Microorganisms

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 contrast, a mixed methanogenic culture that cometabolized 3-chlorophenol exhibited a significantly lower f(e) value, 0.012.

J Cutan Pathol, 1999 Jul, 26(6), 271 - 8
Identification of mycobacterial DNA in cutaneous lesions of sarcoidosis; Li N et al.; Sarcoidosis is a multisystemic granulomatous disease of uncertain etiology . Recently, mycobacterial DNA especially Mycobacterium tuberculosis and Mycobacterium avium complex were detected in lung tissue and bronchial lavage fluid from patients with sarcoidosis by polymerase chain reaction (PCR) assays in 30% to 50% cases . Moreover, cell wall-defective form (CWDF) acid-fast bacteria have been isolated from skin lesions of patients with sarcoidosis which were later confirmed as M . avium complex by PCR assays . CWDF acid-fast bacteria were also found to grow from the blood of 95% patients with active sarcoidosis demonstrating a mycobacterial origin similar to M . tuberculosis . In view of these reports, we investigated 20 cases of cutaneous sarcoidosis using PCR/restriction enzyme pattern analysis (PCR/REPA) to detect mycobacterial DNA from paraffin-embedded skin biopsy samples . The method involves restriction enzyme analysis of nested PCR products obtained with primers encoding for the 65-KDa protein common to all mycobacteria . Using three restriction enzymes, the mycobacterial DNA from PCR product was differentiated to the species level . All the 20 cases had clinical and histologic evidence of sarcoidosis . Special stains for fungi (PAS) and mycobacteria (Fite) were negative and no foreign body was identified on polaroscopic examination in any of the cases . The cell lysates of M . tuberculosis, Mycobacterium bovis, Mycobacterium avium-intracellulare, Mycobacterium kansasii and Mycobacterium marinum from Centers for Disease Control (CDC) were used as standard control for PCR/REPA . Eight cases of foreign body granuloma, seven normal skin samples from the margin of surgical excisions and 5 cases of dermatitis were used as negative controls, and 4 cases of cutaneous tuberculosis were used as positive controls . Mycobacterial DNA was detected by PCR in 16 of the 20 cases of sarcoidosis . PCR/REPA subtyped 8 of these to M . tuberculosis complex (2 cases), M . avium-intracellulare (4 cases), M . kansasii (2 cases) while the other 8 cases were non-tuberculous mycobacteria . All four cases of cutaneous tuberculosis were positive by PCR and had a typical M . tuberculosis PCR/REPA pattern . Mycobacterial DNA was not detected in any of the negative controls . Our results demonstrated that mycobacterial DNA is present in 80% of cutaneous lesions of sarcoidosis and these mycobacteria may play a role in the pathogenesis of sarcoidosis.

Med Pr, 1999, 50(2), 163 - 77
{Chromium carcinogenicity}; Trocha M et al.; Chromium belongs to the group of trace elements hich are essential in numerous functions of the human body . Chromium deficiency may be responsible for various dysfunctions, whereas exposure to chromium at higher concentrations is toxic and may lead to the occurrence of neoplastic diseases . Epidemiological studies of chromium exposure proved its carcinogenity, and thus the IARC recognised Cr(VI) and its compounds as one of ascertained carcinogens . Some findings of these studies were reviewed in the first part of this work . The second part presents some molecular aspects of chromium carcinogenity which are still the subject of medical research . The direct and indirect effects of chromium and its compounds on DNA are analysed as are the relationships between the level of chromium oxidation and carcinogenity, and between the presence of reductants and the kind of DNA damage . Methods for the assessment of chromium mutagenity and genotoxicity are also discussed, and special attention is paid to tests of mutation in bacteria and yeast as well as to sister chromatid exchange (SCE) test.

Am J Respir Crit Care Med, 1999 Sep, 160(3), 942 - 9
Surfactant proteins A and D in premature baboons with chronic lung injury (Bronchopulmonary dysplasia) . Evidence for an inhibition of secretion; Awasthi S et al.; Surfactant proteins A and D (SP-A and SP-D) are believed to participate in the pulmonary host defense and the response to lung injury . In order to understand the effects of prematurity and lung injury on these proteins, we measured the amounts of SP-A and SP-D and their mRNAs in three groups of animals: (1) nonventilated premature baboon fetuses; (2) neonatal baboons delivered prematurely at 140 d gestation age (ga) and ventilated with PRN O(2); (3) animals of the same age ventilated with 100% O(2) to induce chronic lung injury . In nonventilated fetuses, tissue and lavage SP-A were barely detectable in baboons of 125 and 140 d ga, but they equaled or exceeded adult SP-A concentrations (g/g lung dry wt) at 175 d (term gestation, 185 d) . In contrast, SP-D was readily detectable in tissue and lavage at 125 and 140 d ga . When the baboons of 140 d ga were ventilated for 10 d with 100% oxygen to produce chronic lung injury, the tissue concentration of SP-A was five times greater than that of normal adults; SP-D 16-times greater . Despite the sizable tissue pools of SP-A and SP-D, however, lavage SP-A was only 7% of that of normal adults and lavage SP-D just equaled the amount in normal adults . Nevertheless, because SP-D is normally in much lower concentration than is SP-A, their total comprised less than 12% of the SP-A and SP-D found in the lavage of a healthy adult . The results indicate that in chronic lung injury, SP-A is significantly reduced in the alveolar space . SP-D concentration in lavage is about equal to that in normal adults, possibly because of the 16-fold excess in tissue, but the total collectin pool in lavage is still significantly reduced . Because these collectins may bind and opsonize bacteria and viruses, decrements in their amounts may present additional risk to those premature infants who require prolonged periods of ventilatory support.

Anticancer Res, 1999 May-Jun, 19(3A), 2127 - 31
Synthesis and antiproliferative activity of 2-triazenothiophenes; Diana P et al.; A series of 2-triazenothiophene derivatives was prepared and tested to evaluate their biological activity . Two compounds inhibited the proliferation of leukemia, lymphoma and solid tumor-derived cell lines at micromolar concentrations, whereas none of the compounds were active against HIV-1 . Compound 3c inhibited DNA, RNA and protein synthesis, and was also effective against KB cells resistant to etoposide and vincristine . The compounds were inactive against fungi and bacteria.

Curr Biol, 1999 Aug 26, 9(16), 915 - 8
Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon; Emmanouilidou E et al.; Regulated exocytosis involves the Ca(2+)-triggered fusion of secretory vesicles with the plasma membrane, by activation of vesicle membrane Ca(2+)-binding proteins {1} . The Ca(2+)-binding sites of these proteins are likely to lie within 30 nm of the vesicle surface, a domain in which changes in Ca2+ concentration cannot be resolved by conventional fluorescence microscopy . A fluorescent indicator for Ca2+ called a yellow 'cameleon' (Ycam2) - comprising a fusion between a cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13 and an enhanced yellow-emitting GFP - which is targetable to specific intracellular locations, has been described {2} . Here, we generated a fusion between phogrin, a protein that is localised to secretory granule membranes {3}, and Ycam2 (phogrin-Ycam2) to monitor changes in Ca2+ concentration ({Ca2+}) at the secretory vesicle surface ({Ca2+}gd) through alterations in fluorescence resonance energy transfer (FRET) between the linked cyan and yellow fluorescent proteins (CFP and YFP, respectively) in Ycam2 . In both neuroendocrine PC12 and MIN6 pancreatic beta cells, apparent resting values of cytosolic {Ca2+} and {Ca2+}(gd) were similar throughout the cell . In MIN6 cells following the activation of Ca2+ influx, the minority of vesicles that were within approximately 1 microm of the plasma membrane underwent increases in {Ca2+}(gd) that were significantly greater than those experienced by deeper vesicles, and greater than the apparent cytosolic {Ca2+} change . The ability to image both global and compartmentalised {Ca2+} changes with recombinant targeted cameleons should extend the usefulness of these new Ca2+ probes.

Dermatol Surg, 1999 Jun, 25(6), 492 - 3
Prefilled syringes: safe and effective; Melman D et al.; BACKGROUND: Preloaded syringes are time savers, but questions have arisen anecdotally about the risk of infection from this procedure and the possible loss of potency, especially when performed with buffered syringes . OBJECTIVE: To show that preloaded syringes do not develop colonies of bacterial organisms and to confirm that anesthetic potency is maintained for at least 2 weeks . METHODS: Thirty-six syringes were stored for a period of 2 weeks on a shelf in our clinical procedure area with no protection from heat or light . The majority of these were then cultured for bacteria and fungi and one of them was used on one of the authors to determine the potency of the anesthetic . RESULTS: Preloaded syringes do not appear to be prone to the development of bacterial contamination for at least a 2-week period and potency of the anesthetic is maintained . CONCLUSION: Preloaded syringes are time savers and are a safe modality for use in the practicing dermatology office.

Zentralbl Bakteriol, 1999 Jul, 289(3), 355 - 64
Adherence to and accumulation of S . epidermidis on different biomaterials due to extracellular slime production . In vitro comparison of a slime-producing strain (Rp 62 A) and its isogenic slime negative mutant (M7); Konig DP et al.; In an in vitro study, the bacterial adherence of a slime-producing strain (RP 62 A) was compared with its isogenic slime-negative mutant (M7) . Standardized biomaterial discs were incubated under growth conditions in tryptic soy broth containing either strain RP 62 A or M7 . After 24 h of incubation, the attached bacteria were removed by sonication and the colony-forming units were counted after plating of serial dilutions . We observed a significantly increased adherence and accumulation of the slime-producing strain (RP 62 A) . In contrast to the slime negative mutant (M7) (p = 0.0001) . The highest colony counts were found for the slime-producing strain on polyethylene and polymethylmethacrylate . The slime-negative mutant lacked the ability of accumulation . Our in-vitro results show the relevance of slime production by S . epidermidis for in-vitro colonisation of biomaterials, with a preference for polyethylene and polymethylmethacrylate.

Zentralbl Bakteriol, 1999 Jul, 289(3), 301 - 18
Persistence of Borrelia garinii and Borrelia afzelii in patients with Lyme arthritis; Hulinska D et al.; We repeatedly detected DNA of Borrelia garinii or B . afzelii and Borrelia-like structures in the blood, joint fluid or in the synovium of 10 patients with Lyme arthritis by means of the polymerase chain reaction and immunoelectron microscopy at 2-4-month intervals in the course of two years . All samples were analyzed using primers which amplified the 16S rRNA gene sequence of Borrelia burgdorferi sensu lato and nucleotide sequences for the OspA gene . No cross hybridization occurred with DNA from human cells and with DNA from other bacteria . Capture and labelling with monoclonal antibodies of aggregated antigens, membranes and flagellae were evident in the blood of 7 patients, in 4 synovial membranes and 2 synovial fluids . Borreliae were found in blood capillaries, in collagen and in clusters surrounding inflammatory cells in the synovium of patients with recurrent infections who carried IgM and IgG antibodies to OspA and to 83 kDa core protein . After significant improvement for several weeks after treatment, arthritis recurred in six patients . Synoviocyte hyperplasia, inflammatory infiltration and concentric adventitial fibroplasia were seen in the synovium of the patients with persisting borreliae . Only two patients were infected with B . afzelii, the others with B . garinii.

Insect Mol Biol, 1999 Aug, 8(3), 399 - 408
Phylogeny of the arthropod endosymbiont Wolbachia based on the wsp gene; Van Meer MM et al.; Bacteria of the genus Wolbachia (Rickettsiae) are widespread in arthropods and can induce cytoplasmic incompatibility (CI), thelytoky (T) or feminization (F) in their host . Recent research on the wsp gene of mainly CI inducing Wolbachia has shown that this gene evolves at a much faster rate than previously sequenced genes such as 16S or ftsZ . As a result this gene appears to be very useful in subdividing the Wolbachia and twelve groups have been distinguished to date . Here we extend the Wolbachia wsp data set with fifteen T-Wolbachia, one F-Wolbachia and three other CI-Wolbachia strains . The results showed: (i) the addition of seven groups; (ii) no relation between host phenotype and Wolbachia phylogenetic position; and (iii) possible horizontal Wolbachia transfer between the moth Ephestia kuehniella and its parasitoid Trichogramma spp.

Insect Mol Biol, 1999 Aug, 8(3), 329 - 37
An easter-like serine protease from Anopheles gambiae exhibits changes in transcript abundance following immune challenge; Paskewitz SM et al.; The nucleotide and deduced amino acid sequence of a serine protease (AgSp14D1) from the human malaria vector, Anopheles gambiae, is presented . The gene product is a 360 amino acid protein that contains two domains and has the highest sequence similarity to the Drosophila melanogaster serine protease easter and to prophenol oxidase activating enzyme (pPAE) from Manduca sexta . The catalytic domain is at the carboxy terminus and has the conserved serine, histidine and aspartic acid residues found in serine proteases as well as six cysteines common to invertebrate enzymes . The amino terminus contains critical cysteines that define a clip (=disulphide knot) domain which places this gene product in a subfamily of regulatory serine proteases that includes not only easter and pPAE but also the Drosophila proteins masquerade, stubble and snake as well as proclotting enzyme and factor B from the horseshoe crab . In situ hybridization to the polytene chromosomes detects a single band at 14D and Southern analysis with a probe from the 5' end of the gene confirms the single copy status of this gene . Northern analysis reveals changes in transcript abundance during development and following blood feeding . Interestingly, this analysis also shows an increase in transcript levels following wounding or injection of bacteria.

Helicobacter, 1999 Sep, 4(3), 162 - 9
Role of Hpn and NixA of Helicobacter pylori in susceptibility and resistance to bismuth and other metal ions; Mobley HL et al.; BACKGROUND: Helicobacter pylori produces Hpn, a 60-amino acid, histidine-rich protein that avidly binds nickel and zinc ions, and NixA, a high-affinity nickel transporter in the cytoplasmic membrane . We tested the hypothesis that Hpn and NixA govern susceptibility to metal ions in H . pylori . MATERIALS AND METHODS: Hpn-negative mutants of four H . pylori strains were constructed by standard allelic exchange techniques to yield isogenic Hpn+/Hpn-deficient pairs . A metal concentration that inhibited growth by 50% (IC50) was calculated for Ni2+, Zn2+, Cu2+, and Co2+ by comparing OD600 of cultures in metal-supplemented and control media . RESULTS: Among all four pairs of isogenic strains, the tolerance for Ni2+ was reduced significantly (p <.001) in the Hpn mutants; the mean IC50 value for wild-type strains was 1.9 mM; for the mutant, it was 0.8 mM . In contrast, growth inhibition by Zn2+ was identical within the fours pairs, as was Cu2+ and Co2+ tolerance in one pair tested . We also found that deletion of the hpn gene increases susceptibility to therapeutic forms of bismuth by testing a mutant and wild-type pair with ranitidine bismuth citrate, bismuth citrate, and four antibiotics . Minimal inhibitory concentrations of ranitidine bismuth citrate dropped from 9.2 to 2.3 microg/ml, and those of bismuth citrate dropped from 7.4 to 3.2 microg/ml (p <.05 for both comparisons), while susceptibility to the antibiotics was unaffected . Disruption of the nixA gene encoding the specific Ni2+ transport protein of H . pylori did not change susceptibility to bismuth . CONCLUSION: We concluded that bacteria lacking Hpn, cultured in vitro, are more susceptible than is the wild type to bismuth and Ni2+.

Clin Exp Immunol, 1999 Sep, 117(3), 596 - 604
Spleen autotransplantation provides restoration of functional splenic lymphoid compartments and improves the humoral immune response to pneumococcal polysaccharide vaccine; Leemans R et al.; After splenectomy, patients have an increased risk of overwhelming post-splenectomy infection (OPSI) or sepsis involving encapsulated bacteria such as pneumococcus . The value of spleen autotransplantation after splenectomy because of trauma has long been questioned . Much attention has been given to the restoration of mononuclear phagocyte system (MPS) function, which appeared to be similar to that of splenectomized individuals . The presence of specific anti-pneumococcal antibodies may enhance phagocytosis of opsonized bacteria by other parts of the MPS, as present in the liver . Therefore, in the present study we have evaluated the restoration of the humoral immune response after spleen autotransplantation, especially to pneumococcal capsular polysaccharides (PPS) . Wistar rats were divided into three groups which were operated as follows: splenectomy, splenectomy followed by autotransplantation, and sham operation . After 12 weeks the rats were vaccinated with 23-valent pneumococcal vaccine . Blood samples were taken after 3 days, 3 and 6 weeks for anti-PPS IgM and IgG ELISA against types 3, 4, 6, 9, 14 and 23 . In addition, immunohistological studies were performed on the autotransplants . Significant antibody titre rises were found in a main proportion of the autotransplanted rats, comparable to sham-operated rats . Splenectomized rats showed as well a significantly lower increase in immunoglobulin levels, as significant differences in the proportion of rats showing a minimum two-fold increase of antibody level, considered to represent an adequate response . The titres were highest 3 days after vaccination . Immunohistochemical studies demonstrated structurally functional autotransplants, including an intact marginal zone . Considering this significant anti- pneumococcal antibody response, spleen autotransplants can be expected to enable an improved humoral response to PPS, and to contribute to protection against OPSI after splenectomy.

Proc Natl Acad Sci U S A, 1999 Aug 31, 96(18), 10134 - 9
Rotational symmetry of the C ring and a mechanism for the flagellar rotary motor; Thomas DR et al.; FliG, FliM, and FliN, key proteins for torque generation, are located in two rings . The first protein is in the M ring and the last two are in the C ring . The rotational symmetries of the C and M rings have been determined to be about 34 (this paper) and 26 (previous work), respectively . The mechanism proposed here depends on the symmetry mismatch between the rings: the C ring extends 34 levers, of which 26 can bind to the 26 equivalent sites on the M ring . The remaining 8 levers bind to proton-pore complexes (studs) to form 8 torque generators . Movement results from the swapping of stud-bound levers with M ring-bound levers . The model predicts that both the M and C rings rotate in the same direction but at different speeds.

Proc Natl Acad Sci U S A, 1999 Aug 31, 96(18), 10056 - 61
Mutational analysis of a higher plant antenna protein provides identification of chromophores bound into multiple sites; Bassi R et al.; The chromophore-binding properties of the higher plant light-harvesting protein CP29 have been studied by using site-directed mutagenesis of pigment-binding residues . Overexpression of the apoproteins in bacteria was followed by reconstitution in vitro with purified pigments, thus obtaining a family of mutant CP29 proteins lacking individual chromophore-binding sites . Biochemical characterization allowed identification of the eight porphyrins and two xanthophyll-binding sites . It is shown that the four porphyrin-binding sites (A1, A2, A4, and A5) situated in the central, twofold-symmetrical domain of the protein are selective for Chl-a, whereas the four peripheral sites (A3, B3, B5, and B6) have mixed Chl-a-Chl-b specificity . Within a site, porphyrin coordination by glutamine increases affinity for Chl-b as compared with glutamate . Xanthophyll site L1 is occupied by lutein, whereas site L2 can bind violaxanthin or neoxanthin . The protein is relatively stable when site L2 site is empty, suggesting that xanthophylls can be exchanged during operation of xanthophyll cycle-dependent photoprotection mechanism . Differential absorption spectroscopy allowed determination of transition energy levels for individual chromophores, thus opening the way to calculation of energy-transfer rates between Chl in higher plant antenna proteins.

Pharm Res, 1999 Aug, 16(8), 1300 - 8
Controlled DNA delivery systems; Luo D et al.; PURPOSE: Genes are of increasing interest as pharmaceuticals, but current methods for long-term gene delivery are inadequate . Controlled release systems using biocompatible and/or biodegradable polymers offer many advantages over conventional gene delivery approaches . We have characterized systems for controlled delivery of DNA from implantable polymer matrices (EVAc: poly (ethylene-co-vinyl acetate)) and injectable microspheres (PLGA and PLA: poly (D, L-lactide-co-glycolide) copolymer and poly (L-lactide), respectively) . METHODS: Herring sperm DNA and bacteria phage lambda DNA were encapsulated as a model system . Released DNA concentration was determined by fluoroassays . Agarose electrophoresis was used to determine the dependence of release rate on DNA size . The Green Fluorescent Protein (GFP) gene was used to determine the integrity and functionality of released DNA . RESULTS: Both small and large DNA molecules (herring sperm DNA, 0.1-0.6 kb; GFP, 1.9 kb; lambda DNA, 48.5 kb) were successfully encapsulated and released from EVAc matrices, and PLGA or PLA microspheres . The release from DNA-EVAc systems was diffusion-controlled . When co-encapsulated in the same matrix, the larger lambda DNA was released more slowly than herring sperm; the rate of release scaled with the DNA diffusion coefficient in water . The chemical and biological integrity of released DNA was not changed . CONCLUSIONS: These low cost, and adjustable, controlled DNA delivery systems, using FDA-approved biocompatible/biodegradable and implantable/injectable materials, could be useful for in vivo gene delivery, such as DNA vaccination and gene therapy.

Wien Klin Wochenschr, 1999 Jul 30, 111(14), 539 - 48
{Intestinal ischemic reperfusion syndrome: pathophysiology, clinical significance, therapy}; Schwarz B et al.; Gut ischemia-reperfusion injury is a serious condition in intensive care patients . Activation of immune cells within the huge endothelial surface area of gut microcirculation may initiate a systemic inflammatory response with secondary injury to distant organs . Translocation of bacteria and toxins through a leaky gut mucosa may amplify or perpetuate systemic inflammation, leading to multiple organ dysfunction syndrome and death in critically ill patients . Gut ischemia promotes regional production of inflammatory mediators, expression of cell adhesion molecules on endothelial and immune cell surfaces and increases the procoagulatory properties of vascular endothelial cells . During reperfusion, gut injury may be amplified by increased production of oxygen radicals and exhaustion of endogenous antioxidant defence mechanisms . Although several therapeutic strategies to interrupt the pathophysiology of ischemia-reperfusion have been shown to be beneficial in animal experiments, none of these interventions has gained clinical relevance . After initial hemodynamic and respiratory stabilisation of critically ill patients, strategies to prevent secondary gut injury by increasing splanchnic oxygen delivery or augment mucosal cell regeneration may be the only therapeutic options for intensive medical specialists at the present time . Early enteral nutrition and treatment with specific vasoactive drugs may reduce morbidity and costs of treatment in certain critically ill patients . However definitive evidence of a reduction in mortality with these therapies has still not be provided.

Structure Fold Des, 1999 Aug 15, 7(8), 909 - 17
Protein, lipid and water organization in bacteriorhodopsin crystals: a molecular view of the purple membrane at 1.9 A resolution; Belrhali H et al.; BACKGROUND: Bacteriorhodopsin (bR) from Halobacterium salinarum is a proton pump that converts the energy of light into a proton gradient that drives ATP synthesis . The protein comprises seven transmembrane helices and in vivo is organized into purple patches, in which bR and lipids form a crystalline two-dimensional array . Upon absorption of a photon, retinal, which is covalently bound to Lys216 via a Schiff base, is isomerized to a 13-cis,15-anti configuration . This initiates a sequence of events - the photocycle - during which a proton is transferred from the Schiff base to Asp85, followed by proton release into the extracellular medium and reprotonation from the cytoplasmic side . RESULTS: The structure of bR in the ground state was solved to 1.9 A resolution from non-twinned crystals grown in a lipidic cubic phase . The structure reveals eight well-ordered water molecules in the extracellular half of the putative proton translocation pathway . The water molecules form a continuous hydrogen-bond network from the Schiff-base nitrogen (Lys216) to Glu194 and Glu204 and includes residues Asp85, Asp212 and Arg82 . This network is involved both in proton translocation occurring during the photocycle, as well as in stabilizing the structure of the ground state . Nine lipid phytanyl moieties could be modeled into the electron-density maps . Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of single crystals demonstrated the presence of four different charged lipid species . CONCLUSIONS: The structure of protein, lipid and water molecules in the crystals represents the functional entity of bR in the purple membrane of the bacteria at atomic resolution . Proton translocation from the Schiff base to the extracellular medium is mediated by a hydrogen-bond network that involves charged residues and water molecules.

Structure Fold Des, 1999 Aug 15, 7(8), 903 - 8
A model for the incorporation of metal from the copper chaperone CCS into Cu,Zn superoxide dismutase; Falconi M et al.; BACKGROUND: Recent studies have identified the human copper chaperone CCS as the presumed factor responsible for copper incorporation into superoxide dismutase (SOD) . A lack of knowledge of the chaperone's three-dimensional structure has prevented understanding of how the copper might be transferred . RESULTS: The three-dimensional structure of CCS was homology modelled using the periplasmic protein from the bacterial mercury-detoxification system and the structure of one subunit of the human SOD dimeric enzyme as templates . On the basis of the three-dimensional model, a mechanism for the transfer of copper from CCS to SOD is proposed that accounts for electrostatic acceptor recognition, copper storage and copper-transfer properties . CONCLUSIONS: The proposed model identifies a path for copper transfer based on the presence of different metal sites characterized by sulphur ligands . Such a model permits the development of strategies able to interfere with copper incorporation in SOD, providing a possible way to prevent or arrest degeneration in the fatal motor neuron disorder amyotrophic lateral sclerosis.

Vet Rec, 1999 Jul 31, 145(5), 123 - 9
Survey of infectious agents involved in acute respiratory disease in finishing pigs; Loeffen WL et al.; Outbreaks of respiratory disease constitute a major health problem in herds of finishing pigs and their aetiology often remains unclear . In this study, 16 outbreaks of respiratory disease with acute clinical signs in finishing pigs were investigated to determine which infectious agents were involved . From each herd four diseased and two clinically healthy pigs were examined pathologically and for the presence of viruses, bacteria and mycoplasmas . In addition, paired blood samples from 10 groupmates of the diseased pigs were tested for antibodies against commonly known causal agents of respiratory disease . A clear diagnosis was possible in 12 of the 16 outbreaks . Seven were due to an infection with influenza virus and five were due to an infection with Actinobacillus pleuropneumoniae . A combination of influenza virus and A pleuropneumoniae may have caused one other outbreak, but no clear cause could be established for the other three outbreaks.

J Biol Chem, 1999 Sep 3, 274(36), 25827 - 32
Cobalt-dependent transcriptional switching by a dual-effector MerR-like protein regulates a cobalt-exporting variant CPx-type ATPase; Rutherford JC et al.; CoaR associates with and confers cobalt-dependent activation of the coaT operator-promoter . A CoaR mutant (Ser-Asn-Ser) in a carboxyl-terminal Cys-His-Cys motif bound the coaT operator-promoter but did not activate expression in response to cobalt, implicating thiolate and/or imidazole ligands at these residues in an allosteric cobalt binding site . Deletion of 1 or 2 nucleotides from between near consensus, but with aberrant (20 base pairs) spacing, -10 and -35 elements enhanced expression from the coaT operator-promoter but abolished activation by cobalt-CoaR . It is inferred that cobalt effects a transition in CoaR that underwinds the coaT operator-promoter to realign promoter elements . In the absence of cobalt, CoaR represses expression (approximately 50%) . CoaR is a fusion of ancestral MerR (mercury-responsive transcriptional activator)- and precorrin isomerase (enzyme of vitamin B(12) biosynthesis)-related sequences . Expression from the coaT operator-promoter was enhanced in a partial mutant of cbiE (encoding an enzyme preceding precorrin isomerase in B(12) biosynthesis), revealing that this pathway "inhibits" coaT expression . Disruption of coaT reduced cobalt tolerance and increased cytoplasmic (57)Co accumulation . coaT-mediated restoration of cobalt tolerance has been used as a selectable marker.

J Biol Chem, 1999 Sep 3, 274(36), 25281 - 4
The proteolipid of the A(1)A(0) ATP synthase from Methanococcus jannaschii has six predicted transmembrane helices but only two proton-translocating carboxyl groups; Ruppert C et al.; The proteolipid, a hydrophobic ATPase subunit essential for ion translocation, was purified from membranes of Methanococcus jannaschii by chloroform/methanol extraction and gel chromatography and was studied using molecular and biochemical techniques . Its apparent molecular mass as determined in SDS-polyacrylamide gel electrophoresis varied considerably with the conditions applied . The N-terminal sequence analysis made it possible to define the open reading frame and revealed that the gene is a triplication of the gene present in bacteria . In some of the proteolipids, the N-terminal methionine is excised . Consequently, two forms with molecular masses of 21,316 and 21,183 Da were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry . The molecular and biochemical data gave clear evidence that the mature proteolipid from M . jannaschii is a triplication of the 8-kDa proteolipid present in bacterial F(1)F(0) ATPases and most archaeal A(1)A(0) ATPases . Moreover, the triplicated form lacks a proton-translocating carboxyl group in the first of three pairs of transmembrane helices . This finding puts in question the current view of the evolution of H(+) ATPases and has important mechanistic consequences for the structure and function of H(+) ATPases in general.

J Bacteriol, 1999 Sep, 181(17), 5234 - 41
Analysis of BvgA activation of the pertactin gene promoter in Bordetella pertussis; Kinnear SM et al.; Bordetella pertussis, the causative agent of whooping cough, regulates expression of its virulence factors via a two-component signal transduction system encoded by the bvg regulatory locus . It has been shown by activation kinetics that several of the virulence factors are differentially regulated . fha is transcribed at 10 min following an inducing signal, while ptx is not transcribed until 2 to 4 h after the inducing signal . We present data indicating that prn is transcribed at 1 h, an intermediate time compared to those of fha and ptx . We have identified cis-acting sequences necessary for expression of prn in B . pertussis by using prn-lac fusions containing alterations in the sequence upstream of the prn open reading frame . In vitro transcription and DNase I footprinting analyses provided evidence to support our hypothesis that BvgA binds to this sequence upstream of prn to activate transcription from the promoter . Our genetic data indicate that the region critical for prn activation extends upstream to position -84 . However, these data do not support the location of the prn transcription start site as previously published . We used a number of methods, including prn-lac fusions, reverse transcriptase PCR, and 5' rapid amplification of cDNA ends, to localize and identify the bvg-dependent 5' end of the prn transcript to the cytosine at -125 with respect to the published start site.

Scand J Clin Lab Invest, 1999 Jul, 59(4), 279 - 87
Phospholipase A2 in serum and colonic mucosa in ulcerative colitis; Haapamaki MM et al.; Group II phospholipase A2 is involved in the pathogenesis of various inflammatory diseases and in the host defence against bacteria . The enzyme is expressed in the epithelial cells of colonic mucosa in ulcerative colitis . In this study, we measured the concentration of group II phospholipase A2 in serum and colonic mucosa of patients with ulcerative colitis of different severity and of control patients without any inflammatory disease . The activity of ulcerative colitis was assessed by endoscopy . The concentration of group II phospholipase A2 was measured with an immunoassay . The concentrations of group II phospholipase A2 in serum and colonic mucosa were significantly higher in patients with active and inactive ulcerative colitis than in controls . However, the group II phospholipase A2 levels did not separate patients with different disease activity . The concentration of group II phospholipase A2 in colonic mucosa corresponded with the mucosal inflammatory activity (higher in active colonic areas) intra-individually, but not between different patients with ulcerative colitis . Serum group II phospholipase A2 values were above the normal reference range more often than the values of 11 standard laboratory blood tests widely used for the follow-up of inflammatory activity in ulcerative colitis . These results indicate that the concentration of group II phospholipase A2 is increased in serum and colonic mucosa of patients with ulcerative colitis . The clinical value of the measurement of group II phospholipase A2 in the follow-up of ulcerative colitis remains to be clarified.

Ann Acad Med Stetin, 1999, Suppl 47, 1 - 89
{Factors that modify de- and remineralization in dental enamel from the aspect of caries susceptibility}; Buczkowska-Radlinska J; The course of caries depends on an equilibrium between de- and remineralization of enamel . Epidemiologic studies performed during recent years in Poland have demonstrated a high incidence and severity of caries in children and teenagers . Therefore, the actual program of caries prevention covering the whole population of children and teenagers has not performed to the expectations, possibly because the program protocol does not discriminate between children susceptible and resistant to caries . The aims of this work included: 1 . Analysis of tooth groups and surfaces as to their susceptibility to caries; 2 . Evaluation of the influence of de- and remineralization modifying factors such as: a) dental plaque and sugars, b) caries-causing bacteria, c) enamel resistance to acids, d) biochemical properties of saliva--on the susceptibility to caries; 3 . Evaluation of the uptake of fluorides from chewing gum by enamel in relationship to caries susceptibility . An initial clinical examination of permanent teeth was performed in 292 children aged 12 years . Subsequently, two groups were formed: one with 45 children free from caries and another with 77 children at high risk of caries . Control examinations of permanent teeth in both groups were carried out after one and three years from the initial examination . The clinical examination included a check-up of secondary dentition, counting of erupted teeth, sealed teeth, evaluation of oral hygiene status, gingivae, abnormalities in mineralization and measurement of the rate of microdemineralization . The biochemical analysis of saliva was done to measure the content of fluorides, calcium, phosphorus and magnesium, as well as pH . Microsamples of enamel were collected using acid biopsy and the content of calcium and fluorine in the superficial and underlying layer was assayed . The thickness of both layers obtained by biopsy was also measured . Commercial test kits--Dentocult SM and Dentocult LB, served to determine the amount of caries-causing bacteria in saliva . Additionally, the children and their mothers responded to a questionnaire on the diet, previous diseases, therapies with antibiotics and health habits . The results were analysed using Statgraphics software . Differences between means were checked using Student t-test in its classical form or as modified by B . L . Welch . Linear correlation indices were calculated and Spearman rank correlation test was performed . On the basis of the results obtained the following conclusions were drawn: 1 . 12-year old children at high risk of caries need intense dental care due to: a) extensive involvement of masticatory surfaces of molar teeth and contact surfaces of premolar teeth, b) treatment inappropriate to actual needs in this group of children . 2 . Effective programs to improve the knowledge and health of children at high risk to caries should be introduced due to the following facts: a) oral hygiene was unsatisfactory in the examined children, b) children consumed large quantities of sweets . 3 . Disturbances in the mineralization of enamel and the use of antibiotics in childhood were without influence on the susceptibility of teeth to caries . 4 . The content of fluorides, calcium and phosphorus in the saliva of children susceptible to caries could have been below the level necessary to maintain an equilibrium between de- and remineralization of enamel . 5 . Susceptibility of teeth to caries could be due to differences in the chemical composition and structure of enamel, as it appears from the content of fluorides and calcium, depth of enamel biopsy and uptake of fluorides from chewing gum . 6 . Chewing of fluoride-containing gums is a supportive measure in caries prevention, particularly recommended in children at high risk of caries . 7 . Caries prevention should be individualized and matched to caries risk.

J Cell Biochem, 1999 Oct 1, 75(1), 147 - 59
Flotillin 2 is distinct from epidermal surface antigen (ESA) and is associated with filopodia formation; Hazarika P et al.; ECS-1, a monoclonal antibody (MoAb) raised to cultured human keratinocytes, stains the intercellular glycocalyx with a pemphigus-like pattern and recognizes a 35-kDa epidermal surface antigen (ESA) on Western blotting of keratinocyte extracts . When ECS-1 MoAb was used to screen a keratinocyte expression library, a unique cDNA was identified that predicted a 42-kDa globular protein of unknown function . This putative ESA was conserved between mice and humans and was encoded by a gene on chromosome 17q11-12 in linkage with neurofibromin . Homology between the cDNA sequence has been reported with flotillin 1, a caveolae associated protein, as well as Reggie 1 and 2, neuronal proteins expressed during axonal regeneration present in activated GPI-anchored cell adhesion molecules in non-caveolar-associated micropatches . In order to determine whether the cDNA predicted protein and ECS-1 antigen were identical, we compared ECS-1 with the immunoreactivity of a new antibody raised to the cDNA fusion protein in epidermis and cultured cells . The cDNA fusion protein was expressed in bacteria and in cos cells with his, FLAG, and EGFP reporter tags and by stable transfection as an EGFP fusion protein . The fusion protein and native protein of 42 kDa were detected by the new antibody, but not by the original ECS-1 . Thus, the ECS-1 antigen, ESA (35 kDa), is clearly distinct from the protein predicted by the cDNA (renamed flotillin 2) . Stable transfection of ESA/flotillin 2 fusion protein in cos cells induced filopodia formation and changed epithelial cells to a neuronal appearance . Thus, the function of flotillin 2 may resemble that of the goldfish optic nerve neuronal regeneration proteins, Reggie 1 and 2 .

Biochem Biophys Res Commun, 1999 Aug 27, 262(2), 549 - 56
trans-sialidase of Trypanosoma cruzi: location of galactose-binding site(s); Chuenkova M et al.; Trypanosoma cruzi expresses a trans-sialidase on its surface, which catalyzes the transfer of sialic acid from mammalian host glycans to its own surface glycoproteins . It has been proposed that the enzyme consists of three domains prior to a long C-terminal repeating sequence that is not required for enzyme activity . The first of these domains shares significant sequence identity with bacterial sialidases which catalyse the hydrolysis of sialic acid . Here we report the sequence of the N-terminal domains of the TS19y trans-sialidase gene, which was expressed in bacteria with the same specific activity as natural enzyme of T . cruzi . Various deletion mutants of TS19y, without the C-terminal tandem repeat, have been cloned and expressed and their trans-sialidase and sialidase activities measured . These experiments show that all three N-terminal domains are required for full trans-sialidase activity, though only the first is necessary for sialidase activity . Some transferase activity is observed, however, even with the shortest construct comprising the first N-terminal domain . Deletion mutants to probe the role of the N-terminal residues of the first domain suggest that the first 33 residues are also required for trans-sialidase activity, but not for sialidase activity . Molecular modelling of the first N-terminal domain of TS19y based on our structures of bacterial sialidases and site-directed mutations suggests the location of a galactose-binding site within this domain .

J Pept Res, 1999 Aug, 54(2), 168 - 73
Convenient preparation of {Orn(Tfa)2}- and {Orn(Boc)2, Orn(Tfa)2}gramicidin S, versatile unsymmetrically protected derivatives of gramicidin S; Yamada K et al.; Treatment of gramicidin S (GS) with trifluoroacetic anhydride afforded a derivative in which only one of the two Orn side chains was trifluoroacetylated in 72% yield, furnishing the first efficient method for the preparation of a monoprotected derivative of GS . The mono(Tfa) derivative {Orn(Tfa)2'}GS was treated with di-tert-butyl dicarbonate to yield dually protected derivative {Orn(Boc)2,Orn(Tfa)2'}GS from which another monoprotected derivative {Orn(Boc)2}GS was prepared in high yield . These unsymmetrically protected GS derivatives are versatile starting materials for the preparation of various other GS derivatives . As an example of application of the unsymmetrically protected derivatives, a dimeric GS derivative was prepared via a singly p-nitrobenzenesulfonyl(NBS)-activated derivative {Orn(Boc)2,Orn(NBS)2'}GS.

Korean J Intern Med, 1999 Jul, 14(2), 9 - 14
Effect of eradication of Helicobacter pylori on the benign gastric ulcer recurrence--a 24 month follow-up study; Kim N et al.; OBJECTIVES: To evaluate the effect of eradication of Helicobacter pylori (H . pylori) on the recurrence of benign gastric ulcer (BGU) in the patients with BGU . METHODS: This study was performed for 40 H . pylori-positive BGU patients cured of BGU and H . pylori eradicated, and for 25 H . pylori-positive patients (non-eradicated group) who were not treated with H . pylori eradication regimen or H . pylori was not eradicated . Four different methods--CLOtest, microscopy of Gram stained mucosal smear, culture and histology of modified Giemsa staining--were taken for identifying colonization of H . pylori before treatment, and 4 weeks after completion of triple therapy . For the control group in which triple therapy was not tried, follow-up gastroscopy was done to confirm the healing of the ulcer . To detect BGU recurrence, the gastroscopy was performed at 6, 12, 18, and 24 months after therapy . RESULTS: In the non-eradicated group, the BGU recurrence rate was 16% within 6 months, 40% within 1 year, 56% within 18 months and 60% within 2 years . The respective recurrence rates in the 40 patients in whom the bacteria had been eradicated were 0%, 7.5%, 10% and 10% (4 patients), respectively . Among the four BGU-recurred patients in whom H . pylori had been eradicated, one patient was found to have BGU recurring with H . pylori positive again in one year, and another two patients had NSAIDs ingestion history . CONCLUSION: The eradication of H . pylori in patients with BGU reduces the recurrence of BGU . In addition, the major causes of BGU recurrence look like NSAIDs ingestion and reinfection of H . pylori.

Appl Microbiol Biotechnol, 1999 Jul, 52(1), 127 - 30
Bioassays for risk assessment of coal conversion products; Schacht S et al.; Traditional as well as biotechnological processing of coal leads to complex mixtures of products . Besides chemical and physical characterization, which provides the information for product application, there is a need for bioassays to monitor properties that are probably toxic, mutagenic or cancerogenic . Investigations carried out focused on the selection, adaptation and validation of bioassays for the sensitive estimation of toxic effects . Organisms like bacteria, Daphnia magna and Scenedesmus subspicatus, representing different complexities in the biosphere, were selected as test systems for ecotoxicological and mutagenicity studies . The results obtained indicate that bioassays are, in principle, suitable tools for characterization and evaluation of coal-derived substances and bioconversion products . Using coal products, coal-relevant model compounds and bioconversion products, data for risk assessment are presented.

Semin Pediatr Surg, 1999 Aug, 8(3), 148 - 54
Nitric oxide and intestinal barrier failure; Nadler EP et al.; The systemic inflammatory response syndrome (SIRS) is a leading cause of morbidity and mortality in adults and children . Various proinflammatory mediators have been implicated in the pathogenesis of SIRS; however, their mechanisms of action are poorly defined . Recent evidence suggests that nitric oxide (NO) plays a regulatory role in gut barrier function . Sustained upregulation of NO production in the intestine can lead to intestinal epithelial injury through the formation of peroxynitrite . Peroxynitrite can nitrate mitochondrial proteins and inhibit cellular respiration . The resultant changes in mitochondrial function lead to activation of the caspase cascade, subsequent DNA fragmentation, and enterocyte apoptosis . Enterocyte apoptosis results in a transient "bare area" in the intestinal epithelium where bacteria can attach and then penetrate the lamina propria . Bacteria that successfully escape the immune system may in turn incite a systemic inflammatory response.

Arch Microbiol, 1999 Sep, 172(3), 175 - 81
Purification of cold-shock-like proteins from Stigmatella aurantiaca - molecular cloning and characterization of the cspA gene; Stamm I et al.; Prominent low-molecular-weight proteins were isolated from vegetative cells of the myxobacterium Stigmatella aurantiaca and were found to be members of the cold-shock protein family . A first gene of this family (cspA) was cloned and sequenced . It encodes a protein of 68 amino acid residues that displays up to 71% sequence identity with other bacterial cold-shock(-like) proteins . A cysteine residue within the RNP-2 motif is a peculiarity of Stigmatella CspA . A cspA::(Deltatrp-lacZ) fusion gene construct was introduced into Stigmatella by electroporation, a method that has not been used previously for this strain . Analysis of the resultant transformants revealed that cspA transcription occurs at high levels during vegetative growth at 20 and 32 degrees C, and during fruiting body formation.

Drug Metab Dispos, 1999 Sep, 27(9), 966 - 71
N-acetylation of the heterocyclic amine batracylin by human liver; Stevens GJ et al.; Batracylin (8-aminoisoindolo{1,2-b}quinazolin-12(10 H)-one; BAT) is a heterocyclic amine that exhibits antitumor activity in a number of in vivo and in vitro models . The acetyl product has been implicated in BAT toxicity in animals, cells, and bacteria . The ability of human N-acetyltransferase (NAT) to form this product was investigated . Nine human liver samples were analyzed for NAT1 and NAT2 genotypes . Seven of the samples possessed at least one NAT1*4 allele . Three samples contained one or more NAT2*4 allele and were classified as rapid acetylators . The remaining six had two alleles associated with the slow phenotype . NAT activities were evaluated with BAT, sulfamethazine (SMZ), a preferential substrate for human NAT2, and p-aminobenzoic acid, a substrate for NAT1 . BAT activities in the nine donor samples ranged from 14.9 to 0.56 nmol/min/mg . The mean apparent K(m) values in rapid acetylators for BAT, SMZ, and p-aminobenzoic acid were 6.59 +/- 3.21, 278 +/- 69.4, and 31.2 +/- 12.5 microM, respectively . The apparent K(m) values for slow acetylators did not differ from the rapid acetylator phenotype . However, a significant difference in the apparent V(max) for BAT and SMZ was observed between rapid and slow acetylators . Comparing the apparent intrinsic clearance (V(max)/K(m)) for BAT and SMZ, a significant correlation (r(2) = 0.97, p <.001) was observed . These data demonstrate that BAT N-acetylation is similar to SMZ, and suggests that BAT is a preferential substrate for human NAT2 . Thus, rapid acetylators would be more likely to develop toxicity when exposed to this drug.

Med Hypotheses, 1999 Jun, 52(6), 529 - 38
Cysteine, glutathione (GSH) and zinc and copper ions together are effective, natural, intracellular inhibitors of (AIDS) viruses; Sprietsma JE; Sufficient essential nutrients such as methionine, cysteine, copper, selenium, zinc and vitamins C and E are indispensable for the maintenance of optimal (immune) cell functions . Parasitic organisms such as protozoa, fungi, bacteria and viruses also depend on these essential nutrients for their multiplication and functioning . An evolutionarily developed optimal distribution of available nutrients between host (cells) and parasitic organisms normally prevents diseases, the nature of which will depend on genetic and environmental factors . The way in which the right amount of cysteine, glutathione (GSH), and copper and zinc ions made available in the right place at the right time and in the right form can prevent an unchecked multiplication of (AIDS) viruses in a more passive or active way forms the basis for the AIDS zinc-deficiency hypothesis (A-Z hypothesis) presented in this article . Zinc and copper ions stimulate/inhibit/block in a concentration-dependent way the (intracellular) activation of essential protein-splitting enzymes such as HIV proteases . Zinc and copper ions as 'passive' virus inhibitors . Apart from this, zinc ions directly or indirectly regulate, via zinc finger protein molecular structures, the activities of virus-combating Th-1 cells such as cytotoxic T-cells (CTLs) . Zinc ions as regulators of the active, virus-combating Th-1 cells . Zinc and copper ions that remain available in sufficient amounts via cysteine/GSH are effective natural inhibitors/combaters of (AIDS) viruses and thereby prevent the development of chronic virus diseases that can lead to AIDS, autoimmune diseases, (food) allergies and/or cancer . A safe, relatively inexpensive and extensively tested medicine such as N-acetylcysteine (NAC) can help in supplying extra cysteine . The anti-HIV peptide T22, synthesized on the basis of two natural peptides from the Tachypleus tridentatus and Limnus polyphemus crabs, appears to be able to serve as supplier/carrier molecule of cysteine and zinc and/or to hinder the entry of HIVs into cells by way of the CD4 receptor.

Epidemiol Infect, 1999 Jun, 122(3), 521 - 8
PCR-RFLP of outer membrane proteins gene of Dichelobacter nodosus: a new tool in the epidemiology of footrot; Ghimire SC et al.; Currently only phenotypic epidemiological markers, serogrouping and virulence testing of Dichelobacter nodosus, are available for investigating footrot outbreaks in small ruminants . These methods have limitations in tracing the source of infection . In this study, a genotypic marker, PCR-RFLP of outer membrane protein gene, was used to characterize D . nodosus . The technique was evaluated in a controlled experiment involving two strains of bacteria . PCR-RFLP was found to be highly specific in differentiating isolates obtained from recipient animals infected with different strains . Subsequently, this technique was used to characterize isolates obtained from field cases of footrot in Nepal . A total of 11 patterns was recognized among 66 Nepalese D . nodosus isolates representing four different serogroups . PCR-RFLP also discriminated isolates with similar phenotypic characteristics . However, all isolates which, phenotypically, were virulent were represented by only two patterns irrespective of their serogroups . It is suggested that PCR-RFLP described here could be a useful epidemiological marker in the study of footrot.

Microb Pathog, 1999 Aug, 27(2), 105 - 17
Genetic organization of the lipopolysaccharide O-antigen biosynthetic locus of Leptospira borgpetersenii serovar Hardjobovis; Kalambaheti T et al.; Leptospiral LPS plays a critical role in immunity to leptospirosis and forms the basis for serological classification of Leptospira . However, neither the structure of leptospiral LPS nor the genetics of its biosynthesis have been elucidated . A probe derived from the rhamnose biosynthetic genes of L . interrogans serovar Copenhageni was used to identify the rfb locus of L . borgpetersenii serovar Hardjobovis . Chromosome walking and sequence analysis revealed an rfb locus spanning 36.7 kb, which consists of 31 ORFs transcribed in the same direction . Clusters of genes were identified which encode proteins related to enzymes involved in the biosynthesis of activated sugars including rhamnose . Additional ORFs in the locus encode glycosyltransferases for the assembly of the O-antigen subunit and integral membrane proteins for the transport of O-antigen subunits through the membrane and assembly into LPS.

Nature, 1999 Aug 12, 400(6745), 661 - 4
Genome complexity, robustness and genetic interactions in digital organisms; Lenski RE et al.; Digital organisms are computer programs that self-replicate, mutate and adapt by natural selection . They offer an opportunity to test generalizations about living systems that may extend beyond the organic life that biologists usually study . Here we have generated two classes of digital organism: simple programs selected solely for rapid replication, and complex programs selected to perform mathematical operations that accelerate replication through a set of defined 'metabolic' rewards . To examine the differences in their genetic architecture, we introduced millions of single and multiple mutations into each organism and measured the effects on the organism's fitness . The complex organisms are more robust than the simple ones with respect to the average effects of single mutations . Interactions among mutations are common and usually yield higher fitness than predicted from the component mutations assuming multiplicative effects; such interactions are especially important in the complex organisms . Frequent interactions among mutations have also been seen in bacteria, fungi and fruitflies . Our findings support the view that interactions are a general feature of genetic systems.

Gene Ther, 1999 Jul, 6(7), 1291 - 7
Mouse adenovirus (MAV-1) expression in primary human endothelial cells and generation of a full-length infectious plasmid; Nguyen T et al.; Using RT-PCR, we show that mouse adenovirus type I (MAV-1) is capable of infecting and expressing in various cell types, specifically human endothelial cells . The capability of MAV-1 to infect and express in human endothelial cells makes it a potentially useful alternative to the use of human adenoviruses type 2/5 (Ad2/5) in virus-based gene therapy, although presently MAV-1 can only be produced at lower titers than Ad2/5 . In this report, we present methods for the purification of MAV-1 DNA and use of this DNA along with a modified bacteria-based homologous recombination protocol to generate a full-length plasmid clone of MAV-1 DNA . Using various transfection procedures, we show that this plasmid MAV-1 DNA can generate plaques of MAV-1 virus, albeit at low efficiencies (about 0 . 2 p.f.u./microg DNA) . Furthermore, the construction of an MAV-1 plasmid along with its capability to express in human cells justifies the full development of MAV-1 into a system of gene therapy.

Braz J Med Biol Res, 1999 Aug, 32(8), 961 - 6
Enhanced mucosal re-epithelialization induced by short chain fatty acids in experimental colitis; Aguilar-Nascimento JE et al.; The short chain fatty acids (SCFA) are the best nutrients for the colonocytes . Glucose is poorly used as a fuel but may be transformed into SCFA by colonic bacteria . The aim of this study was to investigate the effect of SCFA or glucose on experimental colitis . Colitis was induced in 30 Wistar rats by colonic instillation of 4% acetic acid . Five days later they were randomized to receive twice a day colonic lavage containing saline (controls, N = 10), 10% hypertonic glucose (N = 10) or SCFA (N = 10) until day 8 when they were killed . At autopsy, the colon was removed and weighed and the mucosa was evaluated macro- and microscopically and stripped out for DNA assay . Data are reported as mean +/- SD or median {range} as appropriate . All animals lost weight but there was no difference between groups . Colon weight was significantly lower in the SCFA group (3.8 +/- 0.5 g) than in the control (5.3 +/- 2.1 g) and glucose (5.2 +/- 1.3 g) groups (P<0.05) . Macroscopically, the severity of inflammation was less in SCFA (grade 2 {1-5}) than in control (grade 9 {4-10}) and glucose-treated (grade 9 {2-10}) animals (P<0.01) . Microscopically, ulceration of the mucosa was more severe in the glucose and control groups than in the SCFA group . The DNA content of the mucosa of SCFA-treated animals (8.2 {5.0-20.2} mg/g of tissue) was higher than in glucose-treated (5.1 {4.2-8.5} mg/g of tissue; P<0.01) and control (6.2 {4.5-8.9} mg/g of tissue; P<0.05) animals . We conclude that SCFA may enhance mucosal re-epithelialization in experimental colitis, whereas hypertonic glucose is of no benefit.

J Gastrointest Surg, 1998 Nov-Dec, 2(6), 518 - 25
Intestinal microcirculation and gut permeability in acute pancreatitis: early changes and therapeutic implications; Hotz HG et al.; Translocation of bacteria from the intestine causes local and systemic infection in severe acute pancreatitis . Increased intestinal permeability is considered a promoter of bacterial translocation . The mechanism leading to increased gut permeability may involve impaired intestinal capillary blood flow . The aim of this study was to evaluate and correlate early changes in capillary blood flow and permeability of the colon in acute rodent pancreatitis of graded severity . Edematous pancreatitis was induced by intravenous cerulein; necrotizing pancreatitis by intravenous cerulein and intraductal glycodeoxycholic acid . Six hours after induction of pancreatitis, the permeability of the ascending colon was assessed by the Ussing chamber technique; capillary perfusion of the pancreas and colon (mucosal and subserosal) was determined by intravital microscopy . In mild pancreatitis, pancreatic capillary perfusion remained unchanged (2.13 c 0.06 vs . 1.98 +/-0.04 nl x min(-1) x cap(-1) {control}; P = NS), whereas mucosal (1.59 +/-0.03 vs . 2.28 +/-0.03 nl x min(-1) x cap((-1)){control}; P <0.01) and subserosal (2.47 +/-0.04 vs . 3.74 +/-0.05 nl x min(-1) x cap((-1)){control}; P <0.01) colonic capillary blood flow was significantly reduced . Severe pancreatitis was associated with a marked reduction in both pancreatic (1.06 +/-0.03 vs . 1.98 +/-0.04 nl x min(-1) x cap(-1) {control}; P <0 . 01) and colonic (mucosal: 0.59 +/-0.01 vs . 2.28 +/-0.03 nl x min(-1) x cap((-1)){control}, P <0.01; subserosal: 1.96 +/-0.05 vs . 3.74 +/-0.05 nl x min(-1) x cap(-1) {control}, P <0.01) capillary perfusion . Colon permeability tended to increase with the severity of the disease (control: 147 +/-19 nmol x thr(-1) x cm(-2); mild pancreatitis: 158 +/-23 nmol x hr(-1) x cm(-2); severe pancreatitis: 181 +/-33 nmol x hr(-1) x cm(-2); P = NS) . Impairment of colonic capillary perfusion correlates with the severity of pancreatitis . A decrease in capillary blood flow in the colon, even in mild pancreatitis not associated with significant protease activation and acinar cell necrosis or impairment of pancreatic capillary perfusion, suggests that colonic microcirculation is especially susceptible to inflammatory injury . There was no significant change in intestinal permeability in the early stage of pancreatitis, suggesting a window of opportunity for therapeutic interventions to prevent the later-observed increase in gut permeability, which could result in improved intestinal microcirculation.

Am J Perinatol, 1999, 16(4), 181 - 3
Second-trimester abortion caused by Capnocytophaga sputigena: case report; Alanen A et al.; Intra-amniotic infection is often the cause of a second-trimester abortion . The bacterial species involved include bacteria with low pathogenicity like Ureaplasma urealyticum and various Mycoplasma species . In this case we describe an intra-amniotic infection caused by Capnocytophaga sputigena, often found in the normal bacterial flora of the oral cavity, but not in the vagina . Oral sex during pregnancy was the most probable source of the infection . The aborted fetus showed signs of pneumonia upon histologic examination . The bacterial species was identified using broad-spectrum 16S rDNA polymerase chain reaction (PCR) directly from the amniotic fluid and after bacterial culture . Amniotic fluid glucose was below detection level, confirming the presence of an intra-amniotic infection.

Tohoku J Exp Med, 1999 Jan, 187(1), 37 - 42
Endotoxin contamination in isolation of lamina propria mononuclear cells; Fukushima K et al.; Because the beginning of extraction of lamina propria mononuclear cells is to obtain mucosal tissues that are exposed to luminal bacteria, the contaminated endotoxin in this step and/or the enzymes for mucosal digestion may activate mucosal macrophages and other cells . To address this issue, endotoxin levels in isolation solutions were evaluated during the extraction of lamina propria mononuclear cells from 8 control, 7 Crohn's disease and 8 ulcerative colitis specimens . Endotoxin levels were measured using Toxicolor system based on the limulus tests . Endotoxin levels were consistently below 500 pg/ml, and more importantly, these in enzyme digestion solutions were comparable among control, Crohn's disease, and ulcerative colitis . Therefore, comparative experiments using lamina propria mononuclear cells from these mucosae can be appropriately carried out, at least as far as in a comparable amount of contaminated endotoxin . However, careful consideration is required for the comparative and functional study using peripheral blood and lamina propria mononuclear cells.

Hum Cell, 1999 Mar, 12(1), 3 - 10
Measuring respiration of cultured cell with oxygen electrode as a metabolic indicator for drug screening; Amano Y et al.; New trend in methods for assessing pharmacological action to bacteria and cell is to measure their metabolic activities induced, while the conventional methods used population growth . We focused on respiration volume as an indicator of cell metabolism, and developed inexpensive disposable oxygen electrode sensor and multi-channel dissolved oxygen meters (DOX-10 and DOX-96KB) . Using these instruments, cytotoxicity was measured for 48 hrs and the method showed superior features to conventional methods in its handiness of one step assay, and excellent adaptability to automated systems . Total usability of this oxygen electrode method is being evaluated in bacterial drug susceptibility test, anticancer drug susceptibility test, and alternatives to animal experiment.

Infect Immun, 1999 Sep, 67(9), 4912 - 6
Role of complement receptors in uptake of Mycobacterium avium by macrophages in vivo: evidence from studies using CD18-deficient mice; Bermudez LE et al.; Mycobacterium avium is an intracellular pathogen that has been shown to invade macrophages by using complement receptors in vitro, but mycobacteria released from one cell can enter a second macrophage by using receptors different from complement receptors . Infection of CD18 (beta(2) integrin) knockout mice and the C57 BL/6 control mice led to comparable levels of tissue infection at 1 day, 2 days, 1 week, and 3 weeks following administration of bacteria . A histopathological study revealed similar granulomatous lesions in the two mouse strains, with comparable numbers of organisms . In addition, transmission electron microscopy of spleen tissues from both strains of mice showed bacteria inside macrophages . Our in vivo findings support the hypothesis that M . avium in the host is likely to use receptors other than CR3 and CR4 receptors to enter macrophages with increased efficiency.

Infect Immun, 1999 Sep, 67(9), 4661 - 7
Dominance of immunoglobulin G2c in the antiphosphorylcholine response of rats infected with Trichinella spiralis; Peters PJ et al.; The antibody response to the L1 stage of Trichinella spiralis has been described as biphasic . Worms resident in the intestine during the first week of infection stimulate an antibody response against a subset of larval proteins . L1 larvae in the muscle at the end stage of infection stimulate a second antibody response against tyvelose-bearing glycoproteins . Antityvelose antibodies protect rats against challenge infection with larvae . The aim of this study was to characterize the rat B-cell response against larval antigens during the intestinal phase of T . spiralis infection and to test the antiparasitic effects of such antibodies . Strain PVG rats were infected orally with 500 larvae . Antibodies specific for phosphorylcholine-bearing proteins of L1 larvae first appeared in serum 9 days postinfection . Absorption experiments showed that the majority of antilarval antibodies produced in rats 16 days after infection with T . spiralis were specific for phosphorylcholine-bearing proteins . A fraction of these antibodies bound to free phosphorylcholine . Immunoglobulin G2c (IgG2c) producing cells in the mesenteric lymph node dominated this early antibody response . IgG2c is associated with T-independent immune responses in the rat; however, a comparison of athymic rats with euthymic controls suggested that only a small fraction of the phosphorylcholine-related antibody response against T . spiralis was T independent . Phosphorylcholine is a common epitope in antigens of bacteria and nematode parasites and has been shown to be a target of protective immunity in certain bacteria . A monoclonal IgG2c antibody was prepared from infected rats and shown to be specific for phosphorylcholine . Monoclonal phosphorylcholine-specific IgG2c failed to protect rats against intestinal infection with T . spiralis . Therefore, our findings do not support a role for phosphorylcholine-bearing antigens in immune defense against T . spiralis; however, the potency of the immune response induced suggests an immunomodulatory role for the lymphocytes involved.

Infect Immun, 1999 Sep, 67(9), 4578 - 85
Analysis of antigenic structure and human immune response to outer membrane protein CD of Moraxella catarrhalis; Murphy TF et al.; Moraxella catarrhalis is an important cause of otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease (COPD) . Outer membrane protein CD (OMP CD) is a 45-kDa protein which is a potential vaccine antigen to prevent infections caused by M . catarrhalis . Eight monoclonal antibodies were used to study the antigenic structure of the OMP CD molecule by assaying recombinant peptides corresponding to the sequence of the protein . This approach identified two surface-exposed epitopes, including one near the amino terminus (amino acids 25 to 44) and one in the central region of the molecule (amino acids 261 to 331) . Assays with serum and sputum supernatants of adults with COPD revealed variable levels of antibodies to OMP CD among individuals . To determine which portions of the OMP CD molecule were recognized by human antibodies, three human serum samples were studied with six recombinant peptides which span the sequence of OMP CD . All three sera contained immunoglobulin G antibodies which recognized exclusively the peptide corresponding to amino acids 203 to 260 by immunoblot assay . Adsorption experiments with whole bacteria established that some of the human antibodies are directed at surface-exposed epitopes on OMP CD . We conclude that OMP CD is a highly conserved molecule which contains at least two separate epitopes which are exposed on the bacterial surface . While individual adults with COPD show variability in the immune response to OMP CD, a specific region of the OMP CD molecule (amino acids 203 to 260) is important as a target of the human immune response.

Infect Immun, 1999 Sep, 67(9), 4490 - 8
Legionella pneumophila invasion of MRC-5 cells induces tyrosine protein phosphorylation; Susa M et al.; After uptake and intracellular multiplication of Legionella pneumophila in MRC-5 lung fibroblasts, important cytoskeletal filament structures, like actin, tubulin, or vimentin, and a cell membrane-associated fibronectin were rearranged during early infection, resulting in a loss of cell adhesion and collapse of the cytoskeleton . Dysregulation of the cellular phosphorylation and dephosphorylation cascade may contribute to the observed changes and may support intracellular survival and multiplication of L . pneumophila . We therefore studied expression of phosphoproteins during intracellular growth of L . pneumophila . By using an anti-tyrosine phosphoprotein antibody we showed that proteins phosphorylated on tyrosine residues accumulated progressively during late infection exclusively around or in phagosomes filled with bacteria . In contrast, expression of serine/threonine phosphoproteins did not change . To discern the origin of phosphorylated proteins, the host cells were treated with cycloheximide, an inhibitor of eukaryotic protein synthesis . The newly synthesized proteins were labeled metabolically with {(35)S}methionine-cysteine and immunoprecipitated with a phosphotyrosine-specific antibody . Sodium dodecyl sulfate gel electrophoresis gave evidence for synthesis of at least three protein clusters (160 to 200, 35 to 60, and 19 to 28 kDa) of Legionella origin that were phosphorylated on tyrosine residues 24 h after infection . Treatment of infected host cells with genistein, a tyrosine kinase inhibitor, revealed that tyrosine protein phosphorylation was not important for bacterial uptake but contributed to intracellular growth of L . pneumophila . Bacterial tyrosine phosphoproteins and the observed intracellular structural changes may be important to understanding the process involved in intracellular growth of L . pneumophila.

Infect Immun, 1999 Sep, 67(9), 4427 - 34
Intracellular growth in Acanthamoeba castellanii affects monocyte entry mechanisms and enhances virulence of Legionella pneumophila; Cirillo JD et al.; Since Legionella pneumophila is an intracellular pathogen, entry into and replication within host cells are thought to be critical to its ability to cause disease . L . pneumophila grown in one of its environmental hosts, Acanthamoeba castellanii, is phenotypically different from L . pneumophila grown on standard laboratory medium (BCYE agar) . Although amoeba-grown L . pneumophila displays enhanced entry into monocytes compared to BCYE-grown bacteria, the mechanisms of entry used and the effects on virulence have not been examined . To explore whether amoeba-grown L . pneumophila differs from BCYE-grown L . pneumophila in these characteristics, we examined entry into monocytes, replication in activated macrophages, and virulence in mice . Entry of amoeba-grown L . pneumophila into monocytes occurred more frequently by coiling phagocytosis, was less affected by complement opsonization, and was less sensitive to microtubule and microfilament inhibitors than was entry of BCYE-grown bacteria . In addition, amoeba-grown L . pneumophila displays increased replication in monocytes and is more virulent in A/J, C57BL/6 Beige, and C57BL/6 mice . These data demonstrate for the first time that the intra-amoebal growth environment affects the entry mechanisms and virulence of L . pneumophila.

Infect Immun, 1999 Sep, 67(9), 4376 - 82
Truncated surface protective antigen (SpaA) of Erysipelothrix rhusiopathiae serotype 1a elicits protection against challenge with serotypes 1a and 2b in pigs; Imada Y et al.; Erysipelothrix rhusiopathiae is a causal agent of swine erysipelas, which is of economic importance in the swine industry by virtue of causing acute septicemia, chronic arthritis, and endocarditis . However, little is known about the genetic properties of its protective antigens . Recently, a surface protective antigen (SpaA) gene was identified from serotype 2 in a mouse model . We cloned spaA from virulent strain Fujisawa (serotype 1a) and determined that the N-terminal 342 amino acids without C-terminal repeats of 20 amino acids have the ability to elicit protection in mice . Fusions of 342 amino acids of Fujisawa SpaA and histidine hexamer (HisSpa1.0) protected pigs against challenge with both serotype 1 and serotype 2, the most important serotypes in the swine industry . Pigs immunized with HisSpa1.0 reacted well with both HisSpa1.0 and intact SpaA by enzyme-linked immunosorbent assay and immunoblotting . Serum collected at the time of challenge from a pig immunized with HisSpa1 . 0 markedly enhanced the in vitro phagocytic and killing activity of pig neutrophils against the bacteria . DNA sequences of protective regions of spaA genes from five strains of serotypes 1 and 2 were almost identical . The full DNA sequences also seemed to be conserved among strains of all 12 serotype reference strains harboring the spaA gene by restriction fragment length polymorphism analysis of PCR products . These results indicates that SpaA is a common protective antigen of serotypes 1 and 2 of E . rhusiopathiae in swine and will be a useful tool for development of new types of vaccines and diagnostic tools for effective control of the disease.

Food Chem Toxicol, 1999 May, 37(5), 545 - 52
Toxicological studies on procyanidin B-2 for external application as a hair growing agent; Takahashi T et al.; Procyanidin B-2 {epicatechin-(4beta --> 8)-epicatechin} is one of condensed tannin that exists widely in plants . We have reported previously that procyanidin B-2 possesses hair epithelial cell growth-promoting activity and stimulates anagen induction in hair cycle progression . To evaluate the safety of topical procyanidin B-2 as a hair growing agent, we examined the mutagenicity, acute subcutaneous injection, primary irritation, skin sensitization, and eye irritation of this compound . Mutagenicity tests using bacteria showed procyanidin B-2 to be non-mutagenic . Chromosomal aberration tests using CHL cells indicated that procyanidin B-2 caused polyploidy but no structural aberrations . In micronucleus tests for mutagenicity using mice, procyanidin B-2 was negative . Acute subcutaneous injection study using rats revealed no symptoms of significant injury . The lethal dose of procyanidin B-2 is greater than 2000 mg/kg (subcutaneous injection) . Primary irritation tests using rabbits indicated that procyanidin B-2 containing preparation shows no primary irritation . In the guinea pig maximization test, there was no evidence of sensitization to procyanidin B-2 . In primary ocular irritation tests using rabbits, procyanidin B-2 containing preparation and vehicle showed slight irritation of conjunctivae which is assumed to be caused by ethanol . It is suggested that topical procyanidin B-2 is safe and acceptable from the series of toxicological tests.

Nucleic Acids Res, 1999 Aug 15, 27(16), 3310 - 7
Site-directed mutagenesis of motif III in PcrA helicase reveals a role in coupling ATP hydrolysis to strand separation; Dillingham MS et al.; Motif III is one of the seven protein motifs that are characteristic of superfamily I helicases . To investigate its role in the helicase mechanism we have introduced a variety of mutations at three of the most conserved amino acid residues (Q254, W259 and R260) . Biochemical characterisation of the resulting proteins shows that mutation of motif III affects both ATP hydrolysis and single-stranded DNA binding . We propose that amino acid residue Q254 acts as a gamma-phosphate sensor at the nucleotide binding pocket transmitting conformational changes to the DNA binding site, since the nature of the charge on this residue appears to control the degree of coupling between ATPase and helicase activities . Residues W259 and R260 both participate in direct DNA binding interactions that are critical for helicase activity.

Equine Vet J, 1999 Jul, 31(4), 296 - 303
Histopathological findings in equine sinonasal disorders; Tremaine WH et al.; Biopsies collected from 79 referred cases of equine sinonasal disease, including 27 horses with primary sinusitis, 10 with secondary dental sinusitis, 19 with sinus cysts, 11 with progressive ethmoid haematomata (PEH), 4 with false nostril epidermal inclusion cysts, 4 with sinonasal polyps, 3 with sinonasal mycosis and from 2 control animals were examined histologically . Observations were made on epithelial type and integrity, cellular inflammatory response, fibroplasia and presence of potential pathogens . Chronic inflammatory changes including mucosal thickening, ulceration and significant fibroplasia, were found in the sinus mucosa with most sinus disorders, similar to those found in human chronic sinusitis . Bacteria were variably present on sinusitis mucosae but their aetiological significance was unclear . The presence of apparently irreversible changes including fibroplasia in some of these sinusitis cases may explain their poor or delayed response to treatment . Sinus cysts had histological similarities to human mucocoeles . Progressive ethmoid haematomata showed recent and older haemorrhage, as did sinus cysts (and occasionally some chronic sinusitis sections), but support for a common aetiology between sinus cysts and PEH was absent.

FEBS Lett, 1999 Jul 30, 456(1), 119 - 25
The evolution of starch-binding domain; Janecek S et al.; Amylolytic enzymes belonging to three distinct families of glycosidases (13, 14, 15) contain the starch-binding domain (SBD) positioned almost exclusively at the C-terminus . Detailed analysis of all available SBD sequences from 43 different amylases revealed its independent evolutionary behaviour with regard to the catalytic domains . In the evolutionary tree based on sequence alignment of the SBDs, taxonomy is respected so that fungi and actinomycetes form their own separate parts surrounded by bacteria that are also clustered according to taxonomy . The only known N-terminal SBD from Rhizopus oryzae glucoamylase is on the longest branch separated from all C-terminal SBDs . The 3-dimensional (3-D) structures of fungal glucoamylase and bacterial CGTase SBDs are compared and used to discuss the interesting SBD evolution.

Zhonghua Zheng Xing Shao Shang Wai Ke Za Zhi, 1997 Jul, 13(4), 255 - 8
{The relationship between abnormalities of cell-mediated immunity and gut origin endotoxemia in a rat model of thermal injury}; Yao Y et al.; This study was conducted to determine the relationship between abnormalities of cell-mediated immunity and gut-derived endotoxemia in rats following burns . Animals were subjected to a 40% full-thickness scald injury, and randomly divided into control and selective decontamination of the digestive tract (SDD) treated groups . It was found that thermal injury resulted in marked reductions in splenocyte proliferative response to concanavalin A or phytohemagglutinin, interleukin 2 (IL-2) production, and T helper/suppressor (Th/Ts) cells ratio . Prophylactic treatment with SDD significantly reduced the incidence of endotoxemia, prevented suppressive mitogenic response and inadequacy in IL-2 production (P < 0.05-0.01), but did not affect the abnormal ratio of Th/Ts in blood (P > 0.05) . We conclude that bacteria/endotoxin translocation from the gastrointestinal tract appears to be involved in cellular immune dysfunction after thermal injury . Pretreatment with SDD might attenuate systemic immunosuppression by preventing translocation events.

Electrophoresis, 1999 Jul, 20(10), 2071 - 6
Expression and localisation of stanniocalcin 1 in rat bladder, kidney and ovary; Worthington RA et al.; Bony fish use the glycoprotein hormone stanniocalcin (STC) to counteract hypercalcaemia . This is achieved through dual mechanisms involving gill calcium uptake inhibition and stimulation of renal inorganic phosphate reabsorption . Human STC (hSTC-1) shows considerable homology with both rat and mouse STC (mSTC) and their mRNA is expressed in a wide range of tissues . In fish, STC is produced by endocrine glands known as the corpuscles of Stannius but in mammals the widespread expression is suggestive of a paracrine rather than an endocrine role . In order to determine the distribution and strucutral characteristics of hSTC-1, the recombinant protein was expressed in bacteria, purified by metal-ion affinity chromatography, and a study was made of the likely epitopes for raising an antibody . This novel hSTC-1 antibody was used to test the purification protocol . Since the role of mammalian STC is largely unknown, the specific distribution of STC needed to be addressed . To test the specificity of the antibody, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting was undertaken in homogenised rat bladder, ovary and kidney.

J Clin Periodontol, 1999 Aug, 26(8), 531 - 40
Granulocyte elastase activity and PGE2 levels in gingival crevicular fluid in relation to the presence of subgingival periodontopathogens in subjects with untreated adult periodontitis; Jin LJ et al.; This study aimed to determine the association between the levels of granulocyte elastase and prostaglandin E2 (PGE2) in GCE and the concomitant presence of periodontopathogens in untreated adult periodontitis (AP) . GCF and subgingival plaque were sampled by paper strips and paper points respectively, from various periodontal sites in 16 AP subjects . Granulocyte elastase activity in GCF was analyzed with a low molecular weight substrate specific for granulocyte elastase, pGluProVal-pNA, and the maximal rate of elastase activity (MR-EA, mAbs/min/site) was calculated . PGE2 levels in GCF were determined by radioimmunoassay . 5 species-specific DNA probes were used to detect the presence of A . actinomyceterncomitans (A.a., ATCC 43718), B . forsythus (B.f, ATCC 43037), P . gingivalis (P.g., ATCC 33277), P . intermedia (P.i., ATCC 33563), and T . denticola (T.d., ATCC 35405), with a sensitivity of 10(3) cells/paper point . No A.a . was detectable from all sites sampled . The predominant combination of species detected was B.f., P.g., P.i . & T.d . and it was significantly higher at periodontitis sites (68%) than at healthy (7%) or gingivitis sites (29%) (p<0.05) . Overall, MR-EA values were strongly correlated with PGE2 levels (r=0.655, p<0.001), especially at these periodontitis sites co-infected by B.f., P.g., P.i . & T.d . (r=0.722, p<0.001) . The periodontitis sites co-infected by the 4 species were observable from 15 subjects . These sites were sub-grouped into 8 subjects with a high MR-EA and 7 subjects with a low MR-EA . The PGE2 levels in the high MR-EA group were significantly higher than in the low MR-EA group (p<0.05) . No significant differences in clinical or bacterial data were found between the two groups . While within the high MR-EA group, similar results were found between the paired periodontitis sites in each subject with highest and lowest MR-EA values . This study shows that the local host response to bacterial challenge in untreated periodontal pockets is diverse in terms of the intensity of inflammatory response measured by granulocyte elastase and PGE2 levels in GCE A more thorough evaluation of the risk for active periodontal disease may involve the combined approaches to the test of the dynamic bacteria-host relations.

Immunol Rev, 1999 Jun, 169, 209 - 23
Inflammatory disease in HLA-B27 transgenic rats; Taurog JD et al.; A spontaneous inflammatory disease in rats transgenic for HLA-B27 resembles the B27-associated human spondyloarthropathies . Colitis and arthritis, the two most important features, require T cells, gut bacteria, and high expression of B27 in bone marrow-derived cells . Control rats with HLA-B7 remain healthy . Most rats with HLA-Cw6 (associated with psoriasis vulgaris) remain healthy; a minority develop mild and transient disease . Rats with a mutant B27 with a Cys67-->Ser substitution resemble wild-type B27 transgenics, but with a lower prevalence of arthritis . A similar phenotype is seen in B27 rats co-expressing a viral peptide that binds B27 . Disease-prone LEW but not F344 B27 rats develop high serum IgA levels concurrent with disease progression . Colitis is associated with high interferon-gamma, arthritis with high interleukin-6 . Disease is similar in B27 LEW, F344, and PVG rats, but the DA background is protective . Conclusions: The spondyloarthropathy-like disease in rats is specific for HLA-B27 but does not require Cys67 . Arthritis but not colitis is particularly sensitive to B27 peptide-binding specificity . Genetic background exerts a strong influence, but some phenotypic differences exist between permissive strains that do not influence disease susceptibility . The data favor a role for B27 peptide presentation in arthritis, but other mechanisms to explain the role of B27 have not been excluded.

Nature, 1999 Aug 5, 400(6744), 554 - 7
2-Methylhopanoids as biomarkers for cyanobacterial oxygenic photosynthesis; Summons RE et al.; Oxygenic photosynthesis is widely accepted as the most important bioenergetic process happening in Earth's surface environment . It is thought to have evolved within the cyanobacterial lineage, but it has been difficult to determine when it began . Evidence based on the occurrence and appearance of stromatolites and microfossils indicates that phototrophy occurred as long ago as 3,465 Myr although no definite physiological inferences can be made from these objects . Carbon isotopes and other geological phenomena provide clues but are also equivocal . Biomarkers are potentially useful because the three domains of extant life-Bacteria, Archaea and Eukarya-have signature membrane lipids with recalcitrant carbon skeletons . These lipids turn into hydrocarbons in sediments and can be found wherever the record is sufficiently well preserved . Here we show that 2-methyl-bacteriohopanepolyols occur in a high proportion of cultured cyanobacteria and cyanobacterial mats . Their 2-methylhopane hydrocarbon derivatives are abundant in organic-rich sediments as old as 2,500 Myr . These biomarkers may help constrain the age of the oldest cyanobacteria and the advent of oxygenic photosynthesis . They could also be used to quantify the ecological importance of cyanobacteria through geological time.

EMBO J, 1999 Aug 16, 18(16), 4498 - 504
The crystal structure of the complex of replication protein A subunits RPA32 and RPA14 reveals a mechanism for single-stranded DNA binding; Bochkarev A et al.; Replication protein A (RPA), the eukaryote single-stranded DNA-binding protein (SSB), is a heterotrimer . The largest subunit, RPA70, which harbours the major DNA-binding activity, has two DNA-binding domains that each adopt an OB-fold . The complex of the two smaller subunits, RPA32 and RPA14, has weak DNA-binding activity but the mechanism of DNA binding is unknown . We have determined the crystal structure of the proteolytic core of RPA32 and RPA14, which consists of the central two-thirds of RPA32 and the entire RPA14 subunit . The structure revealed that RPA14 and the central part of RPA32 are structural homologues . Each subunit contains a central OB-fold domain, which also resembles the DNA-binding domains in RPA70; an N-terminal extension that interacts with the central OB-fold domain; and a C-terminal helix that mediate heterodimerization via a helix-helix interaction . The OB-fold of RPA32, but not RPA14, possesses additional similarity to the RPA70 DNA-binding domains, supporting a DNA-binding role for RPA32 . The discovery of a third and fourth OB-fold in RPA suggests that the quaternary structure of SSBs, which in Bacteria and Archaea are also tetramers of OB-folds, is conserved in evolution . The structure also suggests a mechanism for RPA trimer formation.

Curr Opin Struct Biol, 1999 Aug, 9(4), 476 - 83
Structure of the respiratory NADH:ubiquinone oxidoreductase (complex I)
Grigorieff N.
Three-dimensional structures of NADH:ubiquinone oxidoreductase (or complex I) from the respiratory chain of mitochondria and bacteria have been recently studied by electron microscopy . The low-resolution structures all reveal a characteristic L shape for complex I; however, some of the differences among these structures may have important implications for the location of the functional elements of complex I, for example, the ubiquinone-binding site.

Microbiol Immunol, 1999, 43(5), 485 - 90
Detection of Treponema socranskii associated with human periodontitis by PCR; Sakamoto M et al.; A PCR technique was used to detect and identify Treponema socranskii associated with periodontitis . A species-specific forward primer was designed for a variable region within its 16S rRNA gene and was used in conjunction with a conserved reverse primer . This primer pair was tested for specificity against 44 oral bacterial strains . Sensitivity was determined using a serial dilution of T . socranskii cells . Amplification products were obtained from all T . socranskii strains tested, but not from other oral bacteria associated with periodontal disease . The detection limit of PCR was 5 T . socranskii cells per PCR . T . socranskii was detected by PCR in subgingival plaque and saliva samples from patients with periodontitis.

Annu Rev Nutr, 1999, 19, 357 - 77
Vitamin B12 deficiency in the elderly; Baik HW et al.; Vitamin B12 deficiency is estimated to affect 10%-15% of people over the age of 60, and the laboratory diagnosis is usually based on low serum vitamin B12 levels or elevated serum methylmalonic acid and homocysteine levels . Although elderly people with low vitamin B12 status frequently lack the classical signs and symptoms of vitamin B12 deficiency, e.g . megaloblastic anemia, precise evaluation and treatment in this population is important . Absorption of crystalline vitamin B12 does not decline with advancing age . However, compared with the younger population, absorption of protein-bound vitamin B12 is decreased in the elderly, owing to a high prevalence of atrophic gastritis in this age group . Atrophic gastritis results in a low acid-pepsin secretion by the gastric mucosa, which in turn results in a reduced release of free vitamin B12 from food proteins . Furthermore, hypochlorhydria in atrophic gastritis results in bacterial overgrowth of the stomach and small intestine, and these bacteria may bind vitamin B12 for their own use . The ability to absorb crystalline vitamin B12 remains intact in older people with atrophic gastritis . The 1998 recommended daily allowance for vitamin B12 is 2.4 micrograms, but elderly people should try to obtain their vitamin B12 from either supplements or fortified foods (e.g . fortified ready-to-eat breakfast cereals) to ensure adequate absorption from the gastrointestinal tract . Because the American food supply is now being fortified with folic acid, concern is increasing about neurologic exacerbation in individuals with marginal vitamin B12 status and high-dose folate intake.

Annu Rev Nutr, 1999, 19, 63 - 90
Regulation of gene expression by dietary fat; Jump DB et al.; Dietary fat is an important macronutrient for the growth and development of all organisms . In addition to its role as an energy source and its effects on membrane lipid composition, dietary fat has profound effects on gene expression, leading to changes in metabolism, growth, and cell differentiation . The effects of dietary fat on gene expression reflect an adaptive response to changes in the quantity and type of fat ingested . Specific fatty acid-regulated transcription factors have been identified in bacteria, amphibians, and mammals . In mammals, these factors include peroxisome proliferator-activated receptors (PPAR alpha, -beta, and -gamma), HNF4 alpha, NF kappa B, and SREBP1c . These factors are regulated by (a) direct binding of fatty acids, fatty acyl-coenzyme A, or oxidized fatty acids; (b) oxidized fatty acid (eicosanoid) regulation of G-protein-linked cell surface receptors and activation of signaling cascades targeting the nucleus; or (c) oxidized fatty acid regulation of intracellular calcium levels, which affect cell signaling cascades targeting the nucleus . At the cellular level, the physiological response to fatty acids will depend on (a) the quantity, chemistry, and duration of the fat ingested; (b) cell-specific fatty acid metabolism (oxidative pathways, kinetics, and competing reactions); (c) cellular abundance of specific nuclear and membrane receptors; and (d) involvement of specific transcription factors in gene expression . These mechanisms are involved in the control of carbohydrate and lipid metabolism, cell differentiation and growth, and cytokine, adhesion molecule, and eicosanoid production . The effects of fatty acids on the genome provide new insight into how dietary fat might play a role in health and disease.

Immunology, 1999 Jul, 97(3), 490 - 6
Depletion of CD8+ cells abolishes memory in acquired immunity against Chlamydia pneumoniae in BALB/c mice; Penttila JM et al.; The importance of T cells in Chlamydia pneumoniae infection in mice was assessed by comparing wild-type BALB/c mice with nude mice and mice depleted in vivo of either CD4+ or CD8+ T cells . Whereas wild-type mice cleared the primary infection in 3 weeks, nude mice were only able to restrict the infection and could not clear it during the observation period of 56 days . Nude mice exhibited a greater number of macrophages in their lungs and the pulmonary cells secreted a higher level of tumour necrosis factor-alpha (TNF-alpha) than wild-type mice . Depletion of CD4+ cells did not change the overall infection kinetics of the primary infection . However, depletion of CD8+ cells resulted in a slightly impaired clearance of the bacteria in the late stages of primary infection . To assess the role of the two T-cell subsets in the acquired immunity that develops during primary infection in wild-type BALB/c mice, in vivo depletions were performed during reinfection . Prior to reinfection, immunocompetent wild-type mice were infected and natural immunity was allowed to form . During reinfection, depletion of CD4+ cells did not have any effect on infection kinetics, whereas depletion of CD8+ cells abolished the protection, reverting the infection kinetics and bacterial load to the same levels found in wild-type mice during primary infection . These results show that T cells are necessary for clearing C . pneumoniae infection in mice . Furthermore, whereas neither of the two main T-cell subsets, separately, were essential for clearance of primary infection, the induced protective immunity was strongly CD8 dependent.

Immunology, 1999 May, 97(1), 9 - 17
Natural polyreactive immunoglobulin A antibodies produced in mouse Peyer's patches; Shimoda M et al.; To understand the biological function of natural immunoglobulin A (IgA) antibodies in Peyer's patches (PP), we generated IgA monoclonal antibody (mAb) clones from the PP of normal, unimmunized, specific pathogen-free BALB/c mice and examined their reactivities by enzyme-linked immunosorbent assay (ELISA) . Many of these antibodies reacted with more than one antigen examined, suggesting that they were polyreactive Abs . Two mAbs agglutinated several different strains of commensal bacteria isolated from mice . To examine the genetic features of these polyreactive mAbs, the VH genes of seven different IgA mAbs were sequenced . The VH genes from the VGAM, J558 and 7183 families were compared with sequence from the mAbs with distinct VDJ rearrangements . One of the mAbs that agglutinated bacteria was encoded by a germline VH gene, but the VH region of the other polyreactive mAbs contained between seven and 11 mutated sites . No indication of antigenic selection was observed in the pattern of these mutated sites . Our results show that polyreactive IgA Abs are present in PP as a part of the normal B-cell repertoire . These polyreactive Abs may establish a natural immune homeostasis, and function as a polyreactive sensor to detect pathogenic invasion and to control immune response in the gut.

Mol Microbiol, 1999 Aug, 33(4), 766 - 77
Control of DNA topology during thermal stress in hyperthermophilic archaea: DNA topoisomerase levels, activities and induced thermotolerance during heat and cold shock in Sulfolobus; Lopez-Garcia P et al.; Plasmid topology varies transiently in hyperthermophilic archaea during thermal stress . As in mesophilic bacteria, DNA linking number (Lk) increases during heat shock and decreases during cold shock . Despite this correspondence, plasmid DNA topology and proteins presumably involved in DNA topological control in each case are different . Plasmid DNA in hyperthermophilic archaea is found in a topological form from relaxed to positively supercoiled in contrast to the negatively supercoiled state typical of bacteria, eukaryotes and mesophilic archaea . We have analysed the regulation of DNA topological changes during thermal stress in Sulfolobus islandicus (kingdom Crenarchaeota), which harbours two plasmids, pRN1 and pRN2 . In parallel with plasmid topological variations, we analysed levels of reverse gyrase, topoisomerase VI (Topo VI) and the small DNA-binding protein Sis7, as well as topoisomerase activities in crude extracts during heat shock from 80 degrees C to 85-87 degrees C, and cold shock from 80 degrees C to 65 degrees C . Quantitative changes in reverse gyrase, Topo VI and Sis7 were not significant . In support of this, inhibition of protein synthesis in S . islandicus during shocks did not alter plasmid topological dynamics, suggesting that an increase in topoisomerase levels is not needed for control of DNA topology during thermal stress . A reverse gyrase activity was detected in crude extracts, which was strongly dependent on the assay temperature . It was inhibited at 65 degrees C, but was greatly enhanced at 85 degrees C . However, the intrinsic reverse gyrase activity did not vary with heat or cold shock . These results suggest that the control of DNA topology during stress in Sulfolobus relies primarily on the physical effect of temperature on topoisomerase activities and on the geometry of DNA itself . Additionally, we have detected an enhanced thermoresistance of reverse gyrase activities in cultures subject to prolonged heat shock (but not cold shock) . This acquired thermotolerance at the enzymatic level is abolished when cultures are treated with puromycin, suggesting a requirement for protein synthesis.

Genome Res, 1999 Aug, 9(8), 689 - 710
Evolution of aminoacyl-tRNA synthetases--analysis of unique domain architectures and phylogenetic trees reveals a complex history of horizontal gene transfer events; Wolf YI et al.; Phylogenetic analysis of aminoacyl-tRNA synthetases (aaRSs) of all 20 specificities from completely sequenced bacterial, archaeal, and eukaryotic genomes reveals a complex evolutionary picture . Detailed examination of the domain architecture of aaRSs using sequence profile searches delineated a network of partially conserved domains that is even more elaborate than previously suspected . Several unexpected evolutionary connections were identified, including the apparent origin of the beta-subunit of bacterial GlyRS from the HD superfamily of hydrolases, a domain shared by bacterial AspRS and the B subunit of archaeal glutamyl-tRNA amidotransferases, and another previously undetected domain that is conserved in a subset of ThrRS, guanosine polyphosphate hydrolases and synthetases, and a family of GTPases . Comparison of domain architectures and multiple alignments resulted in the delineation of synapomorphies-shared derived characters, such as extra domains or inserts-for most of the aaRSs specificities . These synapomorphies partition sets of aaRSs with the same specificity into two or more distinct and apparently monophyletic groups . In conjunction with cluster analysis and a modification of the midpoint-rooting procedure, this partitioning was used to infer the likely root position in phylogenetic trees . The topologies of the resulting rooted trees for most of the aaRSs specificities are compatible with the evolutionary "standard model" whereby the earliest radiation event separated bacteria from the common ancestor of archaea and eukaryotes as opposed to the two other possible evolutionary scenarios for the three major divisions of life . For almost all aaRSs specificities, however, this simple scheme is confounded by displacement of some of the bacterial aaRSs by their eukaryotic or, less frequently, archaeal counterparts . Displacement of ancestral eukaryotic aaRS genes by bacterial ones, presumably of mitochondrial origin, was observed for three aaRSs . In contrast, there was no convincing evidence of displacement of archaeal aaRSs by bacterial ones . Displacement of aaRS genes by eukaryotic counterparts is most common among parasitic and symbiotic bacteria, particularly the spirochaetes, in which 10 of the 19 aaRSs seem to have been displaced by the respective eukaryotic genes and two by the archaeal counterpart . Unlike the primary radiation events between the three main divisions of life, that were readily traceable through the phylogenetic analysis of aaRSs, no consistent large-scale bacterial phylogeny could be established . In part, this may be due to additional gene displacement events among bacterial lineages . Argument is presented that, although lineage-specific gene loss might have contributed to the evolution of some of the aaRSs, this is not a viable alternative to horizontal gene transfer as the principal evolutionary phenomenon in this gene class.

Prog Biophys Mol Biol, 1999, 72(1), 1 - 17
Genome-based structural biology; Frishman D et al.; Spectacular achievements in whole genome sequencing open up new possibilities for structural research . Protein structures can now be studied in their natural genomic context . On the other hand, structure prediction algorithms can be improved using species-specific tendencies in folding patterns . Finally, efficient strategies to select targets for structure determination can be devised . In this review we consider new computational approaches and results in protein structure analysis stemming from the availability of complete genomes.

Nucleic Acids Res, 1999 Sep 1, 27(17), 3389 - 401
Did DNA replication evolve twice independently?
Leipe DD, Aravind L, Koonin EV.
DNA replication is central to all extant cellular organisms . There are substantial functional similarities between the bacterial and the archaeal/eukaryotic replication machineries, including but not limited to defined origins, replication bidirectionality, RNA primers and leading and lagging strand synthesis . However, several core components of the bacterial replication machinery are unrelated or only distantly related to the functionally equivalent components of the archaeal/eukaryotic replication apparatus . This is in sharp contrast to the principal proteins involved in transcription and translation, which are highly conserved in all divisions of life . We performed detailed sequence comparisons of the proteins that fulfill indispensable functions in DNA replication and classified them into four main categories with respect to the conservation in bacteria and archaea/eukaryotes: (i) non-homologous, such as replicative polymerases and primases; (ii) containing homologous domains but apparently non-orthologous and conceivably independently recruited to function in replication, such as the principal replicative helicases or proofreading exonucleases; (iii) apparently orthologous but poorly conserved, such as the sliding clamp proteins or DNA ligases; (iv) orthologous and highly conserved, such as clamp-loader ATPases or 5'-->3' exonucleases (FLAP nucleases) . The universal conservation of some components of the DNA replication machinery and enzymes for DNA precursor biosynthesis but not the principal DNA polymerases suggests that the last common ancestor (LCA) of all modern cellular life forms possessed DNA but did not replicate it the way extant cells do . We propose that the LCA had a genetic system that contained both RNA and DNA, with the latter being produced by reverse transcription . Consequently, the modern-type system for double-stranded DNA replication likely evolved independently in the bacterial and archaeal/eukaryotic lineages.

Science, 1999 Aug 13, 285(5430), 1080 - 4
Coordinate regulation of RAG1 and RAG2 by cell type-specific DNA elements 5' of RAG2; Yu W et al.; RAG1 and RAG2 are essential for V(D)J recombination and lymphocyte development . These genes are thought to encode a transposase derived from a mobile genetic element that was inserted into the vertebrate genome 450 million years ago . The regulation of RAG1 and RAG2 was investigated in vivo with bacterial artificial chromosome (BAC) transgenes containing a fluorescent indicator . Coordinate expression of RAG1 and RAG2 in B and T cells was found to be regulated by distinct genetic elements found on the 5' side of the RAG2 gene . This observation suggests a mechanism by which asymmetrically disposed cis DNA elements could influence the expression of the primordial transposon and thereby capture RAGs for vertebrate evolution.

Exp Toxicol Pathol, 1999 Jul, 51(4-5), 369 - 74
Homology modelling of human cytochromes P450 involved in xenobiotic metabolism and rationalization of substrate selectivity; Lewis DF; Molecular modelling of human cytochrome P450 (CYP) isoforms is described, based on amino acid sequence homology with a unique bacterial P450 (CYP102) of known crystal structure . It is found that for the human hepatic P450s involved in the metabolism of xenobiotics, ie . CYPIA2, CYP 1A6, CYP2B6, CYP2C9, CYP2C 19, CYP2D6, CYP2E1 and CYP3A4, there is a satisfactory agreement between specific substrate characteristics and topographical features of the putative active sites, including complementarity with key amino acid residues in the P450 haem environments . A combination of homology model interactions with substrates and certain molecular properties of the compounds themselves provides a methodology for the evaluation of potential P450 selectivity in new chemical entities (NCEs).

J Microbiol Methods, 1999 Aug, 37(2), 177 - 82
Helicobacter pylori interactions with human gastric mucin studied with a resonant mirror biosensor; Hirmo S et al.; Helicobacter pylori, a human pathogen colonizing the gastrointestinal tract, is first interacting with mucus glycoproteins to penetrate the gastric mucus layer and then attach to specific epithelial cell targets . An optical biosensor technique based on the resonant mirror was used to study H . pylori interactions with human gastric mucin . The mucin preparation was immobilized on the sensor surface to set up the experimental model, close to the in vivo situation . Both sialylated and sulphated oligosaccharides interfered with the H . pylori binding to the immobilized mucin by reducing or abolishing the binding of the bacteria . Furthermore, the displacement of the bacteria from immobilized mucin by highly sulphated glycosaminoglycans was observed.

Pharmazie, 1999 Jul, 54(7), 503 - 5
Synthesis and biological action of 5-oxo-1,2,4-triazine derivatives; Modzelewska-Banachiewicz B et al.; In this study, the biological activity of the 5-oxo-1,2,4-triazine derivatives 12-17 obtained in a reaction of N3-substituted amidrazones 1-6 with dimethyl acethylenedicarboxylate (DMAD) has been examined.

Crit Rev Eukaryot Gene Expr, 1999, 9(2), 107 - 58
Nuclear snRNA and nuclear function (discovery of 5' cap structures in RNA); Ro-Choi TS; In view of the fact that eukaryotic gene expression starts in the nucleus, it is important to have a thorough understanding of nuclear macromolecular structure and function . Newly discovered snRNAs are eukaryotic cell specific and have unique subnuclear compartmental localizations . There are over 200 nucleolar-specific RNAs that include some abundant U3, U8, and U13 RNAs . Extranucleolar-nuclear-specific RNAs (snRNA) are 4.5S RNA I, II, III, 5S RNA III, U1, U2, U4, U5, and U6, in addition to over 500 different RNA species reported up to now . In particular, some snoRNAs and snRNAs have trimethylguanosine cap structures that are not present in bacteria . They have crucial roles in gene expression, such as transcription (U3 snoRNA), processing (U3, U8, U13, U14, U22, and 7-2/MRP), methylation (U14-16, U18, U20-21, and U24-63), pseudouridylation (E2, E3, U19, U23, and U64-72), and hnRNA splicing (U1, U2, U4, U5, and U6 snRNA).

Arch Environ Health, 1999 May-Jun, 54(3), 172 - 9
Indoor endotoxin and glucan in association with airway inflammation and systemic symptoms; Wan GH et al.; Indoor bioaerosols (i.e., bacteria, fungi, endotoxin, and beta-1,3-glucan) were determined in daycare centers, office buildings, and domestic environments in the Taipei area . In addition, we used a questionnaire survey to determine associations between indoor dampness, bioaerosols, and airway inflammation and systemic symptoms . We demonstrated that the median levels of indoor bacteria and fungi were the highest in daycare centers, followed by those in homes and office buildings . Similar patterns were observed for endotoxin and beta-1,3-glucan . The prevalences of airway inflammation and systemic symptoms were higher for females in office buildings than for employees in daycare centers; all symptoms were more prevalent in females than males . With respect to the relationship between bioaerosol exposure and airway inflammation and systemic symptoms, we found a strong association between beta-1,3-glucan and lethargy/fatigue.

Eur J Nutr, 1999 Jun, 38(3), 107 - 17
Immunological aspects of the potential role of dietary carbohydrates and lectins in human health; Kilpatrick DC; BACKGROUND: Little is known regarding the immunobiology of dietary carbohydrate intake and its relevance to human health, although foodstuffs contain many simple and complex carbohydrates . SYNOPSIS: Lectins, immunoglobulins, viruses, bacteria and host cells interact with each other forming a delicate equilibrium within the alimentary canal which may be perturbed by saccharide intake . The ways in which these components may interact at different sites within the alimentary canal are discussed, as are the possible influences on mucosal immunity and the induction of oral tolerance . The possible systemic influences of absorbed saccharides at loci remote from the gut are considered in terms of inhibition of dietary and endogenous lectins, inhibition of bacterial attachment, and alteration of leukocyte homing behaviour . Finally, possible means by which dietary carbohydrates might modify various specific diseases are considered . CONCLUSIONS: It is probable that dietary carbohydrates can alter the equilibria between lectins, secretory IgA and micro-organisms in the alimentary canal, and this consideration could be exploited to promote health . The possible effects of dietary saccharides on allergy/oral tolerance or on recognition events at gut-remote sites warrant further investigation.

Chronobiol Int, 1999 Jul, 16(4), 377 - 414
Role of posttranscriptional regulation in circadian clocks: lessons from Drosophila; Edery I; Incredible progress has been made in the last few years in our understanding of the molecular mechanisms underlying circadian clocks . Many of the recent insights have been gained by the isolation and characterization of novel clock mutants and their associated gene products . As might be expected based on theoretical considerations and earlier studies that indicated the importance of temporally regulated macromolecular synthesis for the manifestation of overt rhythms, daily oscillations in the levels of "clock" RNAs and proteins are a pervasive feature of these timekeeping devices . How are these molecular rhythms generated and synchronized? Recent evidence accumulated from a wide variety of model organisms, ranging from bacteria to mammals, points toward an emerging trend; at the "heart" of circadian oscillators lies a cell autonomous transcriptional feedback loop that is composed of alternatively functioning positive and negative elements . Nonetheless, it is also clear that to bring this transcriptional feedback loop to "life" requires important contributions from posttranscriptional regulatory schemes . For one thing, there must be times in the day when the activities of negative-feedback regulators are separated from the activities of the positive regulators they act on, or else the oscillatory potential of the system will be dissipated, resulting in a collection of molecules at steady state . This review mainly summarizes the role of posttranscriptional regulation in the Drosophila melanogaster time-keeping mechanism . Accumulating evidence from Drosophila and other systems suggests that posttranscriptional regulatory mechanisms increase the dynamic range of circadian transcriptional feedback loops, overlaying them with appropriately timed biochemical constraints that not only engender these loops with precise daily periods of about 24 h, but also with the ability to integrate and respond rapidly to multiple environmental cues such that their phases are aligned optimally to the prevailing external conditions.

Przegl Lek, 1999, 56(3), 237 - 41
{Alcohol dehydrogenase (ADH): structure and role in ethanol metabolism by the human gastrointestinal tract}; Szczepanek M; Drinking of alcohol beverages is highly spread custom all over the world . It can lead to serious medical and social consequences . This review describes the types of human alcohol dehydrogenase in the gastrointestinal tract and its role in metabolism of ethanol as well as a role of bacterial alcohol dehydrogenase in gastrointestinal tract.

Przegl Lek, 1999, 56(3), 227 - 30
{Defense mechanisms in the peritoneum}; Szczepanik M et al.; There are many specialised defence mechanisms connected with immunity of peritoneal cavity . These are absorbtion of bacteria and their toxins from peritoneum, phagocytosis, opsonization, activation of the complement and separation of infection in the peritoneal cavity . A very important role in defence mechanisms of peritoneal cavity play GALT and PALT . Among many cells of the immune system mastocytes and gamma delta T cells have important role in induction and regulation of immune mechanisms in the peritoneal cavity . Lymphocytes T gamma delta release many cytokines and chemokines what allows them to play their protecting role during peritonitis . Released cytokines (especially IFN-gamma) activate macrophages to produce and secret many proinflammatory cytokines and factors . On the other hand mast cells play their role in defence of peritoneal cavity via TNF-alpha and histamine release and inhibition of fibrynolysis.

Biochemistry, 1999 Aug 10, 38(32), 10442 - 8
Determination of nonligand amino acids critical to {4Fe-4S}2+/+ assembly in ferredoxin maquettes; Mulholland SE et al.; The prototype ferredoxin maquette, FdM, is a 16-amino acid peptide which efficiently incorporates a single {4Fe-4S}2+/+ cluster with spectroscopic and electrochemical properties that are typical of natural bacterial ferredoxins . Using this synthetic protein scaffold, we have investigated the role of the nonliganding amino acids in the assembly of the iron-sulfur cluster . In a stepwise fashion, we truncated FdM to a seven-amino acid peptide, FdM-7, which incorporates a cluster spectroscopically identical to FdM but in lower yield, 29% relative to FdM . FdM-7 consists solely of the . CIACGAC . consensus ferredoxin core motif observed in natural protein sequences . Initially, all of the nonliganding amino acids were substituted for either glycine, FdM-7-PolyGly (.CGGCGGC.), or alanine, FdM-7-PolyAla (.CAACAAC.), on the basis of analysis of natural ferredoxin sequences . Both FdM-7-PolyGly and FdM-7-PolyAla incorporated little {4Fe-4S}2+/+ cluster, 6 and 7%, respectively . A systematic study of the incorporation of a single isoleucine into each of the four nonliganding positions indicated that placement either in the second or in the sixth core motif positions,.CIGCGGC . or.CGGCGIC., restored the iron-sulfur cluster binding capacity of the peptides to the level of FdM-7 . Incorporation of an isoleucine into the fifth position,.CGGCIGC., which in natural ferredoxins is predominantly occupied by a glycine, resulted in a loss of {4Fe-4S} affinity . The substitution of leucine, tryptophan, and arginine into the second core motif position illustrated the stabilization of the {4Fe-4S} cluster by bulky hydrophobic amino acids . Furthermore, the incorporation of a single isoleucine into the second core motif position in a 16-amino acid ferredoxin maquette resulted in a 5-fold increase in the level of {4Fe-4S} cluster binding relative to that of the glycine variant . The protein design rules derived from this study are fully consistent with those derived from natural ferredoxin sequence analysis, suggesting they are applicable to both the de novo design and structure-based redesign of natural proteins.

Luminescence, 1999 Jul-Aug, 14(4), 189 - 92
Practical enzymology course based on bioluminescence; Kratasyuk VA et al.; We describe our experience with laboratory courses in enzymology based on the phenomenon of bioluminescence . The soluble and immobilized enzymes of luminous bacteria are used and the practical enzymological course consists of four main courses: (1) training in measuring the activities of soluble and immobilized enzymes; (2) the investigation of kinetic characteristics (kinetic constants) and enzyme-substrate and enzyme-inhibitor interactions in the bacterial bioluminescent reaction; (3) The testing of physico-chemical characteristics of enzymes (pH, temperature, ion strength, etc.); (4) the effect of inhibitors on enzymes . Training is possible in groups of about ten persons . Our practice work has been introduced in the biological, pedagogical and physical departments of Krasnoyarsk State University . Students of the pedagogical department have created a popular and interesting series of laboratory works for high school children aged 14-17 years .

Nature, 1999 Jul 29, 400(6743), 480 - 3
Proton translocation by cytochrome c oxidase; Verkhovsky MI et al.; Cell respiration in mitochondria and some bacteria is catalysed by cytochrome c oxidase, which reduces O2 to water, coupled with translocation of four protons across the mitochondrial or bacterial membrane . The enzyme's catalytic cycle consists of a reductive phase, in which the oxidized enzyme receives electrons from cytochrome c, and an oxidative phase, in which the reduced enzyme is oxidized by O2 . Previous studies indicated that proton translocation is coupled energetically only to the oxidative phase, but this has been challenged . Here, with the purified enzyme inlaid in liposomes, we report time-resolved measurements of membrane potential, which show that half of the electrical charges due to proton-pumping actually cross the membrane during reduction after a preceding oxidative phase . pH measurements confirm that proton translocation also occurs during reduction, but only when immediately preceded by an oxidative phase . We conclude that all the energy for proton translocation is conserved in the enzyme during its oxidation by O2 . One half of it is utilized for proton-pumping during oxidation, but the other half is unlatched for this purpose only during re-reduction of the enzyme.

J Exp Biol, 1999 Sep, 202(Pt 17), 2245 - 57
Roots as a site of hydrogen sulfide uptake in the hydrocarbon seep vestimentiferan Lamellibrachia sp; Julian D et al.; Vestimentiferan tubeworms have no mouth or gut, and the majority of their nutritional requirements are provided by endosymbiotic bacteria that utilize hydrogen sulfide oxidation to fix CO(2) into organic molecules . It has been assumed that all vestimentiferans obtain the sulfide, O(2) and CO(2) needed by the bacteria across the plume (gill) surface, but some live in locations where very little sulfide is available in the sea water surrounding the plume . We propose that at least some of these vestimentiferans can grow a posterior extension of their body and tube down into the sea-floor sediment, and that they can use this extension, which we call the 'root', to take up sulfide directly from the interstitial water . In this study of the vestimentiferan Lamellibrachia sp., found at hydrocarbon seeps in the Gulf of Mexico at depths of approximately 700 m, we measured seawater and interstitial sulfide concentrations in the hydrocarbon seep habitat, determined the structural characteristics of the root tube using transmission electron microscopy, characterized the biochemical composition of the tube wall, and measured the sulfide permeability of the root tube . We found that, while the sulfide concentration is less than 1 (micro)mol l(-)(1) in the sea water surrounding the gills, it can be over 1.5 mmol l(-)(1) at a depth of 10-25 cm in sediment beneath tubeworm bushes . The root tube is composed primarily of giant (&bgr;)-chitin crystallites (12-30 % of total mass) embedded in a protein matrix (50 % of total mass) . Root tubes have a mean diameter of 1.4 mm, a mean wall thickness of 70 (micro)m and can be over 20 cm long . The tubeworm itself typically extends its body to the distal tip of the root tube . The root tube wall was quite permeable to sulfide, having a permeability coefficient at 20 degrees C of 0 . 41x10(-)(3 )cm s(-)(1), with root tube being 2.5 times more permeable to sulfide than trunk tube of the same diameter . The characteristics of the root suggest that it reaches down to the higher sulfide levels present in the deeper sediment and that it functions to increase the surface area available for sulfide uptake in a manner analogous to a respiratory organ.

J Periodontol, 1999 Jul, 70(7), 711 - 23
Relationship of stress, distress and inadequate coping behaviors to periodontal disease; Genco RJ et al.; BACKGROUND: The association of stress, distress, and coping behaviors with periodontal disease was assessed . METHODS: A cross-sectional study of 1,426 subjects between the ages of 25 and 74 years in Erie County, New York, was carried out to assess these relationships . Subjects were asked to complete a set of 5 psychosocial questionnaires which measure psychological traits and attitudes including discrete life events and their impact; chronic stress or daily strains; distress; coping styles and strategies; and hassles and uplifts . Clinical assessment of supragingival plaque, gingival bleeding, subgingival calculus, probing depth, clinical attachment level (CAL) and radiographic alveolar crestal height (ACH) was performed, and 8 putative bacterial pathogens from the subgingival flora measured . RESULTS: Reliability of subjects' responses and internal consistencies of all the subscales on the instruments used were high, with Cronbach's alpha ranging from 0.88 for financial strain to 0.99 for job strain, uplifts, and hassles . Logistic regression analysis indicated that, of all the daily strains investigated, only financial strain was significantly associated with greater attachment and alveolar bone loss (odds ratio, OR = 1.70, 95% CI = 1.09 to 2.65 and OR = 1.68, 95% CI = 1.20 to 2.37, respectively) after adjusting for age, gender, and cigarette smoking . When coping behaviors were evaluated, it was found that those with more financial strain who were high emotion-focused copers (a form of inadequate coping) had a higher risk of having more severe attachment loss (OR = 2.24, 95% CI = 1.15 to 4.38) and alveolar bone loss (OR = 1.91, 95% CI = 1.15 to 3.17) than those with low levels of financial strain within the same coping group, after adjustment for age, gender, and cigarette smoking . Similar results were found among the low problem-focused copers for AL (OR = 2.21, 95% CI = 1.11 to 4.38) and ACH (OR = 2.12, 95% CI = 1.28 to 3.51) . However, subjects with high levels of financial strain who reported high levels of problem-based coping (considered adequate or good coping) had no more periodontal disease than those with low levels of financial strain, suggesting that the effects of stress on periodontal disease can be moderated by adequate coping behaviors . CONCLUSIONS: We find that psychosocial measures of stress associated with financial strain and distress manifest as depression, are significant risk indicators for more severe periodontal disease in adults in an age-adjusted model in which gender (male), smoking, diabetes mellitus, B . forsythus, and P . gingivalis are also significant risk indicators . Of considerable interest is the fact that adequate coping behaviors as evidenced by high levels of problem-based coping, may reduce the stress-associated risk . Further studies also are needed to help establish the time course of stress, distress, and inadequate coping with respect to the onset and progression of periodontal disease, and the mechanisms that explain this association.

Indoor Air, 1999 Sep, 9(3), 146 - 64
The indoor environment of a modern museum building, the Sainsbury Centre for Visual Arts, Norwich, UK; Brimblecombe P et al.; A multi-disciplinary approach was used to investigate the indoor environment of a modern museum building, and its suitability for the conservation of the collection therein . Climate, gaseous and particulate pollution and the concentrations of bacteria were measured in summer and winter campaigns . While the environment overall was found to be an acceptable one, a number of drawbacks were highlighted, the most serious of these being the large temperature and humidity fluctuations that occurred in the summer.

Eur J Histochem, 1999, 43(2), 121 - 33
Histomorphological and histochemical characteristics of the intestine of the Senegal sole, Solea senegalensis; Arellano J et al.; The Senegal sole, Solea senegalensis has been exploited extensively in aquaculture from different countries; at present an intensive production of larvae and adults is being achieved with some nutritional problems . Since this species displays very different life styles and feeding habits at different stages of its life history (larvae, metamorphosis, adults), and because digestive mucins are implicated in different physiological processes including: increase of digestive efficiency, promotion of macromolecules-absorption, buffering of intestinal fluid, prevention of proteolytic damage to the epithelium and defence against bacteria, etc., we studied the histomorphological aspects, as well as the histochemical distribution of carbohydrates, (PAS, Alcian Blue), proteins (Bromophenol Blue), lipids (Oil Red O, Black Sudan B) and glycoproteins (Horseradish peroxidase-conjugated lectins) in the intestinal epithelium of adult Solea senegalensis specimens . Our data are compared with those obtained in larvae and adults of this and other fish species . Primary and secondary folds, microvilli of the intestinal enterocytes, as well as mucous or goblet cells were observed with a scanning electron microscope . Enterocytes and mucocytes of the intestine of adult Solea senegalensis were characterized by a rich supply of sugar and oligosaccharides . Carbohydrates (glycogen and mucins), proteins and lipids were present in cytoplasm and microvilli--brush border--of the enterocytes, which contain GalNAc, GlcNAc, Man, Glc and sialic acid-N-acetyl-D-galactosamine glycoconjugates . Intestinal mucous cells were strongly or weakly stained with Alcian Blue (pH 2.5 and 1) . PAS reactivity was intense in numerous goblet cells, but some cells were PAS unreactive or weakly stained . Some goblet cells were positive for Bromophenol Blue but numerous cells were unstained; thus many proteins and possibly lipids may be conjugated with sugars . A similar reactivity to WGA and to neuraminidase-WGA was identified in some intestinal goblet, which were Con A unreactive, indicating the absence of Man and/or Glc and NANA glycoconjugates . GalNAc residues were only scarcely present in glycoproteins of some goblet cells.

J Immunol, 1999 Aug 15, 163(4), 2073 - 80
Anergy, IFN-gamma production, and apoptosis in terminal infection of mice with Mycobacterium avium; Gilbertson B et al.; We have followed the course of experimental infection of mice with Mycobacterium avium over an extended period, assessing bacterial numbers and T cell responsiveness . When mice were infected intranasally, bacteria spread to the spleen and liver, but remained in highest numbers in the lungs . Both CD4+ and CD8+ T cells, assayed at any time from 6-28 wk after infection, produced IFN-gamma . After initial rapid growth, bacterial numbers slowly increased from approximately 107 at 6 wk to more than 5 x 108 at 28 wk, indicating that the resistance mechanisms so generated were not adequate to contain the infection . During infection, apoptosis of both CD4+ and CD8+ T cells, measured immediately ex vivo by staining with Annexin V, increased steadily . With some individual exceptions, there was a close correlation between apoptosis of CD4+ cells and level of IFN-gamma production by cultured spleen cells . By 34 wk postinfection, there was an abrupt cessation of IFN-gamma production . No IL-4 was detected, ruling out a switch to Th2 profile . Subsequently, bacterial numbers increased still further to >5 x 109 per lung, and the mice lost body weight and would have died if not killed for experimental or humane reasons . The possibility that T cells exposed over this prolonged period to extremely high doses of Ag may become tolerant by a process of terminal differentiation is discussed.

J Bacteriol, 1999 Aug, 181(16), 4955 - 60
Transcript cleavage, attenuation, and an internal promoter in the Rhodobacter capsulatus puc operon; LeBlanc H et al.; The stoichiometry of the structural proteins of the photosynthetic apparatus in purple photosynthetic bacteria is achieved primarily by complex regulation of the levels of mRNA encoding the different proteins, which has been studied in the greatest detail in the puf operon . Here we investigated the transcriptional and posttranscriptional regulation of the puc operon, which encodes the peripheral light harvesting complex LHII . We show that, analogous to the puf operon, a primary transcript encoding five puc genes is rapidly processed to generate more stable RNA subspecies . Contrary to previous hypotheses, translational coupling and regulation of puc transcription by puc gene products were found not to occur . A putative RNA stem-loop structure appears to attenuate transcription initiated at the puc operon major promoter . We also found that a minor pucD-internal promoter contributes to the levels of a message that encodes the LHII 14-kDa gamma (PucE) protein.

J Bacteriol, 1999 Aug, 181(16), 4734 - 40
An unspliced group I intron in 23S rRNA links Chlamydiales, chloroplasts, and mitochondria; Everett KD et al.; Chlamydia was the only genus in the order Chlamydiales until the recent characterization of Simkania negevensis Z(T) and Parachlamydia acanthamoebae strains . The present study of Chlamydiales 23S ribosomal DNA (rDNA) focuses on a naturally occurring group I intron in the I-CpaI target site of 23S rDNA from S . negevensis . The intron, SnLSU . 1, belonged to the IB4 structural subgroup and was most closely related to large ribosomal subunit introns that express single-motif, LAGLIDADG endonucleases in chloroplasts of algae and in mitochondria of amoebae . RT-PCR and electrophoresis of in vivo rRNA indicated that the intron was not spliced out of the 23S rRNA . The unspliced 658-nt intron is the first group I intron to be found in bacterial rDNA or rRNA, and it may delay the S . negevensis developmental replication cycle by affecting ribosomal function.

J Biol Chem, 1999 Aug 13, 274(33), 22949 - 56
Biosynthesis of KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) . Identification and characterization of a KDN-9-phosphate synthetase activity from trout testis; Angata T et al.; Although the deaminoneuraminic acid or KDN glycotope (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) is expressed in glycoconjugates that range in evolutionary diversity from bacteria to man, there is little information as to how this novel sugar is synthesized . Accordingly, biosynthetic studies were initiated in trout testis, an organ rich in KDN, to determine how this sialic acid is formed . These studies have shown that the pathway consists of the following three sequential reactions: 1) Man + ATP --> Man-6-P + ADP; 2) Man-6-P + PEP --> KDN-9-P + P(i); 3) KDN-9-P --> KDN + P(i) . Reaction 1, catalyzed by a hexokinase, is the 6-O-phosphorylation of mannose to form D-mannose 6-phosphate (Man-6-P) . Reaction 2, catalyzed by KDN-9-phosphate (KDN-9-P) synthetase, condenses Man-6-P and phosphoenolpyruvate (PEP) to form KDN-9-P . Reaction 3, catalyzed by a phosphatase, is the dephosphorylation of KDN-9-P to yield free KDN . It is not known if a kinase specific for Man (Reaction 1) and a phosphatase specific for KDN-9-P (Reaction 3) may exist in tissues actively synthesizing KDN . In this study, the KDN-9-P synthetase, an enzyme that has not been previously described, was identified as at least one key enzyme that is specific for the KDN biosynthetic pathway . This enzyme was purified 50-fold from rainbow trout testis and characterized . The molecular weight of the enzyme was estimated to be about 80,000, and activity was maximum at neutral pH in the presence of Mn(2+) . N-Acetylneuraminic acid 9-phosphate (Neu5Ac-9-P) synthetase, which catalyzes the condensation of N-acetyl-D-mannosamine 6-phosphate and phosphoenol-pyruvate to produce Neu5Ac-9-P, was co-purified with the KDN-9-P synthetase . Substrate competition experiments revealed, however, that syntheses of KDN-9-P and Neu5Ac-9-P were catalyzed by two separate synthetase activities . The significance of these studies takes on added importance with the recent discovery that the level of free KDN is elevated in human fetal cord but not matched adult red blood cells and in ovarian cancer cells (Inoue, S., Lin, S-L., Chang, T., Wu, S-H., Yao, C-W., Chu, T-Y., Troy, F . A., II, and Inoue, Y . (1998) J . Biol . Chem . 273, 27199-27204) . This unexpected finding emphasizes the need to understand more fully the role that free KDN and KDN-glycoconjugates may play in normal hematopoiesis and malignancy.

J Biol Chem, 1999 Aug 13, 274(33), 23591 - 8
Analysis of estrogen response element binding by genetically selected steroid receptor DNA binding domain mutants exhibiting altered specificity and enhanced affinity; Chusacultanachai S et al.; To analyze the role of amino acids in the steroid receptor DNA binding domain (DBD) recognition helix in binding of the receptor to the estrogen response element (ERE), we adapted the powerful P22 challenge phage selection system for use with a vertebrate protein . We used the progesterone receptor DNA binding domain and selected for mutants that gained the ability to bind to the ERE . We used a mutagenesis protocol based on degenerate oligonucleotides to create a large and diverse pool of mutants in which 10 nonconsensus amino acids in the DNA recognition helix of the progesterone receptor DNA binding domain were randomly mutated . After a single cycle of modified P22 challenge phage selection, 37 mutant proteins were identified, all of which lost the ability to bind to the progesterone response element . In gel mobility shift assays, approximately 70% of the genetically selected mutants bound to the consensus ERE with a >4-fold higher affinity than the naturally occurring estrogen receptor DBD . In the P-box region of the DNA recognition helix, the selected mutants contained the amino acids found in the wild-type estrogen receptor DBD, as well as other amino acid combinations seen in naturally occurring steroid/nuclear receptors that bind the aGGTCA half-site . We also obtained high affinity DBDs with Trp(585) as the first amino acid of the P-box, although this is not found in the known steroid/nuclear receptors . In the linker region between the two zinc fingers, G597R was by far the most common mutation . In transient transfections in mammalian cells using promoter interference assays, the mutants displayed enhanced affinity for the ERE . When linked to an activation domain, the transfected mutants activated transcription from ERE-containing reporter genes . We conclude that the P-box amino acids can display considerable variation and that the little studied linker amino acids play an important role in determining affinity for the ERE . This work also demonstrates that the P22 challenge phage genetic selection system, modified for use with a mammalian protein, provides a novel, single cycle selection for steroid/nuclear receptor DBDs with altered specificity and greatly enhanced affinity for their response elements.

J Anim Sci, 1999 Jul, 77(7), 1919 - 29
Effects of saturation and esterification of fat sources on site and extent of digestion in steers: digestion of fatty acids, triglycerides, and energy; Elliott JP et al.; Five steers (mean BW 526 kg) fitted with ruminal, duodenal, and ileal cannulas were used in a 5 x 6 Youden square design with 14-d periods . Diets contained chopped alfalfa hay, corn silage, and concentrate (25:35:40, DM basis) . Treatments were 1) control (no added fat); 2) tallow (T), iodine value (IV) = 51.5; 3) partially hydrogenated tallow (PHT), IV = 30.7; 4) hydrogenated tallow (HT), IV = 6.9; 5) blend (1: 1) of HT and hydrogenated free fatty acids (HTHFA), IV = 9.0; and 6) hydrogenated free fatty acids (HFA), IV = 11.2 . Fats replaced cornstarch in the control diet to supply 5% added fatty acids . Intake was restricted to 90% of ad libitum; DMI was similar among diets (mean 9 kg/d) . Total fatty acid intake averaged 170, 500, 506, 525, 489, and 491 g/d for treatments 1 to 6, respectively . Flows of total C16, total C18, and total fatty acids to the duodenum were increased by supplemental fat; flows of total C18 and total fatty acids were greater than their intake for all treatments . Flow of total fatty acids associated with ruminal bacteria accounted for 50 and 17% of the total duodenal fatty acid flow for the control and fat-supplemented diets, respectively . Digestibility of total fatty acids entering the small intestine (74, 71, 62, 39, 53, and 63% for treatments 1 to 6, respectively) was greater for the control diet than for fat-supplemented diets and decreased as either saturation (T < PHT < HT) or esterification (HFA < HTHFA < HT) increased . Digestibilities of fatty acids in the total tract followed similar patterns . Ruminal lipolysis of dietary triglycerides decreased linearly as the degree of saturation of fat sources increased . Small intestinal disappearance of triglycerides (89, 75, 51, 44, 64, and 73% of duodenal flow for treatments 1 to 6, respectively) decreased linearly as either saturation or esterification increased . Flows and digestion of gross energy followed patterns similar to those for fatty acids and triglycerides . Resistance to ruminal and small intestinal lipolysis is a major factor contributing to the poor digestibility of highly saturated triglycerides.

Pol Merkuriusz Lek, 1999 May, 6(35), 273 - 5
{Sick building syndrome: three case reports}; Bogacka E; SBS--sick building syndrome--is a set multi-organ symptoms related to long-term staying in "sick buildings" . These are modern, energy saving, air tight buildings with reduced ventilation . As a result of such construction, harmful, air-borne substances issued by interior decoration materials, air-conditioning systems and working people are cumulated . The study presents three cases of allergic patients whose original allergic illnesses got aggravated as an effect of: 1) staying in a freshly redecorated room, 2) staying in a fully air-conditioned room, 3) long-term exposition to bacteria and fungi allergens developing in old, used up filters of a car air-condition system.

Urol Int, 1999, 62(1), 40 - 3
Retroperitoneoscopic management of infected cysts in adult polycystic kidney disease; Hemal AK et al.; Conservative measures are the mainstay of therapy in adult polycystic kidney disease (APKD) . Pain, infection and obstructive uropathy are the major indications for intervention . Chronic pain has been treated with narcotic analgesics, needle aspiration of dominant cysts, and open renal cyst decortication . Laparoscopic cyst decortication, by either transperitoneal or retroperitoneal access, is a new emerging option with similar efficacy to open surgery and less morbidity . Cyst infection in these patients responds poorly to commonly used antibiotics . Patients with refractory cyst infection may even require nephrectomy . Herein, we present 2 cases with APKD that were treated by retroperitoneoscopic decortication for painful and infected cysts . Both patients showed prompt and sustained improvement in symptoms, with minimal morbidity and short convalescence . This approach has not hitherto been described for infected cysts in APKD . The retroperitoneoscopic route should be preferred in the presence of infected cysts so as to prevent intraperitoneal contamination.

Int Arch Allergy Immunol, 1999 Jul, 119(3), 205 - 11
Pertussis adjuvant prolongs intestinal hypersensitivity; Kosecka U et al.; BACKGROUND: Immediate hypersensitivity reactions are a hallmark of allergic disease, and result in the clinical features of food allergy, hayfever, and atopic asthma . The mechanism by which an individual becomes sensitized to an ingested or airborne allergen is not clear, however exposure to bacteria or bacterial products that act as adjuvants may be a contributing factor . The purpose of this study was to examine the role of pertussis toxin (PT) in inducing intestinal hypersensitivity reactions, particularly the ability of the adjuvant to prolong the sensitization . METHODS: Rats were sensitized to ovalbumin (OA) by injection of OA alone or with 50 ng PT . Secretory responses to OA challenge and nerve stimulation were assessed in jejunal tissues mounted in Ussing chambers . RESULTS: Jejunal segments from rats sensitized to OA alone responded to antigen challenge with ion secretion, but sensitization was transient in that specific IgE titers and responses to luminal antigen disappeared by 14 days . In contrast, co-administration of 50 ng PT with OA resulted in long-lasting sensitization . Secretory responses to both luminal and serosal OA challenge were present 8 months after primary immunization . Enhanced secretory responses to nerve stimulation, increased mucosal mast cell numbers, as well as elevated IgE titers were also induced and may have contributed to the overall responsiveness of the intestine to antigen challenge . CONCLUSIONS: Our findings indicate nanogram quantities of PT, when administered with a food protein, result in long-term sensitization to the antigen, and altered intestinal neuroimmune function . These data suggest that exposure to bacterial pathogens may prolong the normally transient immune responsiveness to inert food antigens.

Electrophoresis, 1999 Jun, 20(8), 1762 - 7
New applications of low-C0tDNA as a DNA fingerprint probe; Leung FC; New applications of low-C0t DNA are reported as probes for genetic identification and genome characterization . These fast and intermediately reannealing fractions have sometimes either been discarded in genomic library construction to enhance the probability of finding single copy genes, or they are used as resources for identifying individual repetitive sequences . In addition, they are used as blockers to enhance hybridization signals . C0t-1 DNA serves as a probe for DNA fingerprinting of human yeast artificial chromosomes . We have isolated low-C0t DNA from bacteria, fungus, plant, mussel, chicken, rat and fish from the sheared genomic DNA of the respective species . Low-C0t DNA is labeled to generate DNA fingerprints and for in situ hybridization . Individual specific DNA fingerprint profiles are observed and species-specific DNA fragments can be identified in bacteria, fungus, plants (Ginseng and Amaranthus) and mussel . When low-C0t DNA probes from rat, chicken and fish were employed, only smear profiles and no distinct DNA banding patterns were evident . In these species, individual clones can be used as a probe for DNA fingerprinting containing repetitive sequences after subcloning . The advantage of this approach is to quickly develop a useful probe for DNA fingerprinting for genetic identification and analysis without sequencing knowledge a priori . This represents an innovative approach to the use of these repetitive components of the genome.

Acta Gastroenterol Latinoam, 1999, 29(1), 17 - 20
{Evaluation of a fast urease test for the detection of Helicobacter pylori}; Blanco D et al.; Helicobacter Pylori colonize the gastric mucosa and their adaptation to this environment is related with its high activity urease . This enzyme hydrolyzes the gastric urea, neutralizing the acid environment of the bacteria . Based on that reaction numerous presumptive diagnosis tests, have been developed using a solution of urea (usually 6%) with a pH indicator (usually 0.05% fenol-red); nevertheless, the color changes are so light that some persons do not detect it . For that reason, a modification of that reaction was proposed using a mix of pH indicators (0.05% fenolred and 0.002 bromothymol blue) which induces a color change from light green to deep purple . Also, the reaction of urease was evaluated using only bromothymol blue . The reaction using fenol red as indicator showed the higher values for sensitivity of 58.8% and the specificity of 66.6%; whereas using only bromothymol-blue those values were 46 y 71.4% respectively . The efficiency of the test using fenol-red or the mix of this bromothymol- was 64.2 y 62.2%, respectively; however, the mix of indicators induce a change color easily detected, because of changes from ligh-green to deep-purple.

Gene Ther, 1999 Feb, 6(2), 263 - 70
A novel T7 RNA polymerase autogene for efficient cytoplasmic expression of target genes; Brisson M et al.; Inefficient nuclear transport of plasmid DNA continues to be a problem in nonviral vector-mediated gene transfer . This has made the cytoplasmic expression system an increasingly attractive idea . We have developed a new T7 RNA polymerase autogene for cytoplasmic expression containing both a CMV and a T7 promoter . The pCMV/T7-T7pol autogene does not encounter the problems associated with previously used autogenes . For instance, pCMV/T7-T7pol is easily amplified and purified from bacteria . Furthermore, the CMV promoter is used to drive the first round of synthesis of T7 RNA polymerase, thus negating the use of purified enzyme in the transfection complex . The endogenous T7 RNA polymerase produced from the CMV promoter could then act on the T7 promoter of pCMV/T7-T7pol in an autoregulatory mechanism . pCMV/T7-T7pol induces higher, more sustained levels (> 7 days) of reporter gene expression than that observed with the previously used autogene pT7 AUTO 2C- or with the nuclear expression system pCMV-CAT . This seems to be due to the high levels of T7 RNA polymerase protein that are detected in cells transfected with pCMV/T7-T7pol . This vector also functions as an efficient autogene since at least 50 times more mRNA is transcribed from the cytoplasmic T7 promoter as compared with the nuclear CMV promoter in pCMV/T7-T7pol . Therefore, pCMV/T7-T7pol could replace existing autogenes for regeneration of T7 RNA polymerase and efficient target gene expression.

Minerva Stomatol, 1999 May, 48(5), 181 - 9
{Comparison of different finishing methods for composites and compomers . Profilometric analysis}; Rapisarda E et al.; BACKGROUND: Reconstructions with aesthetical materials neither finished nor polished can be extremely irregular and wrinkled . For this reason they represent an ideal basis for the growing of pigmentation which come from food remainings and the nestle of bacteria on the plaque . The finishing of aesthetical materials is a fundamental step in conservative dentistry . The finishing session regarding the aesthetical restorations should be considered and planned at the moment of their insertion in the prepared hollows . The finishing should not be considered an option, but the conclusion of all the conservative treatment . Purpose of the search is to examine and assess, through a technical equipment measuring the superficial wrinkledness of the materials, the action of 4 systems of finishing and polishing on two aesthetical materials widely used in the daily practice by dental surgeons: a compomer (Compoglass, Vivadent) and a composite (Spectrum, Dentsply) . METHODS: For each of these two materials some little slabs model have been prepared, choosing the universal colour in the colorimetric scale . The two types of filling materials have been compared with 4 systems of finishing and polishing: Sof-Lex Pop-on (3M), Enhance system (Dentsply), Hawe Micro Disc (Howe Neos), Heawe Gommini Polisher (Heawe Neos) . The total working time did not overcome 1 minute . The little slabs have been obtained, pressing the resin between two slides used in microscopy . In the hope to guarantee in the different samples, a uniform thickness, we have used a technical device . On each covering slide we have put a weight of 0.5 kg for 5 minutes . The thickness of the little slabs obtained was 2 mm . Thus, pressed between the two slides, the aesthetical material has been photopolymerized according to the time suggested by Manufacturing Industries . The sizes concerning the wrinkledness of samples subjected to different treatments have been carried out using a pointed profilometer with high sensitivity . (Tencor-P10) . This instrument used in the National Laboratory of Catania, called INFN, is able to graphitize the wrinkledness of a surface "survbeying" it with a diamond ultramicrometric point . RESULTS: All tested systems gradually produce the upper layers of the materials less suitable to resist the assault of plaque bacteria as time passed . The 3M coarse and medium grain Disks are very abrasive and for this reason the surface of the materials is ill-shaped . Those disks with fine and extra fine grain, smooth the tracks left by previous disks . As they have been always used according to their decreasing granulometry, the disks are used only for removing small composite pieces in excess and to improve micromorphology of the restauration . The "Gommini" are less abrasive than Disks . In a few minutes and often with only one step they produce a much regular and polished fillings surface . CONCLUSIONS: The "Gommini" have a preference when the last photopolymerization has left a regular layer, with a very good micromorphology . Actually, "Gommini" do not remove much material, but they continue to smooth the outline of the reconstruction . Disks are not classified as being of first quality in the finishing of composites and compomers.

Am J Infect Control, 1999 Aug, 27(4), 367 - 9
Handwashing before entering the intensive care unit: what we learned from continuous video-camera surveillance; Nishimura S et al.; Handwashing is one of the most important factors in controlling the spread of bacteria and in preventing the development of infections . This simple procedure does not have a high compliance rate . The Association for Professionals in Infection Control and Epidemiology, Inc, guideline recommends that hands must be washed before and after patient contact . In our intensive care unit (ICU), we have made it a rule that everyone should wash their hands before entering the ICU . The purpose of this study was to ascertain the handwashing compliance of all personnel and visitors to the ICU . A ceiling-mounted video camera connected to a time-lapse video cassette recorder recorded each person's actions when they entered the ICU during a 7-day period . Handwashing compliance was assessed for 3 different categories: ICU personnel, non-ICU personnel, and visitors to patients . There were 1030 entries to the ICU during the observation period . ICU personnel complied with handwashing in 71% of entries, non-ICU personnel in 74% of entries, and visitors to patients in 94% of entries . Handwashing compliance by visitors to patients was significantly higher than among personnel (P <.001) . Handwashing compliance among personnel before entering the ICU was low . Continuous effort is needed to raise awareness of the handwashing issue, not only to ensure compliance with APIC recommendations but also in our facility, to ensure that health care personnel wash their hands on entry to the ICU.

Endoscopy, 1999 Jun, 31(5), 348 - 51
A new device for abrasive cytology sampling during upper gastrointestinal endoscopy: experience in infectious and neoplastic diseases; Casco C et al.; BACKGROUND AND STUDY AIMS: The increase in infectious diseases of the gastrointestinal tract related to immunosuppression is becoming an important topic for the endoscopist . To improve the diagnostic efficacy of tissue acquisition while at the same time restricting costs, we have developed a new device for obtaining material from the upper gastrointestinal tract that can also be used in the diagnosis of neoplastic disease . PATIENTS AND METHODS: A total of 90 patients were examined and assigned to two groups according to indications . Group A consisted of 53 symptomatic patients with positive human immunodeficiency virus (HIV) serology with a suspicion of gastrointestinal infection . Group B included 37 patients in whom there was an endoscopic suspicion of neoplasia in the upper gastrointestinal tract . Cell fragments for cytological study were obtained using a device introduced through the endoscopic instrumentation channel (abrasive cytology) . Different staining methods were used to isolate bacteria or diagnose tumors from cell fragments . The findings were compared with those obtained from conventional bioptic histology . RESULTS: Potentially responsible pathogens were isolated in 48 of the 53 patients in Group A, while bioptic histology provided a diagnosis in only 32 patients . In the 37 patients in group B, the cytological diagnosis matched the histological results . The costs of this new technique are similar to those for conventional cytological staining, and the time from sampling to obtaining a final diagnosis is less than one hour . CONCLUSIONS: This new device provides a fast and low-cost method of isolating pathogens and obtaining cell fragments from the gastrointestinal mucosa during routine upper gastrointestinal endoscopy.

Am J Public Health, 1999 Aug, 89(8), 1252 - 4
The role of syringe filters in harm reduction among injection drug users; Caflisch C et al.; OBJECTIVES: Three filters were tested for in situ efficacy in reducing bacterial contamination associated with injection drug use . METHODS: In a self-matched control design with blinded laboratory testing, injection drug users were asked to use 3 filters in random succession when loading their syringes with drug solute . RESULTS: The 0.22-micron filter proved significantly better than both the cigarette filter (relative risk {RR} = 18.0) and the 20-micron filter (RR = 4.5) in rendering syringes bacteria-free . CONCLUSIONS: The 15- to 20-micron syringe filter currently provided injection drug users in Switzerland does not significantly reduce contamination associated with common bacterial infections among users . Filters with pore width 1/100th as large are recommended.

Lett Appl Microbiol, 1999 Jul, 29(1), 52 - 5
Comamonas acidovorans contamination of dental unit waters; Stampi S et al.; A study was carried out to evaluate the extent of the colonization of dental water systems by Comamonas acidovorans and to investigate how the occurrence of these bacteria is related to certain water characteristics . The 152 water samples were collected from the oral rinsing cup, air-water syringe, turbine and supply lines to dental units . Comamonas acidovorans was found most frequently and in greatest quantities in samples taken from water entering the units and in samples with a lower total bacterial count at 22 degrees C, higher temperature, lower content of organic matter and, in general, higher concentrations of residual chlorine.

J Oral Pathol Med, 1999 Aug, 28(7), 297 - 302
Neutrophils in the molar tooth extraction wound in the rat: a transmission electron microscope (TEM) study; McMillan MD; Neutrophils have been ascribed a number of functions from ultrastructural studies of healing wounds . All of the wounds so far examined have been relatively aseptic . This study investigates, by TEM, the structure of neutrophils in healing molar tooth sockets in rats . Prior to epithelial coverage, a dense infiltrate of neutrophils separated the viable wound tissues from the overlying debris and bacteria . The more deeply situated neutrophils contained many granules and only occasional phagosomes . More superficial neutrophils contained fewer granules and phagosomes with engulfed bacteria undergoing lysis . The most superficial neutrophils were degenerate, lacked granules and often contained viable bacteria . There were varying numbers of neutrophils containing granules in the blood clot, granulation tissue, wound epithelium and adjacent tissue . No extracellular neutrophil granules nor extracellular discharge of granules was found . These findings differ from those of previous ultrastructural studies of relatively aseptic healing wounds . Ultrastructurally, the only function of neutrophils in healing tooth extraction wounds appears to be phagocytosis of bacteria, which supports a role in the prevention of infection.

Cell Mol Biol (Noisy-le-grand), 1999 Jun, 45(4), 383 - 91
Arginine decarboxylase in Trypanosoma cruzi, characteristics and kinetic properties; Hernandez S et al.; Polyamines are known to play an essential role in cell growth and differentiation . In animals, putrescine is mainly synthesized from ornithine by ornithine decarboxylase (ODC) . In higher plants and in bacteria putrescine can also be synthesized from arginine by arginine decarboxylase (ADC) . In this paper we report the presence of significant levels of ADC activity in crude extracts of Trypanosoma cruzi, RA strain epimastigotes . ADC activity was detected during a very narrow time range, corresponding to the early logarithmic growth phase . This activity was inhibited by DL-alpha-difluoromethylarginine, a specific irreversible inhibitor of ADC and activated by DL-alpha-difluoromethylornithine, a specific irreversible inhibitor of ODC . The reaction showed an absolute requirement for pyridoxal phosphate, dithiothreitol and Mg++ . The enzyme half life was about 10 hrs., showed maximum activity at pH 7.9 and a Km for arginine of 5 mM . ADC activity was stimulated by fetal-calf-serum and inhibited by spermine, probably through a negative feed-back regulation on the levels of the enzyme . ODC activity was not detected . These results confirm our previous reports on the capability of T . cruzi, RA strain epimastigotes to synthesize putrescine from arginine via agmatine by ADC and point out differences on polyamine metabolism between the parasite and the mammalian host cell.

J Helminthol, 1999 Mar, 73(1), 67 - 71
Attachment tests of Pasteuria penetrans to the cuticle of plant and animal parasitic nematodes, free living nematodes and srf mutants of Caenorhabditis elegans; Mendoza de Gives P et al.; Populations of Pasteuria penetrans isolated from root-knot nematodes (Meloidogyne spp.) and cyst nematodes (Heterodera spp.) were tested for their ability to adhere to a limited selection of sheathed and ex-sheathed animal parasitic nematodes, free living nematodes, including Caenorhabditis elegans wild type and several srf mutants, and plant parasitic nematodes . The attachment of spores of Pasteuria was restricted and no spores were observed adhering to any of the animal parasitic nematodes either with or without their sheath or to any of the free living nematodes including C . elegans and the srf mutants . All spore attachment was restricted to plant parasitic nematodes; however, spores isolated from cyst nematodes showed the ability to adhere to other genera of plant parasitic nematodes which was not the case with spores isolated from root-knot nematodes . The results are discussed in relationship to cuticular heterogeneity.

Trends Biochem Sci, 1999 Aug, 24(8), 295 - 8
Transfer RNAs and pathogenicity islands; Hou YM; The tRNAs are central components in translation . In addition, they are essential for replication of retroviruses: tRNAs bind to viral genomes through their 3'-end sequences and act as primers for initiation of viral replication . Here, I discuss the possibility that tRNAs also play a role in the horizontal transfer of bacterial pathogenicity islands between different pathogens . Such a role would implicate tRNAs in DNA recombination.

Genetics, 1999 Aug, 152(4), 1343 - 51
Genetics of nitrogen regulation in Methanococcus maripaludis; Kessler PS et al.; We have used genetic methods in Methanococcus maripaludis to study nitrogen metabolism and its regulation . We present evidence for a "nitrogen regulon" in Methanococcus and Methanobacterium species containing genes of nitrogen metabolism that are regulated coordinately at the transcriptional level via a common repressor binding site sequence, or operator . The implied mechanism for regulation resembles the general bacterial paradigm for repression, but contrasts with well-known mechanisms of nitrogen regulation in bacteria, which occur by activation . Genes in the nitrogen regulons include those for nitrogen fixation, glutamine synthetase, (methyl)ammonia transport, the regulatory protein GlnB, and ammonia-dependent NAD synthetase, as well as a gene of unknown function . We also studied the function of two novel GlnB homologues that are encoded within the nif gene cluster of diazotrophic methanogens . The phenotype resulting from a glnB null mutation in M . maripaludis provides direct evidence that glnB-like genes are involved in "ammonia switch-off," the post-transcriptional inhibition of nitrogen fixation upon addition of ammonia . Finally, we show that the gene nifX is not required for nitrogen fixation, in agreement with findings in several bacteria . These studies illustrate the utility of genetic methods in M . maripaludis and show the enhanced perspective that studies in the Archaea can bring to known biological systems.

Genetics, 1999 Aug, 152(4), 1299 - 305
Divergence of the hyperthermophilic archaea Pyrococcus furiosus and P . horikoshii inferred from complete genomic sequences; Maeder DL et al.; Divergence of the hyperthermophilic Archaea, Pyrococcus furiosus and Pyrococcus horikoshii, was assessed by analysis of complete genomic sequences of both species . The average nucleotide identity between the genomic sequences is 70-75% within ORFs . The P . furiosus genome (1.908 mbp) is 170 kbp larger than the P . horikoshii genome (1.738 mbp) and the latter displays significant deletions in coding regions, including the trp, his, aro, leu-ile-val, arg, pro, cys, thr, and mal operons . P . horikoshii is auxotrophic for tryptophan and histidine and is unable to utilize maltose, unlike P . furiosus . In addition, the genomes differ considerably in gene order, displaying displacements and inversions . Six allelic intein sites are common to both Pyrococcus genomes, and two intein insertions occur in each species and not the other . The bacteria-like methylated chemotaxis proteins form a functional group in P . horikoshii, but are absent in P . furiosus . Two paralogous families of ferredoxin oxidoreductases provide evidence of gene duplication preceding the divergence of the Pyrococcus species.

Rev Belge Med Dent, 1998, 53(4), 181 - 92
{Periodontal aspects of cementation: materials, technics and their biologic reactions}; De Boever JA et al.; Cementation of crowns and bridges has an influence on the health of marginal periodontal structures . Ideally, the marginal discrepancy should be less than 50 microns which has been considered clinically acceptable . With discrepancies larger than 80 microns, the bleeding tendency and sulcus fluid flow rate increased . A shoulder-bevel preparation results in the smallest marginal discrepancy . Cytotoxicity of cements has been studied in cultures of gingival cells and fibroblasts . Composite has always a more pronounced cytotoxicity as compared to glass-ionomers and zinc phosphate cements . However, large differences exist in cytotoxicity between materials of the same group . Erosion of the cement leads to leakage of toxic materials and the formation of niches colonised by oral plaque bacteria . A number of clinical recommendations are made to minimize the effect of cements and cementation on the periodontal structures.

Recenti Prog Med, 1999 Jul-Aug, 90(7-8), 387 - 91
{Diagnosis of fever of unknown origin: use of Bayes theorem}; Campanella N et al.; Sophisticated tests and time are needed to establish the diagnosis of fever of unknown origin (FUO) . Bayes theorem can guide the clinician to early probabilistic diagnosis . The authors had estimated the a priori probabilities in a previous study yet . In this paper the diagnostic probabilities of four clinical signs (arthralgias, myalgias, splenomegaly, multiple micro-lymphoadenopathies) and the probabilities of abnormalities of some routine laboratory tests (neutrophils count, lymphocytes count, platelets count, hemoglobin, lactic-dehydrogenase, C3 and C4 complement subunits and fibrinogen plasma concentrations, erythrocyte sedimentation rate) and of the positive bacterial cultures, were estimated in the main groups of FUO disease (indefinite, aspecific bacteria infections, non-bacterial or specific bacteria infections, neoplasias, connective tissue diseases, miscellaneous group) . The data suggest that the a priori probability is not affected by the clinical signs . Arthralgias increase the probability of connective tissue diseases . Normal erythrocyte sedimentation rate and plasma fibrinogen concentrations increase the probability of paraneoplastic and indefinite fever . Thrombocytosis increases the probability of paraneoplastic fever . Over 1200 U/l serum lactic-dehydrogenase serum concentrations guide to non-bacterial or specific bacteria infectious diseases and to malignancies . Neutrophilia decreases the probability of indefinite diagnosis, even though it is not specific . The heterogeneity of the miscellaneous group makes the interpretations of the data very hard.

J Biol Chem, 1999 Aug 6, 274(32), 22723 - 8
Polyamine stimulation of the synthesis of oligopeptide-binding protein (OppA) . Involvement of a structural change of the Shine-Dalgarno sequence and the initiation codon aug in oppa mRNA; Yoshida M et al.; We previously suggested that the degree of polyamine stimulation of oligopeptide-binding protein (OppA) synthesis is dependent on the secondary structure and position of the Shine-Dalgarno (SD) sequence of OppA mRNA . To study the structural change of OppA mRNA induced by polyamines and polyamine stimulation of initiation complex formation, four different 130-mer OppA mRNAs containing the initiation region were synthesized in vitro . The structural change of these mRNAs induced by polyamines was examined by measuring their sensitivity to RNase T(1), specific for single-stranded RNA, and RNase V(1), which recognizes double-stranded or stacked RNA . In parallel, the effect of spermidine on mRNA-dependent fMet-tRNA binding to ribosomes was examined . Our results indicate that the secondary structure of the SD sequence and initiation codon AUG is important for the efficiency of initiation complex formation and that spermidine relaxes the structure of the SD sequence and the initiation codon AUG . The existence of a GC-rich double-stranded region close to the SD sequence is important for spermidine stimulation of fMet-tRNA binding to ribosomes . Spermidine apparently binds to this GC-rich stem and causes a structural change of the SD sequence and the initiation codon, facilitating an interaction with 30 S ribosomal subunits.

J Biol Chem, 1999 Aug 6, 274(32), 22437 - 44
TIP49b, a new RuvB-like DNA helicase, is included in a complex together with another RuvB-like DNA helicase, TIP49a; Kanemaki M et al.; We previously reported that TIP49a is a novel mammalian DNA helicase showing structural similarity with the bacterial recombination factor RuvB . In this study, we isolated a new TIP49a-related gene, termed TIP49b, from human and yeast cells . TIP49b also resembled RuvB, thus suggesting that TIP49a and TIP49b are included in a gene family . Like TIP49a, TIP49b was abundantly expressed in the testis and thymus . Enzyme assays revealed that TIP49b was an single-stranded DNA-stimulated ATPase and ATP-dependent DNA helicase . Most of the enzymatic properties of TIP49b were the same as those of TIP49a, whereas the polarity of TIP49b DNA helicase activity (5' to 3') was the opposite to that of TIP49a . TIP49b and TIP49a bound to each other and were included in the same complex of approximately 700 kDa in a cell . We found that TIP49b was an essential gene for the growth of Saccharomyces cerevisiae, as is the TIP49a gene, suggesting that TIP49b does not complement the TIP49a function and vice versa . From these observations, we suggest that TIP49b plays an essential role in the cellular processes involved in DNA metabolism.

FEBS Lett, 1999 Jul 16, 455(1-2), 1 - 7
New and unexpected routes for ultrafast electron transfer in photosynthetic reaction centers; van Brederode ME et al.; In photosynthetic reaction centers, the excitation with light leads to the formation of a charge separated state across the photosynthetic membrane . For the reaction center of purple non-sulphur bacteria, it was previously generally assumed that this primary charge separation could only start with the excitation of the so-called special pair of bacteriochlorophyll molecules located in the heart of the RC . However, recently new and ultrafast pathways of charge separation have been discovered in the bacterial RC that are driven directly by the excited state of the accessory monomeric bacteriochlorophyll present in the active branch of cofactors . These results demonstrate that the route for energy conversion in photosynthesis can be much more flexible than previously thought . We suggest that the existence of multiple charge separation routes is particularly relevant for the mechanism of charge separation in the photosystem II reaction center of higher plants.

Bioorg Med Chem, 1999 Jun, 7(6), 1141 - 4
Self-aggregates of a synthetic cadmium chlorin in solid film as a photosynthetic antenna model; Amakawa M et al.; Self-aggregates of a synthetic cadmium chlorin possessing 3(1)-hydroxyl and 13-carbonyl groups were prepared in dried thin film . The solid film was characterized by visible and infrared absorption spectroscopies at both transmission and reflection modes . The spectra given by the two different modes were essentially the same and resembled those in the extramembranous antennas of green photosynthetic bacteria . The circular dichroism and resonance Raman spectra also supported the similarity between the artificial self-aggregates and the natural systems . Electron diffraction of the aggregated film by transmission electron microscopy indicated the presence of an orderly structure with a 6.4-A interval, which was estimated for the close Cd-Cd distance of the stacking in the self-aggregates . The in vitro self-assembly in the solid state is a good structural model for the in vivo antenna.

Cell Adhes Commun, 1999, 7(2), 73 - 83
Borrelia burgdorferi downregulates ICAM-1 on human synovial cells in vitro; Girschick HJ et al.; Lyme arthritis following infection with Borrelia burgdorferi (B . burgdorferi) is associated with the presence of bacteria in the joint, but the mechanism of persistent infection in the presence of specific antibodies and lymphocytes remains unknown . To investigate how an infection with B . burgdorferi might influence the local immune response in the joint, we examined the expression of cell adhesion molecules, human leucocyte antigens and inducible nitric oxide synthase (iNOS)-1 and -2 in human synovial cells after infection with B . burgdorferi in vitro . Synovial cells are known to influence the function of local immunologic effector cells and play a key role in the pannus formation of erosive arthritis . It has been shown previously that B . burgdorferi can persist in the cytosol of human synovial cells . The expression of the surface molecules ICAM-1, VCAM-1, HLA-class-I and -class-II and the cytosolic production of iNOS-1 and -2 in synovial cells was measured by flow cytometry for up to 5 days after infection with B . burgdorferi . A significant, lasting downregulation of surface ICAM-1 could be demonstrated on synovial cells, whereas no significant changes were seen in the expression of VCAM-1, HLA-class-I and -II, and of iNOS-1 and -2 . To determine the biological significance of this downregulation an in vitro adhesion assay using peripheral blood mononuclear cells was developed . After infection with B . burgdorferi a significantly smaller number of mononuclear cells was adhering to the synovial cell monolayer . Adhesion of peripheral mononuclear cells was shown to be in part mediated by ICAM-1 by using a blocking mononuclear antibody against ICAM-1 . Downregulation of ICAM-1 on synovial cells due to infection with B . burgdorferi might suppress the local immunosurveillance and might help the bacteria to persist in joint cells in vivo.

Appl Environ Microbiol, 1999 Aug, 65(8), 3526 - 33
Culturable populations of Sporomusa spp . and Desulfovibrio spp . in the anoxic bulk soil of flooded rice microcosms; Rosencrantz D et al.; Most-probable-number (MPN) counts were made of homoacetogenic and other bacteria present in the anoxic flooded bulk soil of laboratory microcosms containing 90- to 95-day-old rice plants . MPN counts with substrates known to be useful for the selective enrichment or the cultivation of homoacetogenic bacteria (betaine, ethylene glycol, 2, 3-butanediol, and 3,4,5-trimethoxybenzoate) gave counts of 2.3 x 10(3) to 2.8 x 10(5) cells per g of dry soil . Homoacetogens isolated from the terminal positive steps of these dilution cultures belonged to the genus Sporomusa . Counts with succinate, ethanol, and lactate gave much higher MPNs of 5.9 x 10(5) to 3.4 x 10(7) cells per g of dry soil and led to the isolation of Desulfovibrio spp . Counting experiments on lactate and ethanol which included Methanospirillum hungatei in the medium gave MPNs of 2.3 x 10(6) to 7.5 x 10(8) cells per g of dry soil and led to the isolation of Sporomusa spp . The latter strains could grow with betaine, ethylene glycol, 2, 3-butanediol, and/or 3,4,5-trimethoxybenzoate, but apparently most cells of Sporomusa spp . did not initiate growth in counting experiments with those substrates . Spores apparently accounted for 2 . 2% or less of the culturable bacteria . It appears that culturable Desulfovibrio spp . and Sporomusa spp . were present in approximately equal numbers in the bulk soil . Multiple, phylogenetically-distinct, phenotypically-different, strains of each genus were found in the same soil system.

Appl Environ Microbiol, 1999 Aug, 65(8), 3518 - 25
Optimization of terminal-restriction fragment length polymorphism analysis for complex marine bacterioplankton communities and comparison with denaturing gradient gel electrophoresis; Moeseneder MM et al.; The potential of terminal-restriction fragment length polymorphism (T-RFLP) and the detection of operational taxonomic units (OTUs) by capillary electrophoresis (CE) to characterize marine bacterioplankton communities was compared with that of denaturing gradient gel electrophoresis (DGGE) . A protocol has been developed to optimize the separation and detection of OTUs between 20 and 1, 632 bp by using CE and laser-induced fluorescence detection . Additionally, we compared T-RFLP fingerprinting to DGGE optimized for detection of less abundant OTUs . Similar results were obtained with both fingerprinting techniques, although the T-RFLP approach and CE detection of OTUs was more sensitive, as indicated by the higher number of OTUs detected . We tested the T-RFLP fingerprinting technique on complex marine bacterial communities by using the 16S rRNA gene and 16S rRNA as templates for PCR . Samples from the Northern and Middle Adriatic Sea and from the South and North Aegean Sea were compared . Distinct clusters were identifiable for different sampling sites . Thus, this technique is useful for rapid evaluation of the biogeographical distribution and relationships of bacterioplankton communities.

Tidsskr Nor Laegeforen, 1999 Jun 30, 119(17), 2510 - 4
{Dendritic cells--strong candidates for immunotherapy}; Lund-Johansen F et al.; Why are immune responses primarily directed towards infectious agents, and how can the immune system be manipulated to attack for instance malignant cells? The role of the dendritic cells in the immune system may provide the answers . We present a review of a field in which results from basic science are rapidly applied in clinical trials . We searched the Medline database using the terms dendritic cells combined with ontogeny, subpopulations, vaccine or review . Results from our own experimental work are also described . The cited studies show that dendritic cells take up material from their surroundings and migrate to lymphoid tissue where the material is presented to T-cells . Dendritic cells have the ability to selectively direct immune responses towards potentially harmful agents such as bacteria and viruses . Clinical trials show that vaccines based on the use of dendritic cells induce tumor-specific immunity and clinical remission . Experiments conducted by the authors and others indicate the existence of subpopulations of dendritic cells with specialized functions . Dendritic cells play a central role in the initiation of immune responses and may be used to manipulate the immune system . Their use in the treatment of diseases such as cancer is highly promising.

Biochim Biophys Acta, 1999 Jul 30, 1439(2), 299 - 316
Structural organization of mammalian lipid phosphate phosphatases: implications for signal transduction; Waggoner DW et al.; This article describes the regulation of cell signaling by lipid phosphate phosphatases (LPPs) that control the conversion of bioactive lipid phosphates to their dephosphorylated counterparts . A structural model of the LPPs, that were previously called Type 2 phosphatidate phosphatases, is described . LPPs are characterized by having no Mg(2+) requirement and their insensitivity to inhibition by N-ethylmaleimide . The LPPs have six putative transmembrane domains and three highly conserved domains that define a phosphatase superfamily . The conserved domains are juxtaposed to the proposed membrane spanning domains such that they probably form the active sites of the phosphatases . It is predicted that the active sites of the LPPs are exposed at the cell surface or on the luminal surface of intracellular organelles, such as Golgi or the endoplasmic reticulum, depending where various LPPs are expressed . LPPs could attenuate cell activation by dephosphorylating bioactive lipid phosphate esters such as phosphatidate, lysophosphatidate, sphingosine 1-phosphate and ceramide 1-phosphate . In so doing, the LPPs could generate alternative signals from diacylglycerol, sphingosine and ceramide . The LPPs might help to modulate cell signaling by the phospholipase D pathway . For example, phosphatidate generated within the cell by phospholipase D could be converted by an LPP to diacylglycerol . This should change the relative balance of signaling by these two lipids . Another possible function of the LPPs relates to the secretion of lysophosphatidate and sphingosine 1-phosphate by activated platelets and other cells . These exogenous lipids activate phospholipid growth factor receptors on the surface of cells . LPP activities could attenuate cell activation by lysophosphatidate and sphingosine 1-phosphate through their respective receptors.

J Biochem (Tokyo), 1999 Aug, 126(2), 313 - 9
Stimulation of peroxidase activity by decamerization related to ionic strength: AhpC protein from Amphibacillus xylanus; Kitano K et al.; AhpC protein, purified from Amphibacillus xylanus with a molecular mass of 20.8 kDa, protects cells against oxidation damage . The enzyme catalyses the reduction of hydroperoxides in cooperation with the 55 kDa flavoprotein, A . xylanus NADH oxidase (NADH oxidase-AhpC system) . A . xylanus AhpC has two disulfide linkages between monomers and can act in the homodimer form . Gel-filtration column chromatography and dynamic light scattering (DLS) suggest that A . xylanus AhpC also forms a large oligomeric assembly (10-12 mers) . A . xylanus AhpC was crystallized and X-ray diffraction data were collected to 3.0 A . The self-rotation function revealed fivefold and twofold axes located perpendicularly to each other, suggesting that the molecular assembly of A . xylanus AhpC is composed of ten monomers . The oligomerization of A . xylanus AhpC is affected by ionic strength in the DLS measurements . The H(2)O(2) reductase activity of the A . xylanus NADH oxidase-AhpC system is also affected by ionic strength, and it was found that the decamerization of AhpC might be required for the activation of the NADH oxidase-AhpC system.

Biophys J, 1999 Aug, 77(2), 666 - 81
Disordered exciton model for the core light-harvesting antenna of Rhodopseudomonas viridis; Novoderezhkin V et al.; In this work we explain the spectral heterogeneity of the absorption band ( . Biochim . Biophys . Acta . 1229:373-380), as well as the spectral evolution of pump-probe spectra for membranes of Rhodopseudomonas (Rps.) viridis . We propose an exciton model for the LH1 antenna of Rps . viridis and assume that LH1 consists of 24-32 strongly coupled BChl b molecules that form a ring-like structure with a 12- or 16-fold symmetry . The orientations and pigment-pigment distances of the BChls were taken to be the same as for the LH2 complexes of BChl a-containing bacteria . The model gave an excellent fit to the experimental results . The amount of energetic disorder necessary to explain the results could be precisely estimated and gave a value of 440-545 cm(-1) (full width at half-maximum) at low temperature and 550-620 cm(-1) at room temperature . Within the context of the model we calculated the coherence length of the steady-state exciton wavepacket to correspond to a delocalization over 5-10 BChl molecules at low temperature and over 4-6 molecules at room temperature . Possible origins of the fast electronic dephasing and the observed long-lived vibrational coherence are discussed.

Protein Sci, 1999 Jul, 8(7), 1432 - 44
Relating structure to thermodynamics: the crystal structures and binding affinity of eight OppA-peptide complexes; Davies TG et al.; The oligopeptide-binding protein OppA provides a useful model system for studying the physical chemistry underlying noncovalent interactions since it binds a variety of readily synthesized ligands . We have studied the binding of eight closely related tripeptides of the type Lysine-X-Lysine, where X is an abnormal amino acid, by isothermal titration calorimetry (ITC) and X-ray crystallography . The tripeptides fall into three series of ligands, which have been designed to examine the effects of small changes to the central side chain . Three ligands have a primary amine as the second side chain, two have a straight alkane chain, and three have ring systems . The results have revealed a definite preference for the binding of hydrophobic residues over the positively charged side chains, the latter binding only weakly due to unfavorable enthalpic effects . Within the series of positively charged groups, a point of lowest affinity has been identified and this is proposed to arise from unfavorable electrostatic interactions in the pocket, including the disruption of a key salt bridge . Marked entropy-enthalpy compensation is found across the series, and some of the difficulties in designing tightly binding ligands have been highlighted.

Res Microbiol, 1999 Jun, 150(5), 333 - 41
Detection of Bordetella bronchiseptica by the polymerase chain reaction; Hozbor D et al.; Polymerase chain reaction (PCR) assays were developed that enabled not only discriminative detection of three Bordetella species, B . pertussis, B . parapertussis, and B . bronchiseptica (Bspp PCR), but also specific detection of B . bronchiseptica (Bb PCR) . An upstream sequence of the flagellin gene was used as a target DNA region . This sequence contained differences in B . pertussis, B . parapertussis, and B . bronchiseptica DNA . These species could then be differentiated using two different sets of primers, Bspp and Bb . When oligonucleotide Bspp primers were used, PCR products were obtained from the three species of Bordetella . A fragment of the expected size (164 bp) was amplified using B . bronchiseptica and B . parapertussis DNA, but a fragment with a distinct molecular weight was amplified with B . pertussis DNA (195 bp) . This Bspp PCR was specific and sensitive, but it could not differentiate between B . parapertussis and B . bronchiseptica . When Bb primers were used, a 237-bp PCR product was detected only from B . bronchiseptica DNA . No PCR products were identified after Bb PCR amplification of DNAs either from B . parapertussis isolates or B . pertussis isolates, nor from other respiratory pathogen DNAs tested . This second PCR assay had a sensitivity limit of less than 10 organisms of B . bronchiseptica after detection with a specific probe . The specificity and the sensitivity of the fla PCR assay were evaluated with purified DNA, as was its capacity for detecting the bacteria in human clinical samples and in lungs of infected mice.

Chemosphere, 1999 Aug, 39(4), 665 - 82
Identification of single cultured micro-organisms based on their whole-community fatty acid profiles, using an extended extraction procedure; Zelles L; Fatty acid profiles obtained from single cultured organisms have been used to estimate which taxonomic groups are actually represented . The lipid extraction was modified to liberate fatty acids with ester-linkages, as well as those with non-ester-linkages and classify them in different chemically relevant groups . The discriminatory power of fatty acids, in different chemically relevant fractions and subfractions varied considerably . Saturated fatty acids were least able to predict actual group membership (ca . 75%), while nonester-linked hydroxy fatty acids, which largely go undetected by the simple extraction procedure, gave the highest predictability values (ca . 94%) . The discriminatory power of the method used was enhanced by increasing the number of well-defined fatty acid methyl esters . The estimation capacity of the results was improved, when the fatty acids, which were presumed to be common and widespread, were excluded from the whole-community fatty acid profiles prior to multivariate analysis.

Acta Crystallogr D Biol Crystallogr, 1999 Aug, 55 ( Pt 8), 1449 - 58
Two-wavelength MAD phasing: in search of the optimal choice of wavelengths; Gonzalez A et al.; The multiwavelength anomalous dispersion (MAD) method is increasingly being used to determine protein crystal structures . In theory, data collection at two wavelengths is sufficient for the determination of MAD phases, but three or even more wavelengths are used most often . In this paper, the results of the phasing procedure using only two wavelengths for proteins containing different types of anomalous scatterers are analyzed . In these cases, it is shown that this approach leads to interpretable maps, similar in quality to those obtained with data collected at three wavelengths, provided that the wavelengths are chosen so as to give a large contrast in the real part of the anomalous scattering factor f . The consequences for a rational MAD data-collection strategy are discussed.

Chem Biol Interact, 1999 May 14, 119-120, 513 - 7
Molecular cloning of neuropathy target esterase (NTE); Glynn P et al.; Covalent modification of NTE, a neuronal protein with serine esterase activity, by certain organophosphates (OP) initiates degeneration of long axons in the peripheral and central nervous system . Simple inhibition of NTE esterase activity does not initiate neuropathy; the latter requires aging of the OP bound to the catalytic serine residue so that a negatively-charged species is left attached to the active site . This may indicate that a non-esterase function of NTE is important for axonal maintenance . We have recently cloned NTE and shown that it is unrelated to any known serine hydrolases but contains a novel C-terminal domain which is conserved from bacteria to man . Furthermore, the catalytic serine is located within this domain at the centre of a helical hydrophobic segment of the polypeptide's secondary structure . The integrity of NTE would be severely compromised by the presence of a negatively-charged organophosphate moiety at this site . Implications for possible higher-order structures and functions for NTE are discussed.

Chem Biol Interact, 1999 May 14, 119-120, 455 - 62
Alteromonas prolidase for organophosphorus G-agent decontamination; Cheng TC et al.; Enzymes catalyzing the hydrolysis of highly toxic organophosphorus compounds (OPs) are classified as organophosphorus acid anhydrolases (OPAA; EC 3.1.8.2) . Recently, the genes encoding OPAA from two species of Alteromonas were cloned and sequenced . Sequence and biochemical analyses of the cloned genes and enzymes have established Alteromonas OPAAs to be prolidases (E.C . 3.4.13.9), a type of dipeptidase hydrolyzing dipeptides with a prolyl residue in the carboxyl-terminal position (X-Pro) . Alteromonas prolidases hydrolyze a broad range of G-type chemical warfare (CW) nerve agents . Efforts to over-produce a prolidase from A . sp.JD6.5 with the goal of developing strategies for long-term storage and decontamination have been successfully achieved . Large-scale production of this G-agent degrading enzyme is now feasible with the availability of an over-producing recombinant cell line . Use of this enzyme for development of a safe and non-corrosive decontamination system is discussed.

Ann Thorac Surg, 1999 Jul, 68(1), 58 - 62
Influence of different autotransfusion devices on the quality of salvaged blood; Reents W et al.; BACKGROUND: Cardiopulmonary bypass causes a systemic inflammatory response and impaired hemostasis . We investigated whether intraoperative blood salvage with the cardiotomy suction contributes to these alterations . Furthermore, an alternative autotransfusion device (Haemonetics cell-saving device) was examined . METHODS: In 10 patients, interleukin-6, interleukin-8, tumor necrosis factor-alpha, thrombin-antithrombin complex, plasmin-antiplasmin complex, free hemoglobin, and the percentage of CD62+ thrombocytes were determined in the systemic circulation during cardiopulmonary bypass, in the cardiotomy suction tube, and in the blood from the cell-saving device . Additionally, bacterial contamination was examined . RESULTS: Median levels of interleukin-6 (52 versus 10 microg/L; p = 0.005), interleukin-8 (26 versus 20 microg/L; p = 0.017), tumor necrosis factor-alpha (24 versus 1 microg/L; p = 0.005), thrombin-antithrombin complex (113 versus 43 microg/L; p = 0.005), plasmin-antiplasmin complex (566 versus 489 microg/L; p = 0.022), and free hemoglobin (61 versus 30 mg/dL; p = 0.005) were higher in the cardiotomy suction tube compared with the systemic circulation . After processing the blood from the cell-saving device, interleukin-8, thrombin-antithrombin complex, and free hemoglobin remained above reference range, and in 90% of the cases bacterial contamination was observed . CONCLUSIONS: Cardiotomy suction additionally contributes to the release of proinflammatory cytokines, activation of coagulation, and hemolysis . Because blood salvage with a Haemonetics cell-saving device led to normalization of some, but not all, parameters and bacterial contamination was common, the alternative use seems at least questionable.

Vet Pathol, 1999 Jul, 36(4), 347 - 51
Necrotizing mycotic vasculitis with cerebral infarction caused by Aspergillus niger in a horse with acute typholocolitis; Tunev SS et al.; An 18-year-old Morgan mare was presented to the Veterinary Medical Teaching Hospital, University of Illinois, with a 10-day history of watery diarrhea, depression, and dysphagia . On admission, the animal was severely dehydrated, depressed, and unable to swallow and had no clinical signs of diarrhea . The respiratory and heart rate and body temperature were within normal limits . Following fluid therapy, the mare developed severe watery diarrhea and continued to be depressed, incoordinated, and dysphagic . The animal died on the fourth day after admission and was sent to the Laboratories of Veterinary Diagnostic Medicine for necropsy . Gross postmortem findings were consistent with an acute cerebral infarction in the right cerebral hemisphere, an acute necrotizing typhlocolitis, multifocal petechial and ecchymotic hemorrhages, enlarged and congested pars intermedia of the pituitary gland, and marked bilateral adrenocortical hyperplasia with multifocal areas of necrosis and hemorrhage . Histologic evaluation of the affected brain demonstrated an area of coagulative necrosis of the gray matter, with hemorrhage, vasculitis, and thrombosis . There were many fungal hyphae 3.5-6.0 microm, pale basophilic, septate, and occasionally branching at 45 degrees present in the arterial walls and throughout the necrotic tissue . Immunohistochemical analysis revealed Aspergillus niger as the etiologic agent responsible for the mycotic vasculitis and infarction in the brain . Bacteria culture and immunohistochemical staining of the colon and cecum failed to demonstrate specific pathogens.

Probl Tuberk, 1999, (2), 20 - 2
{Present-day clinical and social characteristics of newly diagnosed patients with pulmonary tuberculosis}; Khudushina TA et al.; The present clinical and social characteristics of new cases with pulmonary tuberculosis show some features . The clinical characteristics of patients in 1995 to 1997 indicate an increased number of patients with disseminated, frequently bilateral processes and acute tuberculosis which is largely associated with the decline in preventive fluorographic surveys of the population including those who have contacted bacteria-isolating patients . There were 61.3% with pulmonary symptoms among those who visit physicians and 62.1% among those from the foci of tuberculous infection . Social aspects of the study demonstrate that there is a great number of young patients (56.4% of those under 39-49 years) and a high proportion (81.1%) of able-bodied patients among both males and females . There was an increase in the proportion of patients having higher education (43.7%) and a decrease in laboring patients (21.4%) . This is most likely to be associated with the fact that many patients did not work by the profession they had been trained . There was a higher proportion (43.0%) of the patients having different poor working conditions and patterns . In these years, the patients' financial position deteriorated . It was slightly better in the employed than in the unemployed . However, in both groups the bulk of the patients had income per head in the family at the subsistence level or lower (56.2 and 21.6% in the unemployed and employed, respectively . These data were assessed by the guidelines made by the State Statistics Committee of the Russian Federation and the RF Ministry of Labour . It should be noted that the nutritional quality did not correspond to the optimal composition ratio of dietary foods and it was characterized by the high content of less valuable foods, carbohydrates, low levels of protein and vitamins . Poor social aspects in characteristics of patients were undoubtedly to be associated with a greater number of patients with severe, frequently progressive forms of tuberculosis although they had received specific chemotherapy.

Indian J Pathol Microbiol, 1999 Jan, 42(1), 81 - 7
A study of mycotic keratitis in Mumbai; Deshpande SD et al.; A total of 1010 clinically suspected cases of mycotic keratitis were studied from 1988 to 1996 for evidence of fungal infection and for identification of the aetiologic agents of keratitis in Mumbai . Of these 367 cases were reported positive by microscopy and culture . Seventy nine percent of the cases were between the ages 21 and 50 years . Male patients were more often affected than females . Eighty eight percent of patients were farmers or construction workers and 89.92% of cases gave a definite history of antecedent corneal trauma . A single fungal isolate was obtained in 307 cases and multiple isolates in 20 cases . Mixed isolates of bacteria and fungi were grown in 40 cases . The predominant isolate was Aspergillus species in 219 cases, followed by Candida species (36), Fusarium species (33) and Penicillium species (34) . Filamentous fungal isolates from 22 cases remained unidentified . Mycotic keratitis should be suspected in every patient with a corneal lesion and should be ruled out before commencing steroids and antiboitics.

Can J Microbiol, 1999 Apr, 45(4), 312 - 7
Identification of genes unique to Mo-independent nitrogenase systems in diverse diazotrophs; Loveless TM et al.; A number of nitrogen-fixing bacteria were screened using PCR for genes (vnfG and anfG) unique to the V-containing nitrogenase (vnf) and the Fe-only nitrogenase (anf) systems . Products with sequences similar to that of vnfG were obtained from Azotobacter paspali and Azotobacter salinestris genomic DNAs, and products with sequences similar to that of anfG were obtained from Azomonas macrocytogenes, Rhodospirillum rubrum, and Azotobacter paspali DNAs . Phylogenetic analysis of the deduced amino acid sequences of anfG and vnfG genes shows that each gene product forms a distinct cluster . Furthermore, amplification of an internal 839-bp region in anfD and vnfD yielded a product similar to anfD from Heliobacterium gestii and a product similar to vnfD from Azotobacter paspali and Azotobacter salinestris . Phylogenetic analysis of NifD, VnfD, and AnfD amino acid sequences indicates that AnfD and VnfD sequences are more closely related to each other than either is to NifD . The results of this study suggest that Azotobacter salinestris possesses the potential to express the vanadium (V)-containing nitrogenase (nitrogenase 2) and that R . rubrum, Azomonas macrocytogenes, and H . gestii possess the potential to express the Fe-only nitrogenase (nitrogenase 3) . Like Azotobacter vinelandii, Azotobacter paspali appears to have the potential to express both the V-containing nitrogenase and the Fe-only nitrogenase.

Biometals, 1999 Mar, 12(1), 35 - 45
Redox and specific effects of vanadium ions on respiratory enzymes; Mendz GL; The effects of vanadium ions on the activities of enzymes of aerobic and anaerobic respiratory chains were investigated in vitro and in situ employing 1H-, 14N-, 31P- and 51V- nuclear magnetic resonance spectroscopy, electron paramagnetic resonance spectroscopy and spectrophotometry . Vanadate and vanadyl ions produced either non-specific redox or specific activation or inhibition of respiratory enzymes . The oxidants molybdate and chromate and the reductant dithiothreitol were used to distinguish between non-specific and specific effects of vanadium ions on enzyme activities . The results suggested that components of anaerobic respiratory chains were more susceptible to vanadium ions than those of the aerobic respiratory chain.

Reprod Nutr Dev, 1999 May-Jun, 39(3), 399 - 408
Melatonin and 5-methoxytryptamine in non-metazoans; Hardeland R; Melatonin seems to be an almost ubiquitous substance, which has been detected not only in metazoans, but also in all major non-metazoan taxa investigated, including bacteria, dinoflagellates, euglenoids, trypanosomids, fungi, rhodophyceans, pheophyceans, chlorophyceans and angiosperms . Despite its vast abundance, little is known to date about its functions . Its presence is not necessarily associated with circadian rhythmicity, which is evident in yeast . Circadian rhythms of melatonin have been reported in non-metazoans only for several unicellular organisms and in one angiosperm . In dinoflagellates, which have been studied in the most detail, the effects on enzyme activities and on phase shifting are known, but the most spectacular actions concerning the stimulation of bioluminescence, changes in cytoplasmic pH and induction of resting stages, can be related to a metabolite of melatonin, the 5-methoxytryptamine; therefore, melatonin should also be considered as a source of other agonists.

Klin Monatsbl Augenheilkd, 1999 May, 214(5), 291 - 4
{Central corneal diseases}; Frueh BE; BACKGROUND: Central corneal pathologies can lead to an irreversible decrease of best corrected visual acuity if not diagnosed and treated appropriately . This article reviews the differential diagnosis of central corneal opacities in the newborn, of central infectious corneal ulcers, and the therapy of sterile, central keratolysis . MATERIAL AND METHODS: Authors' personal experience and review of the literature . RESULTS: Flow charts for diagnosis and treatment strategy have been elaborated . CONCLUSIONS: Corneal opacities in newborns create an emergency situation . In order to treat successfully and avoid or diminish amblyopia, it is imperative to rule out congenital glaucoma . The aetiology of central corneal ulcers should always be confirmed by positive cultures to be able to treat specifically . When the standard topic therapy fails, one has to consider rare bacteria, parasites, virus, or patients' compliance . The treatment of central sterile keratolysis in rheumatoid arthritis must be intensive and immunosuppression has to be performed early enough in the course of prevent the formation of a descemetocoele or spontaneous corneal perforation.

J Bacteriol, 1999 Aug, 181(15), 4708 - 10
Gene organization of the dnaA region of Wolbachia; Sun LV et al.; The dnaA region of Wolbachia, an intracellular bacterial parasite of insects, is unique . A glnA cognate was found upstream of the dnaA gene, while neither of the two open reading frames detected downstream of dnaA has any homologue in the database . This unusual gene arrangement may reflect requirements associated with the unique ecological niche this agent occupies.

J Bacteriol, 1999 Aug, 181(15), 4461 - 8
Isolation and properties of the complex between the enhancer binding protein NIFA and the sensor NIFL; Money T et al.; In Azotobacter vinelandii, activation of nif gene expression by the transcriptional regulatory enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo . NIFL is a novel redox-sensing flavoprotein which is also responsive to adenosine nucleotides in vitro . Inhibition of NIFA activity by NIFL requires stoichiometric amounts of the two proteins, implying that the mechanism of inhibition is by direct protein-protein interaction rather than by catalytic modification of the NIFA protein . The formation of the inhibitory complex between NIFL and NIFA may be regulated by the intracellular ATP/ADP ratio . We show that adenosine nucleotides promote complex formation between purified NIFA and NIFL in vitro, allowing isolation of the NIFL-NIFA complex . The complex can also be isolated from cell extracts containing coexpressed NIFL and NIFA in the presence of MgADP . Removal of the nucleotide causes dissociation of the complex . Experiments with truncated proteins demonstrate that the amino-terminal domain of NIFA and the C-terminal region of NIFL potentiate the ADP-dependent stimulation of NIFL-NIFA complex formation.

Protein Expr Purif, 1999 Jul, 16(2), 298 - 307
Expression and purification of recombinant human zona pellucida proteins; Harris JD et al.; Recombinant human zona pellucida (rhZP) proteins (minus the N-terminal leader and the C-terminal transmembrane-like domain) were expressed in four different expression systems: bacteria, yeast, insect cells, and Chinese Hamster Ovary (CHO) cells . The recombinant proteins in each system were engineered with a C-terminal six histidine (His6) segment that was used to purify the proteins by metal affinity {either nickel (Ni) or cobalt (Co)} column chromatography . Each of the rhZP proteins was a candidate antigen as an immunocontraceptive vaccine . However, the rhZP proteins produced in bacteria, yeast and insect cell culture could only be purified after being solubilized by strong denaturants . After purification the final products of each of these expression systems required 6 M urea to maintain solubility . However, the rhZP proteins expressed by CHO cells were secreted into the media, and the soluble proteins could be purified to near homogeneity . In this report the expression and purification procedures used to produce and isolate these secreted proteins are described .

J Theor Biol, 1999 Jul 7, 199(1), 11 - 23
The application of a linear algebra to the analysis of mutation rates; Jones ME et al.; Cells and bacteria growing in culture are subject to mutation, and as this mutation is the ultimate substrate for selection and evolution, the factors controlling the mutation rate are of some interest . The mutational event is not observed directly, but is inferred from the phenotype of the original mutant or of its descendants; the rate of mutation is inferred from the number of such mutant phenotypes . Such inference presumes a knowledge of the probability distribution for the size of a clone arising from a single mutation . We develop a mathematical formulation that assists in the design and analysis of experiments which investigate mutation rates and mutant clone size distribution, and we use it to analyse data for which the classical Luria-Delbruck clone-size distribution must be rejected .

Dev Biol, 1999 Aug 1, 212(1), 182 - 90
Fingerprinting of adenylyl cyclase activities during Dictyostelium development indicates a dominant role for adenylyl cyclase B in terminal differentiation; Meima ME et al.; Activation of cAMP-dependent protein kinase (PKA) triggers terminal differentiation in Dictyostelium, without an obvious requirement for the G-protein-coupled adenylyl cyclase, ACA, or the osmosensory adenylyl cyclase, ACG . A third adenylyl cyclase, ACB, was recently detected in rapidly developing mutants . The specific characteristics of ACA, ACG, and ACB were used to determine their respective activities during development of wild-type cells . ACA was highly active during aggregation, with negligible activity in the slug stage . ACG activity was not present at significant levels until mature spores had formed . ACB activity increased strongly after slugs had formed with optimal activity at early fruiting body formation . The same high activity was observed in slugs of ACG null mutants and ACA null mutants that overexpress PKA (acaA/PKA), indicating that it was not due to either ACA or ACG . The detection of high adenylyl cyclase activity in acaA/PKA null mutants contradicts earlier conclusions (B . Wang and A . Kuspa, Science 277, 251-254, 1997) that these mutants can develop into fruiting bodies in the complete absence of cAMP . In contrast to slugs of null mutants for the intracellular cAMP-phosphodiesterase REGA, where both intact cells and lysates show ACB activity, wild-type slugs only show activity in lysates . This indicates that cAMP accumulation by ACB in living cells is controlled by REGA . Both REGA inhibition and PKA overexpression cause precocious terminal differentiation . The developmental regulation of ACB and its relationship to REGA suggest that ACB activates PKA and induces terminal differentiation .

J Colloid Interface Sci, 1999 Jul 15, 215(2), 226 - 243
Microstructure Evolution in Polymer Latex Coatings for Whole-Cell Biocatalyst Application; Huang Z et al.; The microstructure evolution of two poly(vinyl acetate-co-acrylic acid) latex coatings was elucidated by cryogenic scanning electron microscopy (cryo-SEM) and atomic force microscopy . The stages documented are particle suspension, consolidation, deformation, partial coalescence into a coherent film, and rehydration of the latter . Of particular interest is formation of a porous polymeric matrix of desired porosity and permeability of remnant interstices between deformed and partially coalesced particles; the application is to biocatalytic coatings in which viable bacteria are imprisoned in porous latex coatings . Effects of drying condition and time, rehydration behavior of latex, and the presence of glycerol on the microstructure of latex coatings were revealed by time-sectioning and cryofracture techniques of cryo-SEM . Results showed that porosity and permeability can be controlled by choice of drying and rehydration protocols . Evidence showed that glycerol retarded particle deformation, compaction, and coalescence and that substantial amounts of glycerol were expelled to the surface of the coating as drying proceeded . Implications for design of bacteria-laden and bacteria-free coating layers are discussed .

MMWR Morb Mortal Wkly Rep, 1999 Jul 9, 48(26), 563 - 5
Thimerosal in vaccines: a joint statement of the American Academy of Pediatrics and the Public Health Service; Role of the amino-terminal region of the DnaA protein in opening of the duplex DNA at the oriC region; Faculty of Pharmaceutical Sciences, Okayama University, JapanWe report in this paper that the amino acid residues Ile-26 and Leu-40 of the DnaA protein are essential for the DNA replication activity in vitro . Lines of evidence to support this conclusion are as follows . Variants of the DnaA protein containing either an Ile-26-Ser or Leu-40-Ser replacement were unable to support oriC DNA replication in vitro . Though the mutant DnaA proteins retained the capability to bind oriC DNA, they were unable to open the duplex DNA at oriC . Based on these and other results, we conclude that the N-terminal region of the DnaA protein is involved in the oligomerization of this protein, an essential step for the duplex opening activity at oriC.

Mol Microbiol, 1999 Aug, 33(3), 556 - 68
Entamoeba histolytica : a novel cysteine protease and an adhesin form the 112 kDa surface protein; Garcia-Rivera G et al.; Here, we present evidence that a cysteine protease (EhCP112) and a protein with an adherence domain (EhADH112) form the Entamoeba histolytica 112 kDa adhesin . Immunoelectron microscopy and immunofluorescence assays using monoclonal antibodies (mAbAdh) revealed that, during phagocytosis, the adhesin is translocated from the plasma membrane to phagocytic vacuoles . mAbAdh inhibited 54% adherence, 41% phagocytosis, and 35% and 62% destruction of MDCK cell monolayers by live trophozoites and their extracts respectively . We cloned a 3587 bp DNA fragment (Eh112 ) with two open reading frames (ORFs) separated by a 188 bp non-coding region . The ORF at the 5' end (Ehcp112 ) encodes a protein with a cysteine protease active site, a transmembranal segment and an RGD motif . The second ORF (Ehadh112 ) encodes a protein recognized by mAbAdh with three putative transmembranal segments and four glycosylation sites . Northern blot, primer extension and Southern blot experiments revealed that Ehcp112 and Ehadh112 are two adjacent genes in DNA . Ehcp112 and Ehadh112 genes were expressed in bacteria . The recombinant peptides presented protease activity and inhibited adherence and phagocytosis, respectively, and both were recognized by mAbAdh . The EhCP112 and EhADH112 peptides could be joined by covalent or strong electrostatic forces, which are not broken during phagocytosis.

Mol Microbiol, 1999 Aug, 33(3), 457 - 65
Displacement of cellular proteins by functional analogues from plasmids or viruses could explain puzzling phylogenies of many DNA informational proteins; Forterre P; Comparative genomics has revealed many examples in which the same function is performed by unrelated or distantly related proteins in different cellular lineages . In some cases, this has been explained by the replacement of the original gene by a paralogue or non-homologue, a phenomenon known as non-orthologous gene displacement . Such gene displacement probably occurred early on in the history of proteins involved in DNA replication, repair, recombination and transcription (DNA informational proteins), i.e . just after the divergence of archaea, bacteria and eukarya from the last universal cellular ancestor (LUCA) . This would explain why many DNA informational proteins are not orthologues between the three domains of life . However, in many cases, the origin of the displacing genes is obscure, as they do not even have detectable homologues in another domain . I suggest here that the original cellular DNA informational proteins have often been replaced by proteins of viral or plasmid origin . As viral and plasmid-encoded proteins are usually very divergent from their cellular counterparts, this would explain the puzzling phylogenies and distribution of many DNA informational proteins between the three domains of life.

Infect Immun, 1999 Aug, 67(8), 4264 - 7
Fluorescent labels influence phagocytosis of Bordetella pertussis by human neutrophils; Weingart CL et al.; To explore the role of neutrophil phagocytosis in host defense against Bordetella pertussis, bacteria were labeled extrinsically with fluorescein isothiocyanate (FITC) or genetically with green fluorescent protein (GFP) and incubated with adherent human neutrophils in the presence or absence of heat-inactivated human immune serum . In the absence of antibodies, FITC-labeled bacteria were located primarily on the surface of the neutrophils with few bacteria ingested . However, after opsonization, about seven times more bacteria were located intracellularly, indicating that antibodies promoted phagocytosis . In contrast, bacteria labeled intrinsically with GFP were not efficiently phagocytosed even in the presence of opsonizing antibodies, suggesting that FITC interfered with a bacterial defense . Because FITC covalently modifies proteins and could affect their function, we tested the effect of FITC on adenylate cyclase toxin activity, an important extracellular virulence factor . FITC-labeled bacteria had fivefold-less adenylate cyclase toxin activity than did unlabeled wild-type bacteria or GFP-expressing bacteria, suggesting that FITC compromised adenylate cyclase toxin activity . These data demonstrated that at least one extracellular virulence factor was affected by FITC labeling and that GFP is a more appropriate label for B . pertussis.

Infect Immun, 1999 Aug, 67(8), 4223 - 30
Attenuated host resistance against Mycobacterium bovis BCG infection in mice lacking osteopontin; Nau GJ et al.; Expression of the cytokine osteopontin (OPN) is elevated in granulomas caused by Mycobacterium tuberculosis . We tested the hypothesis that OPN contributes to host protection in a mouse model of mycobacterial infection . When infected with Mycobacterium bovis BCG, mice lacking a functional OPN gene had more severe infections characterized by heavier bacterial loads and a delayed clearance of the bacteria . The OPN-null mice had greater granuloma burdens consistent with the elevated bacterial load . The ability of osteopontin to facilitate the clearance of mycobacteria was most pronounced early after infection and appeared to be independent of known mediators of resistance to infection by mycobacteria: antigen-specific T-cell immunity, gamma interferon production, and nitric oxide production . BCG grew more rapidly in macrophages derived from OPN-null mice than in those from wild-type mice, demonstrating that the null phenotype was due to an intrinsic macrophage defect . These results indicate that osteopontin augments the host response against a mycobacterial infection and that it acts independently from other antimycobacterial resistance mechanisms.

Infect Immun, 1999 Aug, 67(8), 4041 - 7
Early acidification of phagosomes containing Brucella suis is essential for intracellular survival in murine macrophages; Porte F et al.; Brucella suis is a facultative intracellular pathogen of mammals, residing in macrophage vacuoles . In this work, we studied the phagosomal environment of these bacteria in order to better understand the mechanisms allowing survival and multiplication of B . suis . Intraphagosomal pH in murine J774 cells was determined by measuring the fluorescence intensity of opsonized, carboxyfluorescein-rhodamine- and Oregon Green 488-rhodamine-labeled bacteria . Compartments containing live B . suis acidified to a pH of about 4.0 to 4.5 within 60 min . Acidification of B . suis-containing phagosomes in the early phase of infection was abolished by treatment of host cells with 100 nM bafilomycin A(1), a specific inhibitor of vacuolar proton-ATPases . This neutralization at 1 h postinfection resulted in a 2- to 34-fold reduction of opsonized and nonopsonized viable intracellular bacteria at 4 and 6 h postinfection, respectively . Ammonium chloride and monensin, other pH-neutralizing reagents, led to comparable loss of intracellular viability . Addition of ammonium chloride at 7 h after the beginning of infection, however, did not affect intracellular multiplication of B . suis, in contrast to treatment at 1 h postinfection, where bacteria were completely eradicated within 48 h . Thus, we conclude that phagosomes with B . suis acidify rapidly after infection, and that this early acidification is essential for replication of the bacteria within the macrophage.

Infect Immun, 1999 Aug, 67(8), 3763 - 7
Genetic basis for lipopolysaccharide O-antigen biosynthesis in bordetellae; Preston A et al.; Bordetella bronchiseptica and Bordetella parapertussis express a surface polysaccharide, attached to a lipopolysaccharide, which has been called O antigen . This structure is absent from Bordetella pertussis . We report the identification of a large genetic locus in B . bronchiseptica and B . parapertussis that is required for O-antigen biosynthesis . The locus is replaced by an insertion sequence in B . pertussis, explaining the lack of O-antigen biosynthesis in this species . The DNA sequence of the B . bronchiseptica locus has been determined and the presence of 21 open reading frames has been revealed . We have ascribed putative functions to many of these open reading frames based on database searches . Mutations in the locus in B . bronchiseptica and B . parapertussis prevent O-antigen biosynthesis and provide tools for the study of the role of O antigen in infections caused by these bacteria.

Front Biosci, 1999 Jul 15, 4, e42 - 6
Mechanisms of alcohol-induced hepatotoxicity: studies in rats; Thurman RG et al.; Alcohol treatment results in increases in the release of endotoxin from gut bacteria and membrane permeability of the gut to endotoxin, or both . Females are more sensitive to these changes . Elevated levels of endotoxin activate Kupffer cells to release substances such as eicosanoids, TNF-alpha and free radicals . Prostaglandins increase oxygen uptake and most likely are responsible for the hypermetabolic state in the liver . The increase in oxygen demand leads to hypoxia in the liver, and on reperfusion, alpha-hydroxyethyl free radicals are formed which lead to tissue damage in oxygen-poor pericentral regions of the liver lobule.

Contact Dermatitis, 1999 Jul, 41(1), 1 - 13
An update of the risk assessment for methylchloroisothiazolinone/methylisothiazolinone (MCI/MI) with focus on rinse-off products; Fewings J et al.; Methylchloroisothiazolinone/methylisothiazolinone (MCI/MI) has been widely used during the last 20 years for the preservation of aqueous systems in cosmetics, toiletries and in various industrial applications . MCI/MI has a broad spectrum of activity against fungi and bacteria at very low concentrations . The allergic contact potential of MCI/MI has been known for many years . This paper provides a review of pre-clinical and clinical experimental studies as well as experience from dermatology clinics worldwide . This forms the basis for an update of the risk assessment for the use of MCI/MI in rinse-off products . The scientific data indicate that the actual sensitization rate observed with a contact allergen is extremely dependent on dose and type of exposure . This review of the data leads to the conclusion that, under normal use conditions, within the current permitted/ recommended use concentrations for MCI/MI of up to 15ppm, the risk of primary sensitization from the use of rinse-off products is negligible, and elicitation of allergic contact dermatitis in MCI/MI-sensitized individuals rare, after exposure to MCI/MI-preserved rinse-off products.

Ann N Y Acad Sci, 1999 May 18, 870, 293 - 300
Dynamic evolution of genomes and the concept of genome space; Bellgard MI et al.; A new era in the elucidation of genome evolution has been heralded with the availability of numerous genome sequences . With these data, it has been possible to study evolutionary processes at a greater level of detail in order to characterize features such as gene shuffling, genome rearrangements, base bias composition, and horizontal gene transfer . In this paper, we discuss the evolutionary implications of significant rearrangements within genomes as well as characteristic genomic regions that have been conserved across genomes . This is based on our analysis of orthologous and paralogous genes . We argue that genome plasticity has most likely contributed substantially to the dynamic evolution of genomes . We also describe the characteristic mosaic features of an archaea genome that is comprised of both bacterial and eukaryal elements . Here we investigate base compositional differences as well as the similarity of this species' genes to either bacteria or eukarya . We conclude that these features can be largely explained by the mechanism of horizontal gene transfer . Finally, we introduce the concept of genome space which is defined as the entire set of genomes of all living organisms . We explain its usefulness to describe as well as to gain deeper insight into the general features of the dynamic genomic evolutionary process.

Ann N Y Acad Sci, 1999 May 18, 870, 146 - 55
Evolution of evolvability; Radman M et al.; Genomic sequence data provide evidence for a common origin of life and for its evolution by genetic variation via mutation and recombination . This paper discusses the fundamental dialectic paradigm of evolution--stability versus variability--at the crossroads of molecular genetics, population genetics, ecology, and the emerging science of experimental evolution . Experimental evolution of molecules, viruses, and bacteria can be used not only to test some basic evolutionary hypotheses but also to create new organisms for applications in biotechnology, agriculture, and medicine.

J Immunol, 1999 Aug 1, 163(3), 1490 - 7
Cellular immune responses are essential for the development of Helicobacter felis-associated gastric pathology; Roth KA et al.; The bacteria Helicobacter pylori is a major human pathogen that infects over half of the world's population . Infection initiates a series of changes in the gastric mucosa, beginning with atrophic gastritis and leading in some patients to peptic ulcer disease, mucosa-associated lymphomas, and gastric adenocarcinoma . Although this cascade of events clearly occurs, little is known about the role of the host immune response in disease progression . We have utilized the C57BL/6 Helicobacter felis mouse model to critically analyze the role of the adaptive immune response in the development of Helicobacter-associated gastric pathology . Infection of B and T cell-deficient RAG-1-/- mice or T cell-deficient TCRbetadelta-/- mice with H . felis resulted in high levels of colonization, but no detectable gastric pathology . Conversely, infection of B cell-deficient microMT mice resulted in severe gastric alterations identical with those seen in immunocompetent C57BL/6-infected mice, including gastric mucosal hyperplasia and intestinal metaplasia . These results demonstrate that the host T cell response is a critical mediator of Helicobacter-associated gastric pathology, and that B cells and their secreted Abs are not the effectors of the immune-mediated gastric pathology seen after H . felis infection . These results indicate that in addition to specific Helicobacter virulence factors, the host immune response is an important determinant of Helicobacter-associated disease.

Am J Surg, 1999 Jun, 177(6), 450 - 3
External compression dressing versus standard dressing after axillary lymphadenectomy; O'Hea BJ et al.; BACKGROUND: Closed-catheter drainage after axillary lymph node dissection (ALND) for breast cancer may constitute a significant inconvenience to the recovering patient, and may also serve as portals of entry for bacteria . Any intervention that could reduce the volume and duration of postoperative drainage would be beneficial . The purpose of this study was to determine whether an external compression dressing after ALND would decrease postoperative drainage, afford earlier drain removal, and reduce subsequent seroma formation . PATIENTS AND METHODS: One hundred thirty-five women undergoing definitive surgical treatment for breast cancer were randomized to receive a compression dressing (n = 66) or standard dressing (n = 69) . They were also stratified for modified radical mastectomy (MRM; n = 74) or breast conservation therapy (BCT; n = 61) . All patients had ALND . The compression dressing consisted of a circumferential chest wrap of two 6-inch Ace bandages, held in place by circumferential Elastoplast bandage, and it was applied by the same nurse . This dressing remained intact until postoperative day 4 . Patients in the standard dressing group (control) were fitted with a front-fastening Surgibra only . Drains were removed when the total daily amount was <50 cc . Postoperative drainage volume, total days with drain, and frequency of seroma formation were recorded for each patient . RESULTS: After 4 days, wound drainage in both groups was nearly identical (compression = 490 cc, standard = 517 cc; P = 0.48) . Total days with drain were also similar (compression = 6.4 days, standard = 6.1 days; P = 0.69) . The compression dressing did not reduce seroma formation . In fact, there was a statistically significant increase in the number of seroma aspirations per patient in the compression group (compression = 2.9, standard = 1.8; P <0.01) . The increase in seroma aspirations was more significant in MRM patients (compression = 3.1, standard = 1.7; P <0.01) than in BCT patients (compression = 2.6, standard = 1.8; P = 0.20) . CONCLUSIONS: External compression dressing fails to decrease postoperative drainage and may increase the incidence of seroma formation after drain removal . Thus, routine use of a compression dressing to reduce postoperative drainage after ALND for breast cancer is not warranted.

Microbios, 1999, 97(388), 153 - 63
Coccoid forms of Helicobacter pylori can be viable; Ren Z et al.; Controversy exists as to whether the coccoid form of Helicobacter pylori can exist in a viable form . Conversion of helical to coccoid morphology occurs in culture over several days . In this study, the morphology was correlated with parameters of genetic integrity in the reference NCTC 11637 strain over 21 days of culture . The capacity to regrow colonies of helical form was demonstrated from a culture where the coccoid form constituted up to 95% and negligible urease activity could be detected . Urease enzyme activity and its mRNA decreased between day 0 and 10 while 26 kD mRNA and 16S rRNA were expressed unchanged for up to 14 and 21 days of culture, respectively . Expression of mRNA for the Cag A gene behaved in a similar fashion to that of urease . No evidence of DNA fragmentation was detected . These data suggest that a viable form of non-urease producing H . pylori exists after short to intermediate culture and that some if not all of these viable bacteria have coccoid morphology.

Exp Cell Res, 1999 Aug 1, 250(2), 524 - 34
Effects of mutations in the cytoplasmic domain of integrin beta(1) to talin binding and cell spreading; Kaapa A et al.; Integrins are transmembrane proteins linking the extracellular matrix or certain cell-cell contacts to the cytoskeleton . To study integrin-cytoskeleton interactions we wanted to relate talin-integrin interaction to integrin function in cell spreading and formation of focal adhesions . For talin-binding studies we used fusion proteins of glutathione S-transferase and the cytoplasmic domain of integrin beta(1) (GST-cytobeta(1)) expressed in bacteria . For functional studies chimeric integrins containing the extracellular and transmembrane parts of beta(3) linked to the cytoplasmic domain of beta(1) were expressed in CHO cells as a dimer with the alpha(IIb) subunit . Point mutations in the amino acid sequence N(785)PIY(788) of beta(1) disrupted both the integrin-talin interaction and the ability of the integrin to mediate cell spreading . COOH-terminal truncation of beta(1) at the amino acid position 797 disrupted its ability to mediate cell spreading, whereas the disruption of talin binding required deletion of five more amino acids (truncation at position 792) . A synthetic peptide from this region of beta(1) (W(780)DTGENPIYKSAV(792)) bound to purified talin and inhibited talin binding to GST-cytobeta(1) . The ability of the mutants to mediate focal adhesion formation or to codistribute to focal adhesions formed by other integrins correlated with their ability to mediate cell spreading . These results confirm the previous finding that a talin-binding site in the integrin beta(1) tail resides at or close to the central NPXY motif and suggest that the integrin-talin interaction is necessary but not sufficient for integrin-mediated cell spreading .

Genome Res, 1999 Jul, 9(7), 608 - 28
Comparative genomics of the Archaea (Euryarchaeota): evolution of conserved protein families, the stable core, and the variable shell; Makarova KS et al.; Comparative analysis of the protein sequences encoded in the four euryarchaeal species whose genomes have been sequenced completely (Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Archaeoglobus fulgidus, and Pyrococcus horikoshii) revealed 1326 orthologous sets, of which 543 are represented in all four species . The proteins that belong to these conserved euryarchaeal families comprise 31%-35% of the gene complement and may be considered the evolutionarily stable core of the archaeal genomes . The core gene set includes the great majority of genes coding for proteins involved in genome replication and expression, but only a relatively small subset of metabolic functions . For many gene families that are conserved in all euryarchaea, previously undetected orthologs in bacteria and eukaryotes were identified . A number of euryarchaeal synapomorphies (unique shared characters) were identified; these are protein families that possess sequence signatures or domain architectures that are conserved in all euryarchaea but are not found in bacteria or eukaryotes . In addition, euryarchaea-specific expansions of several protein and domain families were detected . In terms of their apparent phylogenetic affinities, the archaeal protein families split into bacterial and eukaryotic families . The majority of the proteins that have only eukaryotic orthologs or show the greatest similarity to their eukaryotic counterparts belong to the core set . The families of euryarchaeal genes that are conserved in only two or three species constitute a relatively mobile component of the genomes whose evolution should have involved multiple events of lineage-specific gene loss and horizontal gene transfer . Frequently these proteins have detectable orthologs only in bacteria or show the greatest similarity to the bacterial homologs, which might suggest a significant role of horizontal gene transfer from bacteria in the evolution of the euryarchaeota.

FEBS Lett, 1999 Jul 2, 454(1-2), 127 - 30
Nitrite reductase activity is a novel function of mammalian mitochondria; Kozlov AV et al.; Nitrite, which is the major stable degradation product of nitric oxide, exists in all tissues capable of nitric oxide synthesis from L-arginine . The present study provides experimental evidence that nitrite in contact with respiring mitochondria accepts reducing equivalents from the ubiquinone cycle of the respiratory chain . Univalent reduction of nitrite was totally inhibited by myxothiazol . We therefore conclude on the involvement of redox cycling that ubisemiquinone is associated with the bc1 complex . Recycling of nitric oxide degradation products via these electron carriers may become a threat to energy-linked respiration since nitric oxide in direct contact with mitochondria was shown to slow the energy-linked respiration down and to trigger a mitochondrial source for superoxide radicals . Until now, the existence of nitrite reductase activity was only demonstrated in plants and bacteria . In addition, the present observation elucidates the existence of a nitric oxide synthase-independent nitric oxide source.

Braz J Med Biol Res, 1999 May, 32(5), 529 - 38
Heparan sulfates and heparins: similar compounds performing the same functions in vertebrates and invertebrates?
Nader HB, Chavante SF, dos-Santos EA, Oliveira TW, de-Paiva JF, Jeronimo SM, Medeiros GF, de-Abreu LR, Leite EL, de-Sousa-Filho JF, Castro RA, Toma L, Tersariol IL, Porcionatto MA, Dietrich CP.
The distribution and structure of heparan sulfate and heparin are briefly reviewed . Heparan sulfate is a ubiquitous compound of animal cells whose structure has been maintained throughout evolution, showing an enormous variability regarding the relative amounts of its disaccharide units . Heparin, on the other hand, is present only in a few tissues and species of the animal kingdom and in the form of granules inside organelles in the cytoplasm of special cells . Thus, the distribution as well as the main structural features of the molecule, including its main disaccharide unit, have been maintained through evolution . These and other studies led to the proposal that heparan sulfate may be involved in the cell-cell recognition phenomena and control of cell growth, whereas heparin may be involved in defense mechanisms against bacteria and other foreign materials . All indications obtained thus far suggest that these molecules perform the same functions in vertebrates and invertebrates.

Klin Padiatr, 1999 May-Jun, 211(3), 165 - 71
{Clinical, histopathologic and immunohistochemical studies of chronic sialectatic parotitis in childhood and adolescence}; Ussmuller J et al.; Chronic sialectatic parotitis (CSP) causes problems in differential diagnosis and therapy . CSP shows the typical clinical features of chronic recurrent parotitis and will be investigated histopathologically only after ultimative parotidectomy . The etiology and pathogenesis of these unspecific inflammations is still unknown . Therefore no causal therapy is available and a lot of different trials (sialogoga, gland massage, infrared light, antibiotics, antiphlogistics, Trasylol, duct occlusion, duct ligation, gland denervation, radiotherapy) are not successful in the long run . MATERIAL AND METHOD: The salivary gland registry of the University of Hamburg (1965-1996) contains 22 infants and juvenile patients showing very severe courses of CSP . These cases have been investigated clinical (ultrasound, sialography), histopathological (paraffin embedded sections, histomorphometry of the ectatic duct lumina) and immunohistochemical (CK-MNF, AKTIN, KiM4) in a retrospective study to research the pathogenesis of CSP . RESULTS: Recurrent and always very dolent parotid swelling occurs between the age of 3 and 14 years for the first time . The courses vary from 3 months until 25 years . Local findings as well as ultrasound and sialographic features allow no certain differentiation of chronic recurrent parotitis . Conservative therapy fails in each case and leads to the necessity of surgical treatment . Histopathological three different stages of development can be observed: Initial stages show regular lobular architectonic structure of the parotid gland parenchyme with duct ectasies surrounded by slight inflammation of lymphocytes and plasmacells . Advanced stages are characterized by an increase of periductal inflammation and the appearance of lymphfollicels . Nearly complete lymphatic transformation of the parenchyme with destruction of the lobular formation dominates the terminal "immunologic" stage . Some cases show multiple myoepithelial islands within this lymphatic stroma typically observed in benign lymphoepithelial lesions . Whether bacteria nor primary obstructive changes can be observed . The histomorphometric analyses of the average and maximal luminal duct diameters show marked increase of 39% respectively 46% from and- vanced to terminal stages of CSP . Therefore the pathognomonic duct ectasies seem to depend on the progredient inflammation and are not due to a hereditary malformation of the duct system . Immunohistochemical terminal stages show follicular lymphatic hyperplasia (KiM4) expressing overshooting humoral immune reaction of MALT . CONCLUSION: Concerning the pathogenesis CSP corresponds to a immunopathological disorder of MALT and seems to be a prestage of benign lymphoepithelial lesion . Consequently important changes in the diagnosis and therapy of CSP lead to early histopathological investigation to differentiate the stage of inflammation . In stage III conservative parotidectomy should be carried out because spontaneous healing can not be expected . In contrast initial cases should be treated at first by glucocorticoids and immunosuppressives.

Proc Natl Acad Sci U S A, 1999 Jul 20, 96(15), 8545 - 50
Transcription in archaea; Kyrpides NC et al.; Using the sequences of all the known transcription-associated proteins from Bacteria and Eucarya (a total of 4,147), we have identified their homologous counterparts in the four complete archaeal genomes . Through extensive sequence comparisons, we establish the presence of 280 predicted transcription factors or transcription-associated proteins in the four archaeal genomes, of which 168 have homologs only in Bacteria, 51 have homologs only in Eucarya, and the remaining 61 have homologs in both phylogenetic domains . Although bacterial and eukaryotic transcription have very few factors in common, each exclusively shares a significantly greater number with the Archaea, especially the Bacteria . This last fact contrasts with the obvious close relationship between the archaeal and eukaryotic transcription mechanisms per se, and in particular, basic transcription initiation . We interpret these results to mean that the archaeal transcription system has retained more ancestral characteristics than have the transcription mechanisms in either of the other two domains.

Science, 1999 Jul 16, 285(5426), 400 - 2
Unraveling the electronic structure of individual photosynthetic pigment-protein complexes
van Oijen AM, Ketelaars M, Kohler J, Aartsma TJ, Schmidt J.
Low-temperature single-molecule spectroscopic techniques were applied to a light-harvesting pigment-protein complex (LH2) from purple photosynthetic bacteria . The properties of the electronically excited states of the two circular assemblies (B800 and B850) of bacteriochlorophyll a (BChl a) pigment molecules in the individual complexes were revealed, without ensemble averaging . The results show that the excited states of the B800 ring of pigments are mainly localized on individual BChl a molecules . In contrast, the absorption of a photon by the B850 ring can be consistently described in terms of an excitation that is completely delocalized over the ring . This property may contribute to the high efficiency of energy transfer in these photosynthetic complexes.

Mol Microbiol, 1999 Jul, 33(2), 429 - 37
In vitro activation and repression of photosynthesis gene transcription in Rhodobacter capsulatus; Bowman WC et al.; It has been known for over half a century that anoxygenic photosynthetic bacteria maximally synthesize their photosystems in the absence of oxygen . During the last decade, it has become clear that this regulation is largely at the transcriptional level, with photosynthesis genes expressed only under anaerobic conditions . We describe here in vitro reconstitution of activation and repression of three photosynthesis promoters, bch (bacteriochlorophyll biosynthesis), puc (light-harvesting II apoproteins) and puf (reaction centre and light-harvesting I apoproteins) using purified transcription factors and RNA polymerase from Rhodobacter capsulatus . Previous genetic results have indicated that each of these three promoters is differentially regulated by three key regulators: CrtJ acting as a repressor of bch and puc and the two-component regulators RegA/RegB, which are activators of puc and puf . These regulators are distinct from those that mediate oxygen control in enteric bacteria . Our in vitro studies show that these purified regulators directly control the expression of the housekeeping RNA polymerase at these promoters . High-level basal expression of the bch promoter is shown to be repressed by CrtJ . The puc promoter is activated by the RegB-phosphorylated RegA protein and additionally repressed by CrtJ . At the puc promoter, CrtJ effectively competes for promoter binding with RegA, while at the bch promoter, repression appears to be by competition for the RNA polymerase binding site . In contrast to what has been suggested previously, the RegA-activated puf promoter is demonstrated as being recognized by the housekeeping RNA polymerase . We also discuss evidence that RegA approximately P activation of the puc and puf promoters involves recruitment of RNA polymerase by different modes of protein-protein interaction.

J Clin Invest, 1999 Jul, 104(2), 155 - 62
Safety and antitumor activity of recombinant soluble Apo2 ligand; Ashkenazi A et al.; TNF and Fas ligand induce apoptosis in tumor cells; however, their severe toxicity toward normal tissues hampers their application to cancer therapy . Apo2 ligand (Apo2L, or TRAIL) is a related molecule that triggers tumor cell apoptosis . Apo2L mRNA is expressed in many tissues, suggesting that the ligand may be nontoxic to normal cells . To investigate Apo2L's therapeutic potential, we generated in bacteria a potently active soluble version of the native human protein . Several normal cell types were resistant in vitro to apoptosis induction by Apo2L . Repeated intravenous injections of Apo2L in nonhuman primates did not cause detectable toxicity to tissues and organs examined . Apo2L exerted cytostatic or cytotoxic effects in vitro on 32 of 39 cell lines from colon, lung, breast, kidney, brain, and skin cancer . Treatment of athymic mice with Apo2L shortly after tumor xenograft injection markedly reduced tumor incidence . Apo2L treatment of mice bearing solid tumors induced tumor cell apoptosis, suppressed tumor progression, and improved survival . Apo2L cooperated synergistically with the chemotherapeutic drugs 5-fluorouracil or CPT-11, causing substantial tumor regression or complete tumor ablation . Thus, Apo2L may have potent anticancer activity without significant toxicity toward normal tissues.

Curr Opin Rheumatol, 1999 Jul, 11(4), 257 - 64
Immunogenetics, HLA-B27 and spondyloarthropathies; Gonzalez S et al.; HLA-B27 is the strongest HLA molecule associated with a disease . However, the reason only a small fraction of HLA-B27 positive individuals develop spondyloarthropathies is still unknown . Recent advances in genetics support the fact that additional genetic factors influence the disease and that the environmental factors may be ubiquitous . The mechanism of association of HLA-B27 and disease remains unknown, but recent studies reveal some peculiar properties of accessory molecules in antigen presentation of B27 . Furthermore, research has focused on the analysis of HLA-B27-restricted processing and presentation of a bacteria-derived peptide as playing a key role in the development of spondyloarthropathy . Other studies support a more complex interaction between bacteria and HLA-B27 and suggest that other roles unrelated to antigen presentation might contribute to the development of SpA.

Clin Sports Med, 1999 Jul, 18(3), 537 - 48
Nutrition, exercise, and immune system function; Nieman DC; Many components of the immune system exhibit adverse change after prolonged, intense exertion . During this "open window" of impaired immunity (which may last 3-72 h, depending on the immune measure), viruses and bacteria may gain a foothold, increasing the risk for subclinical and clinical infection . The influence of nutritional supplements, primarily zinc, vitamin C, glutamine, and carbohydrate, on the acute immune response to prolonged exercise has been measured in endurance athletes . Vitamin C and glutamine have received much attention, but the data thus far are inconclusive . The most impressive results have been reported with carbohydrate supplementation . Carbohydrate beverage ingestion has been associated with increased plasma glucose levels, an attenuated cortisol and growth hormone response, fewer perturbations in blood immune cell counts, decreased granulocyte and monocyte phagocytosis and oxidative burst activity, and a diminished pro-inflammatory and anti-inflammatory cytokine response . Overall these data indicate that the physiologic stress to the immune system is reduced when endurance athletes use carbohydrate beverages before, during, and after prolonged and intense exertion . The clinical significance of these carbohydrate-induced effects on the endocrine and immune systems awaits further research.

Khirurgiia (Mosk), 1999, (6), 25 - 6
{Helicobacter pylori in patients with complicated peptic ulcer}; Gonchar MG et al.; The results of examination of 167 patients suffering from gastric and duodenal ulcers complicated by bleeding and perforation in a rehabilitation period are presented . High contamination by HP bacteria was established in gastroduodenal bleeding especially in duodenal ulcer (94.45%), as well as direct correlation between the contamination range and blood loss degree . The presence of HP infection during a control check-up examination suggests that the course of anti-ulcer therapy should last not less than 4-6 weeks . In treating patients suffering from ulcer bleeding conservative therapy is preferable . Stomach resection is considered as a method of choice . Lethality makes up 0.94 and 6.25%, respectively.

J Med Assoc Thai, 1999 Mar, 82(3), 213 - 9
Clinical study of 457 cholecystectomy cases in a private hospital; Chunhamaneewat S et al.; This is a retrospective study of 457 cases of cholecystectomized patients, who were admitted to Vichaiyut Hospital from 1970 to 1996 . The ratio of male to female was 1:1.6 and the most common age range was 51-60 years, 45.3 per cent of patients were older than 60 years . Associated or underlying diseases were highly prevalent (81.6%) . Diabetes mellitus, cardiovascular disease and liver disease were the three most common associated diseases . In acute cholecystitis the pathological findings were in accordance with clinical feature in only 46.2 per cent but in chronic or subsided cholecystitis pathology confirmed in 97.5 per cent . Carcinoma of the gallbladder was found in 0.9 per cent . Clinical diagnosis of cholecystitis was incorrect in 1.1 per cent . Multiple gallstones were found in 67.3 per cent, single stone in 23.5 per cent, sand stones in 2.1 per cent and acalculous cholecystitis in 7.1 per cent . Combined gallstones and CBD stones were found in 9.8 per cent . Enteric bacteria were isolated from the bile in 32.5 per cent and in acute cholecystitis similar organisms were isolated from both bile and blood cultures in 12.8 per cent . Morbidity rate of cholecystectomy was 7.6 per cent, the most common complication was perioperative infection in 3.5 per cent . It is interesting to find that atelectasis was recognized only in 2 out of 57 laparoscopic cholecystectomy . Mortality rate was low (0.66%).

Orthop Nurs, 1999 Mar-Apr, 18(2), 11 - 7; quiz 18-9
Necrotizing fasciitis: challenging management of a septic wound; Childs SG; Bacteria are everywhere . Most are not pathogenic unless the right environment or quantity is present . A superficial infection in the medically compromised patient can provide a microenvironment conducive to precipitating wound sepsis, local, and systemic immune responses . Necrotizing fasciitis (NF) is a subtle yet bold infection that presents like cellulitis; however, beneath the surface of the integument, the ravaging products of cellular degradation are fulminant . Large dermal and subcutaneous involvement, creating significant tissue defects, require aggressive assessment and management by all members of the health care team.

Can J Microbiol, 1999 Mar, 45(3), 209 - 16
Degradation of morpholine, piperidine, and pyrrolidine by mycobacteria: evidences for the involvement of a cytochrome P450; Poupin P et al.; Nine bacterial strains that grew on morpholine and pyrrolidine as sole carbon, nitrogen, and energy sources were isolated from three different environments with no known morpholine contamination . One of these strains could also degrade piperidine . These bacteria were identified as Mycobacterium strains . A phylogenetic analysis based on the partial 16S rDNA sequences indicated that the isolated strains clustered within the fast growing group of mycobacteria . When the above-mentioned cyclic amines were used as growth substrates, the synthesis of a soluble cytochrome P450 was induced in all these bacteria . Other laboratory strains, Mycobacterium fortuitum and Mycobacterium smegmatis mc(2)155, were tested for their abilities to degrade morpholine . Neither of them degraded morpholine but could use pyrrolidine and piperidine . The growth of M . fortuitum and M . smegmatis mc(2)155 on these compounds involved a soluble cytochrome P450, suggesting that mycobacterial strains are naturally able to use pyrrolidine and have developed a similar enzymatic pathway to metabolize this amine.

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