Antonie Van Leeuwenhoek, 1998 Jan, 73(1), 69 - 77 Transfer of plasmid RP4 in the spermosphere and rhizosphere of barley seedling; Sorensen SJ et al.; Transfer of plasmid RP4 to indigenous bacteria in bulk soil could only be detected in soil with nutrient amendment . Lack of physiological active donor and recipient cells was apparently one of the limiting factors in un-amended bulk soil . Plasmid transfer was detected both in the spermosphere and rhizosphere of barley seedlings . Transfer occurred from seed coated donor bacteria (i) to introduced recipient bacteria and (ii) to indigenous bacteria present in soil . Plasmid transfer was also detected from donor bacteria introduced to the soil to seed coated recipient bacteria . Transfer efficiencies in the rhizosphere were significantly below the transfer efficiencies obtained in the spermosphere . The transfer efficiencies detected in the barley spermosphere were among the highest reported from any natural environment. Infect Immun, 1998 Jun, 66(6), 2943 - 50 Helicobacter pylori disrupts epithelial barrier function in a process inhibited by protein kinase C activators; Terres AM et al.; Helicobacter pylori colonizes the gastric mucosa, and the infection is related to the development of diverse gastric pathologies, possibly by directly or indirectly affecting epithelial-cell function . We analyzed the influence of the bacteria on transepithelial electrical resistance (TER) on a model tight epithelium, T84, grown to confluence in permeable filters . H . pylori sonicates produced a dramatic decrease in TER after 1 to 2 h of exposure, while sonicates from other bacteria did not induce a significant reduction of TER . The effect induced by sonicates was mimicked by a water-soluble fraction from the bacterial surface, was not reproducible with isolated lipopolysaccharide, and was concomitant with a significant increase in the paracellular permeability of the marker molecule {14C}mannitol . Furthermore, H . pylori sonicates also provoked a significant increase in permeability to {14C}mannitol across rat gastric mucosa in vitro . The sonicate-induced decrease in TER in T84 monolayers was inhibited by the protein kinase C (PKC) activator phorbol myristate acetate . As PKC is directly involved in tight junction regulation, we suggest that H . pylori may induce intracellular signalling events counteracting PKC effects . Following long-term H . pylori stimulation, epithelial monolayers regained baseline resistance values slowly after 24 h . The resistance recovery process was inhibited by cycloheximide, indicating its dependency upon protein synthesis . No association between resistance variation and E-cadherin protein levels was observed . These results indicate that H . pylori alters in vitro the barrier properties of the epithelium, probably by generating cell signalling events counteracting the normal function of PKC . This increased permeability may provide a potential mechanism by which H . pylori antigens can reach the gastric lamina propria, thereby activating the mucosal immune system. Infect Immun, 1998 Jun, 66(6), 2895 - 904 Characterization of protective epitopes in a highly conserved Plasmodium falciparum antigenic protein containing repeats of acidic and basic residues; Sharma P et al.; The delineation of putatively protective and immunogenic epitopes in vaccine candidate proteins constitutes a major research effort towards the development of an effective malaria vaccine . By virtue of its role in the formation of the immune clusters of merozoites, its location on the surface of merozoites, and its highly conserved nature both at the nucleotide sequence level and the amino acid sequence level, the antigen which contains repeats of acidic and basic residues (ABRA) of the human malaria parasite Plasmodium falciparum represents such an antigen . Based upon the predicted amino acid sequence of ABRA, we synthesized eight peptides, with six of these (AB-1 to AB-6) ranging from 12 to 18 residues covering the most hydrophilic regions of the protein, and two more peptides (AB-7 and AB-8) representing its repetitive sequences . We found that all eight constructs bound an appreciable amount of antibody in sera from a large proportion of P . falciparum malaria patients; two of these peptides (AB-1 and AB-3) also elicited a strong proliferation response in peripheral blood mononuclear cells from all 11 human subjects recovering from malaria . When used as carrier-free immunogens, six peptides induced a strong, boostable, immunoglobulin G-type antibody response in rabbits, indicating the presence of both B-cell determinants and T-helper-cell epitopes in these six constructs . These antibodies specifically cross-reacted with the parasite protein(s) in an immunoblot and in an immunofluorescence assay . In another immunoblot, rabbit antipeptide sera also recognized recombinant fragments of ABRA expressed in bacteria . More significantly, rabbit antibodies against two constructs (AB-1 and AB-5) inhibited the merozoite reinvasion of human erythrocytes in vitro up to approximately 90% . These results favor further studies so as to determine possible inclusion of these two constructs in a multicomponent subunit vaccine against asexual blood stages of P . falciparum. Infect Immun, 1998 Jun, 66(6), 2762 - 8 Neither the Bvg- phase nor the vrg6 locus of Bordetella pertussis is required for respiratory infection in mice; Martinez de Tejada G et al.; In Bordetella species, the BvgAS sensory transduction system mediates an alteration between the Bvg+ phase, characterized by expression of adhesins and toxins, and the Bvg- phase, characterized by the expression of motility and coregulated phenotypes in Bordetella bronchiseptica and by the expression of vrg loci in Bordetella pertussis . Since there is no known environmental or animal reservoir for B . pertussis, the causative agent of whooping cough, it has been assumed that this phenotypic alteration must occur within the human host during infection . Consistent with this hypothesis was the observation that a B . pertussis mutant, SK6, containing a TnphoA insertion mutation in a Bvg-repressed gene (vrg6) was defective for tracheal and lung colonization in a mouse model of respiratory infection (D . T . Beattie, R . Shahin, and J . Mekalanos, Infect . Immun . 60:571-577, 1992) . This result was inconsistent, however, with the observation that a Bvg+ phase-locked B . bronchiseptica mutant was indistinguishable from the wild type in its ability to establish a persistent respiratory infection in rabbits and rats (P . A . Cotter and J . F . Miller, Infect . Immun . 62:3381-3390, 1994; B . J . Akerley, P . A . Cotter, and J . F . Miller, Cell 80:611-620, 1995) . To directly address the role of Bvg-mediated signal transduction in B . pertussis pathogenesis, we constructed Bvg+ and Bvg- phase-locked mutants and compared them with the wild type for their ability to colonize the respiratory tracts of mice . Our results show that the Bvg+ phase of B . pertussis is necessary and sufficient for respiratory infection . By constructing a strain with a deletion in the bvgR regulatory locus, we also show that ectopic expression of Bvg- phase phenotypes decreases the efficiency of colonization, underscoring the importance of Bvg-mediated repression of gene expression in vivo . Finally, we show that the virulence defect present in strain SK6 cannot be attributed to the vrg6 mutation . These data contradict an in vivo role for the Bvg- phase of B . pertussis. Infect Immun, 1998 Jun, 66(6), 2625 - 31 Mapping and identification of the major cell wall-associated components of Mycobacterium leprae; Marques MA et al.; Mycobacterium leprae, an obligate intracellular pathogen, can be derived only from host tissue and thus affords the opportunity to study in vivo-expressed products responsible for the particular pathogenesis of leprosy . Despite considerable progress in the characterization of the proteins and secondary gene products of M . leprae, there is little information on the nature of the proteins associated with the cell envelope . M . leprae has been fractionated into its major subcellular components, cell wall, cytoplasmic membrane, and soluble cytosol . A number of biochemical markers, including diaminopimelic acid content, monosaccharide composition, mycolic acid, and glycolipid distribution, were applied to their characterization, and two-dimensional gel electrophoresis was used to map the component proteins . A total of 391 major proteins spots were resolved, and 8 proteins were identified based on their reactivity to a panel of monoclonal antibodies and/or relative pI size . Microsequencing of six protein spots present in the cell wall fraction allowed identification of new proteins, including the protein elongation factor EF-Tu and a homolog for the Mycobacterium tuberculosis MtrA response regulator . These results, together with previous studies, contribute to the progressive knowledge of the composition of the in vivo-expressed proteins of M . leprae. J Exp Biol, 1998 Apr, 201(Pt 8), 1073 - 84 Self-association, cooperativity and supercooperativity of oxygen binding by hemoglobins; Riggs AF; Cooperative ligand binding by tetrameric vertebrate hemoglobins (Hbs) makes possible the delivery of oxygen at higher pressures than would otherwise occur . This cooperativity depends on changes in dimer-dimer interactions within the tetramer and is reflected in a 50 000-fold increase in the tetramer-dimer dissociation constant in human Hb upon oxygenation at pH 7.4, from approximately 2x10(-11)mol l-1 to approximately 10(-6)mol l-1 . Hbs that undergo such ligand-dependent changes in association are widespread in non-vertebrates, where the mechanisms are very different from those in vertebrates . Oligomeric Hbs have been identified in organisms in five phyla (molluscs, echinoderms, annelids, phoronids and chordates) that dissociate to subunits upon oxidation of the heme iron and reassociate with the binding of ferric iron ligands such as CN-, N3- or NO2- . Thus, the valence and ligand state of the heme iron control the stability of a critical subunit interface . The broad distribution of this phenomenon suggests a common mechanism of communication between heme and interface that may be almost universal among non-vertebrate Hbs . This interaction may be similar to that known for the homodimeric Hb of the mollusc Scapharca inaequivalvis . Although muscle tissue Hbs or myoglobins (Mbs) are usually monomeric, with non-cooperative O2 binding, the radular muscles of gastropod molluscs and chitons have homodimeric Mbs that bind O2 cooperatively . Cooperative non-muscle tissue Hbs have also been identified . These include the neural Hb of the nemertean worm Cerebratulus lacteus and the Hb of the diving beetle Anisops assimilis, which exhibit deoxygenation-dependent self-association of monomers that is associated with high Hill coefficients . Calculations suggest that the 2-3 mmol l-1 concentration of Hb on a heme basis in the brain of Cerebratulus should substantially extend the time as an active predator in an anaerobic or hypoxic environment . Oxygen from the Hb of Anisops is delivered to a gas bubble and thereby controls the buoyant density . Many Hbs of amphibians, reptiles, birds and some embryonic mammals exhibit a further 'supercooperativity' of O2 binding which depends on reversible deoxygenation-dependent tetramer-tetramer association to form an assemblage with a very low affinity for O2 . This phenomenon results in steeper O2-binding curves than exhibited by tetramers alone . The increased cooperativity should result in an increase in the amount of O2 delivered to the tissues and should be especially valuable for avian flight muscles. Scanning Microsc Suppl, 1996, 10, 177 - 86; discussion 186-7 Electro-optical imaging of F-actin and endoplasmic reticulum in living and fixed plant cells; Allen NS et al.; Confocal and video micrographs of living and fixed alfalfa roots, onion epithelial and pear pollen cells illustrate the architecture of the cytoskeleton and endoplasmic reticulum in plant cells . Fixation of plant tissues to preserve cytoplasmic structure poses special problems . When possible, emphasis should be placed on the imaging of structures in stained living cells over time . The early events that occur when Nod factors or bacteria elicit nodule formation in alfalfa roots will illustrate several approaches to plant cell fixation, staining and imaging . The first observable events after Nod factor stimulation occur in root hairs and are changes in rates of cytoplasmic streaming, nuclear movements, and changes in the shape of the vacuole . Within ten minutes, the endoplasmic reticulum shifts position towards the tip of the root hair . For comparison, the endoplasmic reticulum localization in pollen tubes and onion epithelial cells will be illustrated . The actin cytoskeleton undergoes a series of changes over a twelve hour period . These changes in the cytoskeleton are spatially and temporally correlated with the observed growth changes of the root hairs . This dynamic change of the actin filament and endoplasmic reticulum and associated secretory vesicles in these root hairs suggests a mechanism for the observed root hair growth changes. Scanning Microsc Suppl, 1996, 10, 97 - 107; discussion 107-9 Atomic force microscopy of DNA, nucleoproteins and cellular complexes: the use of functionalized substrates; Lyubchenko YL et al.; Progress towards rapid and simple characterization of biomolecular samples by scanning probe microscopy is impeded mainly by limitations of the current approach to sample preparation . We are working on approaches based on chemical functionalization of mica . Treatment of mica with aminopropyltriethoxy silane (APTES) makes the surface positively charged (AP-mica) and able to hold DNA in place for imaging, even in water . We have shown that AP-mica is an appropriate substrate for numerous nucleoprotein complexes as well . The AFM images of the complex of DNA with RecA protein are stable and indicate a structural periodicity for this filament . AP-mica holds strongly such large DNA complexes as kinetoplast DNA (kDNA) and is an appropriate substrate for their imaging with AFM . We have further develop this approach for making hydrophobic substrates . Silylation of mica surface with hexamethyldisilazane (Me-mica) allowed us to get AFM images of chlorosomes, an antenna complex isolated from green photosynthetic bacteria . Me-mica may be converted into a positively charged substrate after treatment with water solutions of tetraethylammonium bromide or cetyltrimethylammonium bromide . These activated surfaces show high activity towards binding the DNA molecules. Science, 1998 May 1, 280(5364), 722 - 4 Reductive dechlorination of DDE to DDMU in marine sediment microcosms; Quensen JF 3rd et al.; DDT is reductively dechlorinated to DDD and dehydrochlorinated to DDE; it has been thought that DDE is not degraded further in the environment . Laboratory experiments with DDE-containing marine sediments showed that DDE is dechlorinated to DDMU in both methanogenic and sulfidogenic microcosms and that DDD is dehydrochlorinated to DDMU three orders of magnitude more slowly . Thus, DDD does not appear to be an important precursor of the DDMU found in these sediments . These results imply that remediation decisions and risk assessments based on the recalcitrance of DDE in marine and estuarine sediments should be reevaluated. J Biol Chem, 1998 Apr 17, 273(16), 9517 - 26 Solution and crystal structures of a sperm whale myoglobin triple mutant that mimics the sulfide-binding hemoglobin from Lucina pectinata; Nguyen BD et al.; The bivalve mollusc Lucina pectinata harbors sulfide-oxidizing chemoautotrophic bacteria and expresses a monomeric hemoglobin I, HbI, with normal O2, but extraordinarily high sulfide affinity . The crystal structure of aquomet Lucina HbI has revealed an active site with three residues not commonly found in vertebrate globins: Phe(B10), Gln(E7), and Phe(E11) (Rizzi, M., Wittenberg, J . B., Coda, A., Fasano, M., Ascenzi, P., and Bolognesi, M . (1994) J . Mol . Biol . 244, 86-89) . Engineering these three residues into sperm whale myoglobin results in a triple mutant with approximately 700-fold higher sulfide affinity than for wild-type . The single crystal x-ray structure of the aquomet derivative of the myoglobin triple mutant and the solution 1H NMR active site structures of the cyanomet derivatives of both the myoglobin mutant and Lucina HbI have been determined to examine further the structural origin of their unusually high sulfide affinities . The major differences in the distal pocket is that in the aquomet form the carbonyl of Gln64(E7) serves as a H-bond acceptor, whereas in the cyanomet form the amido group acts as H-bond donor to the bound ligand . Phe68(E11) is rotated approximately 90 degrees about chi2 and located approximately 1-2 A closer to the iron atom in the myoglobin triple mutant relative to its conformation in Lucina HbI . The change in orientation potentially eliminates the stabilizing interaction with sulfide and, together with the decrease in size of the distal pocket, accounts for the 7-fold lower sulfide affinity of the myoglobin mutant compared with that of Lucina HbI. Pancreas, 1998 May, 16(4), 481 - 6 Hydrogen breath test with glucose in exocrine pancreatic insufficiency; Casellas F et al.; The present study was designed to investigate the prevalence of bacterial overgrowth in patients with exocrine pancreatic insufficiency by using the hydrogen breath test with glucose . Thus, in 30 patients with exocrine pancreatic insufficiency (in 15 due to chronic pancreatitis and in 15 associated to primary immunodeficiency), established by quantifying trypsin output before and after stimulation with cerulein using a duodenal perfusion technique, a glucose test was performed by administering 50 g of glucose and quantifying H2 in the breath by gas chromatography . The glucose test was positive in six of 15 patients with chronic pancreatitis but in only one of 15 immunodeficient patients (p < 0.05) . Age, sex, etiology, time of evolution, associated diabetes, pancreatic calcifications, duodenal pH, or duodenal trypsin output did not differ between patients with and those without bacterial overgrowth . Previous gastroduodenal surgery was more common in chronic pancreatitis patients with overgrowth (six of six vs . four of nine; p < 0.05) . Five patients with a positive glucose test were treated with antibiotics for 2 weeks and became negative in two of them . These results suggest that a positive glucose test indicating overgrowth is relatively common in exocrine pancreatic insufficiency due to chronic pancreatic, especially in patients with previous gastroduodenal surgery. Eur J Clin Nutr, 1997 Nov, 51 Suppl 4, S28 - 31 A sustainable solution for dietary iron deficiency through plant biotechnology and breeding to increase seed ferritin control; Theil EC et al.; OBJECTIVES: To stimulate novel sustainable solutions to the problem of the nutritional iron deficiency, we asked: How does Nature insure proper iron nutrition of embryos and neonatal animals? Estimates of iron deficiency world-wide are 30% of the population, with women and children at the greatest risk . Recent studies linking iron deficiency with impeded cognitive development emphasizes the enormity of the impact of iron deficiency . Sustainable solutions to the problem of dietary iron deficiency have been elusive . RESULTS: Data for storage iron was examined in seeds, developing plants, embryos and developing animals . In all cases, the common source of stored iron for development was ferritin . The protein component of ferritin concentrates iron billions of times above the solubility of the free metal ion . High conservation of ferritin sequences in bacteria, plants and animals and the specificity of ferritin bioavailability either added extrinsically or intrinsically enriched in a selected soybean cultivar, showed high efficacy in curing dietary iron deficiency in the rat model . Older data on ferritin were reevaluated in light of contemporary knowledge . CONCLUSIONS: Enhancement of natural seed ferritin content by biotechnology and breeding has the potential for a sustainable solution to the problem of global dietary iron deficiency. Diabetes Res Clin Pract, 1998 Feb, 39(2), 123 - 8 Unusual infections in diabetes; Rajbhandari SM et al.; Infection with rare organisms or at unusual sites occur more frequently in people with diabetes . If not recognised and treated promptly, morbidity and mortality are high in such cases . Here we report cases of necrotising fascitis, malignant otitis externa, Fournier's gangrene and psoas abscess occurring in diabetics that needed intensive treatment with antibiotics, surgical debridement and insulin . Literature reviews suggest that cellular defence mechanisms may be impaired in people with diabetes. Comp Immunol Microbiol Infect Dis, 1998 Jan, 21(1), 1 - 14 New emerging zoonoses: a challenge and an opportunity for the veterinary profession; Chomel BB; The concept of emerging infectious diseases appeared in the late 1980s, when major outbreaks occurred around the globe and surprised many scientists who considered infectious diseases to be maladies of the past or limited to the under-developed world . Several reports identified erosion of the public health infrastructure among the factors contributing to new and re-emerging infectious diseases . As indicated by Morse, "Disease emergence often follows ecological changes caused by human activities such as agriculture or agricultural change, migration, urbanization, deforestation, or dam building" . "Among these new diseases, surprisingly, most emergent viruses and many emergent bacteria are zoonotic" . Several new zoonoses have been recently identified . Many of these diseases were either unknown, because we were not able to isolate the infectious agent or to distinguish them from other clinical syndromes, or discovered accidentally . Much of the recent identification of new pathogens has been based on new molecular biology tools or epidemiological studies . For all these diseases or infections, veterinarians played a key role in their identification, isolation of the causative organisms and understanding of the epidemiology of the infection . The role of the veterinary profession is very important in public health and on the rise again in the U.S.A., as it should be in many other countries . Surveillance, clinical curiosity and awareness, epidemiology and laboratory training are the essential tools and competency that the veterinary profession must use to meet the challenge of new emerging zoonoses. Science, 1998 May 8, 280(5365), 912 - 5 Chaos, persistence, and evolution of strain structure in antigenically diverse infectious agents; Gupta S et al.; The effects of selection by host immune responses on transmission dynamics was analyzed in a broad class of antigenically diverse pathogens . Strong selection can cause pathogen populations to stably segregate into discrete strains with nonoverlapping antigenic repertoires . However, over a wide range of intermediate levels of selection, strain structure is unstable, varying in a manner that is either cyclical or chaotic . These results have implications for the interpretation of longitudinal epidemiological data on strain or serotype abundance, design of surveillance strategies, and the assessment of multivalent vaccine trials. J Clin Microbiol, 1998 Apr, 36(4), 937 - 43 Identification of a new DNA region specific for members of Mycobacterium tuberculosis complex; Magdalena J et al.; The successful use of DNA amplification for the detection of tuberculous mycobacteria crucially depends on the choice of the target sequence, which ideally should be present in all tuberculous mycobacteria and absent from all other bacteria . In the present study we developed a PCR procedure based on the intergenic region (IR) separating two genes encoding a recently identified mycobacterial two-component system named SenX3-RegX3 . The senX3-regX3 IR is composed of a novel type of repetitive sequence, called mycobacterial interspersed repetitive units (MIRUs) . In a survey of 116 Mycobacterium tuberculosis strains characterized by different IS6110 restriction fragment length polymorphisms, 2 Mycobacterium africanum strains, 3 Mycobacterium bovis strains (including 2 BCG strains), and 1 Mycobacterium microti strain, a specific PCR fragment was amplified in all cases . This collection included M . tuberculosis strains that lack IS6110 or mtp40, two target sequences that have previously been used for the detection of M . tuberculosis . No PCR fragment was amplified when DNA from other organisms was used, giving a sensitivity of 100% and a specificity of 100% in the confidence limit of this study . The numbers of MIRUs were found to vary among strains, resulting in six different groups of strains on the basis of the size of the amplified PCR fragment . However, the vast majority of the strains (approximately 90%) fell within the same group, containing two 77-bp MIRUs followed by one 53-bp MIRU. Gene, 1998 Mar 27, 210(1), 25 - 36 Evaluation and mapping of the DNA binding and oligomerization domains of the IE2 regulatory protein of human cytomegalovirus using yeast one and two hybrid interaction assays; Ahn JH et al.; The 86-kDa IE2 nuclear phosphoprotein encoded by the human cytomegalovirus (HCMV) major immediate-early (MIE) gene behaves as both a non-specific transactivator of viral and cellular gene expression and as a specific DNA-binding protein targeted to the cis-repression sequence (CRS) at the cap site of its own promoter/enhancer region . Although the IE2 protein produced in bacteria has been shown to bind to the 14-bp palindromic CRS motif and IE2 synthesized in vitro forms stable dimers in solution through the conserved C-terminus of the protein, there is no direct evidence as yet that the intracellular mammalian forms of IE2 do so . Here, we show that the intact HCMV IE2 protein both binds to CRS DNA and dimerizes in yeast cells . In a one-hybrid assay system, a GAL4/IE2 fusion protein expressed in yeast cells activated target HIS3 expression only when CRS sites were located upstream of the GAL1 minimal promoter, but failed to do so on mutant CRS sites, demonstrating a requirement for sequence-specific DNA-binding by IE2 . Examination of a series of deletion and triple amino acid point mutations in the C-terminal half of IE2 mapped the domains required for DNA-binding in yeast to the entire region between codons 313 and 579, whereas in the previous in-vitro study with truncated bacterial GST fusion proteins, it was mapped to between codons 346 and 579 . Transient co-transfection assays with deleted IE2 effector genes in Vero cells showed that the extra segment of IE2 between codons 313 and 346 is also required for both autoregulation and transactivation activity in mammalian cells . In a two-hybrid assay to study IE2 self-interations, we generated both GAL4 DNA-binding (DB) and activation domain (A)/IE2 fusion proteins and showed that IE2 could also dimerize or oligomerize through the C-terminus of the protein in yeast cells . Domains required for this interaction were all mapped to within the region between codons 388 and 542, which is coincident with the domain mapped previously for dimerization by co-translation and immunoprecipitation in vitro . Comparison of the domains of the IE2 protein required for CRS binding and dimerization in yeast suggests that these activities correlate precisely with requirements for the negative autoregulation function of the IE2 protein in mammalian cells. Biochem J, 1998 Mar 15, 330 ( Pt 3), 1405 - 9 Metabolism of agmatine in macrophages: modulation by lipopolysaccharide and inhibitory cytokines; Sastre M et al.; Agmatine is an amine derived from the decarboxylation of arginine by arginine decarboxylase (ADC) and metabolized to putrescine by agmatinase . While prevalent in bacteria and plants, agmatine and its metabolic enzymes have been recently identified in mammalian tissues . In the present study we sought to determine: (a) whether macrophages (cell line RAW 264.7) express ADC and agmatinase, and (b) if the enzymes are regulated by lipopolysaccharide (LPS), and/or by the inhibitory cytokines transforming growth factor-beta (TGF-beta), interleukin-10 (IL-10) and interleukin-4 (IL-4) . LPS induced a dose-dependent stimulation of agmatinase, while it decreased ADC, the effect in both cases being maximum at 20 h . As expected, LPS dose-dependently stimulated the inducible nitric oxide synthase activity (iNOS) . A strong correlation was observed between the effects of LPS on the agmatine-related enzymes and iNOS . By contrast, exposure to IL-10 and TGF-beta caused a reduction in ADC and agmatinase, whereas IL-4 was ineffective on ADC, but reverted the LPS-induced increase of agmatinase . We conclude that the agmatine pathway may be an alternative metabolic route for arginine in macrophages, suggesting a regulatory role of agmatine during inflammation. Am J Otol, 1998 May, 19(3), 266 - 72 Increased numbers of mast cells in human middle ear cholesteatomas: implications for treatment; Albino AP et al.; HYPOTHESIS: Because many of the biologic phenomena in which mast cells are involved also are observed in human cholesteatoma pathology, the authors hypothesized that mast cells may play a role in this disease . The first test of this hypothesis is to determine whether there are an increased number of mast cells associated with cholesteatomas . BACKGROUND: The molecular and cellular defects that result in the pathologic features observed in acquired and congenital cholesteatomas are unknown . One common feature of cholesteatoma pathogenesis is the presence of bacteria and a numerous inflammatory cytokines expressed by host inflammatory cells . The interactions between inflammatory cells and cholesteatoma epithelium could result in the induction of other aberrant biologic features of cholesteatomas . Thus, it is critical to the understanding of the pathogenesis of cholesteatomas to define the specific role of each cell type involved in this disease . Connective tissue mast cells have a complex retinue of functions mediated via the secretion of a variety of cytokines and proteinases, and many of the biologic phenomena in which mast cells are involved also are observed in cholesteatoma pathology . METHODS: The authors evaluated by immunohistochemistry 36 cholesteatomas of all types (e.g., primary and secondary acquired, recurrent, and congenital) and 23 specimens of normal tissues (e.g., tympanic membrane, canal wall skin, and postauricular skin) for the expression of tryptase, a mast cell-specific protease . RESULTS: Cholesteatomas showed approximately threefold to sevenfold increase in the concentration of mast cells when compared with that of normal tissues . In addition, 19-34% of the mast cells were found within the suprabasal layers of the squamous epithelium of cholesteatoma subgroups, a phenomenon observed only in grossly inflamed tympanic membrane specimens, but not in other control tissues including minimally inflamed tympanic membranes . CONCLUSIONS: The authors conclude from these data that mast cells may represent a previously unrecognized host inflammatory cell, which plays an important role in the development of one or more traits of cholesteatoma pathology. J Dairy Sci, 1998 Apr, 81(4), 1078 - 88 Fatty acid flow to the duodenum and in milk from cows fed diets that contained fat and nicotinic acid; Christensen RA et al.; Four cows fitted with ruminal and duodenal cannulas were used in a 4 x 4 Latin square design; treatments were arranged in a 2 x 2 factorial . Treatments were 1) low fat diet, no nicotinic acid; 2) low fat diet, 12 g/d of nicotinic acid; 3) high fat diet, no nicotinic acid; and 4) high fat diet, 12 g/d of nicotinic acid . Cows were fed for ad libitum intake diets consisting of 35% alfalfa silage, 15% corn silage, and either 50% low fat concentrate or 40% high fat concentrate (tallow supplied 6.25% of concentrate) and 10% whole raw soybeans (dry matter basis) . Intake of gross energy (104 Mcal/d) was not different among treatments . Ruminal and postruminal digestibility of energy was not altered by fat or nicotinic acid . Fatty acid intake and flow to the duodenum were increased by fat but were not affected by nicotinic acid . For all diets, flows to the duodenum of C16:0, C18:0, total C18, and total fatty acids increased, and flows of C16:1, C18:1, C18:2, and C18:3 decreased, compared with their intakes . Biohydrogenation of unsaturated C18 was decreased by fat but was not affected by nicotinic acid . Digestibilities of C18:0, C18:1, C18:2, C18:3, and total fatty acids that flowed to the duodenum were decreased by fat but were not affected by nicotinic acid . The yield of C18:0 in milk was increased, and yields of C6:0 to C16:0 fatty acids were decreased, by fat, but yields were not affected by nicotinic acid. Chin Med J (Engl), 1997 Mar, 110(3), 177 - 9 Detection of chlamydia trachomatis by polymerase chain reaction assay in nonbacterial prostatitis; Guo H et al.; OBJECTIVE: To evaluate the sensitivity and specificity of polymerase chain reaction (PCR) technique in comparison with diethylaminoethyl-dextran (DEAE-dextran)-treated HeLa cell culture method for detection of chlamydia trachomatis in nonbacterial prostatitis . METHODS: Thirty patients had symptoms of prostatitis for at least three months . None of them had evidence of urethritis on urethral Gram stain or recurrent bacteria . Routine localization of bacteria was negative . White blood cell count in expressed prostatic secretion (EPS) was more than 10 per high-power field (10/HPF) . None of these patients had received antibiotics during the six weeks before the study, although all had received multiple courses of antibiotics for treatment of prostatitis syndrome . The EPS specimens from these patients were placed in 0.5-ml Eppendorf tubes and stored at -70 degrees C until they were processed for PCR and DEAE-dextran-treated HeLa cell culture . RESULTS: Six specimens were positive for C . trachomatis by both PCR and culture, and 21 were negative by both tests . There were three specimens with discrepant results, including two that were positive by PCR and negative by culture, and one that was positive by culture and negative by PCR . Comparing PCR technique with culture method, the sensitivity, specificity, positive predictive value and negative predictive value of the former were 85.7%, 91.3%, 75.0% and 95.5% respectively . CONCLUSIONS: PCR analysis of EPS is a highly sensitive and specific noninvasive technique for detection of chlamydia trachomatis . It provides a unique opportunity for early identification of or rapid screening for chlamydia trachomatis infection in patients with nonbacterial prostatitis . The reliability of PCR assay offers clinicians a clear indication for the initiation of treatment of chlamydia trachomatis infection. Chin Med J (Engl), 1997 Jan, 110(1), 30 - 5 The relationship between gut-derived endotoxemia and tumor necrosis factor, neopterin: experimental and clinical studies; Sheng Z et al.; OBJECTIVE: To determine the relationship between gut-derived endotoxemia and tumor necrosis factor (TNF), neopterin/biopterin formation following hemorrhage, trauma and burns . METHODS: Rats were subjected to hemorrhagic shock (30 mmHg, 90-180 min) and 40% III degrees thermal injury . Circulating endotoxin, TNF, biopterin levels and liver TNF mRNA expression were measured in animals following acute insults . Also, the subjects of this study included 35 patients with burn size greater than 30%, and 25 patients with multiple injuries (n = 18) and major surgery (n = 7) . RESULTS: It was found that significant portal and systemic endotoxemia took place in the control animal after hemorrhagic shock and thermal injury, but almost not in the animals that treated by measures aiming at controlling endotoxin/bacteria translocation, including polymyxin B, monoclonal antibody against core lipopolysaccharide, and selective decontamination of the digestive tract (SDD) . Concomitantly, hemorrhage and thermal injury resulted in significant increases in systemic plasma TNF level together with tissue TNF mRNA expression, which were associated with the initial appearance of endotoxin in portal vein . However, anti-endotoxin treatment markedly decreased circulating TNF level as well as peak TNF mRNA expression caused by acute insults . There were also lower serum biopterin values in the SDD-treated group as compared with the control group on day 5 postburn . On the other hand, the results showed that the amounts of plasma endotoxin in patients increased during the early stages following major burns, which was significantly correlated with plasma TNF levels, particularly in patients who developed sepsis and multiple organ failure . Although the presence of early endotoxemia did not influence the alterations in serum neopterin, patients with endotoxemia had much higher neopterin values than those who showed no endotoxemia from the second week onward . CONCLUSION: These results suggest that gut-derived endotoxemia could account, at least in part, for the inflammatory mediators formation and release, which might be involved in the pathogenesis of sepsis and multiple organ dysfunction following severe hemorrhage, trauma and burns. J Antibiot (Tokyo), 1997 Nov, 50(11), 919 - 25 Two new components of the aspochalasins produced by Aspergillus sp; Fang F et al.; Aspergillus sp . FO-4282 was found to produce two new components of the aspochalasins . Their structures were determined by spectroscopic analyses. Zhonghua Nei Ke Za Zhi, 1996 Dec, 35(12), 819 - 23 {A randomized clinical study of sulperazone versus tienam in the treatment of LRTIS}; Li J et al.; The purpose of the clinical study is to evaluate the efficacy and safety of Sulperazone (SPZ) (sulbactam/cefoperazone) comparited with tienam in the treatment of lower resperiatory tract infections . A total of 73 patients enrolled in the study . 61 patients were evaluated for efficacy (SPZ 31, TIM 30) and 63 patients were evaluated for safty (SPZ 31, TIM 32) . Drugs were administered twice a day for 7-14 days, at a daily dose of either 2.0-4.0 g of sulperazone or 2.0-4.0 g tienam . The overall clinical efficacy rates of sulperazone and tienam were 93.5% and 93.3%, respectively . Total pathogens were isolated from 49 of 61 patients prior to treatment . Over two-thirds of the isolates (37/48, 77.1%) were beta-lactammase producing strains . The bacterialogical clearance rate was 92.0% and 91.7% for both groups . The incidence of adverse drug reactions for SPZ and TIM were 3.2% and 10.0%, respectively . The susceptibility rates of the bacteria isolated to five drugs: sulperazone, tienam, ceftazidime, ceftriaxone and cefotaxime were 95.9%, 93.9%, 96.0%, 70.0%, and 80.0% respectively . The susceptibility rate of sulperazoene was significant. Zhonghua Kou Qiang Yi Xue Za Zhi, 1996 Jul, 31(4), 241 - 4 {Expression of the PAC gene of S . mutans in S . lactis}; Ling J et al.; Expression of the pac gene in S . lactis were analyzed by dot immunoblotting with rabbit anti-PAc serum . The amount of PAc produced by S . lactis HL107 (pLF107) was measured . This result indicated that a small amount of rPAc was detected in the culture supernatant and cell homogenate of S . lactis HL107 (pLF107) but not in the control strain S . lactis LM0230 . The amount of rPAc in S.lacits HL107 (pLF107) was six times lower than that in S . mutans Ingbritt . Expression of cloned pac gene in S . lactis was also confirmed in this result. J Biochem (Tokyo), 1998 Apr, 123(4), 680 - 3 Effect of glycerol on the affinity of DnaA protein for ATP in the presence of cardiolipin; Hase M et al.; Acidic phospholipids, such as cardiolipin, decrease the affinity of DnaA protein for adenine nucleotides and can activate the inactive form of DnaA protein in vitro . In this study, we examined the effect of glycerol on the affinity of DnaA protein for ATP in the presence of cardiolipin . High concentrations of glycerol (34%) restored the affinity of DnaA protein for ATP, which was decreased by cardiolipin . Glycerol inhibited the binding of cardiolipin with DnaA protein . Glycerol had little effect on membrane fluidity, which is essential for the interaction between cardiolipin and DnaA protein, whereas it increased the Kd value of DnaA protein for ATP in the absence of cardiolipin . These results suggest that glycerol causes DnaA protein to become insensitive as to the interaction with cardiolipin by changing the conformation of the protein without altering the physical nature of the phospholipid. J Mol Neurosci, 1998 Feb, 10(1), 45 - 51 Tyrosine hydroxylase and tryptophan hydroxylase do not form heterotetramers; Mockus SM et al.; Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) both contain a C-terminal tetramerization domain composed of a leucine heptad repeat embedded within a 4,3-hydrophobic repeat . Previous mutagenesis experiments and X-ray crystallographic studies have demonstrated that these repeats are required for tetramer assembly of the hydroxylase enzymes via coiled-coil interactions . The specificity of these particular C-terminal intersubunit binding motifs was investigated by determining if TH and TPH can form heterotetramers when coexpressed in bacteria . Bacterial cells were contransformed with TH and TPH expression plasmids under kanamycin and ampicillin selection, respectively . Immunoprecipitation of induced bacterial supernatants with a TPH monoclonal antibody demonstrated that, unlike the human TH isoforms, TH and TPH do not form heterotetramers . The data suggest that specificity of oligomerization of the aromatic amino acid hydroxylases may be partially determined by polar amino acids interspersed within the coiled-coil . This finding should be influential in the development of eukaryotic expression systems and ultimately in gene therapy approaches. Histol Histopathol, 1998 Apr, 13(2), 347 - 58 An electron microscopic study of Helicobacter pylori in the surface mucous gel layer; Ogata M et al.; This study describes the distribution of spiral and coccoid forms of Helicobacter (H.) pylori within the surface mucous gel layer (SMGL) on human gastric mucosae . Gastric mucosae infected with H . pylori were obtained from surgically removed stomachs of 14 cases of gastric cancer . The glycocalyx of the spiral and coccoid forms of H . pylori was examined for specific lectin labeling . Coccoid forms were identified in 11 of these cases (78.6%) . The SMGL, which was from 50 to 100 microns in thickness, contained H . pylori throughout its entire thickness . Variously-sized vacuole-like clear areas were present near H . pylori . The glycocalyx on both the spiral and coccoid forms was similar in its staining with the 8 types of lectins tested . However, the staining pattern of the lectins varied among different samples . The numerous fibrillae-like filaments radiated from the surface of the bacteria and appeared to link the bacteria to the surface mucous cells or the surrounding mucus . These fibrillae-like filaments were not specifically stained by the lectin reactions, suggesting absence of sugar molecules. J Microsc, 1998 Mar, 189 ( Pt 3), 236 - 48 Electron microscopy of frozen biological objects: a study using cryosectioning and cryosubstitution; Erk I et al.; Freezing of bulk biological objects was investigated by X-ray cryodiffraction . Freezing at atmospheric pressure of most microscopic biological samples gives rise to large hexagonal crystals and leads to poor structural preservation of these specimens . High-pressure freezing induces the formation of different ices (hexagonal, cubic and a high-pressure form) consisting of crystals having sizes smaller than those formed at atmospheric pressure . With both freezing methods, a cryoprotectant has to be added to the biological object to avoid the formation of ice crystals . However, special cases can be encountered: some biological objects contain large amounts of natural cryoprotectant or have a low water content . In these cases, vitrification can be achieved, especially using high-pressure freezing . Cryo-sectioning can be performed on vitrified samples, and the sections studied by electron cryomicroscopy . Images and electron diffraction patterns having a resolution better than 2 and 0.2 nm, respectively, can be obtained with such sections . Because samples containing crystalline ices cannot be cryosectioned, their structure has to be studied using cryosubstitution and resin embedding . We show that bacteria, yeast, and ciliate and marine worm elytrum have cellular compartments with an organization that has not been described by classical techniques relying on chemical fixation of the tissues . A high-pressure artefact affecting the Paramecium trichocysts is described . Such artefacts are not general; for example, we show that 70% of high-pressure frozen yeast cells survive successive high-pressure freezing and thawing steps. C R Seances Soc Biol Fil, 1997, 191(5-6), 755 - 63 {Repair of oxidized guanine in mammals: OGG1 genes}; Radicella JP et al.; This paper reviews the present state of the studies on the repair of a major oxydative lesion on DNA, the 8-oxo-guanine (8-OxoG) . This modified base has been proved to be highly mutagenic and therefore implicated in the ethiology of several pathologies . The cloning of the yeast OGG1 gene, a functional homolog of the fpg from bacteria, allowed the isolation of the mammalian homologs . These genes code for 8-OxoG DNA glycosylases/lyases, whose biochemical properties are consistent with their postulated role as the main defence against the genetic instability induced by the presence of 8-OxoG in DNA . This, together with the mutator phenotype of the yeast ogg1 mutant strains, make of the human OGG1 a candidate for a cancer predisposition gene . The localization of this gene to chromosome 3p and other evidences discussed in this paper indicate that OGG1 could be a tumor suppressor gene implicated in lung cancer. Trends Microbiol, 1998 Apr, 6(4), 139 - 44 Life in grasses: diazotrophic endophytes; Reinhold-Hurek B et al.; N2-fixing bacteria such as Azoarcus spp., Herbaspirillum spp, and Acetobacter diazotrophicus can infect the interior of gramineous plants without causing symptoms of plant disease but do not survive in soil . Like phytopathogens, they can penetrate into central tissues and spread systemically . There is no evidence for an endosymbiosis in living plant cells; however, the bacteria are physiologically active in the plant apoplast. Am Ind Hyg Assoc J, 1998 Apr, 59(4), 234 - 41 Improved methods for generation, sampling, and recovery of biological aerosols in filter challenge tests; McCullough NV et al.; In preparation for filter efficiency tests and sampler comparison studies, methods of biological aerosol generation, sampling, and filter recovery were modified from previous studies . Methods described include (1) techniques for generating aerosols that reduced nuisance particles to negligible levels and increased the cell culturability of Mycobacterium abscessus by 30%, (2) sampling techniques that lowered the detectable range of biological particle size from 0.65 to 0.45 micron and reduced the sampling flow from the chamber from 28.3 to 1.5 L/min, and (3) development of methods to remove culturable organisms from respirator filter media . These methods were developed for filter challenge tests with M . abscessus and were applied to two other bacteria . They may also have application to a wider variety of organisms and bioaerosol assessments. Zentralbl Chir, 1998, 123(3), 205 - 17 {The intestine as the central organ in the development of multiple organ failure after severe trauma--pathophysiology and therapeutic approaches}; Grotz M et al.; Multiple organ failure is with an incidence of 10-25% and a mortality of 50-70% the most severe complication after severe trauma . Intestinal ischemia and a corresponding impaired gut barrier function is thought to have a high impact on the development of multiple organ failure after severe trauma . Under normal conditions the intestinal wall is a sufficient barrier against bacteria and their products . Gut ischemia is followed by mucosal lesions, the intestinal permeability is increased . Translocating bacteria and bacterial products (endotoxin, peptidoglykan) can lead to a local and/or systemic immun-inflammatory response, which is made responsible for the development of multiple organ failure . Tonometry as a possibility of monitoring intestinal ischemia as well as a tool to estimate the prognosis of multiple trauma patients is still discussed controversially . Dopexamin, which directly influences intestinal ischemia (goal directed therapy) might be a successful treatment option, however until now no clinical study about beneficial effects of dopexamine in severely injured patients is available . Selective gut decontamination showed no clinical benefits in multiple trauma patients . Early enteral nutrition especially with immunomodulating ingredients ("immunonutrition") decreases posttraumatic complications as well as the incidence of MOF . However a reduction of mortality could not be described in severely injured patients so far. Nucleic Acids Symp Ser, 1997, (37), 305 - 6 Aminoacyl-tRNA synthesis in Archaea; Ibba M et al.; The mechanism of aminoacyl-tRNA synthesis differs substantially between Archaea, Bacteria and Eukarya . Sequencing of archaeal genomes has suggested that the asparaginyl-, cysteinyl-, glutaminyl- and lysyl-tRNA synthetases are absent from a number of organisms in this kingdom . The absence of the asparaginyl- and glutaminyl-tRNA synthetases is in agreement with the observation that Asn-tRNA and Gln-tRNA are synthesized by tRNA-dependent transamidation of Asp-tRNA and Glu-tRNA respectively in the archaeon Haloferax volcanii . Biochemical and genetic studies have now shown that while the cysteinyl- and lysyl-tRNA synthetases are present, the enzymes responsible for these activities are unique to Archaea. Allergol Immunopathol (Madr), 1998 Jan-Feb, 26(1), 17 - 22 {Safety and efficacy of OM-85-BV plus amoxicillin/clavulanate in the treatment of subacute sinusitis and the prevention of recurrent infections in children}; Gomez Barreto D et al.; A 6-month double-blind, prospective, randomized, placebo-controlled trial was conducted to establish the safety and efficacy of OM-85-BV in the treatment of subacute sinusitis and in the following prevention of the respiratory tract infections in 56 children from 18 months to 9 years of age . In the subacute phase of the sinusitis the patients were given one OM-85-BV capsule (3.5 mg of bacterial extracts) (n = 26) or placebo (n = 30), daily for ten days; additionally both groups took amoxicillin/clavulanate 40/10 mg/kg daily in three divided doses for 21 days . For the following two months the patients took one OM-85-BV capsule or placebo, ten days a month . In the subacute phase the OM-85-BV group of patients improved sooner (5.56 +/- 4.98 vs 10 +/- 8.49 days) and had a shorter convalescence (15.38 +/- 8.91 vs 20.28 +/- 7.17 days) . During the six month follow-up the patients in the OM-85-BV group had a lower number of infections (1.56 +/- 0.3 vs 2.22 +/- 0.43) and required a lower number of drug treatments (1.47 +/- 0.32 vs 1.94 +/- 0.42) . One patient treated with OM-85-BV presented a mild rash which disappeared three days after the drug discontinuation . We conclude that OM-85-BV is safe at pediatric ages, as well as accelerates the cure and improvement of subacute sinusitis while it lowers the incidence of respiratory infections. Eur J Oral Sci, 1998 Apr, 106(2 Pt 2), 696 - 706 The biocompatibility of non-amalgam dental filling materials; Schmalz G; Non-amalgam filling materials may release substances which have been shown to be toxic in cytotoxicity tests and implantation studies . However, results from systemic toxicity tests do not indicate any unacceptable risk to the patient's general health, but data for non-amalgam dental filling materials are scarce in comparison to amalgam . Although estrogen-like effects of one fissure sealant have been claimed, no conclusions can be drawn at present for the patient from these in vitro data because of the limitation of the test methods and materials used . Some components of composite resins/dentin adhesives and a resin-modified glass ionomer cement were mutagenic mainly in in vitro tests . Due to the limitations of the test systems and the comparatively high concentrations needed to elicit the reactions, no unacceptable risk can yet be derived from those data for the patient . However, a no-touch technique is recommended for the dental personnel . As with amalgam, local reactions of the pulp are not expected with alternative filling materials, if the pulp tissue is not exposed and if bacterial penetration is avoided . The latter requirement is still difficult to fulfill, especially for composite resin systems and related materials in posterior teeth situations . Slight gingival reactions to alternative filling materials and to amalgams are mainly attributed to plaque accumulation . From all these data it can be concluded that, for the time being, it is not possible to rank dental filling materials in respect to their biocompatibility, and it is evident that biocompatibility must be considered to the same extent for both amalgams and commonly used or recommended alternative filling materials. Am J Infect Control, 1998 Apr, 26(2), 139 - 42 Tuberculosis control through respirator wear: performance of National Institute for Occupational Safety and Health-regulated respirators; Willeke K et al.; BACKGROUND: In 1995 the National Institute for Occupational Safety and Health issued new rules for personal respirators . All nine new respirator categories are authorized in health care facilities for the prevention of the transmission of tuberculosis (TB) . The new N95 respirator category is the most frequently used for this purpose . Data are presented on their efficiency for collecting TB-size bacteria and their potential for reaerosolizing collected bacteria . METHODS: All measurements of bacterial penetration were performed with dynamic aerosol size spectrometers at flow conditions corresponding to normal wear and respirator certification conditions . The reaerosolization tests were performed at conditions ranging from normal breathing to violent coughing or sneezing . RESULTS: The tested N95 respirators collected 0.1 to 0.3 microm particles with efficiencies of 95% or higher, as specified by the regulations . TB-size bacteria of 0.8 microm and larger, however, were collected with 99.5% or higher efficiencies; that is, the penetration of these bacteria through the filter material was 0.5% or less, much less than the required maximum penetration of 5% for the smaller particle sizes . No bacteria were reaerosolized during normal exhalation . Some reaerosolization (0.1% or less) was observed only at low humidity and extremely high air flow through the respirator, corresponding to violent coughing or sneezing . CONCLUSIONS: The filter materials of N95 respirators provide good protection against TB bacteria . Thus, a significant number of bacteria can enter the respirator-wearer's breathing space only through spaces where the respirator inadequately seals to the wearer's face . Reentrainment and reaerosolization of mycobacteria is not a problem when normal work practices are observed in health care facilities. Nature, 1998 Apr 16, 392(6677), 734 - 7 Electron currents generated by the human phagocyte NADPH oxidase; Schrenzel J et al.; Electron transport across biological membranes is a well-known feature of bacteria, mitochondria and chloroplasts, where it provides motive forces for vectorial transport processes . In contrast, electron transport is generally not found in the plasma membrane of eukaryotic cells, possibly because it would interfere with electric processes at the plasma membrane . An exception is provided by the phagocyte NADPH oxidase, which generates superoxide (O2.-) through electron transfer from cytosolic NADPH to extracellular oxygen . The enzyme is essential for host defence, and patients with chronic granulomatous disease, who lack the functional enzyme, suffer from severe infections . It has been suggested that electron transfer by the NADPH oxidase might be electrogenic . Here we demonstrate, using the whole-cell patch-clamp technique, the generation of electron currents by the NADPH oxidase in human eosinophil granulocytes . The currents were absent in granulocytes of sufferers of chronic granulomatous disease and under conditions of low oxygen . Generation of electron currents across the plasma membrane of eukaryotic cells has not been observed previously and might be-independently of the generation of superoxide-a physiologically relevant function of the phagocyte NADPH oxidase. Blood Cells Mol Dis, 1998 Jun, 24(2), 83 - 100 Sequence, structural, functional, and phylogenetic analyses of three glycosidase families; Mian IS; Glycosidases, which cleave the glycosidic bond between a carbohydrate and another moiety, have been classified into over 63 families . Here, a variety of computational techniques have been employed to examine three families important in normal and abnormal pathology with the aim of developing a framework for future homology modeling, experimental and other studies . Family 1 includes bacterial and archaeal enzymes as well as lactase phlorizin-hydrolase and klotho, glycosidases implicated in disaccharide intolerance II and aging respectively . A statistical model, a hidden Markov model (HMM), for the family 1 glycosidase domain was trained and used as the basis for comparative examination of the conserved and variable sequence and structural features as well as the phylogenetic relationships between family members . Although the structures of four family 1 glycosidases have been determined, this is the first comparative examination of all these enzymes . Aspects that are unique to specific members or subfamilies (substrate binding loops) as well those common to all members (a beta/alpha)8 barrel fold) have been defined . Active site residues in some domains in klotho and lactase-phlorizin hydrolases differ from other members and in one instance may bind but not cleave substrate . The four invariant and most highly conserved residues are not residues implicated in catalysis and/or substrate binding . Of these, a histidine may be involved in transition state stabilization . Glucosylceramidase (family 30) and galactosylceramidase (family 59) are mutated in the lysosomal storage disorders Gaucher disease and Krabbe disease, respectively . HMM-based analysis, structure prediction studies and examination of disease mutations reveal a glycosidase domain common to these two families that also occurs in some bacterial glycosidases . Similarities in the reactions catalyzed by families 30 and 59 are reflected in the presence of a structurally and functionally related (beta/alpha)8 barrel fold related to that in family 1. Eur J Cardiothorac Surg, 1998 Feb, 13(2), 165 - 9 Pericardial effusion and AIDS: benefits of surgical drainage; Gouny P et al.; OBJECTIVES: During the last few years, AIDS has been the main cause of large pericardial effusions in urban settings . We have therefore had to perform surgical pericardial drainage for diagnostic and/or therapeutic purposes in AIDS patients . This study was designed to establish the diagnostic and therapeutic yield of pericardial drainage for these patients . METHODS: We retrospectively reviewed the data of the 13 AIDS patients with a pericardial effusion, referred to our surgical department between December 1989 and December 1996 for surgical drainage and pericardial biopsy . RESULTS: Cytological studies and searches for bacteria, mycobacteria and parasites were all negative . The histology of the 13 pericardial biopsies disclosed three pericardial locations of a Kaposi's sarcoma (all three patients had a pre-existent extra-cardiac location of this sarcoma) and one pericardial location of an already known immature mediastinal teratoma . In the nine other cases, the lesions were aspecific . Four patients died of multivisceral failure within 30 days of surgery . For the survivors, surgical drainage afforded relief and there were no clinical signs of recurrent effusion . CONCLUSIONS: The cause of pericardial effusion in AIDS is still often unknown, even after pericardial biopsy . Here, aspecific pericarditis was the most common diagnosis . Although the prognosis of such effusion in these patients is known to be poor, surgical drainage provided relief for those who survived the post-operative period. Acta Otolaryngol, 1998 Mar, 118(2), 280 - 3 Preservation of tubal function in patients with nasopharyngeal carcinoma, post-irradiation; Young YH et al.; Nineteen nasopharyngeal carcinoma (NPC) patients were subjected to eustachian tube function testing before and 5 years after irradiation . Tubal patency and clearance function of the eustachian tube showed deterioration if maximum irradiation dosage was more than 70 Gy, whereas dynamic function of the eustachian tube was preserved . Development of middle ear complications in NPC patients post-irradiation was caused by both tubal and inflammatory factors . To preserve tubal function, maximum irradiation dosage to NPC should be limited to 70 Gy . To decrease the inflammatory reaction, firstly, middle ear effusion should be drained by repeated myringotomies instead of grommet insertion, and secondly, sinusitis should be evaluated and treated, because sinusitis can aggravate otitis media with effusion. Acta Otolaryngol, 1998 Mar, 118(2), 264 - 71 Acute pharyngotonsillitis is an infection restricted to the crypt and surface secretion; Ebenfelt A et al.; A commonly accepted hypothesis is that acute pharyngotonsillitis is caused by bacteria which first adhere to the epithelial surface and then invade the tonsillar parenchyma; however, evidence directly supporting this hypothesis is not available . In previous studies on acute pharyngotonsillitis, we found that the secretion in crypts and at the surface was infected in acute pharyngotonsillitis while no bacteria were detected in the parenchyma . Based on these results, we have proposed a new hypothesis stating that the infection is restricted to the crypt and surface secretions in acute pharyngotonsillitis . To evaluate this hypothesis further, in the present study we examined tonsillar tissue and secretion from patients with acute pharyngotonsillitis, recurrent pharyngotonsillitis and healthy tonsils . Surface secretion was studied after sampling by an imprint technique followed by routine histological preparation . Tonsillar tissue was examined by fluorescence microscopy after staining with acridine orange and by transmission electron microscopy . There were high numbers of bacteria and moderate or extensive ongoing phagocytosis in the crypt and surface secretion from patients with acute pharyngotonsillitis . Bacteria, leucocytes and phagocytosis were also present, but to less extent in the secretion from patients with recurrent pharyngotonsillitis and to even less extent in the healthy controls . In none of all the investigated tonsils were bacteria present in the parenchyma . Bacterial adherence to the epithelial surface was only very rarely observed . This study supports the hypothesis that acute pharyngotonsillitis is an infection restricted to the crypt and surface secretion and that bacterial adherence is not of significant importance in the pathogenesis of acute pharyngotonsillitis. Biochemistry, 1998 Apr 14, 37(15), 5060 - 73 Dynamics of the DNA binding domain of the fructose repressor from the analysis of linear correlations between the 15N-1H bond spectral densities obtained by nuclear magnetic resonance spectroscopy; van Heijenoort C et al.; The spectral densities of the backbone and arginine side chain NH bonds of the DNA binding domain of the fructose repressor (FruR) were extensively analyzed in order to extract reliable motions parameters . An accurate measurement of 15N NMR relaxation rates allowed their calculation at three frequencies, zero, omegaN, and omegaH + omegaN, using a reduced matrix approach . Linear correlations were found between J(omegaN) and J(0) and between <J(omegaH)> and J(0) . The analysis of the compatibility between the motions parameters obtained independently from the two correlation lines allowed further development of the linear correlation approach proposed recently {Lefevre, J . F., Dayie, K . T., Peng, J . W., and Wagner, G . (1996) Biochemistry 35, 2674-2686} . The results demonstrate (i) the existence of a concerted motion along the whole backbone with a global correlation time equal to 5.95 ns.rad-1, and (ii) the presence of complex internal movements at an intermediate time scale around 1 ns . The extracted motion parameters have been related to those obtained with the extended Lipari and Szabo approach but are incompatible with those obtained using the usual simple Lipari and Szabo approach . They were correlated to the features of the NMR structure of FruR(1-57)* . Some residues in the turns and in the third helix experience slow motions in the micro- to millisecond time scale . Side-chain motions are not correlated to the backbone dynamics . A direct examination of spectral densities reveals a higher flexibility for the side chains of arginines that are not involved in ionic bridges. Biochim Biophys Acta, 1998 Mar 13, 1370(2), 187 - 91 ERD6, a cDNA clone for an early dehydration-induced gene of Arabidopsis, encodes a putative sugar transporter; Kiyosue T et al.; Previously, we constructed a cDNA library from Arabidopsis plants that were exposed to dehydration stress for 1 h and obtained the ERD6 clone . Here we report that the ERD6 cDNA consists of 1741 bp and encodes a polypeptide of 496 amino acids having a predicted molecular weight of 54,354 . The putative polypeptide of ERD6 is related to those of sugar transporters of bacteria, yeasts, plants and mammals . Hydropathy analysis revealed that ERD6 protein has 12 putative transmembrane domains and a central hydrophilic region . Sequences that are conserved at the ends of the 6th and 12th membrane-spanning domains of sugar transporters are also present in ERD6 . These data suggest that ERD6 encodes a sugar transporter . Genomic Southern blots indicate that the ERD6 gene is a member of a multigene family in the Arabidopsis genome . The expression of the ERD6 gene was induced not only by dehydration but also by cold treatment . Proc Natl Acad Sci U S A, 1998 Apr 14, 95(8), 4469 - 74 Functional copies of a human gene can be directly isolated by transformation-associated recombination cloning with a small 3' end target sequence; Kouprina N et al.; Unique, small sequences (sequence tag sites) have been identified at the 3' ends of most human genes that serve as landmarks in genome mapping . We investigated whether a single copy gene could be isolated directly from total human DNA by transformation-associated recombination (TAR) cloning in yeast using a short, 3' unique target . A TAR cloning vector was constructed that, when linearized, contained a small amount (381 bp) of 3' hypoxanthine phosphoribosyltransferase (HPRT) sequence at one end and an 189-bp Alu repeat at the other end . Transformation with this vector along with human DNA led to selective isolations of the entire HPRT gene as yeast artificial chromosomes (YACs) that extended from the 3' end sequence to various Alu positions as much as 600 kb upstream . These YACs were retrofitted with a NeoR and a bacterial artificial chromosome (BAC) sequence to transfer the YACs to bacteria and subsequently the BACs to mouse cells by using a Neo selection . Most of the HPRT isolates were functional, demonstrating that TAR cloning retains the functional integrity of the isolated material . Thus, this modified version of TAR cloning, which we refer to as radial TAR cloning, can be used to isolate large segments of the human genome accurately and directly with only a small amount of sequence information. Biochemistry, 1998 Apr 7, 37(14), 4722 - 30 Crystal structure of Y34F mutant human mitochondrial manganese superoxide dismutase and the functional role of tyrosine 34; Guan Y et al.; Tyrosine 34 is a prominent and conserved residue in the active site of the manganese superoxide dismutases in organisms from bacteria to man . We have prepared the mutant containing the replacement Tyr 34 --> Phe (Y34F) in human manganese superoxide dismutase (hMnSOD) and crystallized it in two different crystal forms, orthorhombic and hexagonal . Crystal structures of hMnSOD Y34F have been solved to 1.9 A resolution in a hexagonal crystal form, denoted as Y34Fhex, and to 2.2 A resolution in an orthorhombic crystal form, denoted as Y34Fortho . Both crystal forms give structures that are closely superimposable with that of wild-type hMnSOD, with the phenyl rings of Tyr 34 in the wild type and Phe 34 in the mutant very similar in orientation . Therefore, in Y34F, a hydrogen-bonded relay that links the metal-bound hydroxyl to ordered solvent (Mn-OH to Gln 143 to Tyr 34 to H2O to His 30) is broken . Surprisingly, the loss of the Tyr 34 hydrogen bonds resulted in large increases in stability (measured by Tm), suggesting that the Tyr 34 hydroxyl does not play a role in stabilizing active-site architecture . The functional role of the side chain hydroxyl of Tyr 34 can be evaluated by comparison of the Y34F mutant with the wild-type hMnSOD . Both wild-type and Y34F had kcat/Km near 10(9) M-1 s-1, close to diffusion-controlled; however, Y34F showed kcat for maximal catalysis smaller by 10-fold than the wild type . In addition, the mutant Y34F was more susceptible to product inhibition by peroxide than the wild-type enzyme . This activity profile and the breaking of the hydrogen-bonding chain at the active site caused by the replacement Tyr 34 --> Phe suggest that Tyr 34 is a proton donor for O2* - reduction to H2O2 or is involved indirectly by orienting solvent or other residues for proton transfer . Up to 100 mM buffers in solution failed to enhance catalysis by either Y34F or the wild-type hMnSOD, suggesting that protonation from solution cannot enhance the release of the inhibiting bound peroxide ion, likely reflecting the enclosure of the active site by conserved residues as shown by the X-ray structures . The increased thermostability of the mutant Y34F and equal diffusion-controlled activity of Y34F and wild-type enzymes with normal superoxide levels suggest that evolutionary conservation of active-site residues in metalloenzymes reflects constraints from extreme rather than average cellular conditions . This new hypothesis that extreme rather than normal substrate concentrations are a powerful constraint on residue conservation may apply most strongly to enzyme defenses where the ability to meet extreme conditions directly affects cell survival. J Biol Chem, 1998 Apr 10, 273(15), 8749 - 55 Transcriptional activation of heat shock factor HSF1 probed by phosphopeptide analysis of factor 32P-labeled in vivo; Xia W et al.; Mapping of tryptic phosphopeptides of heat shock factor 1 (HSF1) from non-stressed or moderately heat-stressed HeLa cells, labeled in vivo by {32P}orthophosphate, revealed four major phosphopeptides A to D . Heat stress drastically increased phosphopeptide signals . To identify target peptides and amino acids and to correlate phosphorylation and transactivation function, phosphopeptide maps were produced of LexA-human HSF1 chimeras and mutant derivatives thereof, and transactivation activities of original and mutant chimeras were compared . LexA-HSF1 chimeras were previously shown to be regulated identically to HSF1, except that they transactivate promoters with LexA-binding sites instead of hsp promoters . The patterns of phosphopeptides of LexA-HSF1 and endogenous HSF1 were similar . Analysis of single residue substitutions suggested that phosphopeptide C is peptide VKEEPPSPPQSPR (297-309) phosphorylated on Ser-307 but not Ser-303 . Substitution of Ser-307 but not Ser-303 caused deregulation of factor activity . Mapping of several constitutively active chimeras associated unphosphorylated peptide C with the transcriptionally active HSF1 conformation, suggesting that dephosphorylation of this peptide (at Ser-307) may either be an integral step in the activation process or serve to maintain the active conformation of HSF1 . Exploiting this correlation, indirect evidence was obtained that activation domains of HSF1 interact with the distantly located regulatory domain to maintain the factor in an inactive state. Eur J Med Res, 1998 Apr 8, 3(4), 216 - 22 Congress Report: XIX symposium of the International Association for Comparative Research on Leukemia and Related Diseases, Mannheim/Heidelberg, Germany, July 13 - 18, 1997; Hochhaus A et al.; The XIX Symposium of the International Association for Comparative Research on Leukemia and Related Diseases (IACRLRD, President: Prof . Dr . R . Hehlmann) was held in Mannheim and Heidelberg, Germany, July 13 - 18, 1997 . Comparative research in cancer was systematically established in the 1950s . Similarities in morphology, biology and pathology between animal and human leukemias and related diseases and the viral origin of a variety of animal leukemias and related diseases (lymphomas, sarcomas, breast tumors etc.) led to the concept that comparative research should promote the understanding of human leukemias and related diseases . In 1960 the World Health Organization inaugurated the establishment of a World Committee for Comparative Leukemia Research . The first symposium took place in Hannover, Germany, in 1963 . After the fifth symposium in Padova, Italy, in 1971 the International Association for Comparative Research on Leukemia and Related Diseases (IACRLRD) was founded to complement the World Committee and to expand the international effort . The history of the symposium shows the evolution from a meeting on animal leukemia viruses into one dealing with viral and genetic aspects of human and animal leukemia and related diseases . The scientific evolution of the Abelson murine leukemia virus with its abl oncogene in the 1970s to what currently appears as the most reliable marker for human chronic myeloid leukemia is merely one example . Comparative research has reached a new dimension with the the recent advances in sequencing of the genomes of a variety of species and of humans . Many genes identified in the human genome and relevant for disease can be found in the genomes of animal species and even in the genomes of bacteria and of yeast . This reminds us that not just human and animal biology but also pathology must be regarded as a continuum of evolution and that much can be learned from comparing the genetic information of different species . Comparative genome research will allow conclusions to be drawn from principles recognized in animal species which are relevant to human diseases . It is likely that the application of comparative research to genome analysis will provide basic new insights in molecular medicine into the function of living beings for both animal species and humans . The current revolution in genomics is the latest phase in a rich history of medical progress related to the comparative approach . Meetings and organizations that have grown out of IACRLRD, include, at least to some extent: the Meeting of the International Human Retrovirology Association, the Gallo Lab Meeting , the Feline Retrovirus Meeting, the Cold Spring Habor Retrovirus Meeting, international and regional AIDS meetings, and many others . The XIX symposium in Mannheim included five memorial lectures, seven plenary sessions, 18 parallel sessions, two round table discussions and a public forum . In addition, six associated satellite symposia were held . The general meeting, attended by participants from 27 countries, integrated thematically contributions of genetic, cellular, and viral factors toward the development of leukemia and lymphoma and sought unifying concepts in leukemogenesis. Blood, 1998 Apr 15, 91(8), 2914 - 24 Hematopoietic remodeling in interferon-gamma-deficient mice infected with mycobacteria; Murray PJ et al.; Control of intracellular bacterial infections requires interferon-gamma (IFN-gamma) both for establishing a Th1 T-cell response and for activating macrophages to kill the bacteria . Exposure of mice deficient in IFN-gamma to mycobacterial infection produces an immune response characterized by a Th2 T-cell phenotype, florid bacterial growth, and death . We report here that IFN-gamma-deficient mice infected with mycobacteria also undergo a dramatic remodeling of the hematopoietic system . Myeloid cell proliferation proceeds unchecked throughout the course of mycobacterial infection, resulting in a transition to extramedullary hematopoiesis . The splenic architecture of infected IFN-gamma-deficient mice is completely effaced by expansion of macrophages, granulocytes, and extramedullary hematopoietic tissue . These features coincide with splenomegaly, an increase in splenic myeloid colony-forming activity, and marked granulocytosis in the peripheral blood . Systemic levels of cytokines are elevated, particularly interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) . These results suggest that in addition to its central role in cellular immunity, IFN-gamma may be a key cytokine in coordinate regulation of immune effector cells and myelopoiesis . This model should be valuable for deciphering the cross-talk between the immune response and hematopoiesis during bacterial infection and for improving our understanding of the mechanisms that control chronic infections. J Exp Med, 1998 Apr 6, 187(7), 1145 - 50 DNA as an adjuvant: capacity of insect DNA and synthetic oligodeoxynucleotides to augment T cell responses to specific antigen; Sun S et al.; How strong adjuvants such as complete Freund's adjuvant (CFA) promote T cell priming to protein antigens in vivo is still unclear . Since the unmethylated CpG motifs in DNA of bacteria and other nonvertebrates are stimulatory for B cells and antigen-presenting cells, the strong adjuvanticity of CFA could be attributed, at least in part, to the presence of dead bacteria, i.e., a source of stimulatory DNA . In support of this possibility, evidence is presented that insect DNA in mineral oil has even stronger adjuvant activity than CFA by a number of parameters . Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs mimic the effects of insect DNA and, even in soluble form, ODNs markedly potentiate clonal expansion of T cell receptor transgenic T cells responding to specific peptide. AIDS Res Hum Retroviruses, 1998 Apr, 14 Suppl 1, S23 - 5 Urological manifestations of HIV infection; Coburn M; HIV-1 disease is associated with many infectious and neoplastic complications . This is particularly true of the genitourinary tract, where high rates of renal disease, neoplasms, voiding and erectile dysfunction, hematuria, opportunistic genitourinary infections, and epididymo-orchitis are found in HIV-1-infected individuals . Recent experience at Ben Taub General Hospital (Houston, TX) shows that men requiring admission to the urologic service for treatment of genitourinary disorders have high rates of HIV-1 seropositivity, particularly in the setting of epididymo-orchitis, where 21 of 106 (20%) were found to be HIV-1 infected . These men were significantly more likely to require surgical treatment and to harbor resistant bacteria in infected tissues . Importantly, for 15 of 21 HIV-1-infected men, epididymo-orchitis was the first clinical manifestation of HIV-1 disease, and 6 were first found to be HIV-1 seropositive during that admission . Our experience illustrates the critical importance of screening for HIV-1 infection in the setting of genitourinary disease. Berl Munch Tierarztl Wochenschr, 1998 Apr, 111(4), 143 - 5 Current status on the laboratory diagnosis of Ornithobacterium rhinotracheale "ORT" in poultry; Hafez HM; Respiratory disease conditions are one of the most serious groups of diseases affecting poultry . Ornithobacterium rhinotracheale by has recently been recognized in many countries . Clinical signs and lesions are of little value in diagnosis . Accurate diagnosis must be substantiated by isolation and identification of the causative bacteria and/or detection of antibodies using serological examination . In the present paper a review on the current status of ORT laboratory diagnosis, results of serotyping of field isolates, serological surveillance in poultry flocks as well as the role of other avian pathogens in course of ORT infection will be given. J Periodontol, 1998 Mar, 69(3), 337 - 47 Root surface characteristics of primary teeth from children with prepubertal periodontitis; Bimstein E et al.; This study describes the histologic characteristics of root surfaces of primary teeth from children with prepubertal periodontitis (PP) . Fifteen primary teeth from 4 children with PP, and 2 control primary teeth from 2 healthy children were examined . Light microscopy revealed normal root surfaces in the control teeth . In contrast, the PP specimens revealed bacteria inside dentin tubules or covering cementum, a cuticle, or resorbed dentin; normal, wider than normal, or hypoplastic cementum; resorption lacunae with various depths; aplastic root resorption; alternate resorption and repair; and active repair . No cementoclasts were found in the resorption lacunae . Scanning electron microscopy revealed intrabony and suprabony root areas, and a "plaque free zone" (PFZ) . Colonies of filaments were evident at the cemento-enamel junction (CEJ) . The suprabony root surfaces had resorption lacunae, isolated short rods, calculus, colonies of filaments, or colonies composed by an heterogeneous bacterial population . The coronal boundary of the PFZ was the border of a sheet-like structure, which included isolated rods or filaments . At the PFZ, isolated filaments and rods, and a fibril matrix were evident . The apical boundary of the PFZ consisted of bundles of soft tissue remnants or the insertion of the periodontal fibers . The intrabony surfaces were mostly covered by soft tissue, which included isolated filaments and short rods . Resorption lacunae with or without soft tissue were also evident in this area . Crystals of calcium oxalate dihydrate and erythrocytes in distinct forms were found at various root areas . The present findings are different from those previously reported for hypophosphatasia specimens. Invest Ophthalmol Vis Sci, 1998 May, 39(6), 859 - 66 Polymerase chain reaction and restriction fragment length polymorphism mediated detection and speciation of Candida spp causing intraocular infection; Okhravi N et al.; PURPOSE: To determine the usefulness of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis in the identification and speciation of Candida spp that causes ocular infection . METHODS: Oligonucleotide primers based on the cytochrome P450 L1 A1 demethylase gene were used to successfully amplify by PCR a single 1.0-kb and a single 500-bp DNA fragment from C . albicans, C . tropicalis, C . krusei, C . glabrata, C . parapsilosis, and C . pelliculosa genomic DNA . RFLPs within the PCR product were identified after restriction enzyme digestion . RESULTS: The sensitivity of the amplification reaction after two rounds of PCR was 10 fg genomic C . albicans DNA or one copy of the gene . No amplification product was obtained when DNA from C . guilliermondii, Aspergillus fumigatus, Fusarium solani, human leukocytes, or 10 species of bacteria was used as a template . Experiments with spiked normal vitreous demonstrated equal sensitivity as long as the volume of vitreous did not exceed 20% of the total PCR volume . RFLP analysis of the PCR product generated from each species obtained from the first- and second-round amplification products enabled species identification after digestion with specific endonucleases . Application of the technique to four clinical samples was successful . CONCLUSIONS: It is expected that the simplicity of the DNA extraction technique allied with the broad specificity of the outer primers for all ophthalmically relevant Candida spp and the sensitivity of the second-round PCR will aid in the detection of fungal DNA in small intraocular samples . PCR-RFLP analysis has great potential in the rapid detection and identification of Candida spp and in the provision of a useful laboratory tool for the future. Microbiology, 1998 Apr, 144 ( Pt 4), 1033 - 44 Oligopeptide permease in Borrelia burgdorferi: putative peptide-binding components encoded by both chromosomal and plasmid loci; Bono JL et al.; To elucidate the importance of oligopeptide permease for Borrelia burgdorferi, the agent of Lyme disease, a chromosomal locus in B . burgdorferi that encodes homologues of all five subunits of oligopeptide permease has been identified and characterized . B . burgdorferi has multiple copies of the gene encoding the peptide-binding component, OppA; three reside at the chromosomal locus and two are on plasmids . Northern analyses indicate that each oppA gene is independently transcribed, although the three chromosomal oppA genes are also expressed as bi- and tri-cistronic messages . Induction of one of the plasmid-encoded oppA genes was observed following an increase in temperature, which appears to be an important cue for adaptive responses in vivo . The deduced amino acid sequences suggest that all five borrelial oppA homologues are lipoproteins, but the protease-resistance of at least one of them in intact bacteria is inconsistent with outer-surface localization . Insertional inactivation of a plasmid-encoded oppA gene demonstrates that it is not essential for growth in culture. Pathobiology, 1998, 66(1), 24 - 32 Differential regulation of the expression of cytokine-induced neutrophil chemoattractant by mouse macrophages; Crippen TL et al.; The production of cytokine-induced neutrophil chemoattractant (CINC) by functionally diverse mouse bone-marrow-derived macrophages was determined . Studies showed that beta1,3-glucan, IL-beta, TNFalpha and IFNgamma/TNFalpha induced expression and production of CINC in macrophages while neither IFNgamma nor TGFbeta alone induced detectable CINC expression . Pretreatment or simultaneous treatment of macrophages with TGFbeta resulted in suppression of CINC protein production . These studies demonstrate that IFNgamma and TNFalpha, found early during the inflammatory response, induce production of CINC, as well as induce macrophages into a cytocidal state that are capable of killing transformed cells, parasites and bacteria, and recruiting neutrophils . In contrast, TGFbeta, found during reparative stages of the inflammatory response, suppressed production of CINC, while inducing the development of inflammatory macrophages that are capable of producing lysosomal enzymes, enhanced endocytosis and ingestion of particulate matter and function to scavenge debris, debride tissue and stimulate repair. Radiology, 1998 May, 207(2), 491 - 6 Tunneled hemodialysis catheters: use of a silver-coated catheter for prevention of infection--a randomized study; Trerotola SO et al.; PURPOSE: To determine whether silver-coated tunneled hemodialysis catheters reduce infection and to determine the frequency of central venous thrombosis and stenosis with percutaneous placement of right internal jugular vein dialysis catheters by interventional radiologists . MATERIALS AND METHODS: Ninety-one patients were randomly assigned to a treatment (silver-coated catheter; n = 47) or control (identical catheter without silver coating; n = 44) arm . Baseline venography was performed . Catheter tips were cultured and venography was repeated at catheter removal . RESULTS: Mean duration of catheter placement was 92 days . Infection occurred in 11 patients (five in the treatment group, six in the control group) . Tip cultures in 15 patients (eight treatment, seven control) were positive without clinical infection . Infection and colonization rates were slightly but not significantly higher in the treatment group than in the control group . Silver-coated catheters in two (4%) patients were removed due to reaction to the coating . Completion venograms (n = 72) showed new minor abnormalities in four (6%) patients and major abnormalities (stenosis, thrombosis) in three (4%) patients . Permanent venous abnormalities occurred in two (3%) patients . CONCLUSION: Silver coating does not confer a benefit against clinical infection or colonization . Interventional radiologic placement of tunneled dialysis catheters yields a low frequency of permanent central venous thrombosis and stenosis. Gut, 1998 Mar, 42(3), 396 - 401 Gut barrier function in malnourished patients; Welsh FK et al.; BACKGROUND: The integrity of the gastrointestinal mucosa is a key element in preventing systemic absorption of enteric toxins and bacteria . In the critically ill, breakdown of gut barrier function may fuel sepsis . Malnourished patients have an increased risk of postoperative sepsis; however, the effects of malnutrition on intestinal barrier function in man are unknown . AIMS: To quantify intestinal barrier function, endotoxin exposure, and the acute phase cytokine response in malnourished patients . PATIENTS: Malnourished and well nourished hospitalised patients . METHODS: Gastrointestinal permeability was measured in malnourished patients and well nourished controls using the lactulose:mannitol test . Endoscopic biopsy specimens were stained and morphological and immunohistochemical features graded . The polymerase chain reaction was used to determine mucosal cytokine expression . The immunoglobulin G antibody response to endotoxin and serum interleukin 6 were measured by enzyme linked immunosorbent assay . RESULTS: There was a significant increase in intestinal permeability in the malnourished patients in association with phenotypic and molecular evidence of activation of lamina propria mononuclear cells and enterocytes, and a heightened acute phase response . CONCLUSIONS: Intestinal barrier function is significantly compromised in malnourished patients, but the clinical significance is unclear. Int Immunol, 1998 Mar, 10(3), 275 - 83 Cloning and characterization of the guinea pig C5a anaphylatoxin receptor: interspecies diversity among the C5a receptors; Fukuoka Y et al.; The anaphylatoxin C5a receptor (C5aR, CD88 in man) plays a prominent role in mediating inflammatory and host defense processes . Direct evidence of C5aR involvement in host defense mechanisms was demonstrated recently using C5aR knockout mice . Mice deficient in C5aR were unable to clear intrapulmonary-instilled bacteria . The guinea pig system is perhaps unique for exhibiting cross-reactivity with human complement components and its high sensitivity to anaphylatoxins . Therefore, we cloned the guinea pig C5aR from a megakaryocyte cDNA library . The deduced amino acid sequence of guinea pig C5aR is 67% identical to human, 61.6% to dog, 60.2% to mouse and 63.6% to rat C5aR . Transient expression of guinea pig C5aR in COS-7 cells and stable expression on L cell fibroblasts were confirmed by FACS analysis . Competitive binding studies using {125I}C5a and stimulation of calcium mobilization by C5a proved that functional C5aR was expressed on these stably transfected L cells . The N-terminal extracellular region of guinea pig C5aR was five to seven residues shorter than the same region in C5aR from other species and sequence homology was limited to 11% . Other outer membrane loops were also poorly conserved (8-33%) when compared across five species . Transmembrane segments were highly conserved between these various species (46-86%) . Guinea pig C5aR binds human C5a, therefore residues critical for C5a binding have been conserved between these species . Sequence comparison of C5aR from multiple species permits conserved elements of the ligand binding sites to be elucidated. Am J Physiol, 1998 Apr, 274(4 Pt 2), R1058 - 64 Chloroquine inhibits proinflammatory cytokine release into human whole blood; Karres I et al.; Excessive synthesis and release of proinflammatory cytokines during endotoxemia causes severe pathophysiological derangements and organ failure . Because the lysosomotropic agent chloroquine has been effective in the treatment of diseases associated with increased secretion of proinflammatory cytokines such as malaria or rheumatoid arthritis, this study evaluates the potential effect of chloroquine on endotoxin-induced cytokinemia using human whole blood from healthy volunteers . Chloroquine revealed a dose-dependent inhibitory effect on endotoxin-induced secretion of tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 that was associated with reduced cytokine mRNA expression . Moreover, ammonia and methylamine, which react as weak bases like chloroquine, reduced synthesis and secretion of proinflammatory cytokines . These data indicate a potent anti-inflammatory effect of chloroquine on endotoxin-induced synthesis of proinflammatory cytokines that may be due to its weak base effect . Thus chloroquine may be of therapeutic benefit not only, during chronic inflammation but also in diseases that are related to bacteria-induced inflammation. J Clin Microbiol, 1998 May, 36(5), 1392 - 8 Simplified quantitative assay system for measuring activities of drugs against intracellular Legionella pneumophila; Higa F et al.; We developed a new simple assay for the quantitation of the activities of drugs against intracellular Legionella pneumophila . The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death . The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h . The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader . The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-alpha agar plates . Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria . The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L . pneumophila were determined by the colorimetric assay system . The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-alpha broth . The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs . According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study . Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples. J Clin Microbiol, 1998 May, 36(5), 1382 - 7 Comparison of methods of identifying Helicobacter hepaticus in B6C3F1 mice used in a carcinogenesis bioassay; Fox JG et al.; In a long-term rodent bioassay evaluating the carcinogenicity of triethanolamine, there was equivocal evidence of carcinogenic activity in male B6C3F1 mice, based on a marginal increase in the number of hepatocellular adenomas and hepatoblastomas . Interpretation was complicated by the presence of Helicobacter hepaticus in selected silver-stained liver sections which also had histological evidence of karyomegaly and oval cell hyperplasia . An increase in numbers of liver tumors, as evidence of carcinogenic activity, was also noted in female mice . However, H . hepaticus was not considered a complicating factor, because the livers of the female mice did not have histological features compatible with H . hepaticus infection . A retrospective analysis of 51 liver tissue samples from the original carcinogenicity study was conducted to determine the incidence of H . hepaticus infection and to evaluate different diagnostic approaches for assessing the presence of H . hepaticus in livers lacking characteristic lesions . In an initial evaluation of seven mice with liver tumors, argyrophilic bacteria resembling H . hepaticus were observed in liver sections, associated with characteristic liver lesions of hepatocytic karyomegaly and oval cell hyperplasia . Frozen liver tissue was available from four of these mice; all were confirmed to be infected with H . hepaticus by culture and PCR . In a larger subsequent analysis using frozen liver tissues from 44 mice without characteristic hepatic lesions, H . hepaticus-specific DNA was amplified from the livers of 21 of 44 of the mice (47%), compared to 14 of 44 of the mice (32%) having H . hepaticus cultured from their frozen liver tumors . The results of H . hepaticus culture and H . hepaticus-specific PCR concurred (i.e., both positive and negative results) in 84% of the cases . Microscopic detection of immunofluorescence-labeled or silver-stained bacteria in liver sections was relatively insensitive compared to either culture or PCR detection . This study confirms the widespread prevalence of H . hepaticus in mice, its potential to confound experimental results, and the need to include diagnostic testing for H . hepaticus in a murine health monitoring program. Langenbecks Arch Chir Suppl Kongressbd, 1997, 114, 483 - 9 {Pyogenic infections of the skin and skin appendages}; Schumpelick V et al.; In Germany more than 120,000 soft tissue infections are treated in hospital per year . Articles about soft tissue infections caused by Strept . pyogenes, presented in the lay press as "flesh-eating bacteria", accentuate this picture . Among these soft tissue infections, necrotising fasciitis and the associated "toxic shock syndrome" and "toxic shock-like syndrome" remain a challenge to the surgeon . The most important role of surgery is to achieve the right diagnosis and introduce definitive therapy in time . Central to surgical therapy is immediate incision and open drainage . Our own experience in animal models underlines the important role of abscess pressure and physicochemical parameters in the "abscess compartment" for systemic spread . Based on this pathophysiological background, the traditional "ubi pus ibi evacua" has lost nothing of its importance. Pract Periodontics Aesthet Dent, 1997 Aug, 9(6 Suppl), 1 - 5 Clinical efficacy of the Nd:YAG laser for combination periodontitis therapy; Neill ME et al.; Recent results of a limited clinical trial suggest that mechanical root scaling and root planing therapy alone may not be the most effective mode of treatment for patients affected by moderate to severe adult periodontitis . However, scaling and planing combined with laser therapy utilizing a low-powered pulsed Nd:YAG laser have been shown to be successful in the elimination of the bacteria commonly associated with the development of this oral condition . The double-blind, split mouth design study involved 10 human subjects randomly assigned to one of three treatments: 1) scaling and root planing alone, 2) dental laser plus scaling and root planing, and 3) control only . This article presents the clinical results of the trial, which suggest that laser therapy is a viable adjunct to local, nonsurgical therapy such as scaling and planing. Oral Microbiol Immunol, 1998 Feb, 13(1), 51 - 4 Helicobacter pylori adheres selectively to Fusobacterium spp; Andersen RN et al.; Helicobacter pylori strains ATCC 43504 and ATCC 43629 were tested for their ability to coaggregate with 79 strains of bacteria representing 16 genera . All except two of the strains were of human origin, and most of the strains were isolated from the oral cavity . The helicobacters failed to coaggregate with all strains except the fusobacteria . Several coaggregations were partially or completely inhibited by lactose . Strong coaggregation was seen with each of four subspecies of Fusobacterium nucleatum and with Fusobacterium periodonticum ATCC 33693, all of human dental plaque origin . In contrast, the helicobacters failed to coaggregate with non-plaque isolates, Fusobacterium mortiferum ATCC 25557 and Fusobacterium ulcerans ATCC 49185 . Heat treatment of the fusobacteria inactivated their ability to coaggregate, whereas heating of the Helicobacter partners had no effect, suggesting the presence of an adhesin on the fusobacteria and a corresponding receptor on the helicobacters . The potential ability of H . pylori to colonize the oral cavity by adhering selectively to the ubiquitous fusobacteria gives credence to the possibility that dental plaque may serve as a reservoir for this pathogen outside of the stomach. J Bacteriol, 1998 May, 180(9), 2484 - 92 Mutations affecting the alpha subunit of Bordetella pertussis RNA polymerase suppress growth inhibition conferred by short C-terminal deletions of the response regulator BvgA; Stibitz S; The effects of short deletions of the C terminus of the BvgA response regulator protein of the BvgAS two-component system were examined in Bordetella pertussis . When present as a single copy in the chromosome, deletions removing as few as two amino acids conferred a completely Bvg- phenotype . When provided in trans, on the broad-host-range plasmid pRK290, under the control of the native bvgAS promoter, deletions of two or three amino acids conferred a profound growth inhibition which was dependent on the integrity and activity of the wild-type chromosomal bvgAS locus . It is proposed that this phenotype was the result of an inappropriate interaction of the mutant BvgA protein with the RNA polymerase enzyme, specifically the alpha subunit . Mutant strains in which this growth inhibition was relieved were isolated and characterized . Although most of the suppressor mutations affected either the mutant plasmid copy or the wild-type chromosomal bvg locus, three mutations which affected the alpha subunit of B . pertussis RNA polymerase were also isolated . Two of these resulted in increased levels of the alpha subunit, and one caused a substitution of glycine for the aspartic acid residue at position 171, in the N-terminal domain . All three mutations also resulted in a differential phenotype in that expression of fha was essentially normal, but expression of ptx was greatly reduced. J Bacteriol, 1998 May, 180(9), 2418 - 25 Structure and expression of the FlaA periplasmic flagellar protein of Borrelia burgdorferi; Ge Y et al.; The spirochete which causes Lyme disease, Borrelia burgdorferi, has many features common to other spirochete species . Outermost is a membrane sheath, and within this sheath are the cell cylinder and periplasmic flagella (PFs) . The PFs are subterminally attached to the cell cylinder and overlap in the center of the cell . Most descriptions of the B . burgdorferi flagellar filaments indicate that these organelles consist of only one flagellin protein (FlaB) . In contrast, the PFs from other spirochete species are comprised of an outer layer of FlaA and a core of FlaB . We recently found that a flaA homolog was expressed in B . burgdorferi and that it mapped in a fla/che operon . These results led us to analyze the PFs and FlaA of B . burgdorferi in detail . Using Triton X-100 to remove the outer membrane and isolate the PFs, we found that the 38.0-kDa FlaA protein purified with the PFs in association with the 41.0-kDa FlaB protein . On the other hand, purifying the PFs by using Sarkosyl resulted in no FlaA in the isolated PFs . Sarkosyl has been used by others to purify B . burgdorferi PFs, and our results explain in part their failure to find FlaA . Unlike other spirochetes, B . burgdorferi FlaA was expressed at a lower level than FlaB . In characterizing FlaA, we found that it was posttranslationally modified by glycosylation, and thus it resembles its counterpart from Serpulina hyodysenteriae . We also tested if FlaA was synthesized in a spontaneously occurring PF mutant of B . burgdorferi (HB19Fla-) . Although this mutant still synthesized flaA message in amounts similar to the wild-type amounts, it failed to synthesize FlaA protein . These results suggest that, in agreement with data found for FlaB and other spirochete flagellar proteins, FlaA is likely to be regulated on the translational level . Western blot analysis using Treponema pallidum anti-FlaA serum indicated that FlaA was antigenically well conserved in several spirochete species . Taken together, the results indicate that both FlaA and FlaB comprise the PFs of B . burgdorferi and that they are regulated differently from flagellin proteins of other bacteria. Infect Immun, 1998 May, 66(5), 2387 - 92 Virulent Brucella abortus prevents lysosome fusion and is distributed within autophagosome-like compartments; Pizarro-Cerda J et al.; Virulent and attenuated Brucella abortus strains attach to and penetrate nonprofessional phagocytic HeLa cells . Compared to pathogenic Brucella, the attenuated strain 19 hardly replicates within cells . The majority of the strain 19 bacteria colocalized with the lysosome marker cathepsin D, suggesting that Brucella-containing phagosomes had fused with lysosomes, in which they may have degraded . The virulent bacteria prevented lysosome-phagosome fusion and were found distributed in the perinuclear region within compartments resembling autophagosomes. Infect Immun, 1998 May, 66(5), 2256 - 63 Identification and characterization of heparin binding regions of the Fim2 subunit of Bordetella pertussis; Geuijen CA et al.; Bordetella pertussis fimbriae bind to sulfated sugars such as heparin through the major subunit Fim2 . The Fim2 subunit contains two regions, designated H1 and H2, which show sequence similarity with heparin binding regions of fibronectin, and the role of these regions in heparin binding was investigated with maltose binding protein (MBP)-Fim2 fusion proteins . Deletion derivatives of MBP-Fim2 showed that both regions are important for binding to heparin . The role of H2 in heparin binding was confirmed by site-directed mutagenesis in which basic amino acids were replaced by alanine . These studies revealed that Lys-186 and Lys-187 are important for heparin binding of MBP-Fim2, whereas Arg-179 is not required . Peptides derived from H1 and H2 (pepH1 and pepH2) also showed heparin binding activity . Using a series of peptides, in each of which a different basic amino acid was substituted for alanine, we demonstrated that the structural requirements for heparin binding differ significantly among pepH1 and pepH2 peptides . A Pepscan analysis of Fim2 revealed regions outside H1 and H2 which bind heparin and showed that not only basic amino acids but also tyrosines may be important for binding to sulfated sugars . A comparison of the heparin binding regions of Fim2 with homologous regions of Fim3 and FimX, two closely related but antigenically distinct fimbrial subunits, showed that basic amino acids and tyrosines are generally conserved . The major heparin binding regions identified in Fim2 are part of epitopes recognized by human antibodies, suggesting that the heparin binding regions are exposed at the fimbrial surface and are immunodominant . Since B . pertussis fimbriae show weak serological cross-reactivity, the differences in primary structure in the heparin binding regions of Fim2, Fim3, and FimX may affect antibody binding but not heparin binding, allowing the bacteria to evade antibody-mediated immunity by switching the fimbrial gene expressed. Appl Environ Microbiol, 1998 May, 64(5), 1963 - 6 Regulation of lipid synthesis in Bradyrhizobium japonicum: low oxygen concentrations trigger phosphatidylinositol biosynthesis; Tang Y et al.; Lowering oxygen tension in free-living Bradyrhizobium japonicum resulted in a dramatic switch of membrane chemistry in which phosphatidylcholine, the predominant lipid in aerated cultures, was no longer synthesized and phosphatidylethanolamine became the major lipid . Besides this change, phosphatidylinositol, a typical plant lipid rarely found in bacteria, was also synthesized. Genes Dev, 1998 Apr 1, 12(7), 1010 - 21 Molecular genetic analysis of the heterodimeric splicing factor U2AF: the RS domain on either the large or small Drosophila subunit is dispensable in vivo; Rudner DZ et al.; The pre-mRNA splicing factor U2AF (U2 snRNP auxiliary factor) has an essential role in 3' splice site selection . U2AF binds the intron pyrimidine tract between the branchpoint and the 3' splice site and recruits U2 snRNP to the branch site at an early step in spliceosome assembly . Human U2AF is a heterodimer composed of large (hU2AF65) and small (hU2AF35) subunits . Both subunits contain a domain enriched in arginine-serine dipeptide repeats termed an RS domain . The two U2AF RS domains have been assigned essential and independent roles in spliceosome assembly in vitro-the hU2AF65 RS domain is required to target U2 snRNP to the branch site and the hU2AF35 RS domain is necessary for protein-protein interactions with constitutive and alternative splicing factors . We have investigated the functional requirements for the RS domains on the Drosophila U2AF homolog in vivo . In sharp contrast to its essential role in U2 snRNP recruitment in vitro, the RS domain on the Drosophila large subunit homolog (dU2AF50) was completely dispensable in vivo . Prompted by this unexpected result, we analyzed the RS domain on the Drosophila small subunit homolog (dU2AF38) . Despite its requirement for enhancer-dependent splicing activity in vitro, the dU2AF38 RS domain was also inessential in vivo . Finally, we have tested whether the Drosophila U2AF heterodimer requires any RS domain . Flies mutant for both the small and large subunits could not be rescued by dU2AF50deltaRS and dU2AF38deltaRS transgenes . Therefore, in contrast to the separate roles assigned to the U2AF RS domains in vitro, our genetic data suggest that they may have redundant functions in vivo. Genes Dev, 1998 Apr 1, 12(7), 996 - 1009 A coactivator of pre-mRNA splicing; Blencowe BJ et al.; The nuclear matrix antigen recognized by the monoclonal antibody (mAb) B1C8 is a novel serine (S) and arginine (R)-rich protein associated with splicing complexes and is named here SRm160 (SR-related matrix protein of 160 kD) . SRm160 contains multiple SR repeats, but unlike proteins of the SR family of splicing factors, lacks an RNA recognition motif . SRm160 and a related protein SRm300 (the 300-kD nuclear matrix antigen recognized by mAb B4A11) form a complex that is required for the splicing of specific pre-mRNAs . The SRm160/300 complex associates with splicing complexes and promotes splicing through interactions with SR family proteins . Binding of SRm160/300 to pre-mRNA is normally also dependent on U1 snRNP and is stabilized by U2 snRNP . Thus, SRm160/300 forms multiple interactions with components bound directly to important sites within pre-mRNA . The results suggest that a complex of the nuclear matrix proteins SRm160 and SRm300 functions as a coactivator of pre-mRNA splicing. Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3726 - 30 Archaeal translation initiation revisited: the initiation factor 2 and eukaryotic initiation factor 2B alpha-beta-delta subunit families; Kyrpides NC et al.; As the amount of available sequence data increases, it becomes apparent that our understanding of translation initiation is far from comprehensive and that prior conclusions concerning the origin of the process are wrong . Contrary to earlier conclusions, key elements of translation initiation originated at the Universal Ancestor stage, for homologous counterparts exist in all three primary taxa . Herein, we explore the evolutionary relationships among the components of bacterial initiation factor 2 (IF-2) and eukaryotic IF-2 (eIF-2)/eIF-2B, i.e., the initiation factors involved in introducing the initiator tRNA into the translation mechanism and performing the first step in the peptide chain elongation cycle . All Archaea appear to posses a fully functional eIF-2 molecule, but they lack the associated GTP recycling function, eIF-2B (a five-subunit molecule) . Yet, the Archaea do posses members of the gene family defined by the (related) eIF-2B subunits alpha, beta, and delta, although these are not specifically related to any of the three eukaryotic subunits . Additional members of this family also occur in some (but by no means all) Bacteria and even in some eukaryotes . The function