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 .