J Biol Chem, 1999 Oct 8, 274(41), 29572 - 81 c-Jun transactivates the promoter of the human p21(WAF1/Cip1) gene by acting as a superactivator of the ubiquitous transcription factor Sp1; Kardassis D et al.; The cell cycle inhibitor protein p21(WAF1/Cip1) (p21) is a critical downstream effector in p53-dependent mechanisms of growth control and p53-independent pathways of terminal differentiation . We have recently reported that the transforming growth factor-beta pathway-specific Smad3 and Smad4 proteins transactivate the human p21 promoter via a short proximal region, which contains multiple binding sites for the ubiquitous transcription factor Sp1 . In the present study we show that the Sp1-occupied promoter region mediates transactivation of the p21 promoter by c-Jun and the related proteins JunB, JunD, and ATF-2 . By using gel electrophoretic mobility shift assays we show that this region does not contain a binding site for c-Jun . In accordance with the DNA binding data, c-Jun was unable to transactivate the p21 promoter when overexpressed in the Sp1-deficient Drosophila-derived SL2 cells . Coexpression of c-Jun and Sp1 in these cells resulted in a strong synergistic transactivation of this promoter . In addition, a chimeric promoter consisting of six tandem high affinity Sp1-binding sites fused with the CAT gene was transactivated by overexpressed c-Jun in HepG2 cells . The above data propose functional cooperation between c-Jun and Sp1 . Physical interactions between the two factors were demonstrated in vitro by using GST-Sp1 hybrid proteins expressed in bacteria and in vitro transcribed-translated c-Jun . The region of c-Jun mediating interaction with Sp1 was mapped within the basic region leucine zipper domain . In vivo, functional interactions between c-Jun and Sp1 were demonstrated using a GAL4-based transactivation assay . Overexpressed c-Jun transactivated a chimeric promoter consisting of five tandem GAL4-binding sites only when coexpressed with GAL4-Sp1-(83-778) fusion proteins in HepG2 cells . By utilizing the same assay, we found that the glutamine-rich segment of the B domain of Sp1 (Bc, amino acids 424-542) was sufficient for c-Jun-induced transactivation of the p21 promoter . In conclusion, our data support a mechanism of superactivation of Sp1 by c-Jun, which is based on physical and functional interactions between these two transcription factors on the human p21 and possibly other Sp1-dependent promoters. J Biol Chem, 1999 Oct 8, 274(41), 28909 - 15 Subunit interactions in the clathrin-coated vesicle vacuolar (H(+))-ATPase complex; Xu T et al.; The vacuolar (H(+))-ATPases (or V-ATPases) are structurally related to the F(1)F(0) ATP synthases of mitochondria, chloroplasts and bacteria, being composed of a peripheral (V(1)) and an integral (V(0)) domain . To further investigate the arrangement of subunits in the V-ATPase complex, covalent cross-linking has been carried out on the V-ATPase from clathrin-coated vesicles using three different cross-linking reagents . Cross-linked products were identified by molecular weight and by Western blot analysis using polyclonal antibodies raised against individual V-ATPase subunits . In the intact V(1)V(0) complex, evidence for cross-linking of subunits C and E, D and F, as well as E and G by disuccinimidyl glutarate was obtained, while in the free V(1) domain, cross-linking of subunits H and E was also observed . Subunits C and E as well as D and E could be cross-linked by 1-ethyl-3-(dimethylaminopropyl)carbodiimide, while subunits a and E could be cross-linked by 4-(N-maleimido)benzophenone . It was further demonstrated that it is possible to treat the V-ATPase with potassium iodide and MgATP in such a way that while subunits A, B, and H are nearly quantitatively removed, significant amounts of subunits C, D, E, and F remain attached to the membrane, suggesting that one or more of these latter subunits are in contact with the V(0) domain . In addition, treatment of the V-ATPase with cystine, which modifies Cys-254 of the catalytic A subunit, results in dissociation of subunit H, suggesting communication between the catalytic nucleotide binding site and subunit H . Finally, the stoichiometry of subunits F, G, and H were determined by quantitative amino acid analysis . Based on these and previous observations, a new structural model of the V-ATPase from clathrin-coated vesicles is proposed. Protist, 1999 Aug, 150(2), 149 - 62 Ultrastructure of Trimastix pyriformis (Klebs) Bernard et al.: similarities of Trimastix species with retortamonad and jakobid flagellates; O'Kelly CJ et al.; Trimastix pyriformis (Klebs 1893) Bernard et al . 1999, is a quadriflagellate, free-living, bacterivorous heterotrophic nanoflagellate from anoxic freshwaters that lacks mitochondria . Monoprotist cultures of this species contained naked trophic cells with anterior flagellar insertion and a conspicuous ventral groove . Bacteria were ingested at the posterior end of the ventral groove, but there was no persistent cytopharyngeal complex . The posterior flagellum resided in this groove, and bore two prominent vanes . A Golgi body (dictyosome) was present adjacent to the flagellar insertion . The kinetid consisted of four basal bodies, four microtubular roots, and associated fibers and bands . Duplicated kinetids, each with four basal bodies and microtubular root templates, appeared at the poles of the open mitotic spindle . Trimastix pyriformis is distinguishable from other Trimastix species on the basis of external morphology, kinetid architecture and the distribution of endomembranes . Trimastix species are most similar to jakobid flagellates, especially Malawimonas jakobiformis, and to species of the retortamonad genus Chilomastix . Retortamonads may have evolved from a Trimastix-like ancestor through loss of "canonical" (easily seen with electron microscopy) endomembrane systems and elaboration of cytoskeletal elements associated with the cytostome/cytopharynx complex. Protist, 1999 Aug, 150(2), 137 - 47 Messenger RNA in dormant cells of Sterkiella histriomuscorum (Oxytrichiade): indentification of putative regulatory gene transcripts; Tourancheau AB et al.; In the absence of food, the oxytrichid Sterkiella histriomuscorum, like many ciliates, enters into dormancy and transforms into a round and walled encysted cell . When transferred back into a feeding medium, the cyst re-transforms into a vegetative cell in a few hours . This encystment-excystment pathway, which is common to many free-living and parasitic protists, is still poorly understood at the molecular level . In order to identify potential dormant transcripts in the cysts of Sterkiella, we have constructed cDNA libraries from mature cysts . Transcripts have been isolated confirming the presence of a mRNA pool in the dormant cells . The sequence analysis of two cDNA indicates open reading frames which show significant similarities to known proteins involved in mechanisms of regulation: 1) nifR3, an element of the nitrogen regulatory system in bacteria and 2) CROC-1, a newly identified human transcription factor . The two corresponding macronuclear genes represent the first putative regulatory genes isolated in ciliates . From a differential screening of the cDNA library against vegetative cDNA, one cyst-specific (and very abundant) transcript has been isolated but the product has not yet been identified . The possible involvment of these new ciliate genes in the excystment process is discussed. Genetika, 1999 Jun, 35(6), 771 - 6 {Sex ratio and male killing in Siberian populations of Harmonia axyridis (Pass.)}; Zakharov IA et al.; In populations of Harmonia axyridis Pall . from Novosibirsk and Kyzyl, females (three out of 34 studied) that produce exclusively female progeny were found . In one of the families studied, the inheritance of the male-killing trait was monitored over five generations . The male-killing trait was maternally inherited . The beetles of this family were infected with the bacteria that, according to the sequence analysis of the gene fragment for 16S rRNA, belong to the genus Spiroplasma (VI group). Gene Ther, 1999 May, 6(5), 922 - 30 Allele replacement: an application that permits rapid manipulation of herpes simplex virus type 1 genomes; Horsburgh BC et al.; Herpes simplex virus (HSV) is a new platform for gene therapy . We cloned the human herpesvirus HSV-1 strain F genome into a bacterial artificial chromosome (BAC) and adapted chromosomal gene replacement technology to manipulate the viral genome . This technology exploits the power of bacterial genetics and permits generation of recombinant viruses in as few as 7 days . We utilized this technology to delete the viral packaging/cleavage (pac) sites from HSV-BAC . HSV-BAC DNA is stable in bacteria and the pac-deleted HSV-BAC (p45-25) is able to package amplicon plasmid DNA as efficiently as a comparable pac-deleted HSV cosmid set when transfected into mammalian cells . Moreover, the utility of bacterial gene replacement is not limited to HSV, since most herpesviruses can be cloned as BACs . Thus, this technology will greatly facilitate genetic manipulation of all herpesviruses for their use as research tools or as vectors in gene therapy. Eur J Biochem, 1999 Oct, 265(2), 645 - 55 Definition of receptor binding sites on human interleukin-11 by molecular modeling-guided mutagenesis; Tacken I et al.; Interleukin-11 (IL-11) belongs to the interleukin-6 (IL-6)-type subfamily of long-chain helical cytokines including IL-6, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M, and cardiotrophin-1, which all share the glycoprotein gp130 as a signal transducing receptor component . IL-11 acts on cells expressing gp130 and the IL-11 receptor (IL-11R) alpha-subunit (IL-11Ralpha) . The structural epitopes of IL-11 required for the recruitment of the individual receptor subunits have not yet been defined . Based on the structure of CNTF, a three-dimensional model of human IL-11 was built . Using this model, 10 surface exposed amino acid residues of IL-11 were selected for mutagenesis using analogies to the well-characterized receptor recruitment sites of IL-6, CNTF, and LIF . The respective mutants of human IL-11 were expressed as soluble fusion proteins in bacteria . Their biological activities were determined on HepG2 and Ba/F3-130-11alpha cells . Several mutants with substantially decreased bioactivity and one hyperagonistic mutant were identified and further analyzed with regard to recruitment of IL-11Ralpha and gp130 . The low-activity mutant I171D still binds IL-11Ralpha but fails to recruit gp130, whereas the hyperagonistic variant R135E more efficiently engages the IL-11R subunits . The low-activity mutants R190E and L194D failed to bind to IL-11Ralpha . These findings reveal a common mechanism of receptor recruitment in the family of IL-6-type cytokines and offer considerable perspectives for the rational design of IL-11 antagonists and hyperagonists. J Cell Sci, 1999 Oct, 112 ( Pt 19), 3195 - 203 Assessing the role of the ASP56/CAP homologue of Dictyostelium discoideum and the requirements for subcellular localization; Noegel AA et al.; The CAP (cyclase-associated protein) homologue of Dictyostelium discoideum is a phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulated G-actin sequestering protein which is present in the cytosol and shows enrichment at plasma membrane regions . It is composed of two domains separated by a proline rich stretch . The sequestering activity has been localized to the C-terminal domain of the protein, whereas the presence of the N-terminal domain seems to be required for PIP(2)-regulation of the sequestering activity . Here we have constructed GFP-fusions of N- and C-domain and found that the N-terminal domain showed CAP-specific enrichment at the anterior and posterior ends of cells like endogenous CAP irrespective of the presence of the proline rich region . Mutant cells expressing strongly reduced levels of CAP were generated by homologous recombination . They had an altered cell morphology with very heterogeneous cell sizes and exhibited a cytokinesis defect . Growth on bacteria was normal both in suspension and on agar plates as was phagocytosis of yeast and bacteria . In suspension in axenic medium mutant cells grew more slowly and did not reach saturation densities observed for wild-type cells . This was paralleled by a reduction in fluid phase endocytosis . Development was delayed by several hours under all conditions assayed, furthermore, motile behaviour was affected. Microsc Res Tech, 1999 Sep 15, 46(6), 380 - 9 Targeted recombinant aequorins: tools for monitoring {Ca2+} in the various compartments of a living cell; Brini M et al.; In the last decade, the study of Ca2+ homeostasis within organelles in living cells has been greatly enhanced by the utilisation of a recombinant Ca(2+)-sensitive photoprotein, aequorin . Aequorin is a Ca2+ sensitive photoprotein of a coelenterate that, in the past, was widely employed to measure Ca2+ concentration in living cells . In fact, the purified protein was widely used to monitor cytoplasmic {Ca2+} changes in invertebrate muscle cells after microinjection . However, due to the time-consuming and traumatic procedure of microinjection, the role of aequorin in the study of Ca2+ homeostasis remained confined to a limited number of cells (giant cells) susceptible to microinjection . Thus, in most instances, it was replaced by the fluorescent indicators developed by Roger Tsien and coworkers . The cloning of aequorin cDNA {Inouye et al . (1985) Proc . Natl . Acad . Sci . U.S.A . 82:3154-3158} and the explosive development of molecular biology offered new possibilities in the use of aequorin, as microinjection has been replaced by the simpler technique of cDNA transfection . As a polypeptide, aequorin allows the endogenous production of the photoprotein in cell systems as diverse as bacteria, yeast, slime molds, plants, and mammalian cells . Moreover, it is possible to specifically localise it within the cell by including defined targeting signals in the amino acid sequence . Targeted recombinant aequorins represent to date the most specific means of monitoring {Ca2+} in subcellular organelles . In this review, we will not discuss the procedure of aequorin microinjection and its use as purified protein but we will present the new advances provided by recombinant aequorin in the study of intracellular Ca2+ homeostasis, discussing in greater detail the advantages and disadvantages in the use of this probe . Vet Rec, 1999 Aug 28, 145(9), 251 - 4 Serotype and apx genotype profiles of Actinobacillus pleuropneumoniae field isolates in Korea; Min K et al.; A total of 100 field isolates of Actinobacillus pleuropneumoniae isolated from lung tissues of pigs with severe pleuropneumonia were serotyped by slide agglutination and precipitation tests . Polymerase chain reactions for apxICA, apxIICA, apxIIICA, apxIBD and apxIIIBD genes were used to determine their genotype prevalence . Serotypes 2 (56 isolates), 5 (28 isolates) and 6 (11 isolates) were the most common; only two isolates belonged to serotype 7, and three were untyped . Among the 97 isolates identified by serotype, 70 had the same apx genes as their respective serotype reference strains, but 27 did not have any of the apx genes present in the corresponding serotype reference strain . Among these 27 isolates, 10 were serotype 2, 12 were serotype 5, three were serotype 6 and two were serotype 7. Eur J Gastroenterol Hepatol, 1999 Aug, 11 Suppl 2, S75 - 9 Vaccines; Lee A; Extensive research with mice has shown that animals can be protected from or cured of Helicobacter infection by immunization . A therapeutic effect has also been demonstrated in ferrets . The possibility of developing a vaccine against H . pylori-associated diseases that will work in humans has been the stimulus for intense research activity . A major subset of lymphocytes involved in immunity against invading pathogens is the CD4+ T-lymphocytes, divided into Th1 and Th2 phenotypes . Th1 cells are involved in cell-mediated immunity and Th2 cells are involved in antibody formation, particularly IgA and IgG, which are most active at mucosal surfaces and thus most likely to be protective against bacteria trying to colonize that surface . H . pylori can exist for the life of its human host, despite the vigorous immune response that is mounted against it . Most infected humans have a Th1 response, which does not eradicate H . pylori . A Th2 response would logically be more effective . Experiments with mice deficient in the cytokines that drive the Th responses are confusing and indicate that a change in the balance of the Th1/Th2 response may have a therapeutic effect In the next 2-5 years, a number of anti-Helicobacter vaccines will be studied in humans and this will open up a completely new approach to the management of gastroduodenal diseases. Int J Pharm, 1999 Oct 5, 187(2), 251 - 7 Selective drug delivery to the colon using pectin:chitosan:hydroxypropyl methylcellulose film coated tablets; Macleod GS et al.; A study has been carried out to assess the potential of pectin:chitosan:hydroxypropyl methylcellulose (HPMC) (P:C:H) films for colonic drug delivery . Radiolabelled (99mTc) tablets were coated with a 3:1:1, P:C:H film and administered to human volunteers . The gastro-intestinal transit of the tablets was assessed by gamma scintigraphy . The results showed that in all cases (n=4), the tablets were able to pass through the stomach and small intestine intact . Break up of the tablets commenced once they were in the colon, due to degradation of the coat by colonic bacteria . The study has highlighted the potential of this coating system for colonic drug delivery. In Vitro Cell Dev Biol Anim, 1999 Sep, 35(8), 449 - 58 Relation of the slow growth phenotype to neoplastic transformation: possible significance for human cancer; Chow M et al.; Deletions are widely distributed over the genome in the most frequently occurring human cancers and are the most abundant genetic lesion found there . Deletions are highly correlated with the slow growth phenotype of mutated animal and human cells and result in chromosomal transposition when the retained ends are joined . Transpositions are only a minor source of mutation in rapidly multiplying bacteria but are a major cause of mutations in stationary bacteria . The NIH 3T3 line of mouse cells undergoes neoplastic transformation during prolonged incubation in a stationary state and expresses the slow growth phenotype on serial subculture at low density, suggesting a relation between transformation and chromosomal deletions . To further explore the relation between neoplastic transformation and the slow growth phenotype as a surrogate for deletions, two sublines of the NIH 3T3 cells with differing competence for transformation were serially subcultured in the stationary state at confluence and tested at each subculture for transformation and growth rate . Cell death in a fraction of the population and a heritable slowdown in proliferation of most of the survivors became increasingly pronounced with successive rounds of confluence . The reduction in growth rate was not proportional to the degree of transformation of the cultures, but all of the transformed cultures were slow growers at low density . All of the discrete colonies from cloning transformed cultures developed at a lower initial rate than control colonies under optimal conditions for growth, but they continued to grow at later stages, forming multilayered colonies under conditions that inhibited the further growth of the control colonies . The results suggest that prolonged incubation of NIH 3T3 cells in the stationary state results in growth-impairing deletions over a wide range of sites in the genome, but more restricted subsets of such lesions are responsible for neoplastic transformation . These findings provide dynamic, functional support in culture for the histopathological evidence that the quiescent state of cells associated with atrophy and fibrosis plays a significant role in the origin of some cancers in experimental animals and human beings. Proc Natl Acad Sci U S A, 1999 Sep 28, 96(20), 11566 - 71 Three unrelated viral transforming proteins (vIRF, EBNA2, and E1A) induce the MYC oncogene through the interferon-responsive PRF element by using different transcription coadaptors; Jayachandra S et al.; Kaposi sarcoma-associated herpesvirus vIRF is a viral transcription factor that inhibits interferon signaling and transforms NIH 3T3 cells, but does not bind interferon-stimulated response element (ISRE) DNA sequences . Here we show that induction of the MYC protooncogene is required for cell transformation by vIRF, and that vIRF increases MYC transcription up to 15-fold through specific promoter interactions at an ISRE sequence called the plasmacytoma repressor factor (PRF) element . These effects are resistant to cycloheximide but are inhibited by a dominant-negative ISRE-binding protein, indicating that vIRF acts together with a cellular cofactor at the PRF element to directly transactivate MYC . The coadaptor CREB-binding protein (CBP) binds vIRF and synergizes transactivation of MYC, but, unexpectedly, closely related histone acetyltransferases p300 and P/CAF potently suppress vIRF transactivation . On the basis of the prediction that other interferon-inhibiting viral transforming proteins behave similarly, we found that Epstein-Barr virus-induced nuclear antigen 2 (EBNA2) also binds p300/CBP, and that both EBNA2 and adenovirus E1A transactivate MYC through the PRF element . For E1A, P/CAF coactivates MYC, whereas both p300 and CBP suppress E1A transactivation . For EBNA2, both P/CAF and CBP coactivate the MYC promoter, whereas p300 suppresses EBNA2 transactivation . These findings demonstrate that viral transforming proteins can activate as well as inhibit transcription through coadaptor interactions . At some promoters CBP and p300 have previously unrecognized, competitive antagonism to each other . While all three viral proteins target the same promoter element, each has a different coadaptor use profile . These findings are consistent with cellular MYC repression playing a role in innate immunity as well as in control of cell proliferation. Ecotoxicol Environ Saf, 1999 Sep, 44(1), 129 - 36 Effects of heavy metals on methane production in tropical rice soils; Mishra SR et al.; In a laboratory incubation study, the effect of select heavy metals on methane (CH(4)) production in three rice soils was investigated under flooded conditions . Heavy metals behaved differently in their effect on methanogenesis in different soils and methane-producing bacteria . Cd, Cu, and Pb inhibited CH(4) production in all the soils . Zn stimulated CH(4) production in the alluvial soil, but inhibited it in laterite and acid sulfate soils . Cr effectively inhibited CH(4) production in the alluvial soil, but stimulated it in laterite and acid sulfate soils . The stimulatory effect of Zn and the inhibitory effect of Cr on methanogenesis in alluvial soil were attributed to their stimulation or inhibition of methanogenic bacterial population . Mol Immunol, 1999 Jun, 36(9), 575 - 85 The architectural transition of human complement component C9 to poly(C9); DiScipio RG et al.; Several regions of C9 including three cysteine-rich modules homologous to those in thrombospondin (TS), the low density lipoprotein receptor (LDL), the epidermal growth factors (EDGF), as well as two middle sections of the polypeptide chain were expressed in bacteria . Antibodies derived from these segments were used to probe the relative exposure of epitopes in C9 and poly(C9) using ELISAs . The results indicated that the TS and LDL modules are fully exposed in both monomer and polymer; however, the middle region of the polypeptide chain is buried in the monomer but external in the polymer . Using specified conditions, Fab fragments to the TS and LDL modules did not block C9 polymerization, but those to the middle region of the polypeptide chain and to some extent to the EDGF module did so . Immuno-electron microscopy of poly(C9) indicated that the C9 polypeptide chain assumes a 'U' shape, in which the TS and LDL modules are located on the upper rim . The EDGF module is located on the lower edge of the upper rim, and midsection of the polypeptide chain constructs the barrel of the tubule . Computer assisted contrast enhancement of select electron micrograph images of poly(C9) allowed the clear visualization of each subunit . These were seen to have a volute shape . The upper rim is composed of whorls that are apparently not in lateral contact . It is concluded that the TS and LDL modules do not participate directly in polymerization but cover the hydrophobic central region of the polypeptide chain in the monomer . As a consequence of circular polymerization the midsection of the polypeptide chain becomes exposed as each C9 lengths to fashion a volute form . reserved. J Bacteriol, 1999 Oct, 181(19), 5984 - 92 Two family B DNA polymerases from Aeropyrum pernix, an aerobic hyperthermophilic crenarchaeote; Cann IK et al.; DNA polymerase activities in fractionated cell extract of Aeropyrum pernix, a hyperthermophilic crenarchaeote, were investigated . Aphidicolin-sensitive (fraction I) and aphidicolin-resistant (fraction II) activities were detected . The activity in fraction I was more heat stable than that in fraction II . Two different genes (polA and polB) encoding family B DNA polymerases were cloned from the organism by PCR using degenerated primers based on the two conserved motifs (motif A and B) . The deduced amino acid sequences from their entire coding regions contained all of the motifs identified in family B DNA polymerases for 3'-->5' exonuclease and polymerase activities . The product of polA gene (Pol I) was aphidicolin resistant and heat stable up to 80 degrees C . In contrast, the product of polB gene (Pol II) was aphidicolin sensitive and stable at 95 degrees C . These properties of Pol I and Pol II are similar to those of fractions II and I, respectively, and moreover, those of Pol I and Pol II of Pyrodictium occultum . The deduced amino acid sequence of A . pernix Pol I exhibited the highest identities to archaeal family B DNA polymerase homologs found only in the crenarchaeotes (group I), while Pol II exhibited identities to homologs found in both euryarchaeotes and crenarchaeotes (group II) . These results provide further evidence that the subdomain Crenarchaeota has two family B DNA polymerases . Furthermore, at least two DNA polymerases work in the crenarchaeal cells, as found in euryarchaeotes, which contain one family B DNA polymerase and one heterodimeric DNA polymerase of a novel family. J Bacteriol, 1999 Oct, 181(19), 5976 - 83 Regulation of transfer functions by the imp locus of the Streptomyces coelicolor plasmidogenic element SLP1; Hagege JM et al.; SLP1(int) is a 17.2-kb genetic element that normally is integrated site specifically into the chromosome of Streptomyces coelicolor A3(2) . The imp operon within SLP1(int) represses replication of both chromosomally integrated and extrachromosomal SLP1 . During mating with S . lividans, SLP1(int) can excise, delete part of imp, and form a family of autonomously replicating conjugative plasmids . Earlier work has shown that impA and impC gene products act in concert to control plasmid maintenance and regulate their own transcription . Here we report that these imp genes act also on a second promoter, P(opimp) (promoter opposite imp), located adjacent to, and initiating transcription divergent from, imp to regulate loci involved in the intramycelial transfer of SLP1 plasmids . spdB1 and spdB2, two overlapping genes immediately 3' to P(opimp) and directly regulated by imp, are shown by Tn5 mutagenesis to control transfer-associated growth inhibition (i.e., pocking) . Additional genes resembling transfer genes of other Streptomyces spp . plasmids and required for SLP1 transfer and/or postconjugal intramycelial spread are located more distally to P(opimp) . Expression of impA and impC in an otherwise competent recipient strain prevented SLP1-mediated gene transfer of chromosomal and plasmid genes but not plasmid-independent chromosome-mobilizing activity, suggesting that information transduced to recipients after the formation of mating pairs affects imp activity . Taken together with earlier evidence that the imp operon regulates SLP1 DNA replication, the results reported here implicate imp in the overall regulation of functions related to the extrachromosomal state of SLP1. Bioessays, 1999 Oct, 21(10), 866 - 70 Building a cellular switch: more lessons from a good egg; Ferrell JE Jr; Xenopus oocytes mature in response to the steroid hormone progesterone . At the level of a population of oocytes, the response is graded-the higher the concentration of progesterone, the larger the fraction of oocytes that will mature-but at the level of the individual oocyte, the response is all-or-none . The all-or-none character of this cell fate switch is hypothesized to arise out of two properties of the signal transduction machinery that mediates maturation, positive feedback, and ultrasensitivity . This combination of positive feedback plus ultrasensitivity crops up again and again in cellular switches, from the lysis-lysogeny switch in phage-infected bacteria to the action potential in neurons . J Trauma, 1999 Sep, 47(3), 515 - 20 Small bowel perforation: is urgent surgery necessary? Fang JF, Chen RJ, Lin BC, Hsu YB, Kao JL, Kao YC, Chen MF. BACKGROUND: Controversies regarding how urgent bowel perforation should be diagnosed and treated exist in recent reports . The approach for early diagnosis is also debatable . The purposes of this study were to evaluate the relationship between treatment delay and outcome of small bowel perforation after blunt abdominal trauma and to determine the best assessment plan for the diagnosis of this injury . METHODS: One hundred eleven consecutive patients with small bowel perforations caused by blunt abdominal trauma were retrospectively reviewed . The patients were divided into four groups according to the time interval between injury and surgery . Hospital stay, time to resume oral intake, and mortality and morbidity rates were compared between groups . Physical signs, laboratory and computed tomographic findings, and the results of diagnostic peritoneal lavage were analyzed to find the most sensitive and specific test for early diagnosis of small bowel perforation . RESULTS: Delay in surgery for more than 24 hours did not significantly increase the mortality with modern method of treatment; however, complications increased dramatically . Hospital stay and time to resume oral intake increased significantly when surgery was delayed for more than 24 hours . Abdominal tenderness was a common finding, but it was not specific for bowel perforation . Only 40% of the computed tomographic scans were diagnostic for bowel perforations: 50% of them showed suggestive signs, and 10% were considered as negative . Persistence of abdominal signs indicated peritoneal lavage . By using cell count ratio in diagnostic peritoneal lavage and/or increased lavage amylase activity, presence of particulate matter and/or bacteria in the lavage fluid, all patients with intraperitoneal bowel perforation were diagnosed accurately before operation . CONCLUSION: Small bowel perforation has low mortality and complication rates if it is treated earlier than 24 hours after injury . The principle of "rushing to the operation suite" for a stable blunt abdominal trauma patients without detailed systemic examination is not justified . The priority of treatment for the small bowel perforation should be lower than the limb-threatening injuries . Diagnostic peritoneal lavage provides high sensitivity and specificity rates for the diagnosis of small bowel perforation if a specially designed positive criterion is applied. Acta Vet Hung, 1999, 47(3), 319 - 24 Occurrence of acute paralysis virus of the honey bee (Apis mellifera) in a Hungarian apiary infested with the parasitic mite Varroa jacobsoni; Bekesi L et al.; Viruses of the honey bee have been known for a long time; however, recently the attention of scientists and apiculturalists has turned towards the relationship between these viruses and the parasitic mite Varroa jacobsoni . Although clinical symptoms indicated the presence of some of the viruses of bees in Hungary, none have previously been isolated or identified . During July unusual adult bee and brood mortality was observed in some colonies of an apiary in Budapest known to be infested with Varroa jacobsoni . Large amounts of acute paralysis virus (APV) were detected serologically in healthy honey bee pupae killed by the injection of a bacteria-free extract of diseased adult bees . Crystalline arrays of 30 nm particles were seen in ultrathin sections of the tissues of injected pupae and naturally infected adult bees . In spite of the application of acaricide treatments the bee population in several colonies had collapsed by the end of summer and the apiary suffered severe wintering losses. J Mol Biol, 1999 Sep 24, 292(3), 569 - 80 A reductase/isomerase subunit of mitochondrial NADH:ubiquinone oxidoreductase (complex I) carries an NADPH and is involved in the biogenesis of the complex; Schulte U et al.; Respiratory chains of bacteria and mitochondria contain closely related forms of the proton-pumping NADH:ubiquinone oxidoreductase, or complex I . The bacterial complex I consists of 14 subunits, whereas the mitochondrial complex contains some 25 extra subunits in addition to the homologues of the bacterial subunits . One of these extra subunits with a molecular mass of 40 kDa belongs to a heterogeneous family of reductases/isomerases with a conserved nucleotide binding site . We deleted this subunit in Neurospora crassa by gene disruption . In the mutant nuo 40, a complex I lacking the 40 kDa subunit is assembled . The mutant complex I does not contain tightly bound NADPH present in wild-type complex I . This NADPH cofactor is not connected to the respiratory electron pathway of complex I . The mutant complex has normal NADH dehydrogenase activity and contains the redox groups known for wild-type complex I, one flavin mononucleotide and four iron-sulfur clusters detectable by electron paramagnetic resonance spectroscopy . In the mutant complex these groups are all readily reduced by NADH . However, the mutant complex is not capable of reducing ubiquinone . A recently described redox group identified in wild-type complex I by UV-visible spectroscopy is not detectable in the mutant complex . We propose that the reductase/isomerase subunit with its NADPH cofactor takes part in the biosynthesis of this new redox group . Infect Immun, 1999 Oct, 67(10), 5151 - 6 In vivo distribution of Helicobacter felis in the gastric mucus of the mouse: experimental method and results; Schreiber S et al.; We describe a method that permits the collection of very small samples (2 nl) from precisely defined positions within the gastric mucus of anesthetized mice . This method was used to study the in vivo local distribution of bacteria within the mucus of Helicobacter felis-infected mice . A total of 200 samples from 40 mice were analyzed . Each sample was microscopically analyzed, within less than 1 min, as a native preparation . To avoid changes in bacterial location within the mucus after collection and to improve the counting accuracy, bacterial motility was blocked by adjusting the pH inside the collecting pipette to 4.5 . The mucus in a collected sample was subdivided into three layers, an epithelial layer (the first 25 micron of mucus from the tissue-mucus interface), a luminal layer (the last 25 micron to the mucus-lumen interface), and the remaining central mucus layer . The volume of the analyzed segments in the sample was between 4 and 9 pl . The concentration of bacteria inside the epithelial mucus layer was 3,400 per nl, but it was only 50 per nl inside the central mucus layer . The mean distance of H . felis to the epithelial surface was 16 microm . A total of 75% of all H . felis bacteria resided in the mucus zone between 5 and 20 micron from the tissue surface, with no bacteria closer than 5 micron to the epithelial surface . This method permits the study of factors determining the density of colonization and distribution of bacteria along chemical gradients with a high precision. Infect Immun, 1999 Oct, 67(10), 4983 - 7 Binding of Actinobacillus pleuropneumoniae lipopolysaccharides to glycosphingolipids evaluated by thin-layer chromatography; Abul-Milh M et al.; The binding profile of Actinobacillus pleuropneumoniae serotypes 1 and 2 to various glycosphingolipids was evaluated by using thin-layer chromatogram overlay . A . pleuropneumoniae whole cells recognized glucosylceramide (Glcbeta1Cer), galactosylceramide (Galbeta1Cer) with hydroxy and nonhydroxy fatty acids, sulfatide (SO(3)-3Galbeta1Cer), lactosylceramide (Galbeta1-4Glcbeta1Cer), gangliotriaosylceramide GgO3 (GalNAcbeta1-4Galbeta1-4Glcbeta1Cer), and gangliotetraosylceramide GgO4 (Galbeta1-3GalNAcbeta1-4Galbeta1-4Glcbeta1Cer) glycosphingolipids . We observed no binding to globoseries, globotriaosylceramide Gb3, globoside Gb4, or Forssman Gb5 glycosphingolipids or to gangliosides GM1, GM2, GM3, GD1a, GD1b, GD3, and GT1b . The A . pleuropneumoniae strains tested also failed to detect phosphatidylethanolamine or ceramide . Interestingly, extracted lipopolysaccharide (LPS) of serotype 1 and serotype 2 as well as detoxified LPS of serotype 1 showed binding patterns similar to that of whole bacterial cells . Binding to GlcCer, GalCer, sulfatide, and LacCer, but not to GgO3 and GgO4 glycosphingolipids, was inhibited after incubation of the bacteria with monoclonal antibodies against LPS O antigen . These findings indicate the involvement of LPS in recognition of three groups of glycosphingolipids: (i) GlcCer and LacCer, where glucose is probably an important saccharide sequence required for LPS binding; (ii) GalCer and sulfatide glycosphingolipids, where the sulfate group is part of the binding epitope of the isoreceptor; and (iii) GgO3 and GgO4, where GalNacbeta1-4Gal disaccharide represents the minimal common binding epitope . Taken together, our results indicate that A . pleuropneumoniae LPS recognize various saccharide sequences found in different glycosphingolipids, which probably represents a strong virulence attribute. J Leukoc Biol, 1999 Sep, 66(3), 521 - 7 Localization of p21-activated kinase 1 (PAK1) to pseudopodia, membrane ruffles, and phagocytic cups in activated human neutrophils; Dharmawardhane S et al.; Leukocyte chemoattractants are known to stimulate signaling pathways that involve Rho family GTPases . Direct evidence for the regulation of the leukocyte cytoskeleton by Rho GTPases and their effector targets is limited . The p21-activated kinases (PAKs) are specific targets of activated GTP-bound Rac and Cdc42, and have been proposed as regulators of chemoattractant-driven actin cytoskeletal changes in fibroblasts . PAK1 colocalizes with F-actin to cortical actin structures in stimulated fibroblasts, and activated PAK1 mutants induce membrane ruffling and polarized cytoskeletal rearrangements . We investigated whether PAK1 was associated with remodeling of the actin cytoskeleton in activated human neutrophils . We monitored the redistribution of PAK1 and F-actin into the actin cytoskeleton after stimulation of human neutrophils with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) or the particulate stimulus, opsonized zymosan (OZ) . PAK1 exhibited a similar distribution as F-actin in fMLP-stimulated leukocytes, localizing in membrane ruffles and to lamellipodia at the leading edge of polarized cells . Addition of OZ induced phagocytic uptake of this particulate stimulus, and PAK1 re-localized to the F-actin-rich pseudopodia and phagocytic cups associated with this process . Once the OZ was internalized, there was little PAK1 localized around the ingested particles, suggesting that PAK1 may be regulating the cytoskeletal extensions and events required for engulfment of bacteria, but not the subsequent steps of internalization . Localization of PAK1 and F-actin in cytoskeletal structures was abolished by the actin polymerization inhibitor cytochalasin D and the phosphatidylinositol 3-kinase inhibitor wortmannin . Our data suggest that PAK1 may regulate a subset of cytoskeletal dynamics initiated by chemoattractant and phagocytic stimuli in human neutrophils. Behav Res Methods Instrum Comput, 1999 May, 31(2), 370 - 5 A computer-controlled olfactometer for fMRI and electrophysiological studies of olfaction; Lorig TS et al.; A design for an inexpensive and reliable olfactometer is presented . The design has several advantages for fMRI and electrophysiology investigators . These advantages include relatively rapid odorant rise times, computer control, multiple odor administration, and no ferrous materials near the subjects . In addition, the device is contamination resistant, and, because the air is neither warmed nor humidified, it is unlikely to become an incubator for bacteria . The olfactometer is constructed of off-the-shelf chromatography parts that require little modification. Oral Microbiol Immunol, 1999 Jun, 14(3), 165 - 71 Studies on the binding of Treponema pectinovorum to HEp-2 epithelial cells; Walker SG et al.; We developed a radioassay to assess the adherence of the oral treponemes Treponema denticola and Treponema pectinovorum to live HEp-2 epithelial cells . T . pectinovorum bound firmly to the epithelial cell monolayer in a concentration-dependent manner . The results indicated that a subpopulation of T . pectinovorum appeared to bind and that the binding could be influenced by environmental factors . Increasing concentrations of fetal bovine serum inhibited binding, whereas T . pectinovorum membrane vesicles and co-incubation with T . denticola ATCC 35404 increased the number of cells bound to the monolayer . Treatment of T . pectinovorum with periodic acid, but not trypsin or proteinase K, decreased the binding suggesting that a cell surface carbohydrate, such as the O-antigenic component of the lipopolysaccharide, mediates attachment of the bacteria to the epithelial cells . Co-infection of the HEp-2 cells with both T . denticola and T . pectinovorum did not interfere with each other in attachment to the epithelial cell suggesting that they do not compete for the same cellular receptor on the host cell surface . This study demonstrates that T . pectinovorum is capable, in vitro, of forming a tight association with host cells and that this binding could represent an initial step in the pathogenesis of T . pectinovorum. Oral Microbiol Immunol, 1999 Jun, 14(3), 137 - 42 Actinobacillus actinomycetemcomitans may utilize either actin-dependent or actin-independent mechanisms of invasion; Brissette CA et al.; Actinobacillus actinomycetemcomitans is an important pathogen implicated in juvenile and adult periodontal diseases . An important virulence factor of A . actinomycetemcomitans is the ability to invade human oral epithelial cells . A clinical isolate, A . actinomycetemcomitans SUNY 465, has previously been shown to enter epithelial cells by an actin-dependent mechanism . The internalized bacteria are surrounded by an actin halo upon entry . These data are consistent with the mode of entry associated with many enteric pathogens . We tested the effects of cytochalasin D, an inhibitor of the actin microfilament network, on bacterial entry to determine whether this mode of entry was common to other A . actinomycetemcomitans clinical isolates . Cytochalasin D was added prior to infection . A . actinomycetemcomitans SUNY 523 and A . actinomycetemcomitans 4065 exhibited enhanced ability to enter epithelial cells in the presence of cytochalasin D . Immunofluorescent labeling of bacteria and host cell actin confirmed that actin was not being mobilized by the entry of A . actinomycetemcomitans SUNY 523 . Inhibitors of receptor-mediated endocytosis inhibited invasion of A . actinomycetemcomitans SUNY 523 and A . actinomycetemcomitans 4065 . Microtubule effectors did not inhibit invasion of A . actinomycetemcomitans . A . actinomycetemcomitans SUNY 523, but not A . actinomycetemcomitans 4065, was deficient in exit from epithelial cells as determined by the absence of organisms in the assay medium . These data suggest that A . actinomycetemcomitans strains utilize at least two distinct mechanisms for entry into epithelial cells, and that A . actinomycetemcomitans SUNY 523 may be defective in exit and cell-to-cell spread. Rev Soc Bras Med Trop, 1999 Jul-Aug, 32(4), 425 - 38 {The association between human toxocariasis and pyogenic abscesses}; Rayes AA et al.; The association between hepatic abscesses and schistosomiasis mansoni was confirmed by clinical and experimental studies . Other parasites may cause systemic immunologic changes and local structural alterations in the affected organs that can facilitate the seeding of these areas by bacteria . Tropical pyomyositis, pyogenic liver and renal abscesses are frequent diseases in tropical areas . The visceral larva migrans syndrome is caused by the presence, in the human body, of larvae of worms that have other animals as their definitive host, most commonly being caused by Toxocara canis . The larvae migrate to various body organs leading to many inflammatory reactions in the form of granuloma and tissue necrosis . In this review we discuss the possible host-parasite-bacteria interactions that would favour the formation of abscesses in the organs involved by the larva of T . canis and present preliminary results of a clinical and experimental study undertaken during the last four years to define the role of this parasite in the pathogenesis of the abscesses. Dev Biol Stand, 1999, 98, 137 - 40; discussion 167 Experience with vero cells at Pasteur Mérieux Connaught; Montagnon BJ et al.; The Vero cell line has been used by Pasteur-Merieux-Connaught (PMC) since 1982 with the Cell Bank's system to produce, at the 142nd passage, inactivated polio vaccine (IPV), oral polio vaccine (OPV) and rabies vaccines . The safety of the cell line has been regularly validated at the WCB level according to the WHO and European Pharmacopeia requirements for absence of bacteria, fungi, mycoplasma and viruses . Special emphasis was devoted to establishing the absence of simian viruses (SV40, SIV, Retro-D virus, simian CMV) . Reverse Transcriptase (RT) activity was also negative . At low level of passage, the Vero cells are not tumorigenic . Vaccines have been prepared in low passage level Vero cells and, together with the excellent downstream purification, has resulted in excellent safety as attested by pharmacovigilance of more than 100 million doses of IPV during 12 years, more than 20 million doses of rabies vaccine during 10 years and more than one billion of OPV during six years. Eur J Pharm Sci, 1999 Oct, 9(1), 85 - 91 Site dependent bioavailability and metabolism of levosimendan in dogs; Antila S et al.; Site specific bioavailability and metabolism of levosimendan was studied in ten dogs by placing intestinal access port catheters in different parts of the gastrointestinal tract . 14C-labelled levosimendan (0.1 mg/kg) was administered intravenously, by gastric tube and directly through catheters that were placed in the duodenum, jejunum and ileum . Plasma samples were collected and radioactivity in the different organs and tissues was measured . The results of the present study showed that bioavailability of levosimendan was high varying from 71 to 86% after extravascular administration . Metabolite OR-1855 concentrations in the plasma were about 3-4 times higher after administration to the ileum compared to the other administration routes . It can be concluded that the bioavailability of levosimendan is not affected by site specific administration . The bacteria or enzymes responsible for the metabolism of levosimendan are located in the lower parts of the gastrointestinal tract. J Nucl Med, 1999 Sep, 40(9), 1451 - 5 Validation of the lactose-{13C}ureide breath test for determination of orocecal transit time by scintigraphy; Geypens B et al.; The breath test using oral administration of a 13C-labeled substrate, lactose-ureide (LU), to measure orocecal transit time (OCTT) was validated against 99mTc-scintigraphy . Although LU is not absorbed in the human small intestine, colonic bacteria readily metabolize LU, producing 13C-labeled CO2 . The time at which 13CO2 appears in breath corresponds to the OCTT . METHODS: Twenty-two healthy volunteers ingested a meal labeled with 99mTc and 13C-LU . Scintigraphy was performed over 8 h at time intervals of 10 or 15 min . OCTT with scintigraphy was defined as the time at which at least 10% of the label had entered the colon . Breath samples were obtained every 10-15 min for 10 h and measured by isotope ratio mass spectrometry . OCTT was defined as the time of first significant increase above baseline . The results were compared using correlation and Altman-Bland statistics . RESULTS: OCTT results from scintigraphy (mean OCTT = 283+/-53 min) and breath test (mean OCTT = 292+/-58 min) correlated well (r = 0.94) . Altman-Bland statistics showed close agreement between scintigraphy and breath test . No significant difference between male and female subjects was observed . CONCLUSION: The breath test using 13C-LU is a valid alternative to scintigraphy techniques for measuring OCTT. Dermatol Surg, 1999 Aug, 25(8), 666 - 8 Eruptive keratoacanthomas following carbon dioxide laser resurfacing; Gewirtzman A et al.; BACKGROUND: Skin resurfacing with the carbon dioxide (CO2) laser is currently a popular means of improving rhytides and scars . Scarring, hyperpigmentation, hypopigmentation, and infection are among the complications that have been known to occur in some patients treated with the CO2 laser . OBJECTIVE: We wish to communicate a previously unreported complication of CO2 laser resurfacing-multiple eruptive keratoacanthomas . METHOD: We describe a 61-year-old woman who presented with multiple eruptive keratoacanthomas subsequent to CO2 laser resurfacing . Her lesions were cultured for fungus and bacteria . Biopsy specimens of two lesions were taken . RESULTS: Cultures were negative for pathogens . Biopsy specimens revealed atypical squamous epithelial proliferation and changes consistent with eruptive keratoacanthomas . CONCLUSION: Multiple eruptive keratoacanthomas should be considered as a rare complication of CO2 laser resurfacing. J Immunol, 1999 Oct 1, 163(7), 3898 - 906 Mycobacterium tuberculosis inhibits IFN-gamma transcriptional responses without inhibiting activation of STAT1; Ting LM et al.; IFN-gamma activates macrophages to kill diverse intracellular pathogens, but does not activate human macrophages to kill virulent Mycobacterium tuberculosis . We tested the hypothesis that this is due to inhibition of IFN-gamma signaling by M . tuberculosis and found that M . tuberculosis infection of human macrophages blocks several responses to IFN-gamma, including killing of Toxoplasma gondii and induction of FcgammaRI . The inhibitory effect of M . tuberculosis is directed at transcription of IFN-gamma-responsive genes, but does not affect proximal steps in the Janus kinase-STAT pathway, as STAT1alpha tyrosine and serine phosphorylation, dimerization, nuclear translocation, and DNA binding are intact in M . tuberculosis-infected cells . In contrast, there is a marked decrease in IFN-gamma-induced association of STAT1 with the transcriptional coactivators CREB binding protein and p300 in M . tuberculosis-infected macrophages, indicating that M . tuberculosis directly or indirectly disrupts this protein-protein interaction that is essential for transcriptional responses to IFN-gamma . Gamma-irradiated M . tuberculosis and isolated cell walls reproduce the effects of live bacteria, indicating that the bacterial component(s) that initiates inhibition of IFN-gamma responses is constitutively expressed . Although lipoarabinomannan has been found to exert effects on macrophages, it does not account for the inhibitory effects of cell walls . These results indicate that one mechanism for M . tuberculosis to evade the human immune response is to inhibit the IFN-gamma signaling pathway, and that the mechanism of inhibition is distinct from that reported for Leishmania donovani or CMV, in that it targets the interaction of STAT1 with the basal transcriptional apparatus. Vet Pathol, 1999 Sep, 36(5), 471 - 4 Nodular Pneumocystis carinii pneumonia in SIV-infected macaques; Yanai T et al.; Pneumocystis carinii (PC) pneumonia is a frequent manifestation of the acquired immunodeficiency syndrome (AIDS) in humans and macaques . An unusual nodular type of PC pneumonia was observed in two simian immunodeficiency virus (SIV)-inoculated rhesus macaques (Macaca mulatta) . These animals developed clinical signs of simian AIDS, including anorexia, weight loss, dyspnea, and collapse . Grossly, both animals had multifocal tan-white nodules 1-10 mm in diameter scattered throughout the lungs . One animal had similar nodules involving the diaphragm and thoracic wall . The lungs were characterized by severe PC pneumonia with numerous large nodules consisting of foamy material that compressed adjacent tissue . The nodules had central areas of necrosis and lysis of alveolar septa . Varying degrees of necrotizing vasculitis were observed in areas of nodular PC pneumonia . The presence of PC in intra-alveolar spaces and nodular lesions was confirmed by immunohistochemistry . No evidence of other agents, including viral inclusions, bacteria, fungi, and lung mites, was detected . The animal with the most severe nodular PC pneumonia had vascular involvement with extrapulmonary spread to the diaphragm, thoracic wall, and regional lymph nodes . This unusual type of nodular PC pneumonia has been rarely seen in human AIDS patients. Vet Pathol, 1999 Sep, 36(5), 412 - 22 A comparison of the morphologic effects of Serpulina hyodysenteriae or its beta-hemolysin on the murine cecal mucosa; Hutto DL et al.; Studies were carried out to compare the early morphologic changes in the cecal mucosa of mice either infected with Serpulina hyodysenteriae or exposed to the beta-hemolysin of S . hyodysenteriae . Sixty-five 12-24-week-old C3H/HeOuJ mice were infected with S . hyodysenteriae by gastric intubation . Two mice were necropsied every hour for 30 hours following infection . S . hyodysenteriae was isolated from the cecal contents of each mouse at all time points . Macroscopic lesions were first apparent at 14 hours postinfection (PI), and light microscopic lesions were first apparent at 10 hours PI, earlier than has been previously reported . Ultrastructural changes, first evident at 6 hours PI, included disarray and loss of microvilli and terminal web, with dilatation of intercellular spaces . Luminal bacteria were translocated through epithelial cells to the lamina propria, where capillaries exhibited changes indicative of increased permeability . In another experiment, solutions containing between 2,500 and 25,000 hemolytic units of purified S . hyodysenteriae hemolysin were placed within the lumen of surgically closed murine ceca (n = 10); ceca were collected for examination 3 hours following treatment . Ultrastructural changes consisted of loss of microvilli and terminal web and marked vacuolation and exfoliation of epithelial cells . Significant numbers of necrotic and apoptotic epithelial cells were present, and epithelial cells internalized moderate numbers of bacteria . The hemolysin of S . hyodysenteriae induces some of the same early ultrastructural changes in the cecal epithelium of mice as occur following infection with S . hyodysenteriae . Based on the observed bacterial translocation, luminal bacteria also appear to play a unique role in lesion development in this model. Plasmid, 1999 Sep, 42(2), 150 - 3 Structural analysis of plasmid pLQ510 from Moraxella catarrhalis E22; Liu L et al.; The complete nucleotide sequence of plasmid pLQ510 from Moraxella catarrhalis strain E22 has been determined . This plasmid contained 12,082 bp with 38% GC content . Five open reading frames that encoded predicted proteins with homology to plasmid-encoded proteins from other bacteria were identified . A putative origin of replication that contained an AT-rich region followed by four direct repeats and an inverted repeat was identified . Plasmid, 1999 Sep, 42(2), 139 - 43 A versatile bait vector allowing rapid isolation of prey vectors in Gal4-dependent yeast two-hybrid screens; Bannasch D et al.; Two-hybrid screens have been widely used in recent years for identifying and isolating new interaction partners to known proteins . Current Gal4-dependent two-hybrid screens employ mostly bait and library vectors, which both have the ampicillin gene as a selection marker in bacteria . The isolation of the library plasmids takes several days, because library and bait plasmid cannot be separated easily . We have replaced the ampicillin gene by a kanamycin gene in a Gal4 DNA binding domain bait vector . This vector reduces four- to fivefold the time period required for the isolation of library plasmid DNA . In addition we have changed the multicloning site in the modified vector for easy cloning of cDNA inserts . This vector is advantageous not only in standard two-hybrid screens, but also in mass screens that require multiple screening rounds in order to characterize networks of protein-protein interactions . J Hypertens, 1999 Sep, 17(9), 1329 - 37 Involvement of receptor-type tyrosine kinase gene families in cardiac hypertrophy; Akiyama Y et al.; OBJECTIVE: The activation of protein tyrosine kinases (PTKs) has been postulated to be involved in cell differentiation and proliferation . To elucidate the involvement of tyrosine kinase genes in normal and pathological conditions, we analysed the expression patterns of receptor-type PTKs in the normal and hypertensive hypertrophied heart in rats . MATERIALS AND METHODS: Hypertrophied and normal rat hearts were obtained from hypertensive rats; deoxycorticosterone acetate (DOCA)-salt and 2 kidney-1 clip (2K-1C), and their sham-operated rats, respectively . A reverse transcription-polymerase chain reaction (RT-PCR) was performed using degenerated primers which were designed from highly conserved regions in the catalytic domains of receptor-type PTKs . The PCR products were ligated into a sequence vector, and subcloned by transforming bacteria . To compare the expression level of these PTK mRNAs in the normal and hypertrophied heart, we performed semi-competetive RT-PCR and immunohistochemical and Western blot analyses . RESULTS: Nucleotide sequencing of approximately 80 clones of PTKs revealed 10 receptor-type, five nonreceptor-type and two unknown types in the rat heart . Tie-2/Tek, Ryk, insulin-like growth factor-I receptor were abundantly expressed in the rat heart as members of receptor-type PTKs . Immunohistochemistry and RT-PCR demonstrated the presence of platelet-derived growth factor (PDGF)-alpha receptor, PDGF-beta receptor and fibroblast growth factor-3 receptor in both normal and hypertrophied hearts . We also confirmed the presence of Flt-1, KDR/FIk-1, and their ligand vascular endothelial growth factor, c-Met and its ligand hepatocyte growth factor (HGF), and Tie-1, Tie-2/Tek by immunohistochemistry and RT-PCR . The coexpression of cardiac HGF and c-Met in hypertrophied hearts, especially in 2K-1 C rats, was induced more intensively than that in DOCA-salt rats . CONCLUSION: These findings suggest that HGF/c-Met interactions may play an important role in cardiac hypertrophy and remodeling, probably as a result of the activation of the local renin-angiotensin system. Dtsch Med Wochenschr, 1999 Aug 27, 124(34-35), 993 - 7 {Miliary pneumonia after the intravesical BCG therapy of a superficial urothelial carcinoma of the bladder}; Habscheid W et al.; HISTORY AND ADMISSION FINDINGS: A multilocular superficial epithelial carcinoma (T1G3) and carcinoma in situ (Cis G3) of the bladder were resected transurethrally followed by intravesical instillation of BCG . The initial cycle of BCG administration had been free of complication, but then high fever, fatigue, cough and dyspnoea had developed with subsequent BCG maintenance treatment . Physical examination on admission revealed fever, clearly reduced general condition, and increased breath sounds with fine rales in the upper and middle lobes . INVESTIGATIONS: A clearly raised erythrocyte sedimentation rate (86 mm/h) and a CRP level at the upper limit of normal (13.6 mg/dl) indicated marked inflammatory reaction . The chest radiogram showed diffuse miliary opacities . Mycobacteria were not demonstrated in either gastric juice or bronchial secretion . TREATMENT AND COURSE: As BCG-induced miliary pneumonia was diagnosed, triple tuberculostatic treatment was commenced (ethambutol, 1200 mg/d; rifampicin, 600 mg/d; isoniazid, 300 mg/d) . Nonetheless his condition deteriorated further . When prednisolone, 40 mg/d was added the symptoms improved rapidly . The tuberculostatic drugs were continued for 6 months . All symptoms had disappeared after 4 months . CONCLUSION: Miliary pneumonia is a rare complication of intravesical BCG installation of a superficial bladder cancer . As living bacteria cannot be excluded as the cause, triple tuberculostatic treatment must be started at once . If this fails to bring about improvement, additional steroid medication is recommended. Bioinformatics, 1999 Jul-Aug, 15(7-8), 536 - 43 Complete genomes in WWW Entrez: data representation and analysis; Tatusova TA et al.; MOTIVATION: The large amount of genome sequence data now publicly available can be accessed through the National Center for Biotechnology Information (NCBI) Entrez search and retrieval system, making it possible to explore data of a breadth and scope exceeding traditional flatfile views . RESULTS: Here we report recent improvements for completely sequenced genomes from viruses, bacteria, and yeast . Flexible web based views, precomputed relationships, and immediate access to analytical tools provide scientists with a portal into the new insights to be gained from completed genome sequences . AVAILABILITY: Entrez Genomes can be accessed on the World Wide Web at org.html. Toxicol Ind Health, 1999 Aug, 15(5), 458 - 63 Nitrobenzene potential human cancer risk based on animal studies; Holder JW; Inhaled nitrobenzene (NB) in animals produces cancer at eight sites in three rodent strains . B6C3F1 mice respond with mammary gland malignant tumors and male lung and thyroid benign tumors, and F344/N male rats respond with liver malignant tumors and thyroid and kidney benign tumors, while females respond with endometrial polyps . Male Sprague-Dawley male rats (CD strain) respond with liver benign tumors . NB is oxidized to various phenolic metabolites, while also being reduced to nitrosobenzene (NOB), phenylhydroxylamine (PH), related free radicals, and aniline (AN) in the cecum by bacteria and in the body by the microsomes . In reduction, NB first forms the nitroanion free radical, which can react with O2 to form O2*- . Repeated NB dosing produces a persistent redox couple NOB<==>PH in red blood cells that generates met-Hb and expends NAD(P)H . NOB forms activated glutathione conjugates . These biochemical effects may lead to critical redox imbalances and macromolecular binding . Known effects are hemosiderosis, methemoglobinemia, and anemia--and now dispersed cancer in rodents . Based on structural and mechanistic similarities, NB compares with other animal and human carcinogenic nitroarenes and aromatic amines . The cancer hazard evaluation of NB is that it is a probable human carcinogen by any route of exposure . The maximum response is in F344/N male rats which is used for dose-response modelling . The model to estimate the upper 95% confidence limit (UCL95%) of NB human carcinogenicity is a no-threshold, linear low-dose, and multistaged animal model (LMS) . The UCL95% of cancer slope is estimated to be 0.11(6) mg/kg/day (mkd) . At de minimus risk (1:10(6)), the virtually safe dose (VSD) is estimated to be 9.1 ng/kg/day (nkd). Cell Mol Life Sci, 1999 Aug 15, 55(10), 1255 - 77 Herbicide resistance and supersensitivity in photosystem II; Oettmeier W; Resistance to triazine herbicides in higher plants was first observed in 1970 . A mutation in the photosystem II reaction center D1 protein at position Ser264 --> Gly is responsible for this resistance . So far, 37 single mutants, 16 double mutants, 5 triple mutants and 5 deletion/insertion mutants in the D1 protein have been obtained by randomly induced and site-directed mutagenesis in cyanobacteria and algae . The influence of these mutations on the binding affinities of different classes of herbicides will be discussed . Because a sufficiently high resolution X-ray structure of photosystem II does not yet exist, the reaction center of purple photosynthetic bacteria, which is homologous to photosystem II, served as a model . In the bacterial reaction center a total of 25 single and 3 double herbicide-resistant mutants have been generated. J Forensic Sci, 1999 Sep, 44(5), 893 - 96 Experimental forensic and bioanthropological aspects of soft tissue taphonomy: 1 . Factors influencing postmortem tissue desiccation rate; Aturaliya S et al.; Euthanized rats' carcasses were exposed in an environmental chamber to multiple variables including: (1) position, (2) enveloping clothing, and (3) soil interment in an effort to determine the individual variables' effect on postmortem rate of body and visceral organ water loss . Results indicated that body water loss was enhanced by a horizontal position versus vertical, probably because of wider spread of bacteria- and enzyme-laden abdominal fluid secondary to diaphragm digestion with consequent greater tissue digestion and liquefaction . Clothing also accelerated the desiccation rate . Desiccation was about equally as effective by soil interment as by air exposure, though simulating windy conditions by tripling the air flow rate resulted in much more rapid desiccation in the air-exposed specimen . These studies suggest that the single most important factor influencing postmortem body water loss rate is the environment at the skin surface that acts to enhance or impair water removal from the skin surface and thus influences the water concentration gradient between the skin and underlying deeper tissues. Allergol Immunopathol (Madr), 1999 Jul-Aug, 27(4), 213 - 31 {Immunological and inflammatory pathogenesis of asthma: the predominance of ontogenic Th2 and its relation to developing immunological mechanisms during fetal and neonatal stages . Therapeutic implications}; Villarrubia V et al.; From an immunopathogenic vantage point, asthma appears to be a complex allergic/inflammatory disorder involving mechanism in which the specific immunological response shifts toward Th2 responses instead of Th1 responses . As a consequence of this shift, the cytokines IL-4, IL-5, IL-10, IL-13, TNF-alpha and CSF-GM are produced . The actions of these cytokines explain the phenomena of eosinophilic infiltration and mastocytic degranulation that characterize allergic asthma . The authors propose that the process of deviation toward Th2 responses occurs in the fetal stage and is a result of maternal immunological remodeling processes characteristics of pregnancy . In this period, the mother's mechanisms of immune rejection (mediated by Th1 lymphocytes and their cytokines IFN-gamma and IL-2) are detained or slowed, leading to the predominance of the Th-2 circuit . This predisposes the child to the development of an allergic response to a chance encounter with allergens, viruses and/or bacteria and/or parasites that activate the Th2 circuit . Moreover, deficits in the function of the Th1 circuit explain the sensitivity of the newborn to infections by viruses and other intracellular pathogens . Knowledge of these immunopathogenic mechanisms suggests that the future treatment of asthma and other allergic diseases will be based on the use of immunomodulators capable of stimulating Th1 response, thus achieving a) a god state of resistance to infection, and b) reductions of the production of pro-inflammatory cytokines . The experimental results of IL-12 in human beings with AM3 (an inductor of IFN-gamma and IL-12) support the pathogenic hypothesis proposed and open new ways for the treatment of asthma and other allergic diseases. Arch Virol, 1999, 144(8), 1513 - 26 Genome evolution of tobacco mosaic virus populations during long-term passaging in a diverse range of hosts; Kearney CM et al.; The effects of host changes on plant virus genome evolution was studied by nucleotide sequencing . A single tobacco (Nicotiana tabacum cv . Xanthi) plant was inoculated with in vitro transcripts from a plasmid clone of tobacco mosaic tobamovirus (TMV) . This initial viral population was then transferred 11-12 times in parallel populations in 7 plant host species (1-4 replicates each) over a period of 413-515 days . Virion RNA was then isolated, reverse transcribed, amplified, cloned in bacteria, and sequenced . Portions of the coat protein, movement protein, and replicase genes were sequenced . Fourteen unique mutations were detected from a total of 188 clones (35,607 bases) sequenced, indicating a relatively small overall mutation rate of 3.1 x 10(-4) nucleotide substitutions/base-year . A small Ka/Ks value of 0.09 was also found, indicating selection against amino acid changes . Eighty-five percent of the substitutions were transitions . A G'(ST) value of 0.7 for the coat protein gene suggested that host type affected sequence changes in this region of the genome, but chi(2) analysis did not support this conclusion . This is the first study using sequencing to compare representative sample sections of a plant viral genome following a major selective disturbance such as extended passaging in an alternate host. J Mol Evol, 1999 Oct, 49(4), 524 - 37 The archaea monophyly issue: A phylogeny of translational elongation factor G(2) sequences inferred from an optimized selection of alignment positions; Cammarano P et al.; A global alignment of EF-G(2) sequences was corrected by reference to protein structure . The selection of characters eligible for construction of phylogenetic trees was optimized by searching for regions arising from the artifactual matching of sequence segments unique to different phylogenetic domains . The spurious matchings were identified by comparing all sections of the global alignment with a comprehensive inventory of significant binary alignments obtained by BLAST probing of the DNA and protein databases with representative EF-G(2) sequences . In three discrete alignment blocks (one in domain II and two in domain IV), the alignment of the bacterial sequences with those of Archaea-Eucarya was not retrieved by database probing with EF-G(2) sequences, and no EF-G homologue of the EF-2 sequence segments was detected by using partial EF-G(2) sequences as probes in BLAST/FASTA searches . The two domain IV regions (one of which comprises the ADP-ribosylatable site of EF-2) are almost certainly due to the artifactual alignment of insertion segments that are unique to Bacteria and to Archaea-Eucarya . Phylogenetic trees have been constructed from the global alignment after deselecting positions encompassing the unretrieved, spuriously aligned regions, as well as positions arising from misalignment of the G' and G" subdomain insertion segments flanking the "fifth" consensus motif of the G domain (AE varsson, 1995) . The results show inconsistencies between trees inferred by alternative methods and alternative (DNA and protein) data sets with regard to Archaea being a monophyletic or paraphyletic grouping . Both maximum-likelihood and maximum-parsimony methods do not allow discrimination (by log-likelihood difference and difference in number of inferred substitutions) between the conflicting (monophyletic vs . paraphyletic Archaea) topologies . No specific EF-2 insertions (or terminal accretions) supporting a crenarchaeal-eucaryal clade are detectable in the new EF-G(2) sequence alignment. J Mol Evol, 1999 Oct, 49(4), 453 - 60 Respiratory chains in the last common ancestor of living organisms; Castresana J et al.; Sequences in current databases show that a number of proteins involved in respiratory processes are homologous in archaeal and bacterial species . In particular, terminal oxidases belonging to oxygen, nitrate, sulfate, and sulfur respiratory pathways have been sequenced in members of both domains . They include cytochrome oxidase, nitrate reductase, adenylylsulfate reductase, sulfite reductase, and polysulfide reductase . These proteins can be assigned to the last common ancestor of living organisms assuming that the deepest split of the three domains of life occurred between Archaea and Bacteria and that they were not acquired through lateral gene transfer by one of these domains . These molecular data indicate that several of the most important respiratory pathways arose early in evolution and that the last common ancestor of living organisms was not a simple organism in its energetic metabolism . Rather, it may have been able to gain energy by means of at least four electron transport chains, and therefore it may have been prepared to face a wide range of environmental conditions. Rev Environ Health, 1999 Apr-Jun, 14(2), 79 - 89 Bioaerosols in ambient air particulates: a review and research needs; Monn C et al.; Fine (< 2.5 microns) and inhalable (< 10 microns) ambient particles are associated with increased morbidity and mortality . In addition to a variety of organic chemicals, salts, and metals, inhalable ambient particles may contain biological species, such as proteins, lipids, and so on, from plants, bacteria, and fungi . In airborne particles, the total mass of biological species is small, but their allergenic and inflammatory potential is strong . This paper provides an overview of the bioaerosols found in ambient air particles . Pollen grains are the strongest aeroallergens and have a size > 10 microns . Major pollen allergens have also been identified in size fractions smaller than that of intact pollen . Special atmospheric conditions (such as rainfall) or interactions between air pollutants and pollen may produce allergenic fine particles . Endotoxin (LPS), another important biological species of particles, may play a role in proinflammatory effects . In this review, we discuss the possible interactions between pollen and pollutants and suggest several directions for future research. Extremophiles, 1999 Aug, 3(3), 191 - 8 Extrinsic protein stabilization by the naturally occurring osmolytes beta-hydroxyectoine and betaine; Knapp S et al.; Thermodynamic aspects of protein stabilization by two widespread naturally occurring osmolytes, beta-hydroxyectoine and betaine, were studied using differential scanning calorimetry (DSC) and bovine ribonuclease A (RNase A) as a model protein . The osmolyte beta-hydroxyectoine purified from Marinococcus was found to be a very efficient stabilizer . At a concentration of 3M it increased the melting temperature of RNase A (Tm) by more than 12K and gave rise to a stability increase of 10.6kJ/mol at room temperature . The heat capacity difference between the folded and unfolded state (deltaC(p)) was found to be significantly increased . Betaine stabilized RNase A only at concentrations less than 3M . Also, here deltaCp was found to be increased . Calculation of the number of water molecules that additionally bind to unfolded RNase A resulted in surprisingly low numbers for both osmolytes . The significant stabilization of RNase A by beta-hydroxyectoine makes this osmolyte an interesting stabilizer in biotechnological processes in which enzymes are applied in the presence of denaturants or at high temperature. Am J Gastroenterol, 1999 Sep, 94(9), 2423 - 9 Intramucosal acidosis and the inflammatory response in acute pancreatitis; Soong CV et al.; OBJECTIVE: The aim of this study was to assess the host response and diminished bowel perfusion during acute pancreatitis . METHODS: A total of 19 patients admitted with established diagnoses of acute pancreatitis on the basis of clinical findings, elevated serum amylase to more than four times the upper limit or by contrast radiology . Patients were stratified into mild and severe pancreatitis using the Atlanta criteria . Blood samples were obtained from in-dwelling lines or direct venipuncture within 12 h of admission and 24 hourly thereafter for measurements of plasma endotoxin, EndoCab immunoglobulin (Ig)G and IgM antibodies, tumor necrosis factor (TNF), p55 TNF receptor, and IL-6 . A gastric tonometer was inserted in place of a nasogastric tube for intramucosal pH evaluation . RESULTS: Episodes of endotoxaemia were more common and endotoxin concentration significantly higher at presentation in the severe group compared to the mild group of patients . A greater consumption of IgM antibody was found in those with severe disease . The decrease in IgM antibody concentration was shown to be a specific host response, as a fall in concentration of antibodies to a neutral antigen, tetanus toxoid, was not observed . Significantly greater elevations were found in p55 TNF receptor and IL-6 concentrations in the severe group in comparison to those suffering mild pancreatitis . Significant correlations were found between gastric intramucosal pH and EndoCab IgM antibody, p55 TNF receptor, and IL-6 . CONCLUSIONS: These results suggest that endotoxemia, an acute inflammatory response, and a reduction in bowel perfusion may occur in severe acute pancreatitis . The endotoxemia and inflammatory response may be due to the permeation of bacteria and their breakdown products across a disrupted bowel mucosal barrier. JPEN J Parenter Enteral Nutr, 1999 Sep-Oct, 23(5 Suppl), S18 - 9 Management of acute diarrhea in infants; Rhoads M; In 1999, children seen in the emergency room of a developed country for watery diarrhea and dehydration will most likely receive an intravenous infusion of fluid, followed by instructions to give oral rehydration solution (ORS) and clear liquids for a day, followed by half-strength lactose-free formula . In fact, the majority of these children could best be managed with supervised ORS followed by early (within 4-6 h) refeeding of their normal diet, based on large numbers of clinical trials and a meta-analysis . In the next decade, effective therapy in addition to glucose-containing oral rehydration solutions should be available which should reduce diarrheal volume and duration of purging . These include amino acid-supplemented "Super ORSs," ORS with soluble fibers, liquid zinc, and probiotic milks containing bacteria which boost the immune response and reduce stool number . In addition, children wealthy enough to be able to afford the new tetravalent vaccine will be largely protected from dehydrating rotavirus diarrhea, the most common cause of dehydration in infants. Surg Today, 1999, 29(8), 735 - 40 The effect of lymphatic blockage on the amount of endotoxin in portal circulation, nitric oxide synthesis, and the liver in dogs with peritonitis; Guler O et al.; This study was performed to investigate the effect of lymphatic blockage on the amount of endotoxin in portal venous blood, nitric oxide synthesis, the release of aspartate aminotransferase (AST) from the liver, hepatic damage, and survival in an experimental model of dogs with peritonitis . The dogs were divided into a control group (group 1), an unligated thoracic duct peritonitis group (group 2), and a ligated thoracic duct peritonitis group (group 3) . Peritoneal fluid and blood from the portal vein and femoral artery were taken for peritoneal culture, endotoxin, and AST assay, respectively, and liver biopsies were performed to assess for hepatic damage and for nitric oxide assay . There was a higher bacteria count in the peritoneal fluid from group 3 than in that from group 2 (P < 0.0001) . Bacteria grew in all of the blood cultures from the group 2 animals, but growth was seen only in blood cultures from four of the group 3 animals . The levels of endotoxin, nitrite, and AST levels in group 3 were significantly increased in comparison with those in group 2 (P < 0.0001) . Extensive hepatocellular necrosis with hemorrhage was observed in the livers of the group 3 animals, and all of them died within 48 h . The results of this study suggest that the blockage of lymph flow has a negative effect on liver and survival in dogs with peritonitis, and that hepatic damage is directly related to the amount of endotoxin to which the liver is exposed. FEMS Microbiol Lett, 1999 Sep 1, 178(1), 19 - 26 Isolation of two subpopulations of Mycobacterium avium within human macrophages; Bermudez LE et al.; Mycobacterium avium is an intracellular pathogen that is associated with disseminated infection in acquired immunodeficiency syndrome (AIDS) patients . Human monocyte-derived macrophages were infected with M . avium strain 101 and a quinolone (Bay y 3118) was used at 8 micrograms ml-1, a concentration that kills growing bacteria but fails to eliminate static organisms . Infected monolayers were treated with Bay y 3118 for 4 days and viable bacteria obtained from the lysis of macrophages were used to infect other macrophages without passage in media . The procedure was repeated five times, after which seven different subpopulations that failed to grow within macrophages were identified . While the DNA fingerprinting confirmed that all came from the same strain, three protein profiles were observed . Static subpopulations were not killed by cytokine-stimulated macrophages, in contrast to the replicating subpopulation . Three of the static subpopulation strains were shown to be auxotrophic for glutamic acid or methionine . All seven non-duplicating subpopulation strains grew well in complete 7H10 agar . The importance of a static subpopulation of M . avium within macrophages is presently unknown . It is possible, however, that the non-growing bacteria would persist within macrophages. J N Z Soc Periodontol, 1998, (83), 15 - 28 Endodontic management of combined endodontic-periodontal lesions; Abbott P; Endodontic-periodontal lesions can provide many challenges to clinicians . Although there may be difficulties in establishing a correct diagnosis, this is the most important phase of their management as the diagnosis will determine the type and sequence of treatment required . In general, if the root canal system is infected, endodontic treatment should be commenced prior to any periodontal therapy in order to remove the intra-canal infection before any cementum is removed . This avoids several complications and provides a favourable situation for tissue repair . The endodontic treatment can be completed before periodontal treatment is provided except where there is a "combined endodontic-periodontal lesion with communication"--in these cases, the root canals should be medicated until the periodontal treatment has been completed and the overall prognosis has been reassessed as being favourable . The use of non-toxic intra-canal therapeutic medicaments is essential to destroy bacteria and to encourage tissue healing. Plant Physiol, 1999 Sep, 121(1), 301 - 10 Carbon and amino acids reciprocally modulate the expression of glutamine synthetase in Arabidopsis; Oliveira IC et al.; In bacteria and yeast, glutamine synthetase (GS) expression is tightly regulated by the metabolic status of the cell, both at the transcriptional and posttranscriptional levels . We discuss the relative contributions of light and metabolic cues on the regulation of members of the GS gene family (chloroplastic GS2 and cytosolic GS1) in Arabidopsis . These studies reveal that the dramatic induction of mRNA for chloroplastic GS2 by light is mediated in part by phytochrome and in part by light-induced changes in sucrose (Suc) levels . In contrast, the modest induction of mRNA for cytosolic GS1 by light is primarily mediated by changes in the levels of carbon metabolites . Suc induction of mRNA for GS2 and GS1 occurs in a time- and dose-dependent manner . Suc-induced changes in GS mRNA levels were also observed at the level of GS enzyme activity . In contrast, amino acids were shown to antagonize the Suc induction of GS, both at the level of mRNA accumulation and that of enzyme activity . For GS2, the gene whose expression was the most dramatically regulated by metabolites, we used a GS2 promoter-beta-glucuronidase fusion to demonstrate that transcriptional control is involved in this metabolic regulation . Our results suggest that the metabolic regulation of GS expression in plants is controlled by the relative abundance of carbon skeletons versus amino acids . This would allow nitrogen assimilation into glutamine to proceed (or not) according to the metabolic status and biosynthetic needs of the plant . This type of GS gene regulation is reminiscent of the nitrogen regulatory system in bacteria, and suggests an evolutionary link between metabolic sensing and signaling in bacteria and plants. J Virol, 1999 Oct, 73(10), 8527 - 40 Human immunodeficiency virus type 1 Gag polyprotein multimerization requires the nucleocapsid domain and RNA and is promoted by the capsid-dimer interface and the basic region of matrix protein; Burniston MT et al.; The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein directs the formation of virions from productively infected cells . Many gag mutations disrupt virion assembly, but little is known about the biochemical effects of many of these mutations . Protein-protein interactions among Gag monomers are believed to be necessary for virion assembly, and data suggest that RNA may modify protein-protein interactions or even serve as a bridge linking Gag polyprotein monomers . To evaluate the primary sequence requirements for HIV-1 Gag homomeric interactions, a panel of HIV-1 Gag deletion mutants was expressed in bacteria and evaluated for the ability to associate with full-length Gag in vitro . The nucleocapsid protein, the major RNA-binding domain of Gag, exhibited activity comparable to that of the complete polyprotein . In the absence of the nucleocapsid protein, relatively weak activity was observed that was dependent upon both the capsid-dimer interface and basic residues within the matrix domain . The relevance of the in vitro findings was confirmed with an assay in which nonmyristylated mutant Gags were assessed for the ability to be incorporated into virions produced by wild-type Gag expressed in trans . Evidence of the importance of RNA for Gag-Gag interaction was provided by the demonstration that RNase impairs the Gag-Gag interaction and that HIV-1 Gag interacts efficiently with Gags encoded by distantly related retroviruses and with structurally unrelated RNA-binding proteins . These results are consistent with models in which Gag multimerization involves indirect contacts via an RNA bridge as well as direct protein-protein interactions. J Bacteriol, 1999 Sep, 181(18), 5825 - 32 The Caulobacter crescentus CgtA protein displays unusual guanine nucleotide binding and exchange properties; Lin B et al.; The Caulobacter crescentus CgtA protein is a member of the Obg-GTP1 subfamily of monomeric GTP-binding proteins . In vitro, CgtA specifically bound GTP and GDP but not GMP or ATP . CgtA bound GTP and GDP with moderate affinity at 30 degrees C and displayed equilibrium binding constants of 1.2 and 0.5 microM, respectively, in the presence of Mg(2+) . In the absence of Mg(2+), the affinity of CgtA for GTP and GDP was reduced 59- and 6-fold, respectively . N-Methyl-3'-O-anthranoyl (mant)-guanine nucleotide analogs were used to quantify GDP and GTP exchange . Spontaneous dissociation of both GDP and GTP in the presence of 5 to 12 mM Mg(2+) was extremely rapid (k(d) = 1.4 and 1.5 s(-1), respectively), 10(3)- to 10(5)-fold faster than that of the well-characterized eukaryotic Ras-like GTP-binding proteins . The dissociation rate constant of GDP increased sevenfold in the absence of Mg(2+) . Finally, there was a low inherent GTPase activity with a single-turnover rate constant of 5.0 x 10(-4) s(-1) corresponding to a half-life of hydrolysis of 23 min . These data clearly demonstrate that the guanine nucleotide binding and exchange properties of CgtA are different from those of the well-characterized Ras-like GTP-binding proteins . Furthermore, these data are consistent with a model whereby the nucleotide occupancy of CgtA is controlled by the intracellular levels of guanine nucleotides. Pol J Pathol, 1999, 50(2), 93 - 7 The presence of Chlamydia pneumoniae in atherosclerotic plaques--a report of three cases of ischaemic heart disease; Walski M et al.; The report presents three cases of ischaemic heart disease in which Chlamydia pneumoniae infections were detected first serologically, later the bacteria were shown in atherosclerotic plaques with electron microscopy, and finally C . pneumoniae strains were isolated from the tissues. Int J Parasitol, 1999 Jun, 29(6), 869 - 75 Do sheep regulate the size of their mallophagan louse populations? James PJ. Alternatives to chemicals for controlling parasites are required to minimise problems from resistance, residues in animal products and occupational exposure . Utilisation of host response to parasites through selection of resistant types or vaccination is an appealing option . To date most studies have been with haematophagous or invasive parasites which directly contact elements of the host immune system . Sheep lice (Bovicola ovis) feed superficially on the skin of sheep ingesting lipid, scurf, bacteria and loose stratum corneum squames . Evidence is presented that despite their surface feeding habit Bovicola ovis stimulate an immune response in sheep and that this response may play a part in regulating the size of louse populations. Eur J Haematol, 1999 Aug, 63(2), 94 - 102 Induction of cytosolic phospholipase A2 and prostaglandin H2 synthase-2 by lipopolysaccharide in human polymorphonuclear leukocytes; Zaitsu M et al.; Polymorphonuclear leukocytes (PMNs) produce arachidonic acid (AA) metabolites including thromboxane A2 (TXA2) . These cells are the first line of defense against bacterial invasion, which often causes endotoxin shock . TXA2 which plays an important role in the pathogenesis of endotoxin shock is synthesized by three consecutive enzyme activation, cytosolic phospholipase A2 (cPLA2), prostaglandin H2 synthase (PHS type 1 and type 2) and TXA2 synthase . Among them, cPLA2- and PHS-2 activity is known to be transcriptionally and/or posttranscriptionally up-regulated by various bioactive substances including lipopolysaccharide (LPS), a bacterial endotoxin, in many cell types . We investigated the action of LPS on TXA2 synthesis in human PMNs . A23187-stimulated production of thromboxane B2 (TXB2, a stable metabolite of TXA2), assayed by specific radioimmunoassay (RIA), was significantly increased from 566.7+/-44.1 pg/10(6) cells to 966.7+/-44.1 pg/10(6) cells (p<0.05) after 6 h-exposure to LPS at the concentration of 100 ng/ml . Messenger RNA for PHS-2, PHS-1, TXA2 synthase and cPLA2, which was assessed by reverse transcription-polymerase chain reaction (RT-PCR), was expressed in PMNs without LPS stimulation . Although PHS-2 was putatively an inducible enzyme, abundance of mRNA for PHS-2 in PMNs without LPS stimulation was detectable . Messenger RNA abundance for PHS-2 and cPLA2, but not for PHS-1 and TXA2 synthase, was enhanced by LPS-treatment, indicating that the increased production of TXB2 was attributable to the up-regulation of cPLA2 and PHS-2 . We conclude that (1) PHS-2 plays a more important role than PHS-1 in the production of TXA2 in human PMNs and (2) TXA2 synthesis in human PMNs is transcriptionally up-regulated by new induction of cPLA2 as well as PHS-2, when the cells encounter endotoxin producing bacteria. J Infect Dis, 1999 Oct, 180(4), 1230 - 7 Interleukin 10 produced by macrophages inoculated with Mycobacterium avium attenuates mycobacteria-induced apoptosis by reduction of TNF-alpha activity; Balcewicz-Sablinska MK et al.; Normal human macrophages respond to infection with Mycobacterium avium, serovar 4, by producing tumor necrosis factor (TNF)-alpha, which mediates apoptosis, and by elaborating interleukin (IL)-10, a TNF-alpha antagonist . We show that IL-10 down-regulates apoptosis by inhibiting the TNF-alpha production of the inoculated macrophages and by inducing the release of soluble TNF receptor type 2 from the macrophages, which leads to inactivation of TNF-alpha . These experiments suggest that induction of IL-10 production is a virulence factor that creates an intracellular sanctuary for the bacteria that is inaccessible to the defense mechanisms of the host. J Infect Dis, 1999 Oct, 180(4), 1205 - 13 Interaction of Shiga toxins with human brain microvascular endothelial cells: cytokines as sensitizing agents; Ramegowda B et al.; Neurologic abnormalities are among the most serious extraintestinal complications of infection with Shiga toxin (Stx)-producing bacteria . Histopathologic examination of tissues from patients with extraintestinal sequelae suggested that Stxs damage endothelial cells . It is shown here that human brain microvascular endothelial cells (HBMECs) are relatively resistant to purified Stxs (50% cytotoxic doses {CD50s} >/=10 microgram/mL) . Pretreatment of HBMECs with tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, n-butyric acid, or a cAMP analogue resulted in a 103- to 104-fold decrease in CD50 values and a 2- to 4-fold increase in fluoresceinated Stx binding to HBMECs . Treatment of HBMECs with lipopolysaccharides did not significantly alter cytotoxicity or toxin binding . TNF-alpha and IL-1beta treatment was associated with the increased HBMEC expression of the toxin-binding glycolipid globotriaosylceramide . HBMECs did not produce IL-1beta and produced only trace amounts of TNF-alpha when stimulated with purified Stx1 in vitro. J Clin Gastroenterol, 1999 Sep, 29(2), 200 - 2 Rectal ulcers: a rare gastrointestinal manifestation of systemic lupus erythematosus; Amit G et al.; A patient with systemic lupus erythematosus (SLE) developed a rectal ulcer and sepsis from colonic bacteria . At that time she had no other clinical manifestations of SLE . Histopathologic examination of the biopsies taken from the ulcer found evidence of vasculitis . Treatment with high-dose systemic steroids healed the ulcer clinically and endoscopically, but symptoms recurred when steroids were tapered . The patient was referred for surgery . This is a rare but dangerous complication of SLE and can be the only clinical manifestation of the disease. Am J Clin Pathol, 1999 Sep, 112(3), 335 - 42 Autopsy findings in umbilical cord blood transplant recipients; Nuckols JD; Human umbilical cord blood stem cells have been used to reconstitute hematopoiesis in patients with malignant and nonmalignant diseases . The immunologic immaturity of cord blood cells confers peculiar characteristics to these hematopoietic precursors . Autopsy reports from January 1, 1988, through June 30, 1998, were searched for patients who had received an umbilical cord blood transplant (UCBT) . Thirty-two patients (19 male, 13 female) were identified with a mean age at autopsy of 13.0 years with a range from 1 to 52 years . Most patients (24) underwent UCBT for treatment of a malignant neoplasm, while the remainder were treated for immunodeficiencies (4), Lesch-Nyhan syndrome (2), Hurler syndrome (1), and Diamond-Blackfan syndrome (1) . Sixteen patients had at least 1 infectious complication, and 8 patients had multiple infections . Organisms included mycoses (7 patients), viruses (8 patients), bacteria (3 patients), and Toxoplasma (2 patients) . Hemorrhagic complications, such as intra-alveolar hemorrhage and gastrointestinal tract hemorrhage, were found in 24 patients . Other frequent findings at autopsy included diffuse alveolar damage (15 patients), hepatic veno-occlusive disease (11 patients), and acute or chronic graft-vs-host disease (9 patients) . Patients who have received UCBT represent a unique population of immunosuppressed patients . Infectious and hemorrhagic complications frequently are encountered at autopsy, and pathologists performing autopsies on these patients should be alert to unusual pathogens. Mutat Res, 1999 Jul 21, 444(1), 207 - 16 Ames assays and unscheduled DNA synthesis assays on 2, 4-dichlorophenoxyacetic acid and its derivatives; Charles JM et al.; 2,4-dichlorophenoxyacetic acid and several of its derivatives (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants . The genetic toxicity in vitro of 2,4-D and seven of its salts and esters were examined by employing gene mutation in bacteria (Ames test) and induction of DNA damage and repair in rat hepatocytes . In addition, an in vivo unscheduled DNA synthesis (UDS) assay was performed on 2,4-D . There were no indications of genotoxic potential for 2,4-D acid, or any of its derivatives, in these assays . These results are consistent with the reported lack of carcinogenic potential for 2,4-D in both mice and rats. Mol Biochem Parasitol, 1999 Jul 30, 102(1), 43 - 52 A new potent antigen from Echinococcus granulosus associated with muscles and tegument; Fu Y et al.; An immunoscreening of a cDNA library derived from the adult stage of the parasitic platyhelminth Echinococcus granulosus has been carried out with sera from infected dogs . The EgA31 clone encodes a fibrous protein which shares some sequence elements with paramyosins . The corresponding gene is present as a single copy in the genome . As revealed by an antibody obtained against a fusion protein produced in bacteria, the polypeptide has a molecular weight of 66 kDa . This polypeptide is present at all developmental stages studied and is a potent antigen during an infection by the adult stage in the dog and during cyst growth in human patients . By immunohistology, it was shown that it is present in the tegument and subtegumental parenchyma of the adult with a main location in the region of the suckers where it rapidly accumulates at the time of the head evagination . It is also present in the germinal layer of the cyst and on the protoscolex. Clin Infect Dis, 1999 Aug, 29(2), 408 - 13 Risk factors for melioidosis and bacteremic melioidosis; Suputtamongkol Y et al.; A case-control study was conducted in four hospitals in northeastern Thailand to identify risk factors for melioidosis and bacteremic melioidosis . Cases were patients with culture-proven melioidosis, and there were two types of controls (those with infections, i.e., with community-acquired septicemia caused by other bacteria, and those without infection, i.e., randomly selected patients admitted with noninfectious diseases to the same hospitals) . Demographic data, clinical presentations, and suspected risk factors were analyzed . Diabetes mellitus, preexisting renal diseases, thalassemia, and occupational exposure, classified by the soil and water risk assessment, were confirmed to be significant risk factors for melioidosis and bacteremic melioidosis . Only diabetes mellitus was a significant factor associated with bacteremic melioidosis, as compared with nonbacteremia . A significant interaction was found between diabetes mellitus and occupational exposure . Thus, diabetic rice farmers would be the most appropriate population group for targeted control measures such as vaccination in the future. Schweiz Med Wochenschr, 1999 Aug 10, 129(31-32), 1099 - 105 Shared vector-borne zoonoses of the Old World and New World: home grown or translocated? Childs JE, Ellis BA, Nicholson WL, Kosoy M, Sumner JW. Humans inhabiting the Old World and New World share a wide variety of pathogens . Processes that result in the disjunct biogeographic distribution of pathogens with common vertebrate reservoirs or vectors are more difficult to unravel than those influencing the distribution of infections spread only through human-to-human transmission . The origins of species and complexes of tick-borne bacteria are unclear . The agent of Lyme borreliosis may have speciated in the New World following geographical isolation of ticks harboring ancestral spirochetes; the subsequent spread to Europe of B . burgdorferi sensu stricto may have occurred within historical times . Other tick-borne agents, such as the ehrlichiae causing human granulocytic ehrlichiosis, are genetically very similar in the Old World and New World . As the taxonomic distinctions among these related agents of human and veterinary importance appear increasingly blurred, the processes leading to the current discontinuous geographic distributions will also become the source of continuing speculation . Accumulating data suggest an Old World origin for a group of bacteria that include B . elizabethae, a human pathogen first identified from the New World . The potential public health significance of these newly described organisms is undefined, but of international interest as their vertebrate reservoir has been introduced throughout the world. Plant J, 1999 Jul, 19(2), 153 - 162 Suppression of pea nuclear topoisomerase I enzyme activity by pea PCNA; Van Hop D et al.; Proliferating cell nuclear antigen (PCNA), a highly conserved DNA polymerase accessory protein of eukary- otic kingdom, has not been studied thoroughly in bio- chemical terms in plants . We describe the isolation of the cDNA encoding PCNA from the pea cDNA library using the PCR approach . The cDNA was used for expression of pea PCNA in bacteria as a fusion protein (GST.PCNA) with the GST tag at the amino terminal end . The GST.PCNA stimulated the partially purified pea DNA polymerases approximately 30-fold . The stimulation was due to the oligomeric form of GST.PCNA . The pea PCNA interacted with the recombinant type I pea topoiso- merase as well as the native pea nuclear topoisomerase I and repressed the DNA relaxation activities . However, the DNA binding activity of Topo I remained undisturbed in the presence of high amounts of PCNA, thereby signify- ing that the catalysis of Topo I was probably affected by PCNA. Hybridoma, 1999 Jun, 18(3), 251 - 5 Characterization of epitopes on human interleukin-2 using phage displayed-peptide libraries: insights into antibody-peptide interactions; Vispo NS et al.; We have characterized the binding epitopes of two monoclonal antibodies (MAbs) reacting with human Interleukin-2 (IL-2), using a phage display peptide library . The first antibody (CB-IL2.1) recognizes the sequence LSFL, amino acid 72 to amino acid 80, numbered in the IL-2 . The second antibody (CB-IL2.2) binds the sequence TTFM (amino acids 101 to 104) located at the opposite site of the four-helix bundle of IL-2 . Enzyme-linked immunoadsorbent assay (ELISA) and Western blot using different IL-2 protein construct expressed in bacteria and phage display demonstrate the specificities of this antibody . The data presented here show that the antibodies characterized in this study are raised against linear epitopes and suggest that these epitope are accessible from the outside in the native IL-2 molecule. N Y State Dent J, 1999 Jun-Jul, 65(6), 32 - 3 Caries and dentin . Myths & consequences; Hasselgren G et al.; The dentin-pulp complex is sensitive to infection . Restorative treatment should function as a Band-Aid protecting the dentin-pulp complex from bacteria and their byproducts . This article describes different ways of treating the exposed dentin-pulp complex in such a way that bacterial infection can be avoided. Mutagenesis, 1999 Jan, 14(1), 135 - 40 Mutations at G:C base pairs predominate after replication of peroxyl radical-damaged pSP189 plasmids in human cells; Burcham PC et al.; The mutagenicity of peroxyl radicals, important participants in lipid peroxidation cascades, was investigated using a plasmid-based mutational assay system . Double-stranded pSP189 plasmids were incubated with a range of concentrations of the water-soluble peroxyl radical generator 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) . Following replication in human Ad293 cells, the plasmids were screened for supF mutations in indicator bacteria . Exposure to peroxyl radicals caused strand nicking and a decrease in transfection efficiency, which was accompanied by a significant increase in supF mutants . Each of these effects was abolished in the presence of the water-soluble vitamin E analogue Trolox . Automated sequencing of 76 AAPH-induced mutant plasmids revealed that substitutions at G:C base pairs were the most common changes, accounting for 85.5% of all identified mutations . Of these, most comprised G:C-->T:A transversions (53.5%), with lesser contributions by G:C-->A:T transitions (23.9%) and G:C-->C:G transversions (22.5%) . Collectively, these data confirm our previous findings concerning the spectrum of mutations produced upon bacterial replication of peroxyl radical-damaged phage DNA and extend them by showing that such damage has mutagenic consequences during replication in more complex eukaryotic systems. Mund Kiefer Gesichtschir, 1999 Jul, 3(4), 205 - 9 {Visualizing carcinomas of the mouth cavity by stimulating synthesis of fluorescent protoporphyrin IX}; Zenk W et al.; The fluorescence diagnosis based on the aminolavulinic acid-stimulated porphyrin synthesis allows the detection of superficial tumors in a very early stage even when they are very small tumors . A fluorescence diagnosis of tumors in the oral cavity can be simply performed by rinsing with a 0.4% ALA solution for 20 min . This topical application avoids systemic side effects such as skin sensitization . The red fluorescent areas are visible to the naked eye; only a blue light source for fluorescence excitation is necessary and a suitable red filter for observation . In our study on 56 patients suffering from carcinoma of the oral cavity, 96% of the histologically confirmed carcinoma could be visualized via fluorescence . In 3 patients additional tumors were detected via fluorescence that were not visible otherwise . However, many patients show fluorescent areas with no correlation to the histological finding . It was verified that bacteria from the oral cavity also produce PpIX after ALA incubation, which leads to false-positive findings . Reduction of the false-positive findings was achieved by rinsing the oral cavity with hydrogen peroxide and by mechanical plaque reduction . This improves the reliability of a fluorescence-guided biopsy . However, suppression of the bacteria fluorescence is necessary for clinical use of this diagnostic method. FEMS Microbiol Lett, 1999 Aug 15, 177(2), 319 - 26 Comparative analysis of the LPS biosynthetic loci of the genetic subtypes of serovar Hardjo: Leptospira interrogans subtype Hardjoprajitno and Leptospira borgpetersenii subtype Hardjobovis; de la Pena-Moctezuma A et al.; Although Leptospira borgpetersenii subtype Hardjobovis and L . interrogans subtype Hardjoprajitno belong to different species, they are serologically indistinguishable and are therefore classified as serovar Hardjo . Since LPS is the major antigen involved in serological classification, this implies that the LPS of these subtypes is identical . Comparison of the LPS biosynthetic loci (rfb) of the subtypes revealed remarkable similarity, with 32 and 31 origins of replication (orfs) in the Hardjoprajitno and Hardjobovis rfb loci, respectively . The order and orientation of these orfs were identical with the exception of an additional orf in Hardjoprajitno between orfs 4 and 5 and intergenic sequences differing between the subtypes . The Hardjoprajitno rfb locus has been divided into four intercalated regions based on sequence similarity to other leptospiral rfb loci . orfJ1-orfJ14 as well as orfJ21-orfJ22 are more similar to regions of the rfb locus of L . borgpetersenii subtype Hardjobovis . orfJ15-orfJ20 as well as orfJ23-orfJ31 are almost identical to the corresponding orfs in L . interrogans serovar Copenhageni . We propose that the progenitor Hardjoprajitno strain, containing an rfb locus which closely resembled the Copenhageni locus, acquired orfs 1-14 and orfs 21-22 from subtype Hardjobovis resulting in two serologically indistinguishable subtypes of serovar Hardjo which in turn constituted the main bovine-adapted leptospiral serovar. FEMS Microbiol Lett, 1999 Aug 15, 177(2), 231 - 5 Diaminodiphenylsulfone resistance of Mycobacterium leprae due to mutations in the dihydropteroate synthase gene; Kai M et al.; The nucleotide sequence analysis of the dihydropteroate synthase (DHPS) gene of six diaminodiphenylsulfone-resistant Mycobacterium leprae strains revealed that the mutation was limited at highly conserved amino acid residues 53 or 55 . Though the mutation at amino acid residue 55 or its homologous site has been reported in other bacteria, the mutation at residue 53 is the first case in bacteria . This is the first paper which links the mutations in DHPS and sulfonamide resistance in M . leprae . This finding is medically and socially relevant, since leprosy is still a big problem in certain regions. Mol Biol Cell, 1999 Sep, 10(9), 3045 - 59 The conserved core of a human SIR2 homologue functions in yeast silencing; Sherman JM et al.; Silencing is a universal form of transcriptional regulation in which regions of the genome are reversibly inactivated by changes in chromatin structure . Sir2 (Silent Information Regulator) protein is unique among the silencing factors in Saccharomyces cerevisiae because it silences the rDNA as well as the silent mating-type loci and telomeres . Discovery of a gene family of Homologues of Sir Two (HSTs) in organisms from bacteria to humans suggests that SIR2's silencing mechanism might be conserved . The Sir2 and Hst proteins share a core domain, which includes two diagnostic sequence motifs of unknown function as well as four cysteines of a putative zinc finger . We demonstrate by mutational analyses that the conserved core and each of its motifs are essential for Sir2p silencing . Chimeras between Sir2p and a human Sir2 homologue (hSir2Ap) indicate that this human protein's core can substitute for that of Sir2p, implicating the core as a silencing domain . Immunofluorescence studies reveal partially disrupted localization, accounting for the yeast-human chimeras' ability to function at only a subset of Sir2p's target loci . Together, these results support a model for the involvement of distinct Sir2p-containing complexes in HM/telomeric and rDNA silencing and that HST family members, including the widely expressed hSir2A, may perform evolutionarily conserved functions. Appl Environ Microbiol, 1999 Sep, 65(9), 4280 - 4 Detection of Verrucomicrobia in a pasture soil by PCR-mediated amplification of 16S rRNA genes; O'Farrell KA et al.; Oligonucleotide primers were designed and used to amplify, by PCR, partial 16S rRNA genes of members of the bacterial division Verrucomicrobia in DNA extracted from a pasture soil . By applying most-probable-number theory to the assay, verrucomicrobia appeared to contribute some 0.2% of the soil DNA . Amplified ribosomal DNA restriction analysis of 53 cloned PCR-amplified partial 16S rRNA gene fragments and comparative sequence analysis of 21 nonchimeric partial 16S rRNA genes showed that these primers amplified only 16S rRNA genes of members of the Verrucomicrobia in DNA extracted from the soil. Appl Environ Microbiol, 1999 Sep, 65(9), 4049 - 56 Fraction of electrons consumed in electron acceptor reduction and hydrogen thresholds as indicators of halorespiratory physiology; Loffler FE et al.; Measurements of the hydrogen consumption threshold and the tracking of electrons transferred to the chlorinated electron acceptor (f(e)) reliably detected chlororespiratory physiology in both mixed cultures and pure cultures capable of using tetrachloroethene, cis-1, 2-dichloroethene, vinyl chloride, 2-chlorophenol, 3-chlorobenzoate, 3-chloro-4-hydroxybenzoate, or 1,2-dichloropropane as an electron acceptor . Hydrogen was consumed to significantly lower threshold concentrations of less than 0.4 ppmv compared with the values obtained for the same cultures without a chlorinated compound as an electron acceptor . The f(e) values ranged from 0.63 to 0.7, values which are in good agreement with theoretical calculations based on the thermodynamics of reductive dechlorination as the terminal electron-accepting process . In contra