vulnerability of CA3 and CA1 to excitotoxic and ischemic insults, respectively . Finally, we show that glucocorticoids, stress-sensitive steroid hormones which are known to exacerbate the toxicity in kainic acid in CA3 neurons, exacerbate the metabolic effects of this excitotoxin as well; in this case, the steroid manipulation was carried out in rats prior to killing . Thus, this instrument represents a complement to more traditional approaches for assessing metabolism in specific brain regions and it can potentially be used for a broad variety of studies with animals of differing ages and pre-mortem manipulations. J Biochem Biophys Methods, 1997 Mar 27, 34(2), 147 - 54 Quantification of total RNA by ethidium bromide fluorescence may not accurately reflect the RNA mass; Kerkhof L; The fluorescent signal from purified large and small ribosomal RNA subunits stained with ethidium bromide was quantified using agarose gels and image analysis . A significantly smaller fluorescent signal was observed for 23 S rRNA compared to 16 S rRNA when normalized to mass . Therefore, small changes in amounts of 23 S or 16 S rRNA within a sample can have a profound impact on observed bulk fluorescence . A comparison of 23 S/16 S rRNA fluorescence ratios for 4 marine bacteria grown in batch culture indicates that the lower fluorescence of 23 S rRNA is widespread . The 23 S/16 S rRNA fluorescence ratios also suggest that intracellular concentrations of ribosomal subunits do not always adhere to a stoichiometry of 2.0. Carbohydr Res, 1997 Mar 26, 299(1-2), 69 - 76 Structure of the capsular polysaccharide from Alteromonas nigrifaciens IAM 13010T containing 2-acetamido-2,6-dideoxy-L-talose and 3-deoxy-D-manno-octulosonic acid; Gorshkova RP et al.; A capsular polysaccharide was obtained from Alteromonas nigrifaciens IAM 13010T by saline extraction . On the basis of 1H and 13C NMR spectroscopy, including one-dimensional (1D) NOE spectroscopy, 2D rotating-frame NOE spectroscopy (ROESY), and 1H-detected heteronuclear 1H,13C multiple-quantum coherence (HMQC), it was concluded that the polysaccharide contained inter alia an acidic sugar, 3-deoxy-D-manno-octulosonic acid (Kdo), and a rare amino sugar, 2-acetamido-2,6-dideoxy-L-talose (L-6dTalNAc, N-acetylpneumosamine), and has a pentasaccharide repeating unit of the following structure: {equation: see text} J Cell Biol, 1997 Mar 24, 136(6), 1271 - 86 Inactivation of two Dictyostelium discoideum genes, DdPIK1 and DdPIK2, encoding proteins related to mammalian phosphatidylinositide 3-kinases, results in defects in endocytosis, lysosome to postlysosome transport, and actin cytoskeleton organization; Buczynski G et al.; Phosphatidylinositide 3-kinases (PI3-kinases) have been implicated in controlling cell proliferation, actin cytoskeleton organization, and the regulation of vesicle trafficking between intracellular organelles . There are at least three genes in Dictyostelium discoideum . DdPIK1, DdPIK2, and DdPIK3, encoding proteins most closely related to the mammalian 110-kD PI-3 kinase in amino acid sequence within the kinase domain . A mutant disrupted in DdPIK1 and DdPIK2 (delta ddpik1/ddpik2) grows slowly in liquid medium . Using FITC-dextran (FD) as a fluid phase marker, we determined that the mutant strain was impaired in pinocytosis but normal in phagocytosis of beads or bacteria . Microscopic and biochemical approaches indicated that the transport rate of fluid-phase from acidic lysosomes to non-acidic postlysosomal vacuoles was reduced in mutant cells resulting in a reduction in efflux of fluid phase . Mutant cells were also almost completely devoid of large postlysosomal vacuoles as determined by transmission EM . However, delta ddpik1/ddpik2 cells functioned normally in the regulation of other membrane traffic . For instance, radiolabel pulse-chase experiments indicated that the transport rates along the secretory pathway and the sorting efficiency of the lysosomal enzyme alpha-mannosidase were normal in the mutant strain . Furthermore, the contractile vacuole network of membranes (probably connected to the endosomal pathway by membrane traffic) was functionally and morphologically normal in mutant cells . Light microscopy revealed that delta ddpik1/ddpik2 cells appeared smaller and more irregularly shaped than wild-type cells; 1-3% of the mutant cells were also connected by a thin cytoplasmic bridge . Scanning EM indicated that the mutant cells contained numerous filopodia projecting laterally and vertically from the cell surface, and fluorescent microscopy indicated that these filopodia were enriched in F-actin which accumulated in a cortical pattern in control cells . Finally, delta ddpik1/ddpik2 cells responded and moved more rapidly towards cAMP . Together, these results suggest that Dictyostelium DdPIK1 and DdPIK2 gene products regulate multiple steps in the endosomal pathway, and function in the regulation of cell shape and movement perhaps through changes in actin organization. Biochim Biophys Acta, 1997 Mar 20, 1351(1-2), 9 - 12 Nucleotide and corrected amino acid sequence of the functional recombinant rat anaphylatoxin C5a; Rothermel E et al.; For bacterial expression of rat anaphylatoxin C5a, the cDNA was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using rat liver RNA and degenerate primers designed according to the published amino acid sequence {1} . Surprisingly, the amino acid sequence deduced from cDNA differed at positions 55 (N for K), 63 (K for H), 67 (E for N), 68 (S for E) and 69 (H for S) from the published sequence . The overall amino acid composition, however, was unchanged because these 5 amino acids were located at different positions compared to the published sequence . As a consequence, the proposed N-glycosylation site was absent, suggesting O-glycosylation of the mature molecule . Recombinant rat C5a with a 6 histidine tag at the N-terminus was expressed in bacteria, purified and renatured . The peptide was as potent as recombinant human C5a in eliciting lysosomal enzyme release from human granulocytes. Cancer Lett, 1997 Mar 19, 114(1-2), 93 - 5 Preventive role of lyophilic dairy products bulgaricum and biostim; Ivanova T et al.; The purpose of the study was to examine the protective role of lyophilic dairy products bulgaricum and biostim with active bacteria lactic acid strain . Protective effect was determined as a phagocytosis activity and activity of major immunoglobulins . In total 120 men aged 30-35 years were included in three groups (30 controls with placebo) . Daily doses (50 g) of these products were taken during a month . Positive responses from investigation links of immune system and paraspecific resistance were observed . Immunologic response was significantly higher after lyophilic dairy diet and this indicates that bulgaricum and biostim are products with protective role due to specific influence of bacteria lactic acid. Bull Acad Natl Med, 1997 Mar 18, 181(3), 431 - 9 {Helicobacter infections of man and of domestic carnivores: comparative data}; Lecoindre P et al.; The role of Helicobacter pylori in generating of the chronic gastritis and in the maintaining of the gastroduodenal ulcerous disease, has been a major medical discovery of these past years in human gastroenterology . More recently in Man, studies have showed that the gastric tumours (adenocarcinoma, lymphoma) are epidemiologically associated with the H . pylori infection . Although the H . pylori infection is the one of the most frequent in the word, the epidemiologic and ecologic aspects of this infections are still not very well known . Thanks to phylogenic studies using the new molecular biology techniques and to fundamental experimental studies, we know more about helicobacteria in domestic carnivores as well as their morphologic characteristic, their taxonomia and more importantly details concerning their ecological niche . Few clinical studies have been made to this day, but the ones that have been undertaken are interesting in confirming the extensive prevalence of Helicobacter infections in domestic carnivores and in underlining their role in the genesis of the inflammatory gastropathies observed in these species . Recent observations have demonstrated the ubiquitous character of these helicobacteria by showing their presence in the stomach of man, dogs and cats . This ubiquitous character has led some scientists to consider the potential zoonotic risk of the human infection by Helicobacter heilmannii, felis or pylori . Finally, the Helicobacter infection of animals seems to be an interesting model not only in the study of the affections caused by these bacteria, but also in the elaboration of a future vaccine against the H . pylori infection in man. EMBO J, 1997 Mar 17, 16(6), 1137 - 44 Transgenic tobacco expressing a foreign calmodulin gene shows an enhanced production of active oxygen species; Harding SA et al.; A strategy for elucidating specific molecular targets of calcium and calmodulin in plant defense responses has been developed . We have used a dominant-acting calmodulin mutant (VU-3, Lys to Arg115) to investigate the oxidative burst and nicotinamide co-enzyme fluxes after various stimuli (cellulase, harpin, incompatible bacteria, osmotic and mechanical) that elicit plant defense responses in transgenic tobacco cell cultures . VU-3 calmodulin differs from endogenous plant calmodulin in that it cannot be methylated post-translationally, and as a result it hyperactivates calmodulin-dependent NAD kinase . Cells expressing VU-3 calmodulin exhibited a stronger active oxygen burst that occurred more rapidly than in normal control cells challenged with the same stimuli . Increases in NADPH level were also greater in VU-3 cells and coincided both in timing and magnitude with development of the active oxygen species (AOS) burst . These data show that calmodulin is a target of calcium fluxes in response to elicitor or environmental stress, and provide the first evidence that plant NAD kinase may be a downstream target which potentiates AOS production by altering NAD(H)/NADP(H) homeostasis. Mutat Res, 1997 Mar 17, 389(2-3), 157 - 65 Sodium azide induces mitotic recombination in Drosophila melanogaster larvae; Gonzalez-Cesar E et al.; Sodium azide (NaN3), a potent mutagen for bacteria and barley, was tested for somatic mutation and mitotic recombination induction in wing imaginal disc cells of Drosophila melanogaster . Comparisons were made among inversion-free flr3/mwh, inversion-heterozygous TM3, Ser/mwh, and inversion-free, high bioactivation OR(R), flr3/mwh flies . Third instar larvae were exposed chronically for 48 h to sodium azide at 0.5, 0.63, 0.75, 0.88 and 1.0 mM . The frequencies of spots per wing obtained in the three kinds of progeny scored were compared . In inversion-free flies, sodium azide induced large single and total spots at all concentrations tested, and small single and twin spots at 0.75 mM and higher concentrations . In contrast, it failed to increase the frequency of small and large single spots in inversion-heterozygous flies . In high bioactivation flies (which are inversion-free), sodium azide increased the frequency of large single spots at 0.63, 0.88 and 1.0 mM and the frequency of total spots at 0.63 mM . From the absence of genotoxic activity observed in inversion-heterozygous flies it is concluded that sodium azide induces exclusively mitotic recombination in wing somatic cells of Drosophila melanogaster larvae after chronic exposure . This recombinogenic activity is reduced in the presence of high bioactivation capacity. Biochem J, 1997 Mar 15, 322 ( Pt 3), 927 - 35 Identification of csk tyrosine phosphorylation sites and a tyrosine residue important for kinase domain structure; Joukov V et al.; The lack of a conserved tyrosine autophosphorylation site is a unique feature of the C-terminal Src-kinase, Csk, although this protein tyrosine kinase can be autophosphorylated on tyrosine residues in vitro and in bacteria . Here we show that human Csk is tyrosine phosphorylated in HeLa cells treated with sodium pervanadate . Phosphorylation in vivo occurs mainly at Tyr-184 and in vitro mainly at Tyr-304 . A Y304F mutation strongly decreased Csk phosphorylation in vitro, and a Y184F mutation abolished tyrosine phosphorylation in vivo . A catalytically inactive form of Csk was also phosphorylated on Tyr-184 in vivo, suggesting that this is not a site of autophosphorylation . The kinase activity of the Y184F protein was not changed, while the Y304F protein showed one-third of wild-type activity . Three-dimensional modelling of the Csk kinase domain indicated that the Y304F mutation abolishes one of two conserved hydrogen bonds between the upper and the lower lobes in the open conformation of the kinase domain . Phosphopeptide binding studies suggested that phosphorylation of Tyr-184 creates a binding site for low-molecular-mass proteins . Cellular Csk was associated with several phosphoproteins, some of which were interacting with the Csk SH2 domain . Taken together these results indicate that Csk can be phosphorylated in vivo at Tyr-184 by an as yet unknown tyrosine kinase, and that autophosphorylation of Tyr-304 occurs only at abnormally high Csk concentrations in vitro . Furthermore, Tyr-304 is required for the maintenance of the structure of the Csk kinase domain. Eur J Biochem, 1997 Mar 15, 244(3), 840 - 51 Benzoyl-CoA reductase (dearomatizing), a key enzyme of anaerobic aromatic metabolism . A study of adenosinetriphosphatase activity, ATP stoichiometry of the reaction and EPR properties of the enzyme; Boll M et al.; An enzyme was recently described, benzoyl-CoA reductase (dearomatizing), which catalyses the ATP-driven reduction of the aromatic ring of benzoyl-CoA yielding a non-aromatic CoA thioester, ADP and phosphate {Boll, M . & Fuchs, G . (1995) Eur . J . Biochem . 234, 921-933} . The 170-kDa enzyme consists of four different subunits and contains approximately 12 Fe and acid-labile sulfur/mol . Benzoyl-CoA reductase exhibits ATPase activity in the absence of substrate . It is shown that only the reduced form of this iron-sulfur protein has ATPase activity . ATPase activity is reversibly lost when the enzyme is oxidized by thionine; reduction of the enzyme fully restores ATPase and ring-reduction activity . 2 mol ATP are hydrolyzed/2 mol electrons transferred in the course of the reaction . The product ADP acts as competitive inhibitor (Ki = 1.1 mM) for ATP in benzoyl-CoA reduction; ADP inhibits ATPase activity to the same extent as ring-reduction activity . EPR investigation of the dithionite-reduced enzyme suggested the presence of two separate {2Fe-2S} clusters and two interacting {4Fe-4S} clusters . Addition of MgATP to the reduced enzyme resulted in a new isotropic signal at g = 5.15 and a weak signal at g = 12; in controls with MgADP only a minor signal at g = 5.15 was observed . The positions, shapes and temperature dependencies of these MgATP-induced signals are indicative for excited states of a S = 7/2 spin multiplet . The {2Fe-2S} signals were not affected by ATP, but one of the {4Fe-4S} clusters became slowly oxidized . Addition of both benzoyl-CoA and MgATP resulted in a major oxidation of the iron-sulfur clusters accompanied by the appearance of some minor signals of unknown origin in the g = 2.037-1.96 region . Neither the benzoyl-CoA plus MgATP-oxidized nor the thionine-oxidized enzyme showed the ATP-dependent formation of the high-spin signals of the reduced enzyme . At present we hypothesize that the S = 7/2 signal is due to an ATP-induced change of one of the {4Fe-4S} clusters . The data suggest that hydrolysis of MgATP is required to activate the enzyme; in the absence of substrate the energy involved in this activation dissipates . MgATP-driven formation of this excited state of the reduced enzyme rather than transfer of electrons from the reduced enzyme to the aromatic substrate appears to be the rate-limiting step in the catalytic cycle . We suggest that the excited state is required to overcome the high activation energy associated with the loss of the aromatic character and/or to render ring reduction irreversible. J Clin Invest, 1997 Mar 15, 99(6), 1432 - 44 Correcting temperature-sensitive protein folding defects; Brown CR et al.; Recently, we found that different low molecular weight compounds, all known to stabilize proteins in their native conformation, are effective in correcting the temperature-sensitive protein folding defect associated with the deltaF508 cystic fibrosis transmembrane regulator (CFTR) protein . Here we examined whether the folding of other proteins which exhibit temperature-sensitive folding defects also could be corrected via a similar strategy . Cell lines expressing temperature-sensitive mutants of the tumor suppressor protein p53, the viral oncogene protein pp60src, or a ubiquitin activating enzyme E1, were incubated at the nonpermissive temperature (39.5 degrees C) in the presence of glycerol, trimethylamine N-oxide or deuterated water . In each case, the cells exhibited phenotypes similar to those observed when the cells were incubated at the permissive temperature (32.5 degrees C), indicative that the particular protein folding defect had been corrected . These observations, coupled with our earlier work and much older studies in yeast and bacteria, indicate that protein stabilizing agents are effective in vivo for correcting protein folding abnormalities . We suggest that this type of approach may prove to be useful for correcting certain protein folding abnormalities associated with human diseases. J Am Vet Med Assoc, 1997 Mar 15, 210(6), 808 - 10 Multifocal polyostotic aneurysmal bone cysts in a llama; Anderson DE et al.; A 3.3-year-old 125-kg castrated male llama was evaluated because of acute non-weight-bearing lameness on the left hind limb . Physical examination revealed crepitus in the midportion of the femur . On radiographs, a comminuted middiaphyseal fracture was seen . There was also a region of bone lysis with cortical thinning and expansion in the distal metaphysis and epiphysis of the left femur . Multiple small circular lesions were observed in the proximal metaphysis of the left femur, and the proximal portion of the left tibia appeared irregular . The owner elected to pursue treatment, and the fracture was repaired with 2 compression plates . Multiple bone biopsy specimens were obtained and submitted for bacterial culture and histologic examination . Cultures yielded neither bacteria nor fungi . Histologic examination revealed fibrous connective tissue, normal appearing cortical bone, and an absence of medullary structures . The llama was maintained in a hind-limb sling for 14 days after surgery, at which time follow-up radiography revealed a comminuted fracture of the proximal portion of the femur . The llama was euthanatized, and multifocal polyostotic aneurysmal bone cysts were found in the proximal and distal metaphyses of the left femur and tibia . Cysts were lined by fibroblasts or endothelial-like cells. Carbohydr Res, 1997 Mar 13, 298(4), 261 - 77 Synthesis of 2"-oxidized derivatives of 5-deoxy-5-epi-5-fluoro-dibekacin and -arbekacin, and study on structure-chemical shift relationships of urethane(or amide)-type NH protons in synthetic intermediates; Kobayashi Y et al.; Three 2"-modified dibekacin-analogs have been prepared as potential compounds active against resistant bacteria producing 2"-O-phosphotransferases; one is 5-deoxy-5,2"-diepi-5-fluorodibekacin (9) prepared from a suitably protected 2"-O-triflyl derivative through the 2" 3"-cyclic carbamate, and the others are 2"-oxo derivatives (12 and 22, both as the hydrate) of 5-deoxy-5-epi-5-fluoro-dibekacin and -arbekacin prepared through oxidation at C-2" of suitably protected derivatives . Relationships between the t-butoxycarbonyl(= Boc)-NH-shifts of per-N-Boc synthetic intermediates and their structures were studied . It was found that the shifts, measured in pyridine-d5 at 80 degrees C, which spread over a close range (delta 6-7 ppm), are sensitively influenced by nearby and surrounding groups around the BocNH group in respect of electron-withdrawing character, hydrogen bonding (BocNH .. . acceptor), and also solvent effects (BocNH .. . NC5H5). FEBS Lett, 1997 Mar 10, 404(2-3), 129 - 34 Biosynthesis of ketocarotenoids in transgenic cyanobacteria expressing the algal gene for beta-C-4-oxygenase, crtO; Harker M et al.; The ketocarotenoid astaxanthin is produced by a number of marine bacteria and microalgae . It is synthesized from beta-carotene by the addition of two keto groups to carbons C4 and C4' and two hydroxyl groups to C3 and C3' . The gene, crtO, encoding beta-C-4-oxygenase which converts beta-carotene to canthaxanthin was cloned from the green alga Haematococcus plurialis . We transferred crtO to the cyanobacterium Synechococcus PCC7942, which contains a beta-carotene hydroxylase gene and normally accumulates beta-carotene and zeaxanthin . The genetically engineered cyanobacterium produced astaxanthin as well as other ketocarotenoids . The results confirm that crtO can function in cyanobacteria in conjunction with the intrinsic carotenoid enzymes to produce astaxanthin . Specifically, this finding indicates that beta-carotene hydroxylase, which normally converts beta-carotene to zeaxanthin, can also function in the biosynthesis of astaxanthin . These results provide the first evidence of genetic manipulation of a plant-type carotenoid biosynthesis pathway toward the production of novel carotenoids. Cell, 1997 Mar 7, 88(5), 667 - 74 Bipolar localization of the replication origin regions of chromosomes in vegetative and sporulating cells of B . subtilis; Webb CD et al.; To investigate chromosome segregation in B . subtilis, we introduced tandem copies of the lactose operon operator into the chromosome near the replication origin or terminus . We then visualized the position of the operator cassettes with green fluorescent protein fused to the Lac1 repressor . In sporulating bacteria, which undergo asymmetric cell division, origins localized near each pole of the cell whereas termini were restricted to the middle . In growing cells, which undergo binary fission, origins were observed at various positions but preferentially toward the poles early in the cell cycle . In contrast, termini showed little preference for the poles . These results indicate the existence of a mitotic-like apparatus that is responsible for moving the origin regions of newly formed chromosomes toward opposite ends of the cell. Science, 1997 Mar 7, 275(5305), 1471 - 5 Structure of a protein photocycle intermediate by millisecond time-resolved crystallography; Genick UK et al.; The blue-light photoreceptor photoactive yellow protein (PYP) undergoes a self-contained light cycle . The atomic structure of the bleached signaling intermediate in the light cycle of PYP was determined by millisecond time-resolved, multiwavelength Laue crystallography and simultaneous optical spectroscopy . Light-induced trans-to-cis isomerization of the 4-hydroxycinnamyl chromophore and coupled protein rearrangements produce a new set of active-site hydrogen bonds . An arginine gateway opens, allowing solvent exposure and protonation of the chromophore's phenolic oxygen . Resulting changes in shape, hydrogen bonding, and electrostatic potential at the protein surface form a likely basis for signal transduction . The structural results suggest a general framework for the interpretation of protein photocycles. Nature, 1997 Mar 6, 386(6620), 88 - 91 Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy; Bottcher B et al.; Hepatitis B virus, a major human pathogen with an estimated 300 million carriers worldwide, can lead to cirrhosis and liver cancer in cases of chronic infection . The virus consists of an inner nucleocapsid or core, surrounded by a lipid envelope containing virally encoded surface proteins . The core protein, when expressed in bacteria, assembles into core shell particles, closely resembling the native core of the virus . Here we use electron cryomicroscopy to solve the structure of the core protein to 7.4 A resolution . Images of about 6,400 individual particles from 34 micrographs at different levels of defocus were combined, imposing icosahedral symmetry . The three-dimensional map reveals the complete fold of the polypeptide chain, which is quite unlike previously solved viral capsid proteins and is largely alpha-helical . The dimer clustering of subunits produces spikes on the surface of the shell, which consist of radial bundles of four long alpha-helices . Our model implies that the sequence corresponding to the immunodominant region of the core protein lies at the tip of the spike and also explains other properties of the core protein. Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 1979 - 84 sigma54, a vital protein for Myxococcus xanthus; Keseler IM et al.; The rpoN gene encoding the transcription factor sigma54 in Myxococcus xanthus has been cloned using a heterologous rpoN probe . The sequence of the cross-hybridizing DNA confirmed the existence of an ORF 1518 bp long that encodes a well conserved member of the sigma54 family of sigma factors . Low- as well as high-stringency hybridizations detected only a single rpoN gene in the M . xanthus chromosome . In other bacteria, sigma54 is an alternative sigma, and null mutants are viable . However, all attempts to construct a strain containing a null mutation in the M . xanthus rpoN have been unsuccessful . Partial diploids of rpoN+/rpoN null are viable . Recombination experiments with such partial diploids showed the impossibility of constructing, either by segregation or by transduction, a viable null haploid under any of a wide range of growth conditions . The product of the rpoN gene, sigma54, therefore appears to be essential for growth in M . xanthus. Zh Mikrobiol Epidemiol Immunobiol, 1997 Mar-Apr, (2), 17 - 20 {A rapid method for assessing the virulence of Francisella tularensis in vitro}; Pavlovich NV et al.; A rapid and simple method for the in vitro evaluation of the virulence of F . tularensis is proposed . The optimal parameters--the concentrations of bacteria and the substrate (phenolphthalein phosphate), the time incubation in the serum--have been developed and special conditions for strains of different subspecies have been selected . The proposed method makes it possible to greatly reduce the time of obtaining results and to exclude labor-consuming experiments on laboratory animals when working with large collections of F . tularensis laboratory and natural wild strains . Moreover, the method widens the possibilities of experimenters in the evaluation of the effect of some mutation on the virulence of the strain under study. Z Arztl Fortbild Qualitatssich, 1997 Mar, 91(2), 111 - 5 {Etiology of stomach carcinoma--opinions and facts--yesterday and today}; Schramm T et al.; Recent research demonstrated that views on the etiology of gastric cancer, which had been considered true over decades do not correspond with the real situation . Therefore, efforts for primary prevention of gastric cancer could not be successful . The bacteria Helicobacter pylori is considered today to be an important but not the only etiologic factor of this disease . Eradication of this bacteria decreases the risk of gastric cancer. J Zoo Wildl Med, 1997 Mar, 28(1), 97 - 100 Organochlorine pesticides associated with ocular, nasal, or otic infection in the eastern box turtle (Terrapene carolina carolina); Tangredi BP et al.; From May 1987 to September 1994, 19 eastern box turtles (Terrapene carolina carolina) originating from scattered locations on Long Island, New York (USA) were presented with one or more of the following signs: listlessness, ocular and nasal discharge, conjunctivitis, blepharitis, and otitis media . Numerous species of bacteria and yeast were isolated by aerobic culture . Histopathologic findings confirmed chronic active bacterial infections . Toxicologic analyses of livers revealed elevated concentrations of chlordane metabolites in two diseased turtles . A third turtle liver contained residues of endosulfan sulfate . Immunosuppressive effects of low-level exposure to organochlorines, including chlordane and endosulfan, could be involved in the pathogenesis of the observed infections. Baillieres Clin Gastroenterol, 1997 Mar, 11(1), 175 - 93 Ileal pouches: adaptation and inflammation; Merrett MN; Ileal pouch-anal anastomosis (IPAA) has become the operation of choice following proctocolectomy for ulcerative colitis (UC) and familial adenomatous polyposis . Functioning ileal pouch mucosa undergoes histological changes resembling the colon (colonic metaplasia) . The possible role of stasis and luminal factors--bile acids, short-chain fatty acids and bacteria--are discussed . It seems likely that colonic metaplasia is an adaptive response to the new luminal environment in IPAA . Inflammation in the ileal reservoir ('pouchitis') is the most significant late complication in IPAA . It occurs in 20-30% of patients and is virtually confined to those with prior UC . The clinical picture in pouchitis is highly variable; however, it can be easily categorized into three groups . Nevertheless, in most cases it is likely to represent recurrent UC in the ileal pouch . Current treatments and possible preventative strategies for pouchitis have been outlined. Age Ageing, 1997 Mar, 26(2), 87 - 9 The effect of long-term omeprazole on the glucose-hydrogen breath test in elderly patients; Hutchinson S et al.; OBJECTIVE: to test whether omeprazole taken for longer than 1 month causes an increase in the rate of small bowel bacterial overgrowth in elderly subjects . SUBJECTS: 44 elderly people, 22 taking omeprazole, 22 not taking omeprazole or H2 receptor antagonists . MAIN OUTCOME MEASURES: rate of positive glucose-hydrogen breath tests; anthropometric measures and blood tests reflecting malabsorption . RESULTS: there was no difference in the rate of positive tests between those taking omeprazole (45%) and those not taking it (59%) . The omeprazole group had significantly lower serum albumin concentrations . There was no difference in body mass index, mid-arm circumference, arm fold thickness, adjusted calcium concentration or haemoglobin levels . CONCLUSIONS: omeprazole does not cause increased bacterial shall bowel overgrowth in elderly subjects. Biol Chem, 1997 Mar-Apr, 378(3-4), 207 - 10 Feedback inhibition of dihydrodipicolinate synthase enzymes by L-lysine; Blickling S et al.; Dihydrodipicolinate synthase (DHDPS) is the first enzyme unique to the lysine biosynthetic pathway and is feedback regulated by L-lysine in plants and some bacteria . The allosteric binding site has been localized by X-ray crystallography and is in agreement with reported mutations of plant DHDPS enzymes, which confer insensitivity to feedback inhibition . Three possible elements of the mechanism of lysine inhibition are discussed. Biol Chem, 1997 Mar-Apr, 378(3-4), 121 - 30 The proteasome: a macromolecular assembly designed to confine proteolysis to a nanocompartment; Baumeister W et al.; Significant progress has been made over the past few years in elucidating the structural principles and the enzymatic mechanism of the 20S proteasome . As a result, the proteasome has become the prototype of a new family of enzymes, the Ntn hydrolases, as well as a paradigm for macromolecular assemblies that confine their proteolytic activity to an inner nanocompartment . Since access to this nanocompartment is restricted to unfolded substrate polypeptides, the 20S proteasome must be functionally linked to a substrate recognition and unfolding machinery . In eukaryotes this is provided by the 19S 'cap' complex, which associates with the 20S core to form the 26S proteasome, a protease capable of degrading ubiquitinated proteins in an ATP-dependent manner. Hepatogastroenterology, 1997 Mar-Apr, 44(14), 599 - 603 Helicobacter pylori after surgery for duodenal ulcer; Kunzle JE et al.; BACKGROUND/AIMS: The role of Helicobacter pylori as a cause of peptic ulcer is still subject to controversy . The Kock's postulates have not been yet fulfilled; the bacteria can be found in normal persons, and it persists in the stomach after the ulcer is healed . MATERIAL AND METHODS: The authors analyzed 41 persons formerly submitted to surgery (after 8 years and 4 months, as a mean), 31 to highly selective vagotomy, and 10 to truncal or selective vagotomy plus gastroduodenal drainage . All of them were asymptomatic, or had symptoms not related to ulcer relapse . RESULTS: At endoscopy the ulcers were healed in all 41 individuals, and there was evidence of gastritis in three . The histopathological exam showed gastritis in all biopsy specimens . The search of H . pylori by urease method and by Giemsa staining was positive in 40 . CONCLUSION: It was concluded that the gastric acid secretion reduced by vagotomy was the main factor to healing the ulcer, not subordinated to H . pylori. Curr Biol, 1997 Mar 1, 7(3), R179 - 81 Ribosomes: protein synthesis in slow motion; Moore PB; Using image reconstruction methods, electron microscopists can now visualize ribosomes at resolutions so high that the changes in the positions of ribosome-bound tRNAs which occur during protein synthesis can be seen. P R Health Sci J, 1997 Mar, 16(1), 9 - 14 Survival and quality of life in HIV-positive patients treated with a polyantigenic immunomodulator; Colon JI et al.; A polyantigenic immunomodulator (PAI), previously known as polyantigenic vaccine, which consists of a mixture of antigens of inactivated bacteria with antigens of influenza virus in a peanut-oil-arlacel-A-aluminium monoesterate emulsion, increased tumor resistance and induced tumor regression in tumor bearing mice . This report presents clinical and laboratory data that demonstrate the effect of PAI in long term prolongation of disease free state in HIV positive patients . A total of 40 patients, 35 males and 5 females, with a mean age of 41.1 +/- 10.5 years, ranging from 28 to 68 years, HIV positive by (ELISA and Western Blot), with no restriction on the CD4 + T lymphocytes counts, were included in this open study . The PAI regimen was one subcutaneous injection per week for patients with < 400 CD4 + lymphocytes and one monthly injection for patients with CD4 + count > 400 . All patients were monitored at different intervals for lymphocyte counts, clinical condition and treatment toxicity . After a follow up of eight years 81% of the patients were alive and 47% were free of disease . In patients without AIDS, the weight was 153.9 +/- 28 pounds pre-PAI and 164.6 +/- 27 (P = 1.2 x 10(-4); the CD4 + lymphocyte count was 795 +/- 421 pre-PAI and 585 +/- 279 post PAI (P = 0.08) . In patients alive with AIDS, the weight was 159.5 +/- 32 pre-PAI and 163.9 +/- 32 pounds post-PAI (P = 0.8); the CD4 + lymphocyte counts was 491 +/- 255 pre-PAI and 298 +/- 142 post-PAI (P = 0.08); and five AIDS-related infections occurred in five patients . In patients who died the weight was 157.7 +/- 23 pre and 146.8 +/- 30 post (P = 0.10); and the CD4 count was 340.7 +/- 149 pre and 103.4 +/- 88 post (P = 0.0057) . All died with infection . No toxicity with the use of PAI was reported . PAI improves disease free survival and quality of life in HIV + patients. Electrophoresis, 1997 Mar-Apr, 18(3-4), 563 - 7 Characterization of Chlamydia trachomatis l2-induced tyrosine-phosphorylated HeLa cell proteins by two-dimensional gel electrophoresis; Birkelund S et al.; Chlamydia trachomatis is an obligate intracellular bacteria, inducing its own uptake in nonprofessional phagocytes either by phagocytosis or pinocytosis . We have previously shown that C . trachomatis L2 induces tyrosine phosphorylation of eukaryotic proteins upon their entry by phagocytosis . In this paper we characterize the tyrosine-phosphorylated proteins by two-dimensional gel electrophoresis . In immunoblotting with anti-phosphotyrosine antibodies of C . trachomatis L2-infected HeLa cells, but not with uninfected cells, two rows of spots were observed with a molecular mass of 69 and 71 kDa and pI from 5.0 to 5.2 . In addition, a single spot of 100 kDa and pI 6.2 was observed. APMIS, 1997 Mar, 105(3), 199 - 206 Identification of Afipia felis antigens in culture medium: reaction with human sera; Engbaek K et al.; Fourteen protein antigens were identified on SDS-PAGE of Afipia felis culture supernatant . Immunoblotting against 10 monoclonal antibodies obtained from mice infected with live A . felis showed that 4 antibodies reacted with a 56 kDa band and 3 with both 56 kDa and 62 kDa bands . Compared with A . felis sonicate, the reacting proteins in culture supernatant showed an increase in molecular mass of 2-3 kDa, suggesting that they were more glycosylated . Purified antigen obtained by affinity chromatography of culture supernatant on the seven immobilized antibodies was tested against antibodies reacting with the 56 kDa and 62 kDa bands . All eluates contained both components, suggesting that the antibodies were directed against different epitopes of a double antigen held together during the affinity chromatography but cleaved by reduction and SDS-PAGE . The molecular size of the uncleaved protein in culture supernatant was determined by size-exclusion chromatography as > 1000 kDa . Testing of pre- and post-infection rabbit sera in immunoblotting against culture supernatant demonstrated that the 56 kDa and 62 kDa components gave the most prominent specific reactions with post-infection sera . One of fifty human sera submitted for testing for cat-scratch disease and 1 of 50 sera from healthy blood donors reacted with several bands in A . felis culture supernatant, including the 56 kDa and 62 kDa bands. APMIS, 1997 Mar, 105(3), 192 - 8 Production and characterization of mouse monoclonal antibodies against Afipia felis; Engbaek K et al.; A series of 10 monoclonal antibodies reacting with Afipia felis antigens were selected from mice immunized with live organisms of the reference strain ATCC 53690 . Immunoblotting against SDS-PAGE-separated A felis sonicate allowed the antibodies to be classified into three groups: 1) 168-4, -6, -7 and -10 reacted with a 53 kDa antigen, 2) 168-1, -3 and -9 reacted with both 53 kDa and 60 kDa antigens, and 3) 168-2, -5 and -9 reacted with other antigens . Antibodies of group 1 did not cross-react with other Afipia species or 36 unrelated bacteria, whereas those of groups 2 and 3 reacted with other Afipia species and some unrelated bacteria . Immunoblots of crossed immunoelectrophoretic patterns of A . felis sonicate against rabbit antiserum showed that antibodies of groups 1 and 2 bound to the same precipitin arcs . Antibodies of group 1 reacted with a species-specific epitope on the 53 kDa antigen, while those of group 2 reacted with other epitopes shared by the 53 kDa and 60 kDa antigens . The binding of antibodies of group 1 to A . felis sonicate was inhibited by post-infection rabbit serum, whereas no inhibition was observed for antibodies of group 2 . The species-specific epitope of the 53 kDa antigen and the early appearance of antibodies against this epitope after infection suggest that this antigen can be used in a serodiagnostic test for A . felis infection. J Oral Rehabil, 1997 Mar, 24(3), 209 - 15 Scanning electron microscopic examination of different cleaners: surface contaminant removal from dentures; Kulak Y et al.; Dentures were examined by scanning electron microscopy to evaluate removal of surface contaminants . Five complete dentures were obtained during patient appointments . The palatal surface of each denture was divided into eight pieces (1 cm2) then each sample cleaned with Corega, Dentipur, Fittydent, sodium hypochloride, Savlon, Ipanol, brushing methods and one sample was also kept as a control . They were prepared for SEM examination and photographed at x500 . One photograph of each sample was evaluated in random order by three judges for a total of 120 observations . Photographs were compared with one of a clean denture sample . Statistical analysis of the results showed that soaking dentures in sodium hypochloride and Savlon removed significantly more contaminants than any of the other methods used in this study. Ecotoxicol Environ Saf, 1997 Mar, 36(2), 99 - 108 Influence of the energy relationships of organic compounds on their specificity toward aquatic organisms; Genoni GP; Unifying concepts are needed in ecotoxicology to deal with the various aspects of the behavior of substances, such as their mobility in the physical environment, bioaccumulation, toxicity, and specificity . The concept of transformity (the relative amount of energy required to generate a component or a flow in a transformation process) may provide a synthetic approach . Transformity correlates positively with the bioaccumulation tendency and toxicity . Since increasing amounts of energy are required in biological or industrial processes that generate increasingly unusual, complex, and specific substances, transformity and specificity may correlate positively as well . This latter hypothesis was tested for a set of 45 compounds, including simple and chlorinated alkanes, alkenes, alcohols, benzenes, and phenols . Published data on their Gibbs energy of formation (an estimate of transformity) and their acute toxicity to 21 species of aquatic organisms (measured as the EC50 or LC50, the duration of the test depending on the life span of the species) were used . To quantify specificity, the coefficient of variation of the toxicity data for the various species, which expresses the relative variability of the data around the mean, was calculated . There was a significant positive correlation between these two quantities . Thus, transformity may provide a conceptual framework for predicting the specificity of substances . The functional relationship between transformity and specificity could not, however, be established with certainty. Transfusion, 1997 Mar, 37(3), 309 - 12 Evaluation of donor skin disinfection methods; Goldman M et al.; BACKGROUND: Because most bacteria isolated from contaminated platelet concentrates are thought to originate from the donor's skin, the efficacy of four methods of skin disinfection was compared . STUDY DESIGN AND METHODS: Contact plates were used for antecubital skin cultures after they were demonstrated to be easier to use and at least as sensitive as a swab system . One antecubital fossa of each subject was disinfected by a standard method, the use of a povidone-iodine swabstick containing 0.75-percent available iodine followed by the use of a povidone-iodine swabstick containing 1-percent available iodine . The other arm was disinfected with either a 70-percent isopropyl alcohol scrub followed by an ampoule of 2-percent iodine tincture (Group 1; n = 126); a green-soap sponge followed by a 70-percent isopropyl alcohol swab, used for donors who are allergic to iodine (Group 2; n = 30); or a 0.5-percent chlorhexidine gluconate and 70-percent isopropyl alcohol sponge followed by an ampoule of 0.5-percent chlorhexidine gluconate and 70-percent isopropyl alcohol (Group 3; n = 40) . Contact plate cultures were done before and after disinfection, and colonies counted after a 48-hour 37 degrees C incubation period . RESULTS: Similar numbers of bacteria grew from both antecubital fossae of the same subject before disinfection (p = 0.71) . Compared to the standard povidoneiodine method, isopropyl alcohol and tincture of iodine resulted in significantly less bacterial growth (p < 0.001), the green soap and isopropyl alcohol method resulted in significantly more bacterial growth (p < 0.001), and the chlorhexidine gluconate and isopropyl alcohol method resulted in similar amounts of bacterial growth (p > 0.3) . CONCLUSION: Isopropyl alcohol scrub followed by iodine tincture is more efficacious than povidone-iodine as measured by contact plate cultures . For donors who are allergic to iodine, chlorhexidine gluconate and isopropyl alcohol is more efficacious than green soap and isopropyl alcohol. Genomics, 1997 Mar 1, 40(2), 277 - 83 Human retina-specific amine oxidase (RAO): cDNA cloning, tissue expression, and chromosomal mapping; Imamura Y et al.; In search of candidate genes for hereditary retinal disease, we have employed a subtractive and differential cDNA cloning strategy and isolated a novel retina-specific cDNA . Nucleotide sequence analysis revealed an open reading frame of 2187 bp, which encodes a 729-amino-acid protein with a calculated molecular mass of 80,644 Da . The putative protein contained a conserved domain of copper amine oxidase, which is found in various species from bacteria to mammals . It showed the highest homology to bovine serum amine oxidase, which is believed to control the level of serum biogenic amines . Northern blot analysis of human adult and fetal tissues revealed that the protein is expressed abundantly and specifically in retina as a 2.7-kb transcript . Thus, we considered this protein a human retina-specific amine oxidase (RAO) . The RAO gene (AOC2) was mapped by fluorescence in situ hybridization to human chromosome 17q21 . We propose that AOC2 may be a candidate gene for hereditary ocular diseases. Eur J Biochem, 1997 Mar 1, 244(2), 527 - 43 Theoretical approaches to the evolutionary optimization of glycolysis--chemical analysis; Melendez-Hevia E et al.; In the first part of this work {Heinrich, R., Montero, F., Klipp, E., Waddell, T . G . & Melendez-Hevia, E . (1997) Eur . J . Biochem . 243, 191-201} the kinetic and thermodynamic constraints under which an optimal glycolysis must be designed have been analysed . In this second part, we present a chemical analysis of the glycolytic pathway in order to determine if its design is chemically optimized according the possibilities that a glycolytic design can have . Our results demonstrate that glycolysis in modern-day cells (from glucose to lactate) has an optimized design for maximizing the flux of ATP production, and a thermodynamic profile which guarantees a high kinetic efficiency . We also discuss some cases of paleometabolism for this pathway as alternative metabolic pathways, less optimized, that exist in some bacteria . Our analysis relates mainly to metabolism designed under constant chemical affinity (substrates and products of the pathway constant), where the target of optimization can be the flux of ATP production . We also discuss the case of an externally imposed input flux, whose target of optimization is the stoichiometric yield of ATP. Clin Infect Dis, 1997 Mar, 24(3), 309 - 19 Role of colonization of the upper intestinal tract in the pathogenesis of ventilator-associated pneumonia; Bonten MJ et al.; Ventilator-associated pneumonia (VAP) is the most frequent infection in mechanically ventilated patients . Colonization of the gastrointestinal tract, especially the stomach, is believed to be important in the pathogenesis of VAP . However, the literature on this topic is full of contradictory evidence . In the present review, we critically assess the existence and importance of the gastropulmonary route of colonization in the pathogenesis of VAP, and we analyze the differences between studies that show different results . Several preventive regimens (e.g., the use of sucralfate for stress ulcer prophylaxis and modulation of enteral feeding) have been used to prevent gastric colonization and decrease the incidence of VAP . However, none of these regimens has been demonstrated to be unequivocally beneficial . Moreover, recent analyses of the sequences of colonization in patients who develop VAP suggest that gastric colonization may be less important in the pathogenesis of VAP than has been assumed . As a result, the impact of other routes of colonization such as cross-colonization or transfer of rectal bacteria via the skin or other vectors to the respiratory tract may have been underestimated . Knowledge of the relative importance of each of these possible routes of colonization is essential for developing effective measures to prevent VAP. Mol Biochem Parasitol, 1997 Mar, 85(1), 77 - 87 Molecular cloning and characterisation of a developmentally regulated putative metallopeptidase present in a host protective extract of Haemonchus contortus; Redmond DL et al.; Antisera from lambs immunised with the Haemonchus contortus integral membrane protein complex, Haemonchus galactose-containing glycoprotein (H-gal-GP), the lambs being refractory to subsequent challenge, were used to identify several clones from an adult H . contortus lambda gt11 cDNA library . Using gene-specific oligonucleotide primers in conjunction with primers directed to a conserved nematode Spliced Leader (SL) sequence and to the polyA+ tail of mRNA, the remaining 5' and 3' sequences of one of these clones, metallopeptidase-1 (MEP1), were amplified . The 2.4 kb full-length coding sequences was subsequently amplified in a single reaction . Sequence analysis identified MEP1 as encoding a putative zinc metallopeptidase, which shared limited homology with the mammalian type II integral membrane protein neutral endopeptidase (NEP) . Southern blotting indicated that MEP1 belonged to a multigene family . MEP1 was expressed in bacteria as a glutathione-S-transferase (GST) fusion protein, and a specific antiserum raised in sheep . This antiserum recognised several polypeptide components of H-gal-GP . Immunolocalisation studies showed that MEP1 encoded a protein located on the luminal surface of the nematode gut . Both MEP1 mRNA and protein are developmentally regulated with expression being limited to the blood-feeding stages of H . contortus. Mol Microbiol, 1997 Mar, 23(6), 1267 - 79 DNA topology in hyperthermophilic archaea: reference states and their variation with growth phase, growth temperature, and temperature stresses; Lopez-Garcia P et al.; In order to address the dynamics of DNA topology in hyperthermophilic archaea, we analysed the topological state of several plasmids recently discovered in Thermococcales and Sulfolobales . All of these plasmids were from relaxed to highly positively supercoiled in vitro, i.e . they exhibited a significant linking excess compared to the negatively supercoiled plasmids from mesophilic organisms (both Archaea and Bacteria) . In the two archaeal orders, plasmid linking number (Lk) decreased as growth temperature was lowered from its optimal value, i.e . positively supercoiled plasmids were relaxed whereas relaxed plasmids became negatively supercoiled . Growth temperatures above the optimum correlated with higher positive supercoiling in Sulfolobales (Lk increase) but with relaxation of positive supercoils in Thermococcus sp . GE31 . The topological variation of plasmid DNA isolated from cells at different growth phases were found to be species specific in both archaeal orders . In contrast, the direction of topological variation under temperature stress was the same, i.e . a heat shock correlated with an increase in plasmid positive supercoiling, whilst a cold shock induced negative supercoiling . The kinetics of these effects were analysed in Sulfolobales . In both temperature upshift (from 80 to 85 degrees C) and downshift (from 80 to 65 degrees C), a transient sharp variation of Lk occurred first, and then DNA supercoiling progressively reached levels typical of steady-state growth at the final temperature . These results indicate that DNA topology can change with physiological states and environmental modifications in hyperthermophilic archaea. J Vet Med Sci, 1997 Mar, 59(3), 213 - 5 Genetic diversity of the genes encoding the outer membrane lipoprotein (omlA) of Actinobacillus pleuropneumoniae; Osaki M et al.; An allelic variation of the genes encoding the protective outer membrane lipoprotein (omlA) in Actinobacillus pleuropneumoniae was investigated with polymerase chain reaction (PCR)-restriction fragment length polymorphisms . Primers for PCR were selected from a conserved sequence compared between the omlA genes of A . pleuropneumoniae serotypes 1 and 5a . A DNA fragment of 970 bp was amplified from the genomic DNA of all 12 serotypes of A . pleuropneumoniae . The amplified DNA sequence specifically hybridized under a low stringent condition to the cloned omlA gene of A . pleuropneumoniae . Digestion of the amplified DNA with the enzymes either HinfI or VspI yielded specific polymorphic patterns, allowing discrimination of all serotypes into five distinct groups. J Physiol Pharmacol, 1997 Mar, 48(1), 3 - 42 Physiological, immunohistochemical and molecular aspects of gastric adaptation to stress, aspirin and to H . pylori-derived gastrotoxins; Konturek PC; Gastric mucosa is continuously exposed to various aggressive factors such as stress, ulcerogenic drugs including aspirin-like agents, gastrotoxic bacteria, particularly Helicobacter pylori (Hp) and many other exogenous and endogenous irritants . The maintenance of mucosal barrier depends upon the activation of pre-epithelial (mucus-alkaline secretion), epithelial (surface active phospholipids, mucosal cell restitution and proliferation) and post-epithelial (mucosal microcirculation) lines of mucosal defence . The mucosa exposed to aggressive factors develops acute lesions, which usually heal completely within few days, but following repeated exposures to hostile environment it adapts to survive the challenge of noxious agents . This adaptation may be of short term (adaptive cytoprotection) and follows the exposure to "mild" irritants that activate local mucosal biosynthesis of protective prostaglandins (PG) and nitric oxide (NO) and stimulate sensory nerves and mucosal cell migration and proliferation through enhanced expression of growth factors such as EGF, TGF alpha and trefoil peptides . The fact that exogenous PG, NO-donor agents, growth factors and capsaicin, stimulating sensory nerves, protect the mucosa against strong necrotizing agents (direct cytoprotection), supports the notion that endogenous PG, NO, growth factors and sensory nerves are involved in the complex process of adaptive cytoprotection . With repeated insults of ulcerogens such as stress aspirin, Hp-derived gastrotoxins, especially ammonia, a long-term adaptation develops which is mediated mainly by overexpression of EGF and TGF alpha and their common receptor (EGFR) with subsequent increase of mucosal cell proliferation and enhanced healing of mucosal lesions . The failure of mucosal adaptation seems to play a pivotal role in the pathogenesis of gastric lesions and peptic ulcerations. Clin Chest Med, 1997 Mar, 18(1), 65 - 77 The origin and erratic global spread of tuberculosis . How the past explains the present and is the key to the future; Stead WW; Although tuberculosis is a disease known in antiquity, it was not distributed equally or simultaneously throughout the world . Recent genetic studies of the various species of mycobacteria give strong evidence of evolution of M . tuberculosis from saprophytic soil bacteria to M . bovis, which attacks a wide spectrum of lower animals, and then to M . tuberculosis, with the pathogenicity largely limited to humans . The great discrepancies in the time of arrival of this organism to diverse parts of the world, and in its ability to kill the young, account for significant differences in the emergence of innate resistance to tuberculosis in various populations . Innate resistance to particular infections are highly specific, and are derived from whatever scourge one's ancestors had to survive. Proteins, 1997 Mar, 27(3), 410 - 24 A new method for modeling large-scale rearrangements of protein domains; Maiorov V et al.; A method for modeling large-scale rearrangements of protein domains connected by a single- or a double-stranded linker is proposed . Multidomain proteins may undergo substantial domain displacements, while their intradomain structure remains essentially unchanged . The method allows automatic identification of an interdomain linker and builds an all-atom model of a protein structure in internal coordinates . Torsion angles belonging to the interdomain linkers and side chains potentially able to form domain interfaces are set free while remaining torsions, bond lengths, and bond angles are fixed . Large-scale sampling of the reduced torsions conformational subspace is effected with the "biased probability Monte Carlo-minimization" method {Abagyan, R.A., Totrov, M.M . (1994): J . Mol . Biol . 235, 983-1002} . Solvation and side-chain entropic contributions are added to the energy function . A special procedure has been developed to generate concerted deformations of a double-stranded interdomain linker in such a way that the polypeptide chain continuity is preserved . The method was tested on Bence-Jones protein with a single-stranded linker and lysine/arginine/ornithine-binding (LAO) protein with a double-stranded linker . For each protein, structurally diverse low-energy conformations with ideal covalent geometry were generated, and an overlap between two sets of conformations generated starting from the crystallographically determined "closed" and "open" forms was found . One of the low-energy conformations generated in a run starting from the LAO "closed" form was only 2.2 A away from the structure of the "open" form . The method can be useful in predicting the scope of possible domain rearrangements of a multidomain protein. J Can Dent Assoc, 1997 Mar, 63(3), 187 - 92, 194-5 Tobacco smoking and periodontal diseases; Qandil R et al.; There is a growing body of scientific evidence indicating that nicotine contributes to the progression of periodontal disease, and is detrimental to healing following periodontal therapy . Smokers show a higher prevalence and greater severity of periodontal disease than non-smokers . Nicotine has toxic effects on peripheral, circulation, which cause gingival vasoconstriction . As a result, a decreased number of immune cells are available in the gingival tissue, which translates into a weakened defence-reparative system . Nicotine can also depress primary and secondary immune response by reducing the chemotactic and phagocytic activities of leukocytes . Clinically, smoking has been associated with increased pocket depths, calculus deposition,alveolar bone loss, acute necrotizing ulcerative gingivitis, and osteoporosis in postmenopausal women . Despite efforts by various investigators, the precise mechanisms underlying the effects of smoking on periodontal status and wound healing remain unresolved . This paper reviews the relationship between smoking and periodontal disease . It also includes a historical overview of the research, and a review of the effects of smoking on oral bacteria, the immune system and exocrine glands . In addition, risk assessment for periodontal disease, clinical features of smokers' periodontitis, response of smokers' periodontal disease to treatment, and ideas for future research are discussed. Lett Appl Microbiol, 1997 Mar, 24(3), 193 - 7 Specific and sensitive detection of Trichomonas tenax by the polymerase chain reaction; Kikuta N et al.; A polymerase chain reaction (PCR) protocol was developed for specific detection of Trichomonas tenax by using a pair of primers designed for its 18S rRNA gene . The detection was specific for T . tenax, since no amplification was detected with DNAs from Trichomonas vaginalis, which belongs to the same genus as T . tenax, in addition to various species of oral protists, fungi and bacteria, and human leukocytes . This method had a detection limit of 100 fg for T . tenax genomic DNA and could detect T . tenax cells in dental plaque at a concentration of as low as 5 cells per PCR mixture . Direct detection from clinical dental plaque samples was also possible; therefore, the present PCR procedure could provide a simple and rapid detection method of T . tenax in dental plaque. Nephrol Dial Transplant, 1997 Mar, 12(3), 528 - 34 Haemodiafiltration with sorbent-regenerated ultrafiltrate as replacement fluid: a multicenter study; de Francisco AL et al.; BACKGROUND: Uncoated adsorbent charcoal may regenerate the ultrafiltrate suggesting its use as an endogenous substitution fluid . The objective of this study was to assess the safety and the long-term clinical results . METHODS: Thirty-three chronic uraemic patients were dialysed for 1 year using two haemodialysers in series in order to separate convection from diffusion . At the outflow of the convective haemofilter, a cartridge containing 130 g of uncoated charcoal was inserted . The regenerated ultrafiltrate was then infused at the entrance of the diffusive dialyser . Ex vivo and in vitro studies were performed to analyse the adsorption characteristics and the release of aluminium, other trace elements, and microparticles . RESULTS: Passage through the charcoal left urea, phosphate, potassium, calcium, and bicarbonate concentrations unchanged . Creatinine, uric acid and beta 2-microglobulin were almost completely absorbed by the charcoal . Aluminium release was dependent upon time of storage, as inferred from studies on inter-lot variability . Washing with bicarbonate buffer (pH 7.0) allowed reduction of aluminium levels to within the pharmacopoeia requirements for intravenous fluids . No significant pre- or post-charcoal differences were observed for several trace elements such as manganese, selenium, arsenic, cadmium, mercury, lead, chromium and zinc . Copper was completely retained in the charcoal . Regenerated ultrafiltrate infused at the entrance of the diffusive dialyser was free of microparticles, bacteria, and endotoxin . Clinical tolerance was excellent and blood pressure control satisfactory . A significant decrease in serum values of beta 2-microglobulin was observed at 6 and 12 months of treatment . CONCLUSIONS: Reinfusion of ultrafiltrate through an uncoated charcoal cartridge proved to be a safe, well-tolerated and simple technique . Further potential benefits of regenerated ultrafiltrate may also include the maintenance of acid-base balance with reinfusion of endogenous bicarbonate. Methods, 1997 Mar, 11(3), 301 - 12 Study of gene regulation by NF-kappa B and AP-1 in response to reactive oxygen intermediates; Muller JM et al.; Reactive oxygen intermediates (ROIs), such as hydrogen peroxide or superoxide, are an evolutionarily ancient threat to all organisms . Exposure of bacteria to ROIs initiates a genetic program that coordinates the production of novel proteins with protective functions . This genetic response is mediated by regulatory proteins that have the potential to initiate transcription of genes when the levels of the ROIs increase . In plant cells, a variety of viral pathogens increase hydrogen peroxide production, which is required to mount a defensive genetic response . It was suggested that in this case H2O2 is used as a secondary messenger and an immediate-early pathogen signal . In higher vertebrates, two transcription factors, nuclear factor kappa B and activator protein 1, were found to respond to ROIs . Both are well studied: they are induced by a great variety of seemingly unrelated conditions and serve important roles in immune, inflammatory, and other pathogen-related genetic responses . In this article we discuss how the ROI responsiveness of transcription factors can be experimentally studied and summarize evidence to suggest that ROIs have been conserved during evolution as messengers of a general pathogen response. Public Health Rep, 1997 Mar-Apr, 112(2), 98 - 106; discussion 107 Dental sealants . Who needs them? Siegal MD, Farquhar CL, Bouchard JM. Most childhood tooth decay is preventable with a combination of fluoride--which protects the smooth surfaces of a tooth--and dental sealants--which protect tooth surfaces with irregularities called pits and fissures . Sealants are plastic coatings that protect these vulnerable areas, often narrower than a single toothbrush bristle, from decay-causing bacteria and food in the mouth . Yet, 1988-1991 data from the National Health and Nutrition Examination Survey showed that while many children still had cavities, over 80% of which were related to pits and fissures, relatively few children had sealants applied to permanent teeth . As caries has gone from a ubiquitous disease to one affecting only half of children in early elementary school and two-thirds of those who are 15 years of age, dentists must consider how to best target sealants to individual children who are at greatest risk for new disease . Most sealants are placed in private dental offices, but children at greatest risk for problems resulting from tooth decay are least likely to get private care . State and local health departments, therefore, have gone after hard-to-reach children and adolescents through school-based and school-linked sealant programs, often using portable dental equipment . This article focuses on public health strategies for community-based prevention. Protein Sci, 1997 Mar, 6(3), 556 - 68 Crystal structure of the hydroxylase component of methane monooxygenase from Methylosinus trichosporium OB3b; Elango N et al.; Methane monooxygenase (MMO), found in aerobic methanotrophic bacteria, catalyzes the O2-dependent conversion of methane to methanol . The soluble form of the enzyme (sMMO) consists of three components: a reductase, a regulatory "B" component (MMOB), and a hydroxylase component (MMOH), which contains a hydroxo-bridged dinuclear iron cluster . Two genera of methanotrophs, termed Type X and Type II, which differ markedly in cellular and metabolic characteristics, are known to produce the sMMO . The structure of MMOH from the Type X methanotroph Methylococcus capsulatus Bath (MMO Bath) has been reported recently . Two different structures were found for the essential diiron cluster, depending upon the temperature at which the diffraction data were collected . In order to extend the structural studies to the Type II methanotrophs and to determine whether one of the two known MMOH structures is generally applicable to the MMOH family, we have determined the crystal structure of the MMOH from Type II Methylosinus trichosporium OB3b (MMO OB3b) in two crystal forms to 2.0 A resolution, respectively, both determined at 18 degrees C . The crystal forms differ in that MMOB was present during crystallization of the second form . Both crystal forms, however, yielded very similar results for the structure of the MMOH . Most of the major structural features of the MMOH Bath were also maintained with high fidelity . The two irons of the active site cluster of MMOH OB3b are bridged by two OH (or one OH and one H2O), as well as both carboxylate oxygens of Glu alpha 144 . This bis-mu-hydroxo-bridged "diamond core" structure, with a short Fe-Fe distance of 2.99 A, is unique for the resting state of proteins containing analogous diiron clusters, and is very similar to the structure reported for the cluster from flash frozen (-160 degrees C) crystals of MMOH Bath, suggesting a common active site structure for the soluble MMOHs . The high-resolution structure of MMOH OB3b indicates 26 consecutive amino acid sequence differences in the beta chain when compared to the previously reported sequence inferred from the cloned gene . Fifteen additional sequence differences distributed randomly over the three chains were also observed, including D alpha 209E, a ligand of one of the irons. Clin Diagn Lab Immunol, 1997 Mar, 4(2), 229 - 31 Recurrent infectious diseases in human CD53 deficiency; Mollinedo F et al.; We report a familiar syndrome of recurrent heterogeneous infectious diseases, caused by bacteria, fungi, and viruses, which has as its only detectable defect the lack of CD53 antigen expression in neutrophils . All other assays ruled out known causes of recurrent infectious diseases due to either leukocyte adhesion or phagocytosis defects . CD53 belongs to the transmembrane-4 superfamily of proteins, which are a novel group of membrane proteins implicated in growth regulation and cell motility and possibly cell adhesion . We postulate that defects in these membrane proteins can be clinically manifested as complex recurrent infections. Carcinogenesis, 1997 Mar, 18(3), 569 - 74 Activation of the NADPH oxidase in human fibroblasts by mechanical intrusion of a single cell with an ultramicroelectrode; Arbault S et al.; We present here a real-time and single cell study of an oxidative stress mechanism in human fibroblasts . Hydrogen peroxide released by a single normal or SV40-transformed human fibroblast was detected at the surface of an ultramicroelectrode while puncturing the cell membrane with the ultramicroelectrode tip itself or with a micropipette . This mechanical intrusion induced the emission of large quantities (10(-15)-10(-14) mol) of H2O2 by the cell with a very short time delay (<0.5 s) . We show that this H2O2 production was an active neo-production by fibroblasts when the membrane was stressed by the cellular puncture and is a model which could mimic similar effects as particle (virus, bacteria, etc.) intrusion into the cell . Cell incubations in the presence of some inhibitors of the different NADPH oxidase enzymes, using ultramicroelectrode measurements of the short time effects (<20 min) let us believe that an NADPH oxidase-like enzyme may be implicated in this induced-H2O2 generation . Phenylarsine oxide (PAO), a specific NADPH oxidase inhibitor, at concentrations between 0.5-50 microM seemed to quickly kill the transformed cells preferentially to the normal cells, pointing out for the future a possible anti-cancerous chemotherapic use. Carcinogenesis, 1997 Mar, 18(3), 531 - 7 Estimates of the chromium(VI) reducing capacity in human body compartments as a mechanism for attenuating its potential toxicity and carcinogenicity; De Flora S et al.; Estimates of the overall reducing capacity of hexavalent chromium(VI) in some human body compartments were made by relating the specific reducing activity of body fluids, cell populations or organs to their average volume, number, or weight . Although these data do not have absolute precision or universal applicability, they provide a rationale for predicting and interpreting the health effects of chromium(VI) . The available evidence strongly indicates that chromium(VI) reduction in body fluids and long-lived non-target cells is expected to greatly attenuate its potential toxicity and genotoxicity, to imprint a threshold character to the carcinogenesis process, and to restrict the possible targets of its activity . For example, the chromium(VI) sequestering capacity of whole blood (187-234 mg per individual) and the reducing capacity of red blood cells (at least 93-128 mg) explain why this metal is not a systemic toxicant, except at very high doses, and also explain its lack of carcinogenicity at a distance from the portal of entry into the organism . Reduction by fluids in the digestive tract, e.g . by saliva (0.7-2.1 mg/day) and gastric juice (at least 84-88 mg/day), and sequestration by intestinal bacteria (11-24 mg eliminated daily with feces) account for the poor intestinal absorption of chromium(VI) . The chromium(VI) escaping reduction in the digestive tract will be detoxified in the blood of the portal vein system and then in the liver, having an overall reducing capacity of 3300 mg . These processes give reasons for the poor oral toxicity of chromium(VI) and its lack of carcinogenicity when introduced by the oral route or swallowed following reflux from the respiratory tract . In terminal airways chromium(VI) is reduced in the epithelial lining fluid (0.9-1.8 mg) and in pulmonary alveolar macrophages (136 mg) . The peripheral lung parenchyma has an overall reducing capacity of 260 mg chromium(VI), with a slightly higher specific activity as compared to the bronchial tree . Therefore, even in the respiratory tract, which is the only consistent target of chromium(VI) carcinogenicity in humans (lung and sinonasal cavities), there are barriers hampering its carcinogenicity . These hurdles could be only overwhelmed under conditions of massive exposure by inhalation, as it occurred in certain work environments prior to the implementation of suitable industrial hygiene measures. Can J Anaesth, 1997 Mar, 44(3), 300 - 4 Epidural abscess after combined spinal-epidural block; Schroter J et al.; PURPOSE: We report the first case of abscess formation after combined spinal-epidural block (CSE) . Penetration of the dura in CSE may constitute an additional risk of subarachnoid spread of bacteria when post-puncture epidural infection is present . CLINICAL FEATURES: The combination of a spinal and a continuous epidural block (CSE) using a needle through needle technique was used in a 72-yr-old man for reconstructive vascular surgery of the lower limb . On the fourth postoperative day the patient demonstrated back pain, fever, and exudation of pus from the CSE-puncture site . An epidural abscess was diagnosed by magnetic resonance imaging and subsequently an emergency hemiaminectomy was performed . Physical examination and surgery did not show evidence of bacterial spread into the subarachnoid space . CONCLUSION: Epidural abscess formation after CSE may increase the risk of bacterial spread into the subarachnoid space . In this case spontaneous exudation and surgical drainage of abscess material may have prevented intrathecal infection . Rapid diagnosis and treatment of an epidural abscess appears particularly essential after CSE to prevent neurological sequelae. Biotechniques, 1997 Mar, 22(3), 506 - 11 Rapid, universal method to isolate PCR-ready DNA using magnetic beads; Rudi K et al.; A magnetic bead-based system for DNA isolation utilizing monodisperse beads was tested with the aim of producing a general approach for PCR-ready DNA . This commercially available system was originally designed for isolating PCR-ready DNA from human whole blood . We tested diverse organisms belonging to the major groups: bacteria, fungi, algae, vascular plants and vertebrates . Optimization of sample amounts and lysis conditions was done using several types of tissue (fish epithelium, plant leaves, mammalian liver and muscle tissues, fungal fruit-bodies and mycelium) . The standard lysis conditions used for blood could be applied with good results for most bacteria, algae and vertebrates, while plant leaves and fungal fruit-bodies had to be mechanically broken to obtain proper lysis . For vascular plants and some cyanobacteria, lysis by heating to 65 degrees C gave better DNA yields than standard lysis at room temperature . In all cases, DNA suitable for PCR was prepared in less than 30 min . The PCR products yielded 350 to 500 bases of DNA sequence (99% accurate) by direct manual or automated sequencing. Am J Ophthalmol, 1997 Mar, 123(3), 409 - 10 Acanthamoeba infection after radial keratotomy; Friedman RF et al.; PURPOSE: To describe a case of Acanthamoeba infection of the cornea after radial and astigmatic keratotomy . METHODS: A 29-year-old man developed ulcerative keratitis in the right eye 6 weeks after uncomplicated radial and astigmatic keratotomy . RESULTS: Three sets of corneal cultures for bacteria and fungi were negative . Culture on non-nutrient agar grew Acanthamoeba organisms . Clinical improvement occurred after topical antiamebic therapy was instituted . CONCLUSIONS: Incisional keratotomy may predispose the cornea to delayed-onset infectious keratitis . Acanthamoeba should be considered as a possible cause of infection and should be cultured for in refractory cases. Nat Biotechnol, 1997 Mar, 15(3), 244 - 7 Transgenic tobacco expressing Vitreoscilla hemoglobin exhibits enhanced growth and altered metabolite production; Holmberg N et al.; The gene for Vitreoscilla hemoglobin (VHb) has been introduced and expressed in Nicotiana tabaccum (tobacco) . Transgenic tobacco plants expressing VHb exhibited enhanced growth, on average 80-100% more dry weight after 35 days of growth compared to wild-type controls . Furthermore, germination time is reduced from 6-8 days for wild-type tobacco to 3-4 days and the growth phase from germination to flowering was 3-5 days shorter for the VHb-expressing transgenes . Transgenic plants contained, on average, 30-40% more chlorophyll and 34% more nicotine than controls . VHb expression also resulted in an altered distribution of secondary metabolites: In the trangenic tobacco plants anabasine content was decreased 80% relative to control plants. J Rheumatol, 1997 Mar, 24(3), 436 - 41 The effects of traditional antirheumatic herbal medicines on immune response cells; Chang DM et al.; OBJECTIVE: Clinically, some traditional Chinese herbal medicines have been thought to be effective in treating rheumatic diseases such as rheumatoid arthritis and systemic lupus erythematosus . To examine the mechanism by which such herbal remedies might be effective, we investigated the ability of Tripterygium wilfordii Hook-f (TWHf) and tetrandrine (TTD) to affect human immune responsiveness in vitro . METHODS: We measured the ability of these agents to affect cytokine secretion from monocytes or T cells, prostaglandin E2 (PGE2) secretion from monocytes, IgG production from B cells, and the phagocytosis of bacteria by neutrophils . RESULTS: These studies revealed that both TWHf and TTD significantly inhibited interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), IL-6, and IL-8 secretion from monocytes, IgG secretion from B cells, and phagocytosis of bacteria by neutrophils; however, only TWHf inhibited IL-2 and IL-4 production from lymphocytes, and PGE2 secretion from monocytes . CONCLUSION: TWHf and TTD exert a powerful suppressive effect on human immune responses . This action might account for their therapeutic effectiveness in rheumatic diseases, and might support broader and more rigorous clinical trials. Appl Environ Microbiol, 1997 Mar, 63(3), 1139 - 42 Characterization of an insertion sequence, IS12528, from Gluconobacter suboxydans; Kondo K et al.; A novel insertion sequence element, IS12528, was found to be associated with inactivation of the alcohol dehydrogenase by insertion in the adhA gene, which encodes the primary dehydrogenase subunit of the three-component membrane-bound alcohol dehydrogenase complex in Gluconobacter suboxydans . Cloning and sequencing analyses revealed that IS12528 was 905 bp in length and had a terminal inverted repeat of 18 bp . In addition, IS12528 was found to generate a 3-bp duplication (TMA, where M represents C or A) at the inserted site upon transposition . IS12528 encoded one long product of 274 amino acids that was rich in basic amino acids . This protein showed significant homology with putative transposases of the IS1031 family isolated from Acetobacter xylinum, which belongs to another genus of acetic acid bacteria . IS12528-like sequences were distributed in a wide variety of acetic acid bacteria, as determined by Southern hybridization and PCR . These observations suggest that IS12528 is one of the insertion sequences that are responsible for genetic instability leading to deficiencies in various physiological properties in a variety of acetic acid bacteria.
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