Microorganism

Hematology (Am Soc Hematol Educ Program) . 2001;:433-42.
Reducing the risk of blood transfusion; Snyder EL et al.; There are continuing concerns over the safety of the nation's and the world's blood supply . The allogeneic blood supply is tested for antibodies to HIV1/2, HTLVI/II, hepatitis B, hepatitis C (HCV) and syphilis . Testing is also performed for donor ALT (SGOT) levels, for the presence of hepatitis B surface antigen, human immunodeficiency virus (HIV) p24 antigen and, using nucleic acid amplification testing (NAT), for HIV and HCV nucleic acids . Still, there are concerns regarding other pathogenic agents . Dr . Roger Dodd addresses a series of pathogens that are already known to be transmissible by transfusion . These include malaria, Chagas' disease, babesiosis, bacteria and some viral agents . The need for new donor screening assays to protect the integrity and purity of the blood supply must be balanced against the loss of potential donors and the cost of developing and implementing these new screening assays . This issue will be highlighted . Dr . Edward Snyder reviews the status of research into development of systems for pathogen inactivation (PI) of blood and its components . A proactive technology wherein PI reagents such as psoralen, riboflavin, dimethylmethylene blue or inactine are added to blood collection bags could assure multiple log reduction of a variety of pathogens including viruses, bacteria, protozoa and fungi without the need to initially pre-screen the blood for a specific pathogen . Such a program could also cover new pathogens as they enter the blood supply . As a key issue relates to the toxicology of these agents, Dr . Snyder provides data on a novel carcinogenicity assay that uses a heterozygous p53 knock-out mouse model . The criteria likely to be needed for PI technology to be adopted by the transfusion community are summarized.

Mol Microbiol, 2001 Nov, 42(3), 741 - 55
The chromosome partitioning protein, ParB, is required for cytokinesis in Caulobacter crescentus; Mohl DA et al.; In Caulobacter crescentus, the genes encoding the chromosome partitioning proteins, ParA and ParB, are essential . Depletion of ParB resulted in smooth filamentous cells in which DNA replication continued . Immunofluorescence microscopy revealed that the formation of FtsZ rings at the mid-cell, the earliest molecular event in the initiation of bacterial cell division, was blocked in cells lacking ParB . ParB binds sequences near the C . crescentus origin of replication . Cell cycle experiments show that the formation of bipolarly localized ParB foci, and presumably localization of the origin of replication to the cell poles, preceded the formation of FtsZ rings at the mid-cell by 20 min . These results suggest that one major function of ParA and ParB may be to regulate the initiation of cytokinesis in C . crescentus.

Mol Microbiol, 2001 Nov, 42(3), 717 - 27
Roles of the two ClpC ATP binding sites in the regulation of competence and the stress response; Turgay K et al.; MecA targets the competence transcription factor ComK to ClpC . As a consequence, this factor is degraded by the ClpC/ClpP protease . ClpC is a member of the Clp/HSP100 family of ATPases and possesses two ATP binding sites . We have individually modified the Walker A motifs of these two sites and have also deleted a putative substrate recognition domain of ClpC at the C-terminus . The effects of these mutations were studied in vitro and in vivo . Deletion of the C-terminal domain resulted in a decreased binding affinity for MecA, a decreased ATPase activity in response to MecA addition and decreased degradative activity in vitro . In vivo, this deletion resulted in a failure to degrade ComK and in a decrease in thermal resistance for growth . Mutation of the N-terminal Walker A box (K214Q) caused a drastically decreased ATPase activity in vitro, but did not interfere with MecA binding . In vivo, this mutation had no effect on thermal resistance, but had a clpC null phenotype with respect to competence . Mutation of the C-terminal Walker A motif (K551Q) caused essentially the reverse phenotype both in vivo and in vitro . Although binding to MecA was only moderately impaired with 2 mM ATP, this mutant protein displayed no response to 0.2 mM ATP, unlike the wild-type ClpC and the K214Q mutant protein . The ATPase activity of the K551Q mutant protein, induced by the addition of MecA plus ComS, was decreased about 10-fold but was not eliminated . In vivo, the K551Q mutation showed a partial defect with respect to competence and a profound loss of thermal resistance . Sporulation was reduced drastically by the K551Q and less so by the K214Q mutation, but remained unaffected by deletion of the C-terminal domain . Although the evidence suggests that the functions of the two ATP-binding domains overlap, it appears that the N-terminal nucleotide-binding domain of ClpC is particularly concerned with MecA-related functions, whereas the C-terminal domain plays a more general role in the activities of ClpC.

Mol Microbiol, 2001 Nov, 42(3), 603 - 17
Icm/dot-dependent upregulation of phagocytosis by Legionella pneumophila; Hilbi H et al.; Legionella pneumophila is the causative agent of Legionnaires' disease, a severe pneumonia . Dependent on the icm/dot loci, L . pneumophila survives and replicates in macrophages and amoebae within a specialized phagosome that does not fuse with lysosomes . Here, we report that phagocytosis of wild-type L . pneumophila is more efficient than uptake of icm/dot mutants . Compared with the wild-type strain JR32, about 10 times fewer icm/dot mutant bacteria were recovered from HL-60 macrophages in a gentamicin protection assay . The defect in phagocytosis of the mutants could be complemented by supplying the corresponding genes on a plasmid . Using fluorescence microscopy and green fluorescent protein (GFP)-expressing strains, 10-20 times fewer icm/dot mutant bacteria were found to be internalized by HL-60 cells and human monocyte-derived macrophages (HMMPhi) . Compared with icm/dot mutants, wild-type L . pneumophila infected two to three times more macrophages and yielded a population of highly infected host cells (15-70 bacteria per macrophage) that was not observed with icm/dot mutant strains . Wild-type and icmT mutant bacteria were found to adhere similarly and compete for binding to HMMPhi . In addition, wild-type L . pneumophila was also phagocytosed more efficiently by Acanthamoeba castellanii, indicating that the process is independent of adherence receptor(s) . Wild-type L . pneumophila enhanced phagocytosis of an icmT mutant strain in a synchronous co-infection, suggesting that increased phagocytosis results from (a) secreted effector(s) acting in trans.

Immunol Rev, 2001 Aug, 182, 190 - 200
Control of intestinal inflammation by regulatory T cells; Singh B et al.; Transfer of CD4+ T cells to immune-deficient mice in the absence of the CD25+ subset leads to the development of colitis, indicating that regulatory cells capable of controlling a bacteria-driven inflammatory response are present in normal mice . Cells with this function are present in the thymus as well as in the periphery of germ-free mice, suggesting they may be reactive with self-antigen . These cells resemble CD4+CD25+ cells that inhibit organ-specific autoimmunity, suggesting that a similar subset of regulatory T cells may control responses to self and foreign antigens . Development of colitis is dependent on accumulation of activated CD134L+ dendritic cells (DC) in the mesenteric lymph nodes, which is inhibited by CD4+CD25+ cells, indicating that regulatory T cells may control DC activation in vivo . Whilst inhibition of T-cell activation in vitro by CD4+CD25+ cells does not involve interleukin-10 and transforming growth factor-beta, these cytokines are required for the suppression of colitis . It may be that control of responses that activate the innate immune system requires multiple mechanisms of immune suppression . Recently, we identified CD4+CD25+ cells with immune suppressive activity in the thymus and peripheral blood of humans, raising the possibility that dysfunction in this mechanism of immune regulation may be involved in the development of autoimmune and inflammatory diseases.

Eur J Biochem, 2001 Nov, 268(22), 5776 - 82
The tetraheme cytochrome c NrfH is required to anchor the cytochrome c nitrite reductase (NrfA) in the membrane of Wolinella succinogenes; Simon J et al.; The electron-transport chain that catalyzes nitrite respiration with formate in Wolinella succinogenes consists of formate dehydrogenase, menaquinone and the nitrite reductase complex . The latter catalyzes nitrite reduction by menaquinol and is made up of NrfA and NrfH, two c-type cytochromes . NrfA is the catalytic subunit; its crystal structure is known . NrfH belongs to the NapC/NirT family of membrane-bound c-type cytochromes and mediates electron transport between menaquinol and NrfA . It is demonstrated here by MALDI MS that four heme groups are attached to NrfH . A Delta nrfH deletion mutant of W . succinogenes was constructed by replacing the nrfH gene with a kanamycin-resistance gene cartridge . This mutant did not form the NrfA protein, probably because of a polar effect of the mutation on nrfA expression . The nrfHAIJ gene cluster was restored by integration of an nrfH-containing plasmid into the genome of the Delta nrfH mutant . The resulting strain had wild-type properties with respect to growth by nitrite respiration and nitrite reductase activity . A mutant (stopH) that contained the nrfHAIJ locus with nrfH modified by two artificial stop codons near its 5' end produced wild-type amounts of NrfA in the absence of the NrfH protein . NrfA was located exclusively in the soluble cell fraction of the stopH mutant, indicating that NrfH acts as the membrane anchor of the NrfHA complex in wild-type bacteria . The stopH mutant did not grow by nitrite respiration and did not catalyze nitrite reduction by formate, indicating that the electron transport is strictly dependent on NrfH . The NrfH protein seems to be an unusual member of the NapC/NirT family as it forms a stable complex with its redox partner protein NrfA.

Anal Chem, 2001 Nov 1, 73(21), 5150 - 6
Fiber-optic luminescent sensors with composite oxygen-sensitive layers and anti-biofouling coatings; Navarro F et al.; Anti-biofouling polymers containing phosphorylcholine (PC)-substituted methacrylate units have been prepared by copolymerization with dodecyl methacrylate and used to coat luminescent oxygen sensors . Nanometer-sized coatings of such materials are shown to reduce significantly the adhesion of marine bacteria (more than 70%) and thrombocytes (more than 90%) to the surface of tris-(4,7-diphenyl-1,10-phenanthroline)ruthenium(II)-doped silicone layers . A thorough analytical characterization of both the PC-coated and the uncoated dyed films has demonstrated that the anti-biofouling layers do not alter dramatically the performance of the fiber-optic oxygen sensors in aqueous media and are mechanically stable for more than one year of continuous immersion . The slope of the linear calibration plots in the 0-8 mg L(-1) oxygen concentration range (ca . 1.0 L mg(-1)) decreases 8-11% after applying the 50-nm protective layer with no change in the sensor precision (1.1-1.9% RSD, n = 6) . The response time of the 200-microm O2-sensitive layers (1.5-6 min) increases up to 2-fold, depending on the nature of the PC polymer used, but the temperature effect on the sensor response (0.020 L mg(-1) degrees C(-1)) remains essentially unchanged . Oxygen detection limits as low as 0.04 mg L(-1) have been measured with the coated optodes . The novel biofouling-resistant optosensors have been successfully validated against a commercial oxygen electrode and are shown to respond faster than the electrochemical device for large oxygen concentration changes . The biomimetic coatings will be particularly useful for drift-free long-term operation of environmental optosensors and in vivo fiber-optic oxygen analyzers.

Am J Gastroenterol, 2001 Nov, 96(11), 3192 - 4
Management of suppurative pylephlebitis by percutaneous drainage: placing a drainage catheter into the portal vein; Pelsang RE et al.; Persistent infection of the portal vein is a rare entity with significant mortality . We present two cases of infected thombis of the portal vein, one infected with fungus and the other with bacteria, both requiring percutaneous drainage to allow a response to antibiotics . The distinction between bland thrombis, infected thrombis, portal venous air, and pneumobilia will be discussed so that suppurative pylephlebitis can be recognized more easily as drain placement appears to affect a more prompt degree of improvement than antibiotics alone.

Pest Manag Sci, 2001 Nov, 57(11), 1075 - 80
Effect of some granular insecticides currently used for the treatment of maize crops (Zea mays) on the survival of inoculated Azospirillum lipoferum; Revellin C et al.; Four insecticides, carbofuran, chlormephos, terbufos and benfuracarb, currently used on maize (Zea mays) at sowing, were tested for their compatibility with Azospirillum lipoferum strain CRT1 used as an inoculant to improve maize growth and yield . The growth or survival of A lipoferum was studied in the presence of the insecticides: (1) in liquid and solid cultures of the bacteria, (2) when a commercial inoculant (Azogreen-m, Liphatech, Meyzieu, France) was inoculated directly on insecticide granules, (3) when inoculated Azogreen-m granules were mixed with insecticide granules and (4) when inoculated Azogreen-m granules were delivered separately to the seed bed . Of the four insecticides tested, only terbufos had a slight effect on growth of A lipoferum in solid cultures . All the insecticides decreased the survival of A lipoferum when the bacteria were inoculated directly on to the granules, or when inoculated Azogreen-m granules were mixed with an insecticide . We hypothesize that the discrepancies between bacterial culture tests and survival studies might be explained by the conditions of desiccation encountered during inoculation of the granules . Desiccation stress could increase the toxic effect of the insecticides . We therefore suggest including desiccation stress in the biotest used to assess inoculant-pesticide compatibility.

Lung Cancer, 2001 Dec, 34 Suppl 2, S71 - 7
The impact of immune responses on lung cancer and the development of new treatment modalities; Pluygers E et al.; OBJECTIVE: This presentation covers predominantly review data in relation with immune responses initiating and accompanying lung carcinogenesis or- on the contrary-contributing to novel therapeutic developments . Occasionally, personal findings will be considered . RESULTS 1 OF IMMUNE DEFICIENCY: It is known for several decades that cancer incidence (several sites) is increased in subjects receiving immunosuppressive therapy, e.g . to avoid transplant rejection, or suffering from AIDS . We have observed that in areas heavily polluted by industrial activities, resulting in immune deficiency, cancer incidence is increased, notably for lung cancer . On the other hand, neoplastic cells are able to escape the host's immune responses by inducing apoptosis of the effector T lymphocytes . Apoptosis in T-cells is triggered by the interaction of the membrane receptor Fas with its normal ligand Fas L, or an activating antibody . Now lung carcinoma cells have been shown to express Fas L, enabling them to destroy cytolytic T cells . RESULTS 2 OF IMMUNE TREATMENT: It is well over a century ago that interest in the immunotherapy of cancer was aroused by the observation of tumour regressions concomitant with bacterial infection, an observation leading to the development of 'Coley's toxin', a mixture of killed bacteria (presently known to act through the presence of TNF-alpha) . Since these long-standing empirical attempts, a lasting search for immune control of cancer has been initiated, comprising such different approaches as active non-specific immunotherapy, active specific immunotherapy, approaches based on the use of monoclonal antibodies, as well as those depending on cellular immunity and the development of adoptive immunotherapy, and the use of peptide vaccines . These different approaches will be described and their results presented . CONCLUSION: Present state-of-the-art will be discussed and new pathways for development evoked; better understanding of immune mechanisms is opening new avenues for improved treatment efficacy.

Org Lett, 2001 Nov 29, 3(24), 3819 - 22
1-Deoxy-D-xylulose: synthesis based on molybdate-catalyzed rearrangement of a branched-chain aldotetrose; Zhao S et al.; 1-Deoxy-D-xylulose has been prepared in seven steps and approximately 21% overall yield from 2,3-O-isopropylidene-D-erythrono-1,4-lactone . The key reaction involves transformation of a branched-chain aldotetrose to the 1-deoxy-2-ketopentose catalyzed by molybdic acid . Other branched-chain aldotetroses containing bulkier substituents at C2 also engage in the conversion, suggesting routes to protected 2-ketoses and alpha-ketoacids/esters . This synthetic route mimics reactions of the non-mevalonate isoprenoid pathway in plants and bacteria . {reaction: see text}

J Biol Chem, 2002 Feb 1, 277(5), 3504 - 10 Epub 2001 Nov 21.
Membrane topography of the coupling ion binding site in Na+-translocating F1F0 ATP synthase; von Ballmoos C et al.; A carbodiimide with a photoactivatable diazirine substituent was synthesized and incubated with the Na(+)-translocating F(1)F(0) ATP synthase from both Propionigenium modestum and Ilyobacter tartaricus . This caused severe inhibition of ATP hydrolysis activity in the absence of Na(+) ions but not in its presence, indicating the specific reaction with the Na(+) binding c-Glu(65) residue . Photocross-linking was investigated with the substituted ATP synthase from both bacteria in reconstituted 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC)-containing proteoliposomes . A subunit c/POPC conjugate was found in the illuminated samples but no a-c cross-links were observed, not even after ATP-induced rotation of the c-ring . Our substituted diazirine moiety on c-Glu(65) was therefore in close contact with phospholipid but does not contact subunit a . Na(+)in/(22)Na(+)out exchange activity of the ATP synthase was not affected by modifying the c-Glu(65) sites with the carbodiimide, but upon photoinduced cross-linking, this activity was abolished . Cross-linking the rotor to lipids apparently arrested rotational mobility required for moving Na(+) ions back and forth across the membrane . The site of cross-linking was analyzed by digestions of the substituted POPC using phospholipases C and A(2) and by mass spectroscopy . The substitutions were found exclusively at the fatty acid side chains, which indicates that c-Glu(65) is located within the core of the membrane.

Adv Drug Deliv Rev, 2001 Nov 19, 52(3), 245 - 53
Towards synthetic viruses; Zuber G et al.; Gene delivery is too complex to be performed with a single carrier molecule . Synthetic multicomponent vectors are being designed that mimic key properties of viruses . Some solutions, such as diverting cell-anchoring molecules or the endogenous nuclear import machinery from their normal function, are directly copied from bacteria and viruses . Some other solutions are original ones: monomolecular genome condensation via detergent dimerization or endosome disruption by the proton sponge effect are not exploited by natural cell invaders . All these components, however, still have to be assembled into a unique supramolecular system, an 'artificial virus'.

Adv Drug Deliv Rev, 2001 Nov 19, 52(3), 165 - 76
Approaches for generating recombinant adenovirus vectors; Mizuguchi H et al.; Various methods have been developed to facilitate the generation of recombinant adenovirus vectors, and three commercially available methods have been most widely used: the homologous recombination method in E1-complement cell lines, the homologous recombination method in bacteria, and an in vitro ligation method based on simple routine plasmid construction . These methods can insert foreign genes not only into the E1 deletion region, but also into the E3 deletion region, thereby permitting the construction of a binary transgene expression system in which heterologous genes can be inserted into both the E1 and E3 regions . By modifying the latter two methods, fiber-mutant adenovirus vectors can be also constructed in order to modify vector tropism . In this paper, we review recent advances in the construction of first generation adenovirus vectors and fiber-modified adenovirus vectors.

Eur J Surg Suppl, 2001, (586), 66 - 72
Understanding inflammatory bowel disease--the clinician's perspective; Rutgeerts P et al.; The treatment of patients with ulcerative colitis and Crohn's disease is a huge challenge to the clinician mainly because the etiology of IBD is still completely unknown . Pathogenetic mechanisms, in constrast, have partly been elucidated thanks to immunohistochemical investigation of the tissue in IBD and the study of experimental animal models of gut inflammation . There is extensive evidence that at least in Crohn's disease tolerance for commensal bacteria is lost which leads to uncontrolled inflammation mediated by a T-helper 1 response and to tissue destruction of the gut epithelium with inadequate repair . Genetic factors are probably responsible for increased susceptibility and may define disease phenotypes . These insights have led to a dramatic improvement of our therapeutic means with the development of the chimeric monoclonal antibody to TNF . The pathogenetic mechanisms are less clear in UC . The hypothesis is that UC is a T-helper 2 mediated disease . We have a very attractive way to study the early onset of Crohn's disease in the postoperative Crohn's situation . Newer therapeutic approaches should aim at preventing recurrence of Crohn's lesions in the normal postoperative bowel . Such model is also available for UC since there is increasing evidence that pouchitis is recurrent ulcerative colitis in the small bowel . Cooperation between clinicians and basic scientists is the best way to achieve fast progress in understanding IBD.

Eur J Surg Suppl, 2001, (586), 30 - 3
New therapeutic possibilities in inflammatory bowel disease; Kamm MA; Therapeutic approaches in inflammatory bowel disease (IBD) involve targeting the broad inflammatory process, targeting specific inflammatory mediators, changing the interior milieu to diminish antigenic drive, or removing the main mediators of inflammation, the inflammatory cells, completely . Recent developments have occurred in each of these therapeutic areas . New steroids, and better use of existing drugs, such as azathioprine and cyclosporin, have improved therapy . Genetically engineered drugs, such as TNF-alpha antibody, offer the prospect of powerful treatment for patients resistant to standard therapies . The use of probiotics offers an entirely new approach, by changing the lumenal milieu . Understanding how bacteria and epithelial cells interact is likely to bring important rewards in therapy . Finally, immune cell replacement may be a possible therapy for severe disease when all else has failed.

Eur Biophys J, 2001 Oct, 30(6), 421 - 9
Channel activity of a phytotoxin of Clavibacter michiganense ssp . nebraskense in tethered membranes; Michalke A et al.; Solid-supported membranes immobilized on gold electrodes were used to detect and characterize the spontaneously inserting anion-selective protein channel (Clavibacter anion channel, CAC) present in the culture fluid of Clavibacter michiganense ssp . nebraskense . Three different membrane systems varying in the composition of the first chemisorbed monolayer were investigated by means of impedance spectroscopy . Conductance changes of the immobilized lipid membranes were sensitively detected after adding the culture fluid of the bacteria to the solid-supported membranes, indicating that the relative change in conductance is largest if the lipid layer is attached to the surface via a flexible lipid anchor . Variation in the d.c . potential revealed that CAC exhibits a voltage dependence in these tethered membranes which can be described by an exponential function in accordance with previous results obtained from patchclamp measurements and impedance analysis . The addition of an inhibitor that selectively blocks anion channels abolished the channel conductance almost completely, indicating that the increased conductivity can be attributed to the specific insertion of the CAC . A linear dependence of the channel conductance on the chloride concentration was found, which was modulated by the charges of the second lipid monolayer . The results demonstrate that tethered lipid membranes on gold surfaces in conjunction with impedance spectroscopy allows one to monitor and characterize water-soluble spontaneously inserting channels, providing an effective means to probe for bacterial toxins.

Zhonghua Yi Xue Za Zhi, 2001 Aug 25, 81(16), 999 - 1003
{Tumor targeted expression of adenovirus mediated CDglyTK gene regulated by irradiation via Egr-1 promoter}; Wei D et al.; OBJECTIVE: To construct adenoviral vecter of AdEgr-CD/TK in which CDglyTK gene was driven by Egr-1 promoter, and control the tumor targeted expression of CDglyTK gene by gamma irradiation so as to observe the effects of gene-radiotherapy of liver cancer . METHODS: Adenoviral vector of AdEgr-CD/TK was generated through homologous recombination in bacteria . The expression of CDglyTK gene in MM45T . Li cells infected with AdEgr-CD/TK and exposed to different doses of gamma irradiation were analyzed, and the relative survival rate of the cells in presence of prodrugs 5-FC and GCV was tested . In addition, the tumor suppression effects of different treatments were investigated in 64 mice bearing liver cancer by observing the size of tumor at interval of four days . RESULTS: In vitro experiment showed that gamma irradiation markedly and dose-dependently induced CDglyTK expression in MM45T . Li tumor cells, and significantly enhanced the sensitivity of MM45T . Li cells infected with AdEgr-CD/TK to prodrugs 5-FC or GCV resulting in cell being killed (P < 0.01) . A synergetic cytotoxic effect was found when both 5-FC and GCV were added . In vivo experiment revealed that, compared with other control treatments, intratumoral injection of AdEgr-CD/TK combined with intraperitoneal injection of GCV + 5-FC and TLI obviously suppressed tumor growth (P < 0.01), with 30% of the tumor eradicated completely while without causing the increase of whole body cytotoxic effect . CONCLUSION: Tumor targeted expression of CDglyTK gene under the control of irradiation represents a novel strategy for safe and effective gene therapy of cancer and might have wide application in the future.

Zhonghua Nei Ke Za Zhi, 2001 Aug, 40(8), 510 - 3
{The transcription of interleukin-8 in gastric epithelial cells by Helicobacter pylori strains in Chinese}; Li S et al.; OBJECTIVE: To observe the effect of the vac A genotype and the existence of cag pathogenicity island (cag PAI) of Helicobacter pylori (Hp) isolated from Chinese patients on the transcription of IL-8 in gastric epithelial cells . METHODS: The genotype of vac A and the existence of cag PAI of Hp were determined by gene sequencing and Southern blot, respectively; the constructed L5F11 cells were cocultured with the bacteria and the transcription of interleukin-8(IL-8) was indicated by luciferase activity and the cytotoxin activity showed by using HeLa cell vacuolating assay . RESULTS: The ability to induce luciferase by the strains with complete cag PAI (n = 15) was much higher than that by the wild cag negative strain G50{(0.47 +/- 0.09) x 10(6) cpm vs (0.13 +/- 0.02) x 10(6) cpm, P < 0.01} and there was no difference in the luciferase activity induced by the strains with partial cag (n = 4) and by G50{(0.23 +/- 0.08) x 10(6) cpm vs (0.13 +/- 0.02) x 10(6) cpm, P > 0.05}; Also no difference was found in the luciferase activity induced by the strains with vac A s1/m1 (n = 5) and the strains with s1/m2 (n = 14) {(0.29 +/- 0.12) x 10(6) cpm vs (0.53 +/- 0.41) x 10(6) cpm, P > 0.05} although the formers had much higher vacuolating cytotoxin activity than the latters (P < 0.001) . CONCLUSION: The transcription of IL-8 in gastric epithelial cells induced by Hp isolated from Chinese patients was associated with the existence of cag PAI and not with the genotype of vac A.

J Endotoxin Res, 2001, 7(4), 287 - 91
Toll-like receptor-2 transduces signals for NF-kappa B activation, apoptosis and reactive oxygen species production; Aliprantis AO et al.; The innate immune system coordinates the inflammatory response to pathogens . To do so, cells of the innate immune system must rapidly discriminate between self and non-self . All bacteria express membrane-associated lipoproteins . These molecules activate cells of the innate immune system to initiate host defense mechanisms . However, it is currently unknown how the innate immune system recognizes bacterial lipoproteins . Here, we describe that in response to bacterial lipoprotein, human Toll-like receptor-2 activates three different cellular responses: nuclear factor-kappaB dependent transcription, programmed cell death and reactive oxygen species production . We propose that Toll-like receptor-2 fulfils multiple roles in the genesis of the immune response to bacterial pathogens.

Proc Natl Acad Sci U S A, 2001 Nov 20, 98(24), 13814 - 9
Role of positively charged transmembrane segments in the insertion and assembly of mitochondrial inner-membrane proteins; Saint-Georges Y et al.; The biogenesis of membrane oligomeric complexes is an intricate process that requires the insertion and assembly of transmembrane (TM) domains into the lipid bilayer . The Oxa1p family plays a key role in this process in organelles and bacteria . Hell et al . (2001, EMBO J., 20, 1281-1288) recently have proposed that Oxa1p could act as part of a general membrane insertion machinery for mitochondrial respiratory complex subunits . We have previously shown that mutations in the TM domain of Cyt1p can partially compensate for the absence of Oxa1p . Here, we demonstrate that a single amino acid substitution in the TM domain of Qcr9p can bypass Oxa1p in yeast . Qcr9p and Cyt1p are two subunits of the respiratory complex bc1 and their relative roles in the assembly of other respiratory complexes have been investigated . The mutations we have isolated in Cyt1p or Qcr9p introduce positively charged amino acids, and we show that the mutant TM domain of Cyt1p mediates the restoration of complex assembly . We propose that the positive charges introduced in Cyt1p and Qcr9p TM domains promote interactions with negatively charged TM domains of other respiratory complex subunits, allowing the coinsertion of both domains into the membrane, in the absence of Oxa1p . This model argues in favor of a role of Oxa1p in the insertion and the lateral exit of less hydrophobic TM domains from the translocation site into the lipid bilayer.

Proc Natl Acad Sci U S A, 2001 Nov 20, 98(24), 13490 - 5
Magnetite morphology and life on Mars; Buseck PR et al.; Nanocrystals of magnetite (Fe(3)O(4)) in a meteorite from Mars provide the strongest, albeit controversial, evidence for the former presence of extraterrestrial life . The morphological and size resemblance of the crystals from meteorite ALH84001 to crystals formed by certain terrestrial bacteria has been used in support of the biological origin of the extraterrestrial minerals . By using tomographic and holographic methods in a transmission electron microscope, we show that the three-dimensional shapes of such nanocrystals can be defined, that the detailed morphologies of individual crystals from three bacterial strains differ, and that none uniquely match those reported from the Martian meteorite . In contrast to previous accounts, we argue that the existing crystallographic and morphological evidence is inadequate to support the inference of former life on Mars.

J Biol Chem, 2002 Jan 18, 277(3), 2006 - 11 Epub 2001 Nov 20.
A critical role for the Var2 FtsH homologue of Arabidopsis thaliana in the photosystem II repair cycle in vivo; Bailey S et al.; Using a var2-2 mutant of Arabidopsis thaliana, which lacks a homologue of the zinc-metalloprotease, FtsH, we demonstrate that this protease is required for the efficient turnover of the D1 polypeptide of photosystem II and protection against photoinhibition in vivo . We show that var2-2 leaves are much more susceptible to light-induced photosystem II photoinhibition than wild-type leaves . Furthermore, the rate of photosystem II photoinhibition in untreated var2-2 leaves is equivalent to that of var2-2 and wild-type leaves, which have been treated with lincomycin, an inhibitor of the photosystem II repair cycle at the level of D1 synthesis . This is in contrast to untreated wild-type leaves, which show a much slower rate of photosystem II photoinhibition due to an efficient photosystem II repair cycle . The recovery of var2-2 leaves from photosystem II photoinhibition is also impaired relative to wild-type . Using Western blot analysis in the presence of lincomycin we show that the D1 polypeptide remains stable in leaves of the var2-2 mutant under photoinhibitory conditions that lead to D1 degradation in wild-type leaves and that the abundance of DegP2 is not affected by the var2-2 mutation . We conclude, therefore, that the Var2 FtsH homologue is required for the cleavage of the D1 polypeptide in vivo . In addition, we identify a conserved lumenal domain in Var2 that is unique to FtsH homologues from oxygenic phototrophs.

J Bacteriol, 2001 Dec, 183(24), 7408 - 11
NAD(P)-dependent aldehyde dehydrogenases induced during growth of Ralstonia eutropha strain Bo on tetrahydrofurfuryl alcohol; Schrader T et al.; Different aldehyde dehydrogenases (AlDHs) were formed during growth of Ralstonia eutropha Bo on tetrahydrofurfuryl alcohol (THFA) . One of these enzymes, AlDH 4, was purified and characterized as a homodimer containing no prosthetic groups, showing a strong substrate inhibition, and having an N-terminal sequence similar to those of various NAD(P)-dependent AlDHs . The conversion rate of THFA by the quinohemoprotein THFA dehydrogenase was increased by AlDH 4.

J Bacteriol, 2001 Dec, 183(24), 7387 - 91
Stability and subcellular localization of cytadherence-associated protein P65 in Mycoplasma pneumoniae; Jordan JL et al.; The surface protein P65 is a constituent of the Mycoplasma pneumoniae cytoskeleton and is present at reduced levels in mutants lacking the cytadherence accessory protein HMW2 . Pulse-chase studies demonstrated that P65 is subject to accelerated turnover in the absence of HMW2 . P65 was also less abundant in noncytadhering mutants lacking HMW1 or P30 but was present at wild-type levels in mutants lacking proteins A, B, C, and P1 . P65 exhibited a polar localization like that in wild-type M . pneumoniae in all mutants having normal levels of HMW1 and HMW2 . Partial or complete loss of these proteins, however, correlated with severe reduction in the P65 level and the inability to localize P65 properly.

Clin Orthop, 2001 Nov, (392), 377 - 82
Gout-induced arthropathy after total knee arthroplasty: a report of two cases; Archibeck MJ et al.; Gout, although relatively rare in joint replacements, can present as an acute or chronic painful knee or hip arthroplasty . Gout and acute infection of a joint replacement can be difficult to differentiate, with the physical examination and laboratory study results frequently being similar . Both conditions can present with a rapid onset of joint pain, swelling, erythema, and constitutional symptoms, including fevers and malaise . Laboratory findings in both conditions often include an elevated leukocyte count, erythrocyte sedimentation rate, and C-reactive protein level . Negatively birefringent, needle-shaped crystals in the synovial fluid confirm the diagnosis of gout . The mistaken diagnosis of septic arthritis in a joint replacement with crystal-induced synovitis can lead to inappropriate open debridement or component removal . The current study includes a review of the literature and presents two cases of gout after total knee arthroplasty . These cases suggest that in situations of suspected sepsis without synovial fluid crystals, operative intervention is indicated with a presumed diagnosis of septic arthritis . The identification of chalky white or yellow deposits in the synovium or bone is highly suggestive of gout . The definitive diagnosis is made by polarized light histologic evaluation of these tissues . If these deposits are present in the absence of a positive preoperative culture, positive Gram stain for bacteria, or component loosening, component retention is indicated.

Am J Trop Med Hyg, 2001 Nov, 65(5), 603 - 9
Tissue diagnosis of Ehrlichia chaffeensis in patients with fatal ehrlichiosis by use of immunohistochemistry, in situ hybridization, and polymerase chain reaction; Dawson JE et al.; In the United States, human ehrlichiosis is a complex of emerging tick-borne diseases caused by 3 distinct Ehrlichia species: Ehrlichia chaffeensis, Ehrlichia ewingii, and the human granulocytotropic ehrlichiosis agent . Ehrlichioses are characterized by a mild to severe illness, and approximately 4% of cases are fatal . Because these obligate intracellular bacteria are difficult to resolve with routine histologic techniques, their distribution in tissues has not been well described . To facilitate the visualization and detection of ehrlichiae, immunohistochemistry (IHC), in situ hybridization (ISH), and polymerase chain reaction (PCR) assays were developed by use of tissues from 4 fatal cases of E . chaffeensis infection . Evidence of E . chaffeensis via IHC, ISH, and PCR was documented in all 4 cases . Abundant immunostaining and in situ nucleic acid hybridization were observed in spleen and lymph node from all 4 patients . Significantly, in 2 of these patients, serologic evidence of infection was absent . Use of IHC, ISH, and PCR to visualize and detect Ehrlichia in tissues can facilitate diagnosis of ehrlichial infections.

Curr Opin Mol Ther, 1999 Apr, 1(2), 204 - 15
Therapeutic mammalian artificial episomal chromosomes; Vos JM; Two general strategies are being developed to engineer mammalian artificial chromosomes (MACs) as therapeutic vectors: (i) in vitro MAC cloning by enzymatic ligation of the individual MAC components followed by propagation in single cell organisms such as bacteria or yeast; and (ii) in situ MAC assembly by co-introduction of the various MAC elements into an 'incubator' mammalian tissue culture cell and use of it as a 'foster parental' donor cell . Because of their organizational compactness, in vitro built MACs are stuitable for somatic-based human gene therapy . In contrast, the long-term persistence of in situ built MACs can be capitalized on to generate husbandry transgenic animals expressing therapeutic genes . While current MAC systems generally rely on cis-elements exclusively from viral or genomic origin, the next generation of MACs may combine both into chimeric systems . As illustration of the genetic flexibility and technological potential of chimeric MACs, the herpes viral oriP/EBNA1 system, paradigm of a self-replicating and self-segregating episome in human cells is discussed in terms of future therapeutic applications.

J Gen Virol, 2001 Dec, 82(Pt 12), 2935 - 43
The NS5A protein of bovine viral diarrhoea virus interacts with the alpha subunit of translation elongation factor-1; Johnson CM et al.; A cellular protein that interacts with the NS5A polypeptide of bovine viral diarrhoea virus (BVDV) was identified in a yeast two-hybrid screen . The NS5A interactor was identified as the alpha subunit of bovine translation elongation factor 1A (eEF1A) . Cell-free binding studies were performed with chimeric NS5A fused to glutathione S-transferase (GST-NS5A) expressed in bacteria . GST-NS5A bound specifically to both in vitro-translated and mammalian cell-expressed eEF1A . Moreover, purified eEF1A bound specifically to GST-NS5A attached to a solid phase . Conservation of this interaction was then analysed using a set of NS5A proteins derived from divergent BVDV strains encompassing known biotypes and genotypes . NS5A from all BVDV strains tested so far interacted with eEF1A . The conserved association of eEF1A with virus molecules involved in genome replication and the postulated role of pestivirus and hepacivirus NS5A in replication indicate that this interaction may play a role in the replication of BVDV.

J Immunol, 2001 Dec 1, 167(11), 6545 - 51
Fc receptor-mediated immunity against Bordetella pertussis; Rodriguez ME et al.; The relevance of specific Abs for the induction of cellular effector functions against Bordetella pertussis was studied . IgG-opsonized B . pertussis was efficiently phagocytosed by human polymorphonuclear leukocytes (PMN) . This process was mediated by the PMN IgG receptors, FcgammaRIIa (CD32) and FcgammaRIIIb (CD16), working synergistically . Furthermore, these FcgammaR triggered efficient PMN respiratory burst activity and mediated transfer of B . pertussis to lysosomal compartments, ultimately resulting in reduced bacterial viability . Bacteria opsonized with IgA triggered similar PMN activation via FcalphaR (CD89) . Simultaneous engagement of FcalphaRI and FcgammaR by B . pertussis resulted in increased phagocytosis rates, compared with responses induced by either isotype alone . These data provide new insights into host immune mechanisms against B . pertussis and document a crucial role for Ig-FcR interactions in immunity to this human pathogen.

J Biol Chem, 2002 Feb 1, 277(5), 3727 - 32 Epub 2001 Nov 19.
Crystal structure of quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni: structural basis for substrate oxidation and electron transfer; Oubrie A et al.; Quinoprotein alcohol dehydrogenases are redox enzymes that participate in distinctive catabolic pathways that enable bacteria to grow on various alcohols as the sole source of carbon and energy . The x-ray structure of the quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni has been determined at 1.44 A resolution . It comprises two domains . The N-terminal domain has a beta-propeller fold and binds one pyrroloquinoline quinone cofactor and one calcium ion in the active site . A tetrahydrofuran-2-carboxylic acid molecule is present in the substrate-binding cleft . The position of this oxidation product provides valuable information on the amino acid residues involved in the reaction mechanism and their function . The C-terminal domain is an alpha-helical type I cytochrome c with His(608) and Met(647) as heme-iron ligands . This is the first reported structure of an electron transfer system between a quinoprotein alcohol dehydrogenase and cytochrome c . The shortest distance between pyrroloquinoline quinone and heme c is 12.9 A, one of the longest physiological edge-to-edge distances yet determined between two redox centers . A highly unusual disulfide bond between two adjacent cysteines bridges the redox centers . It appears essential for electron transfer . A water channel delineates a possible pathway for proton transfer from the active site to the solvent.

Pharmacotherapy, 2001 Nov, 21(11 Pt 2), 319S - 330S
Pharmacodynamics of antiinfective therapy: taking what we know to the patient's bedside; Rodvold KA; Applied pharmacokinetics has long been a lifeline of clinical pharmacy services . National surveys during the past decade documented clinical pharmacy services and demonstrated that a substantial rate of growth occurred in clinical pharmacokinetic consultations and management of drug therapy protocols . Pharmacodynamic principles of antiinfective agents are rapidly becoming a new paradigm of clinical pharmacy services . beta-Lactams, aminoglycosides, and fluoroquinolones represent the three classes of antiinfective agents that have made the most progress toward the clinical applications of pharmacodynamics . Pharmacodynamic parameters are being used to select and compare agents within an antiinfective class (e.g., fluoroquinolones), make modifications in the dosage (e.g., extended-interval dosing of aminoglycosides) and/or mode of administration (e.g., continuous infusion of beta-lactams), develop in vivo breakpoint determinations, and assess the development of bacterial resistance . In addition, pharmacodynamic parameters have influenced the clinical drug development of new (e.g., linezolid) and older (amoxicillin-clavulanate, fluoroquinolones) antiinfective agents . Further investigations are needed to explore the clinician's use of validated prediction methods and patient-specific pharmacodynamic parameters at the bedside . By linking pharmacokinetic services with pharmacodynamic principles, the opportunity for continued progress toward our assessment and decisions for successful clinical outcomes is possible with old and new antiinfective agents.

Dig Dis Sci, 2001 Nov, 46(11), 2378 - 84
Immunologic influences of hyperthermia in a rat model of obstructive jaundice; Gulluoglu BM et al.; In this study, the effect of hyperthermia on immune response and bacterial translocation from the gut in jaundiced rats was assessed . In hyperthermic (HP; N = 8) and normothermic (NP; N = 8) preconditioning groups, rats were preconditioned by hyperthermia for 15 min at 42 degrees C or 38 degrees C, respectively . After 8 hr, the common bile duct (CBD) of each animal was ligated . In thermal (TT; N = 8) and normothermic treatment groups (NT; N = 8) the CBD of the animals was ligated, and after seven days rats were treated by hyperthermia for 15 min at 42 degrees C and 38 degrees C, respectively . The rats in the preconditioning groups (HP and NP) were killed at day 7 and rats in the treatment groups (TT and NT) were killed 8 hr after they were put in a water bath . Determination of the immunophenotypes of lymphocytes and serum levels of bilirubin was done in serum samples taken just after death . The quantity and identify of translocated bacteria were determined in tissue samples of mesenteric lymph nodes, spleen, and liver . NK cell expression as well as CD4+/CD8+ ratio were elevated in HP group when compared to NP group . CD8+ expression was found to be low in HP group when compared to NP group . CD4+, CD11b+, and B cell expressions were not found to be different between HP and NP groups . All immunologic parameters were similar when TT and NT groups were compared to each other . In the TT group, half of the rats revealed bacterial translocation, whereas in all other groups, we determined translocation in only 1/8 rats . The application of hyperthermia as preconditioning rather than applying it after the establishment of jaundice seemed to be beneficial . Hyperthermic preconditioning led an improvement in immune responses whereas the latter resulted an increase in bacterial translocation with no favorable influence on immune system . Bacterial translocation was unrelated with the immune status.

Nucleic Acids Res, 2001 Nov 15, 29(22), 4509 - 17
RadA protein from Archaeoglobus fulgidus forms rings, nucleoprotein filaments and catalyses homologous recombination; McIlwraith MJ et al.; Proteins that catalyse homologous recombination have been identified in all living organisms and are essential for the repair of damaged DNA as well as for the generation of genetic diversity . In bacteria homologous recombination is performed by the RecA protein, whereas in the eukarya a related protein called Rad51 is required to catalyse recombination and repair . More recently, archaeal homologues of RecA/Rad51 (RadA) have been identified and isolated . In this work we have cloned and purified the RadA protein from the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus and characterised its in vitro activities . We show that (i) RadA protein forms ring structures in solution and binds single- but not double-stranded DNA to form nucleoprotein filaments, (ii) RadA is a single-stranded DNA-dependent ATPase at elevated temperatures, and (iii) RadA catalyses efficient D-loop formation and strand exchange at temperatures of 60-70 degrees C . Finally, we have used electron microscopy to visualise RadA-mediated joint molecules, the intermediates of homologous recombination . Intriguingly, RadA shares properties of both the bacterial RecA and eukaryotic Rad51 recombinases.

Optometry, 2001 Oct, 72(10), 649 - 52
Possible iatrogenic transmission of Creutzfeldt-Jakob disease via tonometer tips: a review of the literature; Walia JS et al.; BACKGROUND: Tonometer tips are used by optometrists to measure intraocular pressures . The recommended procedure of soaking in bleach solution kills bacteria and certain viruses, such as human immunodeficiency virus, herpes simplex virus-1 and herpes simplex virus-2, adenovirus 8, and hepatitis B, from the tip . Conversely, recommendations made in literature to sterilize equipment that may have come in contact with virus-contaminated tissue from patients with Creutzfeldt-Jakob disease have a somewhat tougher requirement . METHODS: Autoclaving for 1 hour at a temperature of at least 120 degrees C (15 psi), or a 1-hour exposure to 0.5% sodium hypochlorite (a 10-fold dilution of household bleach) should provide excellent disinfection . One-hour exposure to 1 N Sodium hydroxide has also been mentioned in the literature . RESULTS: Studies have shown that corneas of guinea pigs with Cruetzfeldt-Jakob disease (C-J disease) are infectious . Infected corneas have been shown to cause transmission via corneal transplants, and via experimental placement of infected guinea pig's cornea into the anterior chamber of uninfected guinea pigs . Many researchers have strongly suggested that C-J disease can be iatrogenically transmitted via applanation tonometer tips . An epidemiologic case-controlled study found statistically significant odds ratio for intraocular pressure testing in the medical history of patients with C-J disease . CONCLUSION: Even though there have not been any proven studies confirming iatrogenic transmission through tonometer tips, optometrists should be cautious if a patient has C-J disease, or manifests symptoms of C-J disease and use alternatives to Goldmann applanation tonometry.

Rocz Akad Med Bialymst, 2000, 45, 78 - 86
Effect of aquatic macrophytes on the quantity of bacterioplankton; Czeczuga B et al.; The authors investigated the effect of aquatic macrophytes on the quantity of bacterioplankton . Among the 7 species of aquatic macrophytes the most pronounced effect on bacterioplankton is exerted by Potamogeton crispus, causing a decrease in its quantity to 53.28% . Polygonum amphibium stimulates the growth of bacteria to 121.96% . Under the influence of macrophytes the spherical to cylindrical ratio changes from 1:1.31 (Lemna minor) to 1:2.07 (Potamogeton crispus).

Rocz Akad Med Bialymst, 2000, 45, 68 - 77
Effect of the cyanobacterium Anabaena spiroides Klebahn on the quantity of bacterioplankton in water of varied trophicity; Czeczuga B et al.; The authors investigated the effect of the cyanobacterium Anabaena spiroides on the quantity of bacterioplankton in water of varied trophicity . The cyanobacterium Anabaena spiroides, introduced to the polluted water of the river Biala has the strongest effect on bacterioplankton--the number of bacteria decreases to 31.78% . The spherical:cylindrical ratio changes in favour of the latter when affected by the cyanobacterium . This is the most pronounced in the river Biala, where spherical:cylindrical changes from 1:0.88 to 1:1.96 . Anabaena spiroides exerts the most significant effect on the quantity of bacterioplankton in lake Sniardwy and pond Fosa after 24 hours, and in the other water bodies after 72 hours following the introduction of the cyanobacterium.

Microb Pathog, 2001 Nov, 31(5), 243 - 53
HpaA shows variable surface localization but the gene expression is similar in different Helicobacter pylori strains; Lundstrom AM et al.; Due to earlier contradictory results regarding the localization of the putative Helicobacter pylori adhesin A (HpaA), we aimed to compare the gene and protein expression and surface localization of HpaA in different H . pylori strains . Five H . pylori strains were cultivated for 11 days and analysed by Northern blot analysis, flow cytometry (FCM), semi-quantitative dot blot, colony blot, immuno-electron microscopy (IEM), and phase-contrast microscopy . The highest transcriptional activity of the hapA gene as observed after 3-4 days of cultivation and two mRNA transcripts of 1600 and 3100 nucleotides, respectively, were detected in all five strains with the hpaA probe . We also showed by reverse transcription-polymerase chain reaction (RT-PCR) that the hpaA gene is co-transcribed with the downstream omp18 gene . The highest total HpaA protein production in bacteria occurred between day 3 and 7, as determined by semi-quantitative dot blot, and was similar in the different strains . The maximal proportion of cells with HpaA on the bacterial surface, detected by FCM, was for strain SS1, 90%; Hel 344, 60%; CCUG 17875, 61%; CCUG 17874, 86% and for strain AH 244 only 35% . By IEM HpaA was detected in all strains both on the bacterial surface and on the flagellar sheath .

Semin Nucl Med, 2001 Oct, 31(4), 286 - 95
Radiopharmaceuticals to image infection and inflammation; Boerman OC et al.; Scintigraphic imaging of infection and inflammation is a powerful diagnostic tool in the management of patients with infectious or inflammatory diseases . Most infectious and inflammatory foci can be visualized accurately with radiolabeled autologous leukocytes . However, the preparation of this radiopharmaceutical is laborious and requires the handling of potentially contaminated blood . A few radiopharmaceuticals are available that could be used instead of radiolabeled leukocytes to scintigraphically visualize infectious and inflammatory foci, such as 67Ga-citrate and 99mTc-labeled antigranulocyte antibody preparations . Various agents labeled with 99mTc are currently developed for this application . Most of these newly developed agents are ligands that bind receptors on white blood cell subpopulations, ie, monoclonal antibodies, chemotactic peptides, and cytokines . Furthermore, agents are developed that potentially could distinguish between infection and nonmicrobial inflammation . In addition, 18F-fluorodeoxyglucose positron emission tomography imaging was proposed to visualize inflammatory foci when a high spatial resolution is required . In this article, the characteristics and diagnostic potential of established and experimental radiopharmaceuticals for infection and inflammation imaging are reviewed.

J Vet Med A Physiol Pathol Clin Med, 2001 Oct, 48(8), 475 - 86
Mycoplasma hyosynoviae arthritis in grower-finisher pigs; Nielsen EO et al.; The aetiology of acute lameness in pigs 3-5 months of age in nine Danish herds with high incidences of lameness was investigated . Eighty-seven acutely lame pigs, that exhibited lameness of varying degree in the hind legs, were selected . Non-lame pigs were matched on pen, sex and weight . The lame pigs had soft fluctuating joint swellings (odds ratio (OR), 7.21; 95% confidence interval (CI), 3.41-15.47) . No indication of suppurative arthritis was observed . Joint infection with Mycoplasma hyosynoviae was found by culture in 20% (17 of 86) of the lame pigs and in 8% (seven of 83) of the non-lame pigs . Lameness and joint infection with M . hyosynoviae were significantly associated . Other ordinary bacteria were not found in any case . Macroscopic osteochondrotic lesions were observed at slaughter in 47% (37 of 78) of the previously lame pigs and in 35% (55 of 158) of an enlarged group without history of lameness . The cubital joints were most frequently affected and a history of hind leg lameness was not statistically associated with osteochondrotic lesions at slaughter (OR, 1.69; 95% CI, (1.94-3.05), or joint infection with M . hyosynoviae at slaughter (OR, 0.88; 95% CI, 0.31-2.40) . Arthritis due to M . hyosynoviae infection was the primary cause of acute and severe lameness in grower-finisher pigs . Moreover, M . byosynoviae was isolated from joints of several pigs without signs of lameness . This suggests that M . hyosynoriae may be present in joints without provoking clinical illness . The mean daily incidence of treatments due to lameness in the herds was 5.4 per 1,000 pigs . Joint disease implied 30-90 min extra labour for surveillance and treatment every day per 1,000 pigs, and 5% of the affected individuals were euthanized due to lameness . The average daily weight gains in the selected pigs until slaughter seemed unaffected by the lameness.

Ann R Australas Coll Dent Surg, 2000 Oct, 15, 141 - 3
Prognosis--the dilemma of modern periodontics; Pearlman B; As periodontics has progressed towards an understanding of the influences of risk factors such as genetics, smoking and stress in the occurrence and severity of periodontal disease, the question of prognosis, so essential to treatment planning, has become even more perplexing to the clinician . A survey of long term clinical practice gives insight into the outcome of therapy relative to initial severity, and modern concepts of bacteria versus host relationships provide directions for greater predictability . However, it is only with observation over time that a more accurate assessment of prognosis can be made, as response to initial therapeutic measures can be determined.

Ann Rheum Dis, 2001 Dec, 60(12), 1141 - 4
Risk factors and prognostic influence of infection in a single cohort of 87 adults with systemic lupus erythematosus; Noel V et al.; OBJECTIVES: To describe infectious complications and analyse their risk factors and prognostic role in adults with systemic lupus erythematosus (SLE) . METHODS: A monocentric cohort of 87 adults with SLE (1960-1997) was studied to determine the risk factors for infection (disease activity evaluated by SLAM and SLEDAI scores, type of organ(s) involved or any biological abnormality, specific treatments) by comparing patients who had suffered at least one infectious episode (n=35; 40%) with non-infected patients (n=52; 60%) . Prognostic indicators were assessed by comparing survivors at 10 years with non-survivors . RESULTS: Of the 57 infectious episodes, 47 (82%) were of bacterial origin, 16 (28%) were pneumonia, and 46 (81%) were community acquired . According to univariate analysis, significant risk factors for infection were: severe flares, lupus glomerulonephritis, oral or intravenous corticosteroids, pulse cyclophosphamide, and/or plasmapheresis . No predictors were identified at the time of SLE diagnosis . Multivariate analyses retained intravenous corticosteroids (p<0.001) and/or immunosuppressants (p<0.01) as independent risk factors for infection, which was the only factor for death after 10 years of evolution (p<0.001) . CONCLUSION: In adults with SLE, infections are common and most often caused by community acquired bacteria . Intravenous corticosteroids and immunosuppressants are independent risk factors for infection, which is the only independent risk factor for death after 10 years of SLE evolution.

Semin Immunol, 2001 Dec, 13(6), 347 - 55
Actin dynamics during phagocytosis; Castellano F et al.; Bacteria, apoptotic cells and other particulate material are taken up through phagocytosis, a conserved cellular function driven by actin polymerization . As reviewed here, small GTPases of the Rho family, their activators and effectors control the local reorganization of the actin cytoskeleton underneath bound particles . Remarkably, the molecular actors and regulatory mechanisms involved during phagocytosis through the FcR or the CR3 receptors are very similar to those underlying the cytoskeletal rearrangements that take place at the leading edge of motile cell and at adhesion sites, respectively .

J Theor Biol, 2001 Nov 7, 213(1), 73 - 88
Regulation of gene expression in flux balance models of metabolism; Covert MW et al.; Genome-scale metabolic networks can now be reconstructed based on annotated genomic data augmented with biochemical and physiological information about the organism . Mathematical analysis can be performed to assess the capabilities of these reconstructed networks . The constraints-based framework, with flux balance analysis (FBA), has been used successfully to predict time course of growth and by-product secretion, effects of mutation and knock-outs, and gene expression profiles . However, FBA leads to incorrect predictions in situations where regulatory effects are a dominant influence on the behavior of the organism . Thus, there is a need to include regulatory events within FBA to broaden its scope and predictive capabilities . Here we represent transcriptional regulatory events as time-dependent constraints on the capabilities of a reconstructed metabolic network to further constrain the space of possible network functions . Using a simplified metabolic/regulatory network, growth is simulated under various conditions to illustrate systemic effects such as catabolite repression, the aerobic/anaerobic diauxic shift and amino acid biosynthesis pathway repression . The incorporation of transcriptional regulatory events in FBA enables us to interpret, analyse and predict the effects of transcriptional regulation on cellular metabolism at the systemic level .

Anim Health Res Rev, 2001 Jun, 2(1), 75 - 82
Classification of Brachyspira spp . isolated from Swedish dogs; Fellstrom C et al.; Brachyspira spp . were isolated from 21 of 32 sampled dogs (66%) in a colony of Swedish beagle dogs with a history of diarrhea and from 3 of 17 Swedish pet dogs (17%) with diarrhea . All Swedish isolates were weakly beta-hemolytic and gave a negative indole reaction . Eighty-eight percent showed negative alpha-galactosidase and hippurate reactions, but a positive beta-glucosidase reaction . Two isolates were hippurate positive with a negative beta-glucosidase reaction . One additional German isolate diverged by showing a positive indole reaction in combination with a positive hippurate reaction . Sequencing of 16S rDNA indicated that the hippurate-positive isolates belonged to the species Brachyspira pilosicoli . Four representative isolates were examined using pulsed-field gel electrophoresis (PFGE) and compared with six reference strains and five porcine isolates of Brachyspira spp . The canine isolates clustered together in the PFGE analysis . Necropsy examination of a culture-positive B . pilosicoli colony-raised beagle dog revealed macro- and microscopical lesions of colitis with numerous spiral-shaped bacteria in the lumens of the crypts, in goblet cells and within the colonic epithelium.

Anim Health Res Rev, 2001 Jun, 2(1), 3 - 17
Comparative pathology and pathogenesis of naturally acquired and experimentally induced colonic spirochetosis; Duhamel GE; Research in the past decade has led to the recognition of Brachyspira (formerly Serpulina) pilosicoli as the primary etiologic agent of colonic spirochetosis (CS), an emerging cause of colitis in humans and animals . Attachment of spirochetes to the epithelial surface of the lower intestine is considered to be the hallmark of CS . However, because B . pilosicoli, B . aalborgi and unclassified flagellated bacteria are found singly or together in humans and non-human primates with CS lesions, attachment of spiral-shaped bacteria may not represent the same etiopathogenetic entity in all hosts . Moreover, North American opossums with CS are infected with B . aalborgi-like spirochetes together with flagellated bacteria, whereas B . pilosicoli is found alone in dogs, pigs, chickens and other species of birds with CS . Conversely, guinea-pigs with CS have unidentified spirochetes that may be B . pilosicoli or B . aalborgi . The pig model of CS suggests that attachment of B . pilosicoli to epithelial cells may be transient . By contrast, persistence of B . pilosicoli in the cecal and colonic crypt lumina, chronic inflammation caused by spirochetal invasion into the subepithelial lamina propria and translocation to extraintestinal sites may be more important than previously thought . This review describes the lesions seen in naturally occurring and experimentally induced CS of animals, and it sets the stage for future research into the pathogenic mechanisms of infection and colitis caused by B . pilosicoli.

J Rheumatol, 2001 Nov, 28(11), 2487 - 93
Use of a peptide based enzyme immunoassay in diagnosis of Chlamydia trachomatis triggered reactive arthritis; Nikkari S et al.; OBJECTIVE: To assess the presence of circulating IgA and IgG antibodies to Chlamydia trachomatis in sera of patients with reactive arthritis (ReA) and other arthritides . METHODS: A peptide based enzyme immunoassay (EIA) was used to study 132 patients divided into 5 groups: C . trachomatis triggered ReA, uroarthritis, enteroarthritis, oligoarthritis, and rheumatoid arthritis (RA) . Followup sera were available from 19 patients . RESULTS: An increased prevalence of C . trachomatis antibodies was observed in patients with ReA triggered by C . trachomatis; 18/23 (78%) had IgA and 19/23 (83%) had IgG antibodies . In patient groups with uroarthritis (n = 12), enteroarthritis (n = 56), oligoarthritis (n = 16), and RA (n = 25), C . trachomatis IgA/IgG antibodies were detected in 58%/75%, 27%/21%, 25%/31%, and 20%/32% of patients, respectively . Both the IgA and IgG antibodies were positive in 74%, 50%, 16%, 25%, and 12% of the patients with C . trachomatis triggered ReA, uroarthritis, enteroarthritis, oligoarthritis, and RA, respectively . Based on positivity of both isotypes the sensitivity of the assay was 74% and specificity 84% . In the followup sera, an association between circulating C . trachomatis-specific antibody concentrations and clinical disease outcome of the arthritis was seen in patients with culture-positive C . trachomatis triggered ReA . CONCLUSION: C . trachomatis species-specific peptide EIA correlates well with conventional diagnosis of primary C . trachomatis infection in patients with ReA . This assay may be a valuable contribution to the diagnosis of C . trachomatis triggered ReA.

Protein Eng, 2001 Sep, 14(9), 615 - 31
Pectin degrading glycoside hydrolases of family 28: sequence-structural features, specificities and evolution; Markovic O et al.; Family 28 belongs to the largest families of glycoside hydrolases . It covers several enzyme specificities of bacterial, fungal, plant and insect origins . This study deals with all available amino acid sequences of family 28 members . First, it focuses on the detailed analysis of 115 sequences of polygalacturonases yielding their evolutionary tree . The large data set allowed modification of some of the existing family 28 sequence characteristics and to draw the sequence features specific for bacterial and fungal exopolygalacturonases discriminating them from the endopolygalacturonases . The evolutionary tree reflects both the taxonomy and specificity so that bacterial, fungal and plant enzymes form their own clusters, the endo- and exo-mode of action being respected, too . The only insect (animal) representative is most related to fungal endopolygalacturonases . The present study brings further: (i) the analysis of available rhamnogalacturonase sequences; (ii) the elucidation of relatedness between the recently added member, the endo-xylogalacturonan hydrolase and the rest of the family; and (iii) revealing the sequence features characteristic of the individual enzyme specificities and the evolutionary relationships within the entire family 28 . The disulfides common for the individual enzyme groups were also proposed . With regard to functionally important residues of polygalacturonases, xylogalacturonan hydrolase possesses all of them, while the rhamnogalacturonases, known to lack the histidine residue (His223; Aspergillus niger polygalacturonase II numbering), have a further tyrosine (Tyr291) replaced by a conserved tryptophan . Evolutionarily, the xylogalacturonan hydrolase is most related to fungal exopolygalacturonases and the rhamnogalacturonases form their own cluster on the adjacent branch.

J Exp Biol, 2001 Oct, 204(Pt 20), 3403 - 9
Phototransduction in Drosophila melanogaster; Hardie RC; As in most invertebrate microvillar photoreceptors, phototransduction in Drosophila melanogaster uses a G-protein-coupled phosphoinositide pathway, whereby hydrolysis of phosphatidyl inositol 4,5-bisphosphate (PIP(2)) by phospholipase C generates inositol 1,4,5-trisphosphate (InsP(3)) and diacyl glycerol (DAG), leading to activation of two classes of Ca(2+)-permeable light-sensitive channel, encoded by the trp and trpl genes . In some invertebrate photoreceptors, excitation is mediated by release of Ca(2+) from intracellular stores by InsP(3); however, in Drosophila melanogaster, recent evidence suggests instead that a lipid messenger, such as DAG, its metabolites and/or the reduction in PIP(2) levels, may mediate excitation . Like vertebrate rods, Drosophila melanogaster photoreceptors generate quantum bumps in response to single photons, but their kinetics is approximately 10-100 times faster, and this reflects a fundamentally different strategy incorporating a threshold, positive and negative feedback by Ca(2+) acting downstream of phospholipase C and a refractory period.

Vet Parasitol, 2001 Nov 22, 101(3-4), 291 - 310
Advances and prospects for subunit vaccines against protozoa of veterinary importance; Jenkins MC; Protozoa are responsible for considerable morbidity and mortality in domestic and companion animals . Preventing infection may involve deliberate exposure to virulent or attenuated parasites so that immunity to natural infection is established early in life . This is the basis for vaccines against theilerosis and avian coccidiosis . Vaccination may not be effective or practical with diseases, such as cryptosporidiosis, that primarily afflict the immune-compromised or individuals with an incompletely developed immune system . Strategies for combating these diseases often rely on passive immunotherapy using serum or colostrums containing antibodies to parasite surface proteins . Subunit vaccines offer an attractive alternative to virulent or attenuated parasites for several reasons . These include the use of bacteria or lower eukaryotes to produce recombinant proteins in batch culture, the relative stability of recombinant proteins compared to live parasites, and the flexibility to incorporate only those antigens that elicit "protective" immune responses . Although subunit vaccines offer many theoretical advantages, our lack of understanding of immune mechanisms to primary and secondary infection and the capacity of many protozoa to evade host immunity remain obstacles to developing effective vaccines . This review examines the progress made on developing recombinant proteins of Eimeria, Giardia, Cryptosporidium, Toxoplasma, Neospora, Trypanosoma, Babesia, and Theileria and attempts to use these antigens for vaccinating animals against the associated diseases.

FEBS Lett, 2001 Nov 9, 508(1), 1 - 4
Inter-domain cross-talk controls the NifA protein activity of Herbaspirillum seropedicae; Monteiro RA et al.; Herbaspirillum seropedicae is an endophytic diazotroph, which colonizes sugar cane, wheat, rice and maize . The activity of NifA, a transcriptional activator of nif genes in H . seropedicae, is controlled by ammonium ions through a mechanism involving its N-terminal domain . Here we show that this domain interacts specifically in vitro with the N-truncated NifA protein, as revealed by protection against proteolysis, and this interaction caused an inhibitory effect on both the ATPase and DNA-binding activities of the N-truncated NifA protein . We suggest that the N-terminal domain inhibits NifA-dependent transcriptional activation by an inter-domain cross-talk between the catalytic domain of the NifA protein and its regulatory N-terminal domain in response to fixed nitrogen.

J Dent Res, 2001 Oct, 80(10), 1875 - 9
Tumor necrosis factor modulates fibroblast apoptosis, PMN recruitment, and osteoclast formation in response to P . gingivalis infection; Graves DT et al.; P . gingivalis is an important oral pathogen, which has been closely linked to periodontal disease as well as lesions of endodontic origin . Both infections are associated with a decrease in fibroblast numbers, formation of an inflammatory infiltrate, and bone resorption . The goal of this study was to investigate the role that the host response plays in the capacity of P . gingivalis to stimulate fibroblast apoptosis, PMN recruitment, and osteoclastogenesis . This was accomplished by the use of an in vivo calvarial model in mice with targeted deletion of TNF receptors p55 and p75 and matched wild-type mice . The results indicate that P . gingivalis induces fibroblast apoptosis in vivo and establish for the first time that this involves the stimulation of a host response . Moreover, bacteria-stimulated PMN recruitment and osteoclastogenesis were also dependent upon the host response . The results suggest that much of the damage caused by P . gingivalis infection, including fibroblast apoptosis, at least under some circumstances, results from stimulation of the host response rather than the direct effect of bacterial products . Furthermore, this may represent a more general mechanism by which bacterial challenge induces apoptosis of matrix-producing cells through the induction of TNF.

J Biol Chem, 2002 Jan 18, 277(3), 2202 - 6 Epub 2001 Nov 08.
Ubiquinone is necessary for Caenorhabditis elegans development at mitochondrial and non-mitochondrial sites; Hihi AK et al.; Ubiquinone (UQ) is a lipid co-factor that is involved in numerous enzymatic processes and is present in most cellular membranes . In particular, UQ is a crucial electron carrier in the mitochondrial respiratory chain . Recently, it was shown that clk-1 mutants of the nematode worm Caenorhabditis elegans do not synthesize UQ(9) but instead accumulate demethoxyubiquinone (DMQ(9)), a biosynthetic precursor of UQ(9) (the subscript refers to the length of the isoprenoid side chain) . DMQ(9) is capable of carrying out the function of UQ(9) in the respiratory chain, as demonstrated by the functional competence of mitochondria isolated from clk-1 mutants, and the ability of DMQ(9) to act as a co-factor for respiratory enzymes in vitro . However, despite the presence of functional mitochondria, clk-1 mutant worms fail to complete development when feeding on bacteria that do not produce UQ(8) . Here we show that clk-1 mutants cannot grow on bacteria producing only DMQ(8) and that worm coq-3 mutants, which produce neither UQ(9) nor DMQ(9), arrest development even on bacteria producing UQ(8) . These results indicate that UQ is required for nematode development at mitochondrial and non-mitochondrial sites and that DMQ cannot functionally replace UQ at those non-mitochondrial sites.

Infect Immun, 2001 Dec, 69(12), 7922 - 6
Limited mycobacterial infection of the liver as a consequence of its microanatomical structure causing restriction of mycobacterial growth to professional phagocytes; Seiler P et al.; Among sites of extrapulmonary growth of Mycobacterium tuberculosis, the liver is the least infected . Our data suggest that this is due to the complete restriction of mycobacterial growth to liver macrophages . Unlike in organs more persistently seeded by M . tuberculosis, in the liver the bacteria do not infect cell types other than professional phagocytes.

Infect Immun, 2001 Dec, 69(12), 7736 - 42
Brucella abortus genes identified following constitutive growth and macrophage infection; Eskra L et al.; The chronicity of Brucella abortus infection in humans and animals depends on the organism's ability to escape host defenses by gaining entry and surviving inside the macrophage . Although no human vaccine exists for Brucella, vaccine development in other bacteria has been based on deletions of selective nutritional as well as regulatory systems . Our goal is to develop a vaccine for Brucella . To further this aim, we have used a green fluorescent protein (GFP) reporter system to identify constitutively and intracellularly induced B . abortus genes . Constitutively producing gfp clones exhibited sequence homology with genes associated with protein synthesis and metabolism (initiation factor-1 and tRNA ribotransferase) and detoxification (organic hydroperoxidase resistance) . Of greater interest, clones negative for constitutively produced gfp in agar were examined by fluorescence microscopy to detect promoter activity induced within macrophages 4 and 24 h following infection . Bacterial genes activated in macrophages 4 h postinfection appear to be involved in adapting to intracellular environmental conditions . Included in this group were genes for detoxification (lactoglyglutathione lyase gene), repair (formamidopyrimidine-DNA glycosylase gene), osmotic protection (K(+) transport gene), and site-specific recombination (xerD gene) . A gene involved in metabolism and biosynthesis (deoxyxylulose 5' phosphate synthase gene) was also identified . Genes activated 24 h following infection were biosynthesis- and metabolism-associated genes (iron binding protein and rhizopine catabolism) . Identification of B . abortus genes that are activated following macrophage invasion provides insight into Brucella pathogenesis and thus is valuable in vaccine design utilizing selective targeted deletions of newly identified Brucella genes.

Infect Immun, 2001 Dec, 69(12), 7729 - 35
Role of complement in Mycobacterium avium pathogenesis: in vivo and in vitro analyses of the host response to infection in the absence of complement component C3; Bohlson SS et al.; We investigated the importance of the host complement system in the pathogenesis of disease mediated by the intramacrophage pathogen Mycobacterium avium . Mycobacteria opsonized with complement are efficiently ingested by macrophages through various complement receptors . Furthermore, unlike other bacteria, mycobacteria can activate both the alternative and classical complement pathways in the absence of specific antibodies . Therefore, to examine the role of complement in the mycobacterial infection process in vivo, mice deficient in complement component C3 were infected with M . avium . Surprisingly, C3-deficient mice infected intravenously with M . avium displayed no difference in bacterial burden or granulomatous response compared to wild-type control mice . C3-sufficient mice and C3-deficient mice were equally susceptible to infection by M . avium regardless of the genotype at the bcg locus, a locus known to confer susceptibility to infection with intracellular pathogens . In vitro studies using mouse bone marrow-derived macrophages resulted in significant M . avium invasion of macrophages in the absence of C3; however, the kinetics of infection were delayed compared to complement-mediated invasion . The data indicate that complement does not play an essential role in mediating M . avium infections in the mouse and suggest either that other invasion mechanisms can compensate for the absence of complement-mediated entry or that complement is not a major mycobacterial opsonin in vivo.

Infect Immun, 2001 Dec, 69(12), 7635 - 41
Pertussis toxin and lipopolysaccharide influence phagocytosis of Bordetella pertussis by human monocytes; Schaeffer LM et al.; The potential of human monocytes to mediate the clearance of Bordetella pertussis infection was examined . Bacteria expressing green fluorescent protein were incubated with adherent peripheral blood monocytes, and phagocytosis was quantified by using fluorescence microscopy . Monocytes internalized only a small percentage of the adherent bacteria . Surface-associated Bvg-regulated virulence factors, including adenylate cyclase toxin and filamentous hemagglutinin, did not affect attachment or phagocytosis . However, 1-h pretreatment with purified pertussis toxin inhibited the ability of monocytes to internalize wild-type bacteria . Mutations affecting the terminal trisaccharide of lipopolysaccharide resulted in reduced internalization without affecting adherence of bacteria to monocytes . Opsonization with human serum played only a modest role in promoting phagocytosis . The viability of internalized bacteria was determined by colony counts following treatment with polymyxin B and gentamicin . Less than 1% of internalized bacteria remained viable . These results suggest that pertussis toxin plays a role in the evasion of monocyte phagocytosis and that these cells represent a potential mediator of the clearance of B . pertussis infection.

Infect Immun, 2001 Dec, 69(12), 7501 - 11
Mycobacterial protein HbhA binds human complement component C3; Mueller-Ortiz SL et al.; Mycobacterium tuberculosis and Mycobacterium avium are facultative intracellular pathogens that are able to survive and replicate in mononuclear phagocytes . Human complement component C3 has previously been shown to mediate attachment and phagocytosis of these bacteria by mononuclear phagocytes . In this study, a C3 ligand affinity blot protocol was used to identify a 30-kDa C3-binding protein in M . tuberculosis and Mycobacterium smegmatis and a 31-kDa C3-binding protein in M . avium . The C3-binding proteins in M . tuberculosis and M . avium localized to the cell membrane fraction and partitioned to the detergent fraction during Triton X-114 phase partitioning . The C3-binding protein from M . tuberculosis was partially purified using a cation exchange column and was shown to bind concanavalin A . The N terminus and an internal fragment of the partially purified C3-binding protein were subjected to amino acid sequence analysis . The resulting amino acid sequences matched the M . tuberculosis heparin-binding hemagglutinin (HbhA) protein . Recombinant full-length HbhA and the C terminus of HbhA fused to maltose-binding protein, but not recombinant HbhA lacking the C-terminal region, bound human C3 . Recombinant full-length HbhA coated on polystyrene beads, was found to enhance the adherence and/or phagocytosis of the coated beads to J774.A1 cells in both the presence and absence of human serum . The presence of complement-sufficient serum increased the adherence of the HbhA-coated beads to the J774.A1 cells in a C3-dependent manner . If HbhA within the bacterial cell membrane functions similarly to isolated HbhA, this protein may enhance the adherence and phagocytosis of M . tuberculosis and M . avium to mononuclear phagocytes through the binding of C3 and interaction with C3 receptors on mononuclear phagocytes.

Infect Immun, 2001 Dec, 69(12), 7349 - 55
Mycobacterium tuberculosis chaperonin 60.1 is a more potent cytokine stimulator than chaperonin 60.2 (Hsp 65) and contains a CD14-binding domain; Lewthwaite JC et al.; Much attention has focused on the Mycobacterium tuberculosis molecular chaperone chaperonin (Cpn) 60.2 (Hsp 65) in the pathology of tuberculosis because of its immunogenicity and ability to directly activate human monocytes and vascular endothelial cells . However, M . tuberculosis is one of a small group of bacteria that contain multiple genes encoding Cpn 60 proteins . We have now cloned and expressed both M . tuberculosis proteins and report that the novel chaperonin 60, Cpn 60.1, is a more potent inducer of cytokine synthesis than is Cpn 60.2 . This is in spite of 76% amino acid sequence similarity between the two mycobacterial chaperonins . The M . tuberculosis Cpn 60.2 protein activates human peripheral blood mononuclear cells by a CD14-independent mechanism, whereas Cpn 60.1 is partially CD14 dependent and contains a peptide sequence whose actions are blocked by anti-CD14 monoclonal antibodies . The cytokine-inducing activity of both chaperonins is extremely resistant to heat . Cpn 60.1 may be an important virulence factor in tuberculosis, able to activate cells by diverse receptor-driven mechanisms.

Heredity, 2001 Aug, 87(Pt 2), 227 - 33
Sex ratio and Wolbachia infection in the ant Formica exsecta; Keller L et al.; Sex allocation data in social Hymenoptera provide some of the best tests of kin selection, parent-offspring conflict and sex ratio theories . However, these studies critically depend on controlling for confounding ecological factors and on identifying all parties that potentially manipulate colony sex ratio . It has been suggested that maternally inherited parasites may influence sex allocation in social Hymenoptera . If the parasites can influence sex allocation, infected colonies are predicted to invest more resources in females than non-infected colonies, because the parasites are transmitted through females but not males . Prime candidates for such sex ratio manipulation are Wolbachia, because these cytoplasmically transmitted bacteria have been shown to affect the sex ratio of host arthropods by cytoplasmic incompatibility, parthenogenesis, male-killing and feminization . In this study, we tested whether Wolbachia infection is associated with colony sex ratio in two populations of the ant Formica exsecta that have been the subject of extensive sex ratio studies . In these populations colonies specialize in the production of one sex or the other . We found that almost all F . exsecta colonies in both populations are infected with Wolbachia . However, in neither population did we find a significant association in the predicted direction between the prevalence of Wolbachia and colony sex ratio . In particular, colonies with a higher proportion of infected workers did not produce more females . Hence, we conclude that Wolbachia does not seem to alter the sex ratio of its hosts as a means to increase transmission rate in these two populations of ants.

Sheng Wu Gong Cheng Xue Bao, 2001 Jul, 17(4), 467 - 70
{Studies on asymmetric synthesis of R-phenylethanolamine by whole cells of Arachnia sp . P163}; Wang JL et al.; Effects of various factors on asymmetric synthesis of R-phenylaminoethanol from aminoacetophenone by the whole cells of Arachnia sp . P163 producing alcohol dehydrogenase for phenylethanol amine was investigated . It found that, although the reduction was inhibited by the substrate and the product, but it has the very high stereoselectivity . The reduction was normaly carried out with 2% glucose for reproduction of coenzyme in the reaction system without oxygen . The conversion yield and ee value of the product achieved 65% and 100%, respectively.

Arch Microbiol, 2001 Nov, 176(5), 323 - 8
Amorphous mineral phases in magnetotactic multicellular aggregates; Lins U et al.; Magnetotactic multicellular aggregates consist of several bacteria that produce iron sulfide magnetosomes through a complex and poorly understood process . We observed new amorphous mineral particles within the cytoplasm of magnetotactic multicellular aggregates . Elemental mapping and electron energy loss spectroscopy detected iron and oxygen, but not sulfur, in these particles . These amorphous particles were about the same size as mature magnetosomes, around 50-70 nm in diameter . No membranes were observed surrounding the amorphous minerals . Partially crystalline inclusions composed of a crystalline core and an amorphous region around them similar in texture to the amorphous particles were also present . The shape of these amorphous regions followed the shape of the crystalline cores they enveloped . These regions also contained oxygen and iron . The crystalline phase, as previously reported, contained sulfur and iron . The presence of independent amorphous particles has not been reported before in magnetotactic multicellular aggregates.

Science, 2001 Nov 9, 294(5545), 1334 - 6
Collaboration between CC- and A-adding enzymes to build and repair the 3'-terminal CCA of tRNA in Aquifex aeolicus; Tomita K et al.; The universal 3'-terminal CCA sequence of all transfer RNAs (tRNAs) is repaired, and sometimes constructed de novo, by the CCA-adding enzyme {ATP(CTP):tRNA nucleotidyltransferase} . This RNA polymerase has no nucleic acid template, yet faithfully builds the CCA sequence one nucleotide at a time using cytidine triphosphate (CTP) and adenosine triphosphate (ATP) as substrates . All previously characterized CCA-adding enzymes from all three kingdoms are single polypeptides with CCA-adding activity . Here, we demonstrate through biochemical and genetic approaches that CCA addition in Aquifex aeolicus requires collaboration between two related polypeptides, one that adds CC and another that adds A.

Annu Rev Phytopathol, 2001, 39, 187 - 224
Pathogen fitness penalty as a predictor of durability of disease resistance genes; Leach JE et al.; Host plant resistance has been used extensively for disease control in many crop species; however, the resistance conferred by many sources is not durable as a result of rapid changes in the pathogen . Although many resistance genes have been identified in plant germplasm, there is no easy way to predict the quality or durability of these resistance genes . In this review, we revisit the hypothesis that resistance genes imposing a high penalty to the pathogen for adaptation will likely be durable . By elucidating the molecular changes involved in pathogen adaptation and the associated fitness cost, a proactive approach may be developed to predict the durability of resistance genes available for deployment.

Annu Rev Biomed Eng, 2000, 2, 339 - 76
Antibody engineering; Maynard J et al.; Antibodies are unique in their high affinity and specificity for a binding partner, a quality that has made them one of the most useful molecules for biotechnology and biomedical applications . The field of antibody engineering has changed rapidly in the past 10 years, fueled by novel technologies for the in vitro isolation of antibodies from combinatorial libraries and their functional expression in bacteria . This review presents an overview of the methods available for the de novo generation of human antibodies, for engineering antibodies with increased antigen affinity, and for the production of antibody fragments . Select applications of recombinant antibodies are also presented.

Cell, 2001 Nov 2, 107(3), 339 - 46
Differential gene expression governed by chromosomal spatial asymmetry; Dworkin J et al.; The activity of the transcription factor sigmaF is confined to one (the forespore) of two cells created by asymmetric division during sporulation in B . subtilis . We show that sigmaF activation is partly governed by the position of the gene for the unstable anti-sigmaF factor SpoIIAB . Because cytokinesis precedes chromosome segregation, most of the chromosome is translocated into the forespore after division . We hypothesize that because spoIIAB enters the forespore late, SpoIIAB lost to proteolysis is temporarily not replenished . Thus, chromosome asymmetry would be translated into the asymmetric distribution of SpoIIAB . Supporting this idea, transposition of spoIIAB to sites present in the forespore at the time of division impaired sporulation when a second pathway that participates in sigmaF activation was disabled.

Anal Biochem, 2001 Nov 15, 298(2), 151 - 62
Detection of epitope-tagged proteins in flow cytometry: fluorescence resonance energy transfer-based assays on beads with femtomole resolution; Buranda T et al.; Epitope tagging of expressed proteins is a versatile tool for the detection and purification of the proteins . This approach has been used in protein-protein interaction studies, protein localization, and immunoprecipitation . Among the most popular tag systems is the FLAG epitope tag, which is recognized by three monoclonal antibodies M1, M2, and M5 . We describe novel approaches to the detection of epitope-tagged proteins via fluorescence resonance energy transfer on beads . We have synthesized and characterized biotinylated and fluorescein-labeled FLAG peptides and examined the binding of FLAG peptides to commercial streptavidin beads using flow cytometric analysis . A requirement of assay development is the elucidation of parameters that characterize the binding interactions between component systems . We have thus compiled a set of Kd values determined from a series of equilibrium binding experiments with beads, peptides, and antibodies . We have defined conditions for binding biotinylated and fluoresceinated FLAG peptides to beads . Site occupancies of the peptides were determined to be on the order of several million sites per bead and Kd values in the 0.3-2.0 nM range . The affinity for antibody attachment to peptides was determined to be in the low nanomolar range (less than 10 nM) for measurements on beads and solution . We demonstrate the applicability of this methodology to assay development, by detecting femtomole amounts of N-terminal FLAG-bacteria alkaline phosphatase fusion protein . These characterizations form the basis of generalizable and high throughput assays for proteins with known epitopes, for research, proteomic, or clinical applications .

Optom Vis Sci, 2001 Oct, 78(10), 732 - 43
The role of fenestrations and channels on the transverse motion of a soft contact lens; Chauhan A et al.; PURPOSE: Ineffectual removal of potentially harmful species from the postlens tear film (POLTF) may lead to adverse responses among extended wearers of soft contact lenses . It is apparently important to remove bacteria, cell debris, and metabolic products from the postlens tear film to the outer tear lake; the flushing or dispersion rate of these species is enhanced by increasing fluid movement in the tear film driven by periodic lens motion . The contact lens moves laterally (up-down) and transversally (in-out) due to the action of the eyelid forces during blinking . Viscous drag in the POLTF resists lens motion . Consequently, any design change in the lens that reduces viscous drag increases motion and improves flushing of unwanted species from the POLTF . We investigate quantitatively the effect of channels cut on the back surface of the lens and fenestrations (holes) drilled through the lens on transverse lens motion . METHODS: We model the lens as a curved solid body with a periodic arrangement of channels/holes . The cornea is treated as a flat surface, and the hydrodynamic equations of motion are solved for Newtonian fluid transport in the POLTF assuming lubrication and creeping flow . POLTF pressure profiles, obtained by solving these equations, are integrated to determine the lens settling velocity in the transverse direction for a given amount of applied lid force . Lens settling velocity is then compared with the same velocity in the absence of channels/fenestrations . Further, we calculate the total transverse motion in a blink for lenses with and without channels/fenestrations to estimate the possible enhancement in transverse motion due to the channels/fenestrations . RESULTS: Variables that affect the fluid mixing in the POLTF are the postlens tear film thickness, lens thickness, channel length, depth and spacing, and the hole diameter, location, and spacing . We study the effect of each of these variables on the enhancement of transverse motion for channels and holes of diameters varying from 0.1 to 2 mm with spacing varying from 1 to 5 mm . CONCLUSIONS: We demonstrate that incorporation of channels and holes reduces viscous resistance and increases transverse lens motion, and thus increases fluid mixing and dispersive flushing from the POLTF . The increase in transverse motion depends strongly on the postlens tear film thickness . Enhancement of the transverse motion varies from a factor of about 2 to 20 depending on the particular lens design and the postlens tear film thickness . Because fluid mixing increases up to the square of the transverse motion, channels/holes are expected to render flushing of the POLTF considerably more effective . We find that channels and/or fenestrations, when appropriately designed, can provide significant improvement in flushing from the POLTF . This work provides a new quantitative tool for the efficient design of channels/holes in soft contact lenses.

Genome Inform Ser Workshop Genome Inform, 2000, 11, 161 - 71
Intrinsic protein disorder in complete genomes; Dunker AK et al.; Intrinsic protein disorder refers to segments or to whole proteins that fail to fold completely on their own . Here we predicted disorder on protein sequences from 34 genomes, including 22 bacteria, 7 archaea, and 5 eucaryotes . Predicted disordered segments > or = 50, > or = 40, and > or = 30 in length were determined as well as proteins estimated to be wholly disordered . The five eucaryotes were separated from bacteria and archaea by having the highest percentages of sequences predicted to have disordered segments > or = 50 in length: from 25% for Plasmodium to 41% for Drosophila . Estimates of wholly disordered proteins in the bacteria ranged from 1% to 8%, averaging to 3 +/- 2%, estimates in various archaea ranged from 2 to 11%, plus an apparently anomalous 18%, averaging to 7 +/- 5% that drops to 5 +/- 3% if the high value is discarded . Estimates in the 5 eucarya ranged from 3 to 17% . The putative wholly disordered proteins were often ribosomal proteins, but in addition about equal numbers were of known and unknown function . Overall, intrinsic disorder appears to be a common, with eucaryotes perhaps having a higher percentage of native disorder than archaea or bacteria.

Microbiology, 2001 Nov, 147(Pt 11), 3127 - 34
In vitro secretion kinetics of proteins from Legionella pneumophila in comparison to proteins from non-pneumophila species; Flieger A et al.; It has been shown that the loss of PilD, a prepilin peptidase necessary for type IV pilus biogenesis and establishment of the type II secretion apparatus is associated with loss of virulence in Legionella pneumophila . L . pneumophila is the species most frequently associated with Legionnaires' disease, but virulence factors unique to this species are not known, so the secretion kinetics of several pilD-dependent enzyme activities, including protease, acid phosphatase, phospholipase A (PLA) and lysophospholipase A (LPLA), of L . pneumophila and non-pneumophila species were compared during growth in BYE broth . Enzyme activity appeared during mid-exponential growth phase and reached maximal levels on entry into stationary growth phase . None of the enzyme activities were unique to L . pneumophila and it did not exclusively secrete the highest amounts of the hydrolytic proteins . However, the timing of PLA and LPLA secretion in L . pneumophila differed compared to other species . PLA activity was secreted prior to LPLA activity in L . pneumophila, which may lead to an accumulation of the cytotoxic agent lysophosphatidylcholine (LPC) . In addition to L . pneumophila, several other Legionella species, including Legionella steigerwaltii and Legionella gormanii, were able to enrich for LPC due to a very potent PLA activity accompanied by only moderate LPLA activity . These species, in contrast to L . pneumophila, have not been shown to multiply within monocytic host cells . Thus none of the secreted enzymic activities investigated were unique to L . pneumophila, nor were they secreted at high concentrations . However, the timing of PLA and LPLA secretion may contribute to pathogenicity.

J Magn Reson, 2001 Nov, 153(1), 69 - 74
Ferromagnetic resonance of horse spleen ferritin: core blocking and surface ordering temperatures; Wajnberg E et al.; In nature, ferritin, an iron-storage molecule, is found in species ranging from bacteria to man . In the past 50 years its chemical, physical, and magnetic properties have been studied, searching to relate function and structure . Horse spleen ferritin has been investigated by EPR at temperatures between 7 and 290 K . These spectra change from an isotropic line at 290 K to an anisotropic one at 19 K, with a behavior consistent with a system of particles that undergoes superparamagnetic relaxation . A blocking temperature of (116+/-9) K is obtained . A new temperature-dependent signal is observed in the low field region at temperatures higher than 80 K . At 7 K no EPR signal appears, suggesting (14+/-5) K as the Neel temperature of surface spins . Analysis of the temperature dependence of the distance between EPR lines extrema, under the view of two theoretical models, allowed the evaluation of magnetic parameters . These parameters are 2K/M=2.7 x 10(3) Oe and MV=1.9 x 10(-17) emu or K/M=1.3 x 10(3) Oe and MV=2.0 x 10(-17) emu, where K is the anisotropy energy per unit volume, M is the sample magnetization, and V is the superparamagnetic core volume . The results are also discussed, and some structural models in the literature are considered .

J Mol Biol, 2001 Nov 9, 313(5), 941 - 54
Comparison of repressor and transcriptional attenuator systems for control of amino acid biosynthetic operons; Elf J et al.; In bacteria, expression from amino acid biosynthetic operons is transcriptionally controlled by two main mechanisms with principally different modes of action . When the supply of an amino acid is in excess over demand, its concentration will be high and when the supply is deficient the amino acid concentration will be low . In repressor control, such concentration variations in amino acid pools are used to regulate expression from the corresponding amino acid synthetic operon; a high concentration activates and a low concentration inactivates repressor binding to the operator site on DNA so that initiation of transcription is down or up-regulated, respectively . Excess or deficient supply of an amino acid also speeds or slows, respectively, the rate by which the ribosome translates mRNA base triplets encoding this amino acid . In attenuation of transcription, it is the rate by which the ribosome translates such "own" codons in the leader of an amino acid biosynthetic operon that decides whether the RNA polymerase will continue into the operon, or whether transcription will be aborted (attenuated) . If the ribosome rate is fast (excess synthesis of amino acid), transcription will be terminated and if the rate is slow (deficient amino acid supply) transcription will continue and produce more messenger RNAs.Repressor and attenuation control systems have been modelled mathematically so that their behaviour in living cells can be predicted and their system properties compared . It is found that both types of control systems are unexpectedly sensitive when they operate in the cytoplasm of bacteria . In the repressor case, this is because amino acid concentrations are hypersensitive to imbalances between supply and demand . In the attenuation case, the reason is that the rate by which ribosomes translate own codons is hypersensitive to the rate by which the controlled amino acid is synthesised.Both repressor and attenuation mechanisms attain close to Boolean properties in vivo: gene expression is either fully on or fully off except in a small interval around the point where supply and demand of an amino acid are perfectly balanced.Our results suggest that repressors have significantly better intracellular performance than attenuator mechanisms . The reason for this is that repressor, but not attenuator, mechanisms can regulate expression from biosynthetic operons also when transfer RNAs are fully charged with amino acids so that the ribosomes work with maximal speed .

J Dent Educ, 2001 Oct, 65(10), 1038 - 45
Inherited risks for susceptibility to dental caries; Shuler CF; Dental caries incidence is affected by host factors that may be related to the structure of dental enamel, immunologic response to cariogenic bacteria, or the composition of saliva . Genetic variation of the host factors may contribute to increased risks for dental caries . This systematic review examined the literature to address the question, "Is the risk for dental decay related to patterns of genetic inheritance?" Numerous reports have described a potential genetic contribution to the risk for dental caries . Studies on twins have provided strong evidence for the role of inheritance . Establishing a basis for a genetic contribution to dental caries will provide a foundation for future studies utilizing the human genome sequence to improve understanding of the disease process . Inherited disorders of tooth development with altered enamel structure increase the incidence of dental caries . Specific genetic linkage has not been determined for all of the syndromes of altered tooth development . Consequently, genetic screens of large populations for genes or mutations associated with increased caries susceptibility have not been done . Altered immune response to the cariogenic bacteria may also increase the incidence of caries . Association between specific patterns of HLA genetic inheritance and dental caries risk is weak and does not provide a predictable basis for predicting future decay rates . The evidence supporting an inherited susceptibility to dental caries is limited . Genetic linkage approaches on well-characterized populations with clearly defined dental caries incidence will be required to further analyze the relationship between inheritance and dental caries.

IUBMB Life, 2001 May, 51(5), 295 - 8
Identification of a transactivation motif in the CLN3 protein; Leung KY et al.; A transactivation motif has been identified in the neurodegenerative disease protein, CLN3 . The C-terminal domain (residues 394-438) of CLN3 can function as a transcriptional activator when fused to the DNA binding domain, LexA . A series of deletion and substitution constructs have been generated to identify the essential region for transactivation . A similar motif is also present in the POU domain transcription factor, nubbin . However, this domain alone does not activate transcription, allowing further localisation of the critical residues in CLN3 required for activity.

IUBMB Life, 2001 May, 51(5), 289 - 93
Partial recovery of light-independent chlorophyll biosynthesis in the chlL-deletion mutant of Synechocystis sp . PCC 6803; Wu Q et al.; A chlL-deletion mutant of Synechocystis sp . PCC 6803 designated as chlL- was unable to make significant amounts of chlorophyll in darkness . However, an apparent pseudorevertant has been generated spontaneously that can synthesize an increased amount of chlorophyll under light-activated heterotrophic growth conditions . Under these conditions, the chlorophyll content in this pseudorevertant was about 20% of that in the wild-type strain and about 4 times more than that in the original and in the recently recreated chlL-deletion mutant . This is paralleled by increased performance of dark-grown cells in terms of chlorophyll fluorescence induction and oxygen evolution rates in the pseudorevertant versus in the original mutant . PCR analysis confirmed that the chlL- -pseudorevertant mutant still lacked the chlL gene . These results imply that the light-independent chlorophyll biosynthesis pathway was partly recovered.

Membr Cell Biol, 2001 Jul, 14(5), 673 - 97
Hydrophobicity and prediction of the secondary structure of membrane proteins and peptides; Klevanik AV; Reliability of the hydropathy method to predict the formation of membrane-spanning alpha-helices by integral membrane proteins and peptides whose structure is known from X-ray crystallography is analysed . It is shown that Kyte-Doolittle hydropathy plots do not predict accurately 22 transmembrane alpha-helices in the reaction centres (RC) of the photosynthetic bacteria Rhodopseudomonas viridis and Rhodobacter sphaeroides (R-26) . The accuracy of prediction for these proteins was improved using an optimised Kyte-Doolittle hydrophobicity scale . However, this hydrophobicity scale did not improve the predictions for the alphabeta-peptides of the B800-850 (LH2) complexes of the photosynthetic bacteria Rhodopseudomonas acidophila and Rhodospirillum molischianum, which were excluded from the optimisation procedure . The best and worst predictions of membrane-spanning alpha-helices for the RC proteins and LH2 peptides, respectively, were obtained with a propensity scale (PRC) calculated from the amino acid sequences and X-ray data for the RC proteins . A propensity scale (PLH) obtained using the amino acid sequences and X-ray data for the alphabeta-peptides of the LH2 complexes did not give an acceptable prediction of the transmembrane segments in the LH2 peptides; moreover, it markedly contradicted the PRC scale . Amino acids have been concluded to have no significant preference to localisation in transmembrane segments . Therefore, the predictive ability of the hydropathy methodology appears to be limited: the number of transmembrane segments can be correctly calculated for the best case only, and the lengths and positions of membrane-spanning alpha-helices in a protein amino acid sequence can not be predicted exactly.

J Periodontol, 2001 Oct, 72(10), 1332 - 9
Non-redundant roles for interleukin-1 alpha and interleukin-1 beta in regulating human IgG2; Ishihara Y et al.; BACKGROUND: Serum concentrations of immunoglobulin G2 (IgG2) are elevated in localized aggressive periodontitis (LAgP) patients, and secretory products of monocytes from LAgP patients enhance IgG2 responses of lymphocytes from healthy subjects . Furthermore, genes regulating production of interleukin (IL)-1 influence the risk for both aggressive periodontitis (AgP) and chronic periodontitis . These observations, and the fact that IgG2 dominates responses to carbohydrates from Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis, prompted the hypothesis that IL-1 alpha, IL-1 beta, and IL-RA may help regulate human IgG2 responses . METHODS: Human peripheral blood leukocytes (PBL) were stimulated in culture with pokeweed mitogen (PWM); the levels of available IL-1 gene products were manipulated; and the effect on IgG2 production was monitored . Manipulations of IL-1 were accomplished by adding specific neutralizing monoclonal antibodies or recombinant IL-1RA, IL-1 alpha, or IL-1 beta . RESULTS: Blocking the IL-1 receptor with IL-1RA or neutralizing IL-1 alpha or IL-1 beta with specific antibody dramatically suppressed IgG2 production (50% to 70%) . Additionally IL-1 alpha did not compensate for neutralized IL-1 beta, and additional IL-1 beta did not compensate for neutralized IL-1 alpha, suggesting the 2 monokines have separate roles in promoting IgG2 . Furthermore, combinations of anti-IL-1 alpha and anti-IL-1 beta were more inhibitory than either antibody alone, and IL-1 alpha and IL-1 beta in combination appeared to work additively in promoting IgG2 . Moreover, PBL cultures from a group of LAgP patients with high IgG2 levels had elevated levels of IL-1 beta . CONCLUSION: IL-1 alpha and IL-1 beta appear to have critical and non-redundant roles in the generation and regulation of potent IgG2 responses, which appear to be important in human responses to carbohydrate-bearing bacteria.

J Bacteriol, 2001 Dec, 183(23), 6951 - 6
The LexA protein from Deinococcus radiodurans is not involved in RecA induction following gamma irradiation; Narumi I et al.; The involvement of LexA in induction of RecA was investigated in Deinococcus radiodurans . As in the wild-type strain, an increase in RecA protein synthesis following gamma irradiation was detected in a lexA disruptant, indicating that LexA is not involved in the induction of RecA in D . radiodurans.

J Bacteriol, 2001 Dec, 183(23), 6801 - 6
Transcriptional regulation of furA and katG upon oxidative stress in Mycobacterium smegmatis; Milano A et al.; The DNA region upstream of katG in Mycobacterium smegmatis was cloned and sequenced . The furA gene, highly homologous to Mycobacterium tuberculosis furA, mapped in this region . The furA-katG organization appears to be conserved among several mycobacteria . The transcription pattern of furA and katG in M . smegmatis upon oxidative stress was analyzed by Northern blotting and primer extension . Although transcription of both furA and katG was induced upon oxidative stress, transcripts covering both genes could not be identified either by Northern blotting or by reverse transcriptase PCR . Specific transcripts and 5' ends were identified for furA and katG, respectively . By cloning M . smegmatis and M . tuberculosis DNA regions upstream of a reporter gene, we demonstrated the presence of two promoters, pfurA, located immediately upstream of the furA gene, and pkatG, located within the terminal part of the furA coding sequence . Transcription from pfurA was induced upon oxidative stress . A 23-bp sequence overlapping the pfurA -35 region is highly conserved among mycobacteria and streptomycetes and might be involved in controlling pfurA activity . Transcription from a cloned pkatG, lacking the upstream pfurA region, was not induced upon oxidative stress, suggesting a cis-acting regulatory role of this region.

J Bacteriol, 2001 Dec, 183(23), 6787 - 93
The global regulators GacA and sigma(S) form part of a cascade that controls alginate production in Azotobacter vinelandii; Castaneda M et al.; Transcription of the Azotobacter vinelandii algD gene, which encodes GDP-mannose dehydrogenase (the rate-limiting enzyme of alginate synthesis), starts from three sites: p1, p2, and p3 . The sensor kinase GacS, a member of the two-component regulatory system, is required for transcription of algD from its three sites during the stationary phase . Here we show that algD is expressed constitutively throughout the growth cycle from the p2 and p3 sites and that transcription from p1 started at the transition between the exponential growth phase and stationary phase . We constructed A . vinelandii strains that carried mutations in gacA encoding the cognate response regulator of GacS and in rpoS coding for the stationary-phase sigma(S) factor . The gacA mutation impaired alginate production and transcription of algD from its three promoters . Transcription of rpoS was also abolished by the gacA mutation . The rpoS mutation impaired transcription of algD from the p1 promoter and increased it from the p2 sigma(E) promoter . The results of this study provide evidence for the predominant role of GacA in a regulatory cascade controlling alginate production and gene expression during the stationary phase in A . vinelandii.

Am J Physiol Cell Physiol, 2001 Dec, 281(6), C1858 - 70
Characteristics of EYFP-actin and visualization of actin dynamics during ATP depletion and repletion; Herget-Rosenthal S et al.; Disruption of the actin cytoskeleton in proximal tubule cells is a key pathophysiological factor in acute renal failure . To investigate dynamic alterations of the actin cytoskeleton in live proximal tubule cells, LLC-PK(10) cells were transfected with an enhanced yellow fluorescence protein (EYFP)-actin construct, and a clone with stable EYFP-actin expression was established . Confluent live cells were studied by confocal microscopy under physiological conditions or during ATP depletion of up to 60 min . Immunoblots of stable transfected LLC-PK(10) cells confirmed the presence of EYFP-actin, accounting for 5% of total actin . EYFP-actin predominantly incorporated in stress fibers, i.e., cortical and microvillar actin as shown by excellent colocalization with Texas red phalloidin . Homogeneous cytosolic distribution of EYFP-actin indicated colocalization with G-actin as well . Beyond previous findings, we observed differential subcellular disassembly of F-actin structures: stress fibers tagged with EYFP-actin underwent rapid and complete disruption, whereas cortical and microvillar actin disassembled at slower rates . In parallel, ATP depletion induced the formation of perinuclear EYFP-actin aggregates that colocalized with F-actin . During ATP depletion the G-actin fraction of EYFP-actin substantially decreased while endogenous and EYFP-F-actin increased . During intracellular ATP repletion, after 30 min of ATP depletion, there was a high degree of agreement between F-actin formation from EYFP-actin and endogenous actin . Our data indicate that EYFP-actin did not alter the characteristics of the endogenous actin cytoskeleton or the morphology of LLC-PK(10) cells . Furthermore, EYFP-actin is a suitable probe to study the spatial and temporal dynamics of actin cytoskeleton alterations in live proximal tubule cells during ATP depletion and ATP repletion.

Trends Immunol, 2001 Nov, 22(11), 618 - 26
Atopic disorders: a vaccine around the corner?
Wohlleben G, Erb KJ.
The incidence and severity of atopic disorders, in particular asthma, is steadily increasing at an alarming rate . Furthermore, no primary prevention measure exists to date . However, recent results obtained from numerous animal studies suggest that primary prevention in humans might be possible in the near future . The most promising approaches include the induction of systemic or local allergen-dependent or -independent T helper 1 (Th1) immune responses, through the use of killed bacteria (or components derived from them), CpG oligodeoxynucleotides or plasmid DNA, and the induction of allergen-specific T-cell tolerance . Here, we review the data showing that animals can be protected from developing allergic Th2 responses by vaccination . Possible future use in humans and potential side-effects of the described vaccination strategies are discussed also.

Eur J Pharmacol, 2001 Oct 19, 429(1-3), 287 - 96
Interactive roles of superoxide and inducible nitric oxide synthase in rat intestinal injury provoked by non-steroidal anti-inflammatory drugs; Evans SM et al.; The role of nitric oxide (NO) formed by inducible NO synthase (iNOS), superoxide and the lipopolysaccharide from luminal bacteria in non-steroidal anti-inflammatory drug-induced intestinal injury was investigated in the rat . Administration (s.c . or p.o.) of indomethacin (10 mg kg(-1)), flurbiprofen (40 mg kg(-1)) or diclofenac (40 mg kg(-1)) increased the vascular leakage of radiolabelled albumin in the jejunum, determined after 24 h, associated with the induction of iNOS, assessed by the conversion of radiolabelled L-arginine . Pre-treatment with ampicillin (200 mg kg(-1) day(-1), p.o.), metronidazole (200 mg kg(-1) day(-1), p.o.), or polymixin B (15 mg kg(-1) day(-1), s.c.), inhibited indomethacin-induced lesion formation, reduced microvascular leakage and prevented the expression of iNOS activity . Administration of the highly selective iNOS inhibitor, GW273629 ((R)-2-amino-4,4-dioxo-6(1-iminioethylamino)-4-thiahexanoic acid; 5 mg kg(-1), s.c.), 18 h after indomethacin, likewise prevented the intestinal lesions and attenuated the microvascular leakage . Superoxide dismutase conjugated with polyethylene glycol (3000 U kg(-1), i.v.), inhibited the indomethacin-induced lesions and microvascular leakage, but not the expression of iNOS activity . These findings suggest that non-steroidal anti-inflammatory drugs compromise mucosal integrity, leading to luminal bacterial translocation . This provokes iNOS induction, leading to microvascular injury involving both NO and superoxide.

J Drug Target, 2001, 9(4), 253 - 66
Studies on the potential of microparticles entrapping pDNA-poly(aminoacids) complexes as vaccine delivery systems; Benoit MA et al.; Poly(D,L-lactide-co-glycolide) (PLGA) microparticles containing plasmid DNA (pDNA) have potential uses as vaccine delivery systems . Nevertheless, the established double emulsion and solvent evaporation method used to produce them is characterised by a low encapsulation efficiency (about 20%) and nicks the supercoiled DNA . The aim of this work was to develop an encapsulation process to optimise the overall encapsulation efficiency and the supercoiled DNA content, to obtain a carrier suitable for mucosal delivery of DNA vaccines . Our strategy was to reduce the global negative charge of DNA which was unfavourable to its incorporation into the polymer by condensing it with cationic poly(aminoacids) which were previously reported to improve cell transfection . In this study, after characterisation of the compaction of DNA plasmid encoding for a Green Fluorescent Protein, we demonstrated that resulting complexes were successfully encapsulated into PLGA microparticles presenting a mean size around 4.5 microns . The preliminary step of complexation enhances the yield of the process by a factor 4.1 and protects the supercoiled form . In a bacteria transformation assay, we demonstrated that extracted pDNA (naked or complexed) remained in a transcriptionally active form after encapsulation . Bovine macrophages in culture phagocytosed microparticles loaded with uncomplexed/complexed with poly(L-lysine) pDNA . The production of the Green Fluorescent Protein demonstrated that these carriers could deliver intact and functional plasmid DNA probably by escaping from lysosomal degradation.

Lett Appl Microbiol, 2001 Nov, 33(5), 329 - 33
Adaptation of an {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide} assay to evaluate the cytotoxicity of the extracellular products of micro-organisms pathogenic to fish; Zorrilla I et al.; AIMS: Adaptation of a colorimetric assay using {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide} (MTT) to evaluate the cytotoxicity of the extracellular products of micro-organisms pathogenic to fish . METHODS AND RESULTS: The optimal conditions for the colorimetric assay were determined and this method was compared with the trypan blue exclusion assay . The protein concentration of extracellular products causing the death of 50% of the cell population (CI50) was determined . CONCLUSIONS: This assay enables quantitative and objective comparison of the cytotoxicity of the extracellular products of micro-organisms pathogenic to fish . It was shown to be more accurate than conventional counting with the trypan blue exclusion assay . SIGNIFICANCE AND IMPACT OF THE STUDY: This method may also be useful for characterizing the cytotoxicity of specific components of extracellular products.

Biochem J, 2001 Nov 15, 360(Pt 1), 1 - 16
Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily; Sheehan D et al.; The glutathione transferases (GSTs; also known as glutathione S-transferases) are major phase II detoxification enzymes found mainly in the cytosol . In addition to their role in catalysing the conjugation of electrophilic substrates to glutathione (GSH), these enzymes also carry out a range of other functions . They have peroxidase and isomerase activities, they can inhibit the Jun N-terminal kinase (thus protecting cells against H(2)O(2)-induced cell death), and they are able to bind non-catalytically a wide range of endogenous and exogenous ligands . Cytosolic GSTs of mammals have been particularly well characterized, and were originally classified into Alpha, Mu, Pi and Theta classes on the basis of a combination of criteria such as substrate/inhibitor specificity, primary and tertiary structure similarities and immunological identity . Non-mammalian GSTs have been much less well characterized, but have provided a disproportionately large number of three-dimensional structures, thus extending our structure-function knowledge of the superfamily as a whole . Moreover, several novel classes identified in non-mammalian species have been subsequently identified in mammals, sometimes carrying out functions not previously associated with GSTs . These studies have revealed that the GSTs comprise a widespread and highly versatile superfamily which show similarities to non-GST stress-related proteins . Independent classification systems have arisen for groups of organisms such as plants and insects . This review surveys the classification of GSTs in non-mammalian sources, such as bacteria, fungi, plants, insects and helminths, and attempts to relate them to the more mainstream classification system for mammalian enzymes . The implications of this classification with regard to the evolution of GSTs are discussed.

J Bioenerg Biomembr, 2001 Jun, 33(3), 213 - 22
The origin of cluster N2 of the energy-transducing NADH-quinone oxidoreductase: comparisons of phylogenetically related enzymes; Yano T et al.; NADH-quinone (Q) oxidoreductase is a large and complex redox proton pump, which utilizes the free energy derived from oxidation of NADH with lipophilic electron/proton carrier Q to translocate protons across the membrane to generate an electrochemical proton gradient . Although its molecular mechanism is largely unknown, recent biochemical, biophysical, and molecular biological studies have revealed that particular subunits and cofactors play an essential role in the energy-coupling reaction . Based on these latest experimental data, we exhaustively analyzed the sequence information available from evolutionarily related enzymes such as {NiFe} hydrogenases . We found significant and conserved sequence differences in the PSST/Nqo6/NuoB, 49kDa/Nqo4/NuoD, and ND1/Nqo8/NuoH subunit homologs between complex I/NDH-1 and {NiFe} hydrogenases . The alterations, especially in the postulated ligand motif for cluster N2 in the PSST/Nqo6/NuoB subunits, appear to be evolutionarily important in determining the physiological function of complex I/NDH-1 . These observations led us to propose a hypothetical evolutionary scheme: during the course of evolution, drastic changes have occurred in the putative cluster N2 binding site in the PSST/Nqo6/NuoB subunit and the progenitors of complex I/NDH-1 have concurrently become to utilize a lipophilic electron/proton carrier such as Q as its physiological substrate . This scheme provides new insights into the structure and function relationship of complex I/NDH-1 and may help us understand its energy-coupling mechanism.

J Parasitol, 2001 Oct, 87(5), 1064 - 70
Lactoferrin-binding proteins of Tritrichomonas foetus; Grab DJ et al.; Tritrichomonas foetus is a common, sexually transmitted, protozoan parasite of cattle . It has an essential requirement for iron, which it obtains from host lactoferrin . However, specific lactoferrin-binding protein receptors have not yet been identified in T . foetus . To differentiate specific and nonspecific binding of lactoferrin, lactoferrin affinity chromatography and Western blotting was used to identify metabolically or surface-labeled T . foetus lactoferrin-binding proteins . Bovine lactoferrin was shown to bind more efficiently than human lactoferrin, and each of these bound much better than bovine transferrin . This is relevant because T . foetus is both species-specific and only infects the mucosal surface of the reproductive tract, which has little transferrin . Whereas the majority of lactoferrin binding was specific, competitive inhibition studies showed that nonspecific, charge-related binding of lactoferrin to T . foetus may also be involved . In the presence of bovine cervical mucus, binding of lactoferrin to T . foetus was diminished, suggesting that mucus has an effect on lactoferrin binding . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of surface biotinylated proteins affinity-purified on lactoferrin-Sepharose showed biotinylated bands at Mr values of 22, 49, 55, 72, and 155 kDa . Because lactoferrin-binding proteins may be susceptible to digestion by T . foetus extracellular cysteine proteinases, it is suspected that the 155-kDa protein is the specific lactoferrin-binding protein and that the lower-Mr lactoferrin-binding molecules may be fragmentation products that contain the lactoferrin-binding site; however, other interpretations are clearly feasible . It is possible that there may be multiple proteins or multimers of the same protein . In summary, the data showed that binding of lactoferrin to T . foetus may be regulated by an interplay of specific receptor interactions as well as by hydrophobic and charge-related interactions.

Recenti Prog Med, 2001 Oct, 92(10), 573 - 7
{Helicobacter pylori and gastric carcinogenesis}; Testino G et al.; Gastric carcinogenesis is a multistep and multifactorial process beginning with chronic gastritis Helicobacter pylori (Hp) induced in most cases . There are some obstacles to an exclusive acceptance of the idea that the relation of Hp with the preneoplastic/neoplastic changes solely develops by means of the chronic gastritis with its atrophic evolution and achlorydria . Hp may be considered a trigger by means of alteration of cellular synetics without any direct influence on genetic alterations . Under the push of an intense proliferation, the expression of typical antigens of the organ is progressively lost, with acquisition of antigens from other organs . Cellular dedifferentiation displayed, with the progressive increase of immature elements that progress to the more or less total disappearance of differentiated gastric cells or differentiating ones, with the formation of metaplastic/dysplastic clones or even neoplastic ones . The bacteria, together with other environmental factors and individual genetic susceptibility, determine the final risk for the development of gastric cancer . Considering that 1-2% of the Hp positive subjects are estimated to develop gastric cancer and that Hp is considered the cause of 75% of gastric cancer, the eradication of the infection, not only in the initial phases but even in those with preneoplastic changes, involves an advantage for the prevention of gastric cancer . For the prevention among the general population testing Hp-positive subjects for serum antibodies against CagA protein might represent an effective way of identifying patients in whom Hp eradication is advisable also in term of gastric cancer prevention.

Cell Mol Life Sci, 2001 Sep, 58(10), 1475 - 90
Cellulose synthesis: mutational analysis and genomic perspectives using Arabidopsis thaliana; Williamson RE et al.; Cellulose microfibrils containing crystalline beta-1,4-glucan provide the major structural framework in higher-plant cell walls . Genetic analyses of Arabidopsis thaliana now link specific genes to plant cellulose production just as was achieved some years earlier with bacteria . Cellulose-deficient mutants have defects in several members of one family within a complex glycosyltransferase superfamily and in one member of a small family of membrane-bound endo-1,4-beta-glucanases . The mutants also accumulate a readily extractable beta-1,4-glucan that has short chains which, in at least one case, are lipid linked . Cellulose could be made by direct extension of the glucan chain by the glycosyltransferase or, as the mutant suggests, by an indirect route which makes lipid-linked oligosaccharides . Models discussed incorporate the known enzymes and lipo-glucan and raise the possibility that different CesA glycosyltransferases may catalyse different steps.

J Photochem Photobiol B, 2001 Sep 1, 62(1-2), 35 - 42
The role of solar UV radiation in the ecology of alpine lakes; Sommaruga R; Solar ultraviolet radiation (UVR, 290-400 nm) is a crucial environmental factor in alpine lakes because of the natural increase of the UVR flux with elevation and the high water transparency of these ecosystems . The ecological importance of UVR, however, has only recently been recognized . This review, examines the general features of alpine lakes regarding UVR, summarizes what is known about the role of solar UVR in the ecology of alpine lakes, and identifies future research directions . Unlike the pattern observed in most lowland lakes, variability of UV attenuation in alpine lakes is poorly explained by differences in dissolved organic carbon (DOC) concentrations, and depends mainly on optical characteristics (absorption) of the chromophoric dissolved organic matter (CDOM) . Within the water column of lakes with low DOC concentrations (0.2-0.4 mg l(-1)), UV attenuation is influenced by phytoplankton whose development at depth (i.e . the deep chlorophyll maximum) causes important changes in UV attenuation . Alpine aquatic organisms have developed a number of strategies to minimize UV damage . The widespread synthesis or bioaccumulation of different compounds that directly or indirectly absorb UV energy is one such strategy . Although most benthic and planktonic primary producers and crustacean zooplankton are well adapted to high intensities of solar radiation, heterotrophic protists, bacteria, and viruses seem to be particularly sensitive to UVR . Understanding the overall impact of UVR on alpine lakes would need to consider synergistic and antagonistic processes resulting from the pronounced climatic warming, which have the potential to modify the UV underwater climate and consequently the stress on aquatic organisms.

Ir Med J, 2001 Jul-Aug, 94(7), 203 - 4
Chronic inflammatory bowel disease and the 'over-clean' environment: rarity in the Irish 'traveller' community; McCormick P et al.; The causes of ulcerative colitis and Crohn's disease are unknown . Current research is focused on genetic factors but environmental factors may also be important . Exposure to infections in early life is believed to reduce the incidence of bronchial asthma . We hypothesised that exposure to enteric bacteria and infections in early life may also reduce the incidence of idiopathic inflammatory bowel disease . Chronic inflammatory bowel disease may be a by-product of our over-clean environment . Social groups with poorer living standards might be expected to have a lower prevalence of this condition . The "traveller" or itinerant community in Ireland is one such group . We therefore attempted to estimate the prevalence of inflammatory bowel disease in the traveller population . Twenty five of 30 gastroenterologists or surgeons in Ireland responded to requests for information . None could recall ever seeing a "traveller" with idiopathic inflammatory bowel disease . While genetic factors cannot be excluded, a low prevalence of idiopathic inflammatory bowel disease would support the environmental hypothesis.

Lik Sprava, 2001 Jul-Aug, (4), 176 - 7
{Particular features of the relationship of Helicobacter pylori and G cells in patients with duodenal ulcer, with special reference to the Helicobacter pylori strain}; Gaidar IuA et al.; Helicobacter pylori positive form of duodenal ulcer (CagA+ and CagA-) was studied in 120 patients with duodenal ulcer (DU) . CagA+ was found in 78 patients (65%); CagA(-)--in 42 (35%) . Diagnostic criteria of symbiosis between Helicobacter pylori and macroorganisms have been established . They describe normal mucous structure, absence of CagA+ and G-cell hyperplasia . It can be important for diagnosis and choice of a suitable mode of treatment to be employed in DU patients.

Mikrobiol Z, 2001 Jul-Aug, 63(4), 20 - 6
{Plesiomonas in sea medium and some hydrobionts}; Puchenkova SG; Seading of some hydrobionts and sea medium objects by plesiomonads has been investigated in different regions; dynamics of the number of these bacteria in the sea water and mussels of the Kerch strait has been traced . More frequent occurrence of plesiomonads in molluscs than in fish as well as in the water medium of the region of mussels cultivation as compared with the neighbouring water areas has been registered . A broad set of tests was used to study biological properties of 63 strains of isolated plesiomonads.

Science, 2001 Nov 2, 294(5544), 1102 - 5
Mammalian TOR: a homeostatic ATP sensor; Dennis PB et al.; The bacterial macrolide rapamycin is an efficacious anticancer agent against solid tumors . In a hypoxic environment, the increase in mass of solid tumors is dependent on the recruitment of mitogens and nutrients . When nutrient concentrations change, particularly those of essential amino acids, the mammalian Target of Rapamycin (mTOR) functions in regulatory pathways that control ribosome biogenesis and cell growth . In bacteria, ribosome biogenesis is independently regulated by amino acids and adenosine triphosphate (ATP) . Here we demonstrate that the mTOR pathway is influenced by the intracellular concentration of ATP, independent of the abundance of amino acids, and that mTOR itself is an ATP sensor.

Hum Mol Genet, 2001 Oct 1, 10(21), 2463 - 8
The phylogenetic distribution of frataxin indicates a role in iron-sulfur cluster protein assembly; Huynen MA et al.; Much has been learned about the cellular pathology of Friedreich's ataxia, a recessive neurodegenerative disease resulting from insufficient expression of the mitochondrial protein frataxin . However, the biochemical function of frataxin has remained obscure, hampering attempts at therapeutic intervention . To predict functional interactions of frataxin with other proteins we investigated whether its gene specifically co-occurs with any other genes in sequenced genomes . In 56 available genomes we identified two genes with identical phylogenetic distributions to the frataxin/cyaY gene: hscA and hscB/JAC1 . These genes have not only emerged in the same evolutionary lineage as the frataxin gene, they have also been lost at least twice with it, and they have been horizontally transferred with it in the evolution of the mitochondria . The proteins encoded by hscA and hscB, the chaperone HSP66 and the co-chaperone HSP20, have been shown to be required for the synthesis of 2Fe-2S clusters on ferredoxin in proteobacteria . JAC1, an ortholog of hscB, and SSQ1, a paralog of hscA, have been shown to be required for iron-sulfur cluster assembly in mitochondria of Saccharomyces cerevisiae . Combining data on the co-occurrence of genes in genomes with experimental and predicted cellular localization data of their proteins supports the hypothesis that frataxin is directly involved in iron-sulfur cluster protein assembly . They indicate that frataxin is specifically involved in the same sub-process as HSP20/Jac1p.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2001, 36(9), 1757 - 66
Enhanced phosphorus removal by glucose fed sequencing batch reactor; Akin BS et al.; In this study an anaerobic/aerobic sequencing batch reactor was used for an enhanced biological phosphorus removal . These conditions were tested by a laboratory scale reactor (14 liter) with a synthetic feed, glucose being the sole carbon source . However, enhanced biological phosphorus removal (EBPR) was not achieved during this study . A series of batch tests were conducted with various substrates; acetic acid, glucose and the mixture of acetic acid and glucose . High phosphate removal was obtained when acetate was the substrate (68.7%) . It was observed that when the acetic acid was used as a sole substrate, the bacteria growing in the alternating anaerobic/aerobic system removed the organic substrate under anaerobic conditions . However, in case of the glucose feed most of the COD removal took place under not truly anaerobic conditions . This also coincides with low phosphate removal (37.8%) . These results are in consistent with the results of the continuous operation . When the acetic acid-glucose mixture was used, the phosphate removal decreased down to 60.8% . This was thought to be due to the glucose that reduced the dependency on poly-P as an energy source.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2001, 36(9), 1747 - 56
Preserved granular sludge for inoculation of new UASB reactors; Xu HL et al.; In this study, granular sludges collected from a mesophilic laboratory-scale UASB reactor were stored for 6 months at 4 degrees C, 24 degrees C (room temperature) and 35 degrees C, respectively . The preservation characteristics of granular sludges (including soluble COD, granular particle size and granular surface morphology) in terms of storage age and temperature were investigated . It was found that 4 degrees C stored UASB granules were more capable of maintaining their granular structures over those stored at 24 degrees C and 35 degrees C . Granulation in UASB reactors seeded with preserved sludge granules reached its post-maturation stage about 40 days ahead of the control, mainly by means of eliminating the embryonic granule formation stage from the granulation process . The results in this study indicated that the preserved granular sludge could be used as an effective inoculum to accelerate new UASB reactor start-up.

Proc Natl Acad Sci U S A, 2001 Nov 6, 98(23), 13408 - 13 Epub 2001 Oct 30.
A paradox resolved: sulfide acquisition by roots of seep tubeworms sustains net chemoautotrophy; Freytag JK et al.; Vestimentiferan tubeworms, symbiotic with sulfur-oxidizing chemoautotrophic bacteria, dominate many cold-seep sites in the Gulf of Mexico . The most abundant vestimentiferan species at these sites, Lamellibrachia cf . luymesi, grows quite slowly to lengths exceeding 2 meters and lives in excess of 170-250 years . L . cf . luymesi can grow a posterior extension of its tube and tissue, termed a "root," down into sulfidic sediments below its point of original attachment . This extension can be longer than the anterior portion of the animal . Here we show, using methods optimized for detection of hydrogen sulfide down to 0.1 microM in seawater, that hydrogen sulfide was never detected around the plumes of large cold-seep vestimentiferans and rarely detectable only around the bases of mature aggregations . Respiration experiments, which exposed the root portions of L . cf . luymesi to sulfide concentrations between 51-561 microM, demonstrate that L . cf . luymesi use their roots as a respiratory surface to acquire sulfide at an average rate of 4.1 micromol x g(-1) x h(-1) . Net dissolved inorganic carbon uptake across the plume of the tubeworms was shown to occur in response to exposure of the posterior (root) portion of the worms to sulfide, demonstrating that sulfide acquisition by roots of the seep vestimentiferan L . cf . luymesi can be sufficient to fuel net autotrophic total dissolved inorganic carbon uptake.

Proc Natl Acad Sci U S A, 2001 Nov 6, 98(23), 13207 - 12 Epub 2001 Oct 30.
Retrotransposition of a yeast group II intron occurs by reverse splicing directly into ectopic DNA sites; Dickson L et al.; Group II introns, the presumed ancestors of nuclear pre-mRNA introns, are site-specific retroelements . In addition to "homing" to unoccupied sites in intronless alleles, group II introns transpose at low frequency to ectopic sites that resemble the normal homing site . Two general mechanisms have been proposed for group II intron transposition, one involving reverse splicing of the intron RNA directly into an ectopic DNA site, and the other involving reverse splicing into a site in RNA followed by reverse transcription and integration of the resulting cDNA by homologous recombination . Here, by using an "inverted-site" strategy, we show that the yeast mtDNA group II intron aI1 retrotransposes by reverse splicing directly into an ectopic DNA site . This same mechanism could account for other previously described ectopic transposition events in fungi and bacteria and may have contributed to the dispersal of group II introns into different genes.

Biochim Biophys Acta, 2001 Oct 30, 1507(1-3), 291 - 310
Daddy, where did (PS)I come from?
Baymann F, Brugna M, Muhlenhoff U, Nitschke W.
The reacton centre I (RCI)-type photosystems from plants, cyano-, helio- and green sulphur bacteria are compared and the essential properties of an archetypal RCI are deduced . Species containing RCI-type photosystems most probably cluster together on a common branch of the phylogenetic tree . The predicted branching order is green sulphur, helio- and cyanobacteria . Striking similarities between RCI- and RCII-type photosystems recently became apparent in the three-dimensional structures of photosystem I (PSI), PSII and RCII . The phylogenetic relationship between all presently known photosystems is analysed suggesting (a) RCI as the ancestral photosystem and (b) the descendence of PSII from RCI via gene duplication and gene splitting . An evolutionary model trying to rationalise available data is presented.

Biochim Biophys Acta, 2001 Oct 30, 1507(1-3), 278 - 90
The antenna reaction center complex of heliobacteria: composition, energy conversion and electron transfer; Neerken S et al.; A survey is given of various aspects of the photosynthetic processes in heliobacteria . The review mainly refers to results obtained since 1995, which had not been covered earlier . It first discusses the antenna organization and pigmentation . The pigments of heliobacteria include some unusual species: bacteriochlorophyll (BChl) g, the main pigment, 8(1) hydroxy chlorophyll a, which acts as primary electron acceptor, and 4,4'-diaponeurosporene, a carotenoid with 30 carbon atoms . Energy conversion within the antenna is very fast: at room temperature thermal equilibrium among the approx . 35 BChls g of the antenna is largely completed within a few ps . This is then followed by primary charge separation, involving a dimer of BChl g (P798) as donor, but recent evidence indicates that excitation of the acceptor pigment 8(1) hydroxy chlorophyll a gives rise to an alternative primary reaction not involving excited P798 . The final section of the review concerns secondary electron transfer, an area that is relatively poorly known in heliobacteria.

Biochim Biophys Acta, 2001 Oct 30, 1507(1-3), 212 - 25
Light-induced spin polarization in type I photosynthetic reaction centres; van der Est A; The use of light-induced spin polarization to study the structure and function of type I reaction centres is reviewed . The absorption of light by these systems generates a series of sequential radical pairs, which exhibit spin polarization as a result of the correlation of the unpaired electron spins . A description of how the polarization patterns can be used to deduce the relative orientation of the radicals is given and the most important structural results from such studies on photosystem I (PS I) are summarized . Quinone exchange experiments which demonstrate the influence of protein-cofactor interactions on the polarization patterns are discussed . The results show that there are significant differences between the binding sites of the primary quinone acceptors in PS I and purple bacterial reaction centres and suggest that pi-pi interactions probably play a more important role in PS I . Studies using spin-polarized EPR transients and spectra to investigate the electron transfer pathway and kinetics are also reviewed . The results from PS I, green-sulphur bacteria and Heliobacteria are compared and the controversy surrounding the role of a quinone in the electron transfer in the latter two systems is discussed.

Biochim Biophys Acta, 2001 Oct 30, 1507(1-3), 180 - 93
Fourier transform infrared spectroscopy of primary electron donors in type I photosynthetic reaction centers; Breton J; The vibrational properties of the primary electron donors (P) of type I photosynthetic reaction centers, as investigated by Fourier transform infrared (FTIR) difference spectroscopy in the last 15 years, are briefly reviewed . The results obtained on the microenvironment of the chlorophyll molecules in P700 of photosystem I and of the bacteriochlorophyll molecules in P840 of the green bacteria (Chlorobium) and in P798 of heliobacteria are presented and discussed with special attention to the bonding interactions with the protein of the carbonyl groups and of the central Mg atom of the pigments . The observation of broad electronic transitions in the mid-IR for the cationic state of all the primary donors investigated provides evidence for charge repartition over two (B)Chl molecules . In the green sulfur bacteria and the heliobacteria, the assignments proposed for the carbonyl groups of P and P(+) are still very tentative . In contrast, the axial ligands of P700 in photosystem I have been identified and the vibrational properties of the chlorophyll (Chl) molecules involved in P700, P700(+), and (3)P700 are well described in terms of two molecules, denoted P(1) and P(2), with very different hydrogen bonding patterns . While P(1) has hydrogen bonds to both the 9-keto and the 10a-ester C=O groups and bears all the triplet character in (3)P700, the carbonyl groups of P(2) are free from hydrogen bonding . The positive charge in P700(+) is shared between the two Chl molecules with a ratio ranging from 1:1 to 2:1, in favor of P(2), depending on the temperature and the species . The localization of the triplet in (3)P700 and of the unpaired electron in P700(+) deduced from FTIR spectroscopy is in sharp contrast with that resulting from the analysis of the magnetic resonance experiments . However, the FTIR results are in excellent agreement with the most recent structural model derived from X-ray crystallography of photosystem I at 2.5 A resolution that reveals the hydrogen bonds to the carbonyl groups of the Chl in P700 as well as the histidine ligands of the central Mg atoms predicted from the FTIR data.

Biochim Biophys Acta, 2001 Oct 30, 1507(1-3), 139 - 60
Iron-sulfur clusters in type I reaction centers; Vassiliev IR et al.; Type I reaction centers (RCs) are multisubunit chlorophyll-protein complexes that function in photosynthetic organisms to convert photons to Gibbs free energy . The unique feature of Type I RCs is the presence of iron-sulfur clusters as electron transfer cofactors . Photosystem I (PS I) of oxygenic phototrophs is the best-studied Type I RC . It is comprised of an interpolypeptide {4Fe-4S} cluster, F(X), that bridges the PsaA and PsaB subunits, and two terminal {4Fe-4S} clusters, F(A) and F(B), that are bound to the PsaC subunit . In this review, we provide an update on the structure and function of the bound iron-sulfur clusters in Type I RCs . The first new development in this area is the identification of F(A) as the cluster proximal to F(X) and the resolution of the electron transfer sequence as F(X)-->F(A)-->F(B)-->soluble ferredoxin . The second new development is the determination of the three-dimensional NMR solution structure of unbound PsaC and localization of the equal- and mixed-valence pairs in F(A)(-) and F(B)(-) . We provide a survey of the EPR properties and spectra of the iron-sulfur clusters in Type I RCs of cyanobacteria, green sulfur bacteria, and heliobacteria, and we summarize new information about the kinetics of back-reactions involving the iron-sulfur clusters.

Cochrane Database Syst Rev . 2001;(4):CD003325.
Removal of nail polish and finger rings to prevent surgical infection; Arrowsmith VA et al.; BACKGROUND: Surgical wound infection may be caused by transfer of bacteria from the hands of the surgical team during operative procedures . Careful surgical scrubbing is therefore performed to reduce the number of bacteria on the skin . The wearing of finger rings and nail polish is thought to reduce the efficacy of the scrub as they are thought to harbour bacteria in microscopic imperfections of nail polish and on the skin beneath finger rings . OBJECTIVES: To assess the effect of removal of finger rings and nail polish by the surgical scrub team, on postoperative wound infection rates . SEARCH STRATEGY: We searched the Cochrane Wounds Group Specialised Trials Register up to November 2000 using the search strategy developed by the Cochrane Wounds Group . We wrote to manufacturers of surgical scrubbing agents for ongoing and unpublished research . Reference lists of articles were searched and relevant journals outside the electronic databases were hand searched . No restriction was placed on literature based on date of publication, language or publication status . SELECTION CRITERIA: Randomised controlled trials evaluating the effect of wearing or removal of finger rings and nail polish by the surgical scrub team on post operative wound infections and number of bacteria on the hands of the surgical scrub team . DATA COLLECTION AND ANALYSIS: The abstracts of studies identified were scanned by all reviewers . All abstracts were checked against a checklist to determine whether they fulfilled the inclusion criteria . Full reports of relevant studies were obtained and checked against the checklist by two reviewers . The full reports of all excluded trials were checked by all reviewers independently to ensure appropriate exclusion . MAIN RESULTS: We found no randomised controlled trials that compared the wearing of finger rings with the removal of finger rings . We found no trials of nail polish wearing / removal that measured patient outcomes, including surgical infection . We found one small randomised controlled trial which evaluated the effect of nail polish on the number of bacterial colony forming units on the hands after pre-operative hand washing (also called surgical scrubbing) . Nurses were allocated to: unpolished nails, freshly applied nail polish (less than two days old), or old nail polish (more than four days old) . Both before and after surgical scrubbing, there was no significant difference in the number of bacteria on the hands . REVIEWER'S CONCLUSIONS: There is no evidence of the effect of removing nail polish or finger rings on the rate of surgical wound infection . There is insufficient evidence of the effect of wearing nail polish on the number of bacteria on the skin . However, the one trial making this comparison trial was too small to exclude anything other than a very large difference in the number of bacteria on the skin.

Environ Int, 2001 Oct, 27(4), 265 - 74
Exploring correlation between redox potential and other edaphic factors in field and laboratory conditions in relation to methane efflux; Singh SN; Methane is primarily a biogenic gas, which is implicated in global warming . Although its production in the anoxic conditions is regulated by several edaphic factors, aquatic macrophytes also influence methane emission by providing aerenchyma to act as chimney for CH4 transport from the sediment to troposphere, by releasing root exudates to the sediment to serve as substrate for methanogenic bacteria and by transporting atmospheric O2 to rhizosphere, which stimulates CH4 consumption . Among the edaphic factors, redox potential (Eh) is the most important, which largely determines the action of methanogenic bacteria . Hence, a study was undertaken first to find out the correlation between CH4 emission and edaphic factors in the field conditions and then to understand the relationship between Eh and other edaphic factors . The field studies revealed that natural wetlands were the major source of CH4 emission, and the vegetation plays an important role in CH4 emission from the water bodies . However, it was very difficult to establish a strong relationship between the CH4 emission and the edaphic factors in the field conditions due to other limiting factors and their constant fluctuations . In this connection, the laboratory experiments exhibited that soil temperature, pH, moisture regime and incubation period were negatively correlated with Eh, which determines the initiation of methanogenic process . However, organic carbon and the water regime over the soil surface did not show any impact on Eh in this study.

Biomed Pharmacother, 2001 Oct, 55(8), 454 - 7
Metabolism of 10-formyldihydrofolate in humans; Baggott JE et al.; The metabolism of 10-formyldihydrofolate is reviewed in this article . It had been the dogma that only tetrahydrofolates participate in enzyme-catalyzed one-carbon transfer reactions, until we showed in 1986 that 10-formyldihydrofolate serves as a substrate for aminoimidazolecarboxamide ribotide (AICAR) transformylase . Our data from studies in humans, cultured cells and bacteria as well as in vitro experiments indicate that the oxidation of 10-formyltetrahydrofolate to 10-formyldihydrofolate takes place, and 1 0-formyldihydrofolate is subsequently converted to dihydrofolate by AICAR transformylase . Dihydrofolate is then reduced to tetrahydrofolate and further metabolized by the well-established enzyme reactions . We believe that a new folate metabolic map is needed which incorporates the oxidation of 10-formyltetrahydrofolate and the utilization of 10-formyldihydrofolate by AICAR transformylase.

Surg Today, 2001, 31(9), 850 - 2
Minilaparotomy wound edge protector (Lap-Protector): a new device; Nakagoe T et al.; Laparoscopic-assisted or minimally invasive surgery involving minilaparotomy is occasionally complicated by infection of the minilaparotomy wound caused by intestinal bacteria . Furthermore, when this procedure is performed to excise colorectal or gastric cancer, tumor recurrence may develop in the minilaparotomy wound . In an attempt to minimize the risk of these complications, we developed a new, easy-to-use device which we named the "Lap-Protector." Minilaparotomy was performed using the Lap-Protector in 28 patients with colon cancer and eight patients with early gastric cancer who underwent minimally invasive surgery between January and September, 1999 . During a median follow-up period of 15.9 (range 12.4-21.0) months, none of the 36 patients showed any sign of wound infection or tumor recurrence in the minilaparotomy wound . These results indicated that the Lap-Protector is a safe and useful device that may help to prevent infections and cancer cell contamination of the minilaparotomy wound.

Eur J Pediatr, 2001 Oct, 160(10), 583 - 91
Flow cytometric immunophenotyping in the diagnosis and follow-up of immunodeficient children; de Vries E et al.; From time to time, paediatricians are confronted with children who might suffer from a primary immunodeficiency disease . For practical purposes, these children can be divided into four main clinical categories: (1) a relatively large group of children with recurrent ear-nose and throat and lower respiratory tract infections, in some cases caused by deficiencies of antibodies or complement; (2) children with failure to thrive, intractable diarrhoea or an opportunistic infection which can be caused by a T-lymphocyte or combined immunodeficiency; (3) children with infections with pyogenic bacteria or fungi as seen in case of granulocyte/monocyte function deficiency; and (4) a small heterogeneous group of children with recurrence of particular infections . Also, acquired immunodeficiency becomes a more common problem in paediatric practice . Flow cytometric immunophenotyping of leucocytes appears to be an efficient and rapid tool in the diagnosis and follow-up of immunodeficient patients, supporting early recognition, before serious infections have compromised the child's general condition . This technique can now be performed in many hospitals . In this review, we give directions for the use of flow cytometric immunophenotyping of leucocytes in the diagnosis and follow-up of immunodeficient children according to the four main clinical categories.

J Air Waste Manag Assoc, 2001 Oct, 51(10), 1436 - 42
An analytical method for the measurement of nonviable bioaerosols; Menetrez MY et al.; Exposures from indoor environments are a major issue for evaluating total long-term personal exposures to the fine fraction (<2.5 microm in aerodynamic diameter) of particulate matter (PM) . It is widely accepted in the indoor air quality (IAQ) research community that biocontamination is one of the important indoor air pollutants . Major indoor air biocontaminants include mold, bacteria, dust mites, and other antigens . Once the biocontaminants or their metabolites become airborne, IAQ could be significantly deteriorated . The airborne biocontaminants or their metabolites can induce irritational, allergic, infectious, and chemical responses in exposed individuals . Biocontaminants, such as some mold spores or pollen grains, because of their size and mass, settle rapidly within the indoor environment . Over time they may become nonviable and fragmented by the process of desiccation . Desiccated nonviable fragments of organisms are common and can be toxic or allergenic, depending upon the specific organism or organism component . Once these smaller and lighter fragments of biological PM become suspended in air, they have a greater tendency to stay suspended . Although some bioaerosols have been identified, few have been quantitatively studied for their prevalence within the total indoor PM with time, or for their affinity to penetrate indoors . This paper describes a preliminary research effort to develop a methodology for the measurement of nonviable biologically based PM, analyzing for mold and ragweed antigens and endotoxins . The research objectives include the development of a set of analytical methods and the comparison of impactor media and sample size, and the quantification of the relationship between outdoor and indoor levels of bioaerosols . Indoor and outdoor air samples were passed through an Andersen nonviable cascade impactor in which particles from 0.2 to 9.0 microm were collected and analyzed . The presence of mold, ragweed, and endotoxin was found in all eight size ranges . The presence of respirable particles of mold and pollen found in the fine particle size range from 0.2 to 5.25 microm is evidence of fragmentation of larger source particles that are known allergens.

Curr Microbiol, 2001 Dec, 43(6), 400 - 2
Possible involvement of a single histidine residue in the P-type Na+-ATPase of a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum; Hirano A et al.; Effect of various inhibitors on the P-type Na+-ATPase of a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum, was examined . The ATPase was extremely sensitive to p-chloromercuriphenylsulfonic acid, a modifier of SH-group . The enzyme was also sensitive to diethylpyrocarbonate, and analysis of the inhibition kinetics by the drug indicated that modification of a single histidine residue per ATPase molecule was sufficient to inactivate the enzyme.

Jpn J Infect Dis, 2001 Aug, 54(4), 131 - 6
A quantitative in vitro assay for detecting biological activity of endotoxin using rabbit peripheral blood; Ochiai M et al.; The pyrogen test or the endotoxin test has been playing a crucial role in detecting endotoxin in parenteral drugs . The current test methods, however, have disadvantages such as requiring a relatively high number of animals or an inadequacy in direct evaluation of in vivo activity . We made an attempt to establish a new in vitro assay method that can overcome the shortcomings of the current assay methods . We standardized the system of tumor necrosis factor-alpha (TNF-alpha) induction from the peripheral blood of rabbits for assaying endotoxin activity . This in vitro assay showed a linear dose-response regression between 0.1 and 5.0 endotoxin units per milliliter of endotoxin and a definite homogeneity of variance by logarithmically transforming the endotoxin and TNF-alpha concentrations in the reaction mixtures at 5 h of incubation at 37 degrees C . The assay showed a definite correlation with the pyrogen test but not with the endotoxin test when endotoxins from various bacteria were tested.

Mol Cell, 2001 Oct, 8(4), 873 - 83
Self-reinforcing activation of a cell-specific transcription factor by proteolysis of an anti-sigma factor in B . subtilis; Pan Q et al.; The transcription factor sigma(F), which is activated in a cell-specific manner during sporulation in B . subtilis, is initially held in an inactive complex by the anti-sigma factor SpoIIAB . The anti-anti-sigma factor SpoIIAA reacts with SpoIIAB.sigma(F) to induce the release of free sigma(F) and free SpoIIAB . We now report that free SpoIIAB is subject to proteolysis and that it is protected from degradation by sigma(F) in the SpoIIAB.sigma(F) complex and by SpoIIAA in an alternative complex . Proteolysis requires residues located near the extreme C terminus of SpoIIAB and is dependent upon the ClpCP protease . The reaction of SpoIIAA with SpoIIAB.sigma(F) and the resulting degradation of newly released SpoIIAB could set up a self-reinforcing cycle that locks on the activation of sigma(F).

Helicobacter, 2001 Sep, 6(3), 244 - 8
Treatment of Helicobacter pylori in children with recurrent abdominal pain; Wewer V et al.; BACKGROUND: The role of Helicobacter pylori remains unclear in children with recurrent abdominal pain (RAP) . In this study children with RAP were included in a double blind treatment study to elucidate whether symptoms disappear in children with a H . pylori infection and RAP, if the bacteria are eradicated . METHODS: Thirty-seven H . pylori-infected children aged 4.9-14.5 years (median 9.8 years) with RAP were included . H . pylori was identified by histology and culture . The children were treated with amoxicillin and metronidazole for 14 days . A re-endoscopy including biopsies for histology and culture was done at least one month after the end of treatment . Simple questions for symptoms were asked and blood for serology was repeated 3 and 6 months after the end of treatment . During the observation period the results of the re-endoscopy and the serology 3 and 6 months after the re-endoscopy were blinded for 23 patients and opened to 14 of the patients according to the choice of the families . RESULTS: The eradication rates were 81% (30/37) in the total group and 74% (17/23) in the blinded group . The IgG antibodies to H . pylori decreased significantly 3 (p =.03) as well as 6 months after end of treatment (p <.001) in children with successful eradication . The number of children with RAP decreased after examination and treatment and the well-being improved after 6 months in almost 95% of the children . However, no correlation was seen between eradication of H . pylori and disappearance of RAP, neither after 3 nor after 6 months' observation in the total group of patients (p =.94 and p =.90) or in the blinded group (p =.42 and p =.65) . CONCLUSIONS: These results do not provide evidence for a causal relationship between RAP and H . pylori.

Environ Microbiol, 2001 Sep, 3(9), 600 - 3
Use of stable isotopes to identify benzoate as a metabolite of benzene degradation in a sulphidogenic consortium; Phelps CD et al.; An enriched sulphidogenic consortium capable of mineralizing benzene was used to study the metabolic pathway of anaerobic benzene degradation . Benzoate was detected in active cultures and benzene was confirmed to be the source of this benzoate by the addition of deuterated benzene (D6) and subsequent detection of deuterated benzoate (D5) in active cultures but not in autoclaved controls . Benzoate was utilized by this culture at 1/12 the rate of benzene, while its presence did not inhibit benzene utilization . The benzene utilization rate was reduced, however, in the presence of 2-fluorobenzoate . When the culture was supplemented with {(13)C}-bicarbonate, the carboxyl group on benzoate was not labelled with {(13)C}-carbon, suggesting that this transformation relies on a more complex set of reactions than simple addition of carbonate.

Aliment Pharmacol Ther, 2001 Nov, 15(11), 1763 - 7
Citric acid-enhanced Helicobacter pylori urease activity in vivo is unrelated to gastric emptying; Shiotani A et al.; BACKGROUND: In a previous study, the use of a citric acid test meal produced a rapid dose-dependent increase in urease activity that was significantly greater than that resulting from a pudding meal, ascorbic acid or sodium citrate . The mechanism was hypothesized to be related to the ability of citric acid to delay gastric emptying and possibly to enhance intragastric distribution of the urea . OBJECTIVE: To compare the effects of sodium citrate, two doses of citric acid and a pudding meal on gastric motor function . METHOD: Eleven normal healthy volunteers were investigated using non-invasive techniques to measure gastric emptying and gastric motility . We evaluated gastric emptying using the Meretek 13Ceebiscuit solid phase gastric emptying breath test, which employs a 340-calorie biscuit containing 200 mg of the edible 13C-blue-green alga Spirulina platensis, after the administration of test meals of pudding, 2 g and 4 g of citric acid and 2 g of sodium citrate . Electrogastrograms (Digitrapper EGG) were also recorded for 30 min before and 180 min after the test meal . RESULTS: Gastric emptying, as assessed by the half-time (T1/2), was delayed similarly with the pudding (136.8 +/- 9 min) and with 4 g of citric acid (144.5 +/- 7 min) (P > 0.7) . Sodium citrate (108.7 +/- 6 min) and 2 g of citric acid (110.1 +/- 6 min) had similar effects on gastric emptying (P=0.986), and were significantly less effective in delaying gastric emptying (P < 0.01) compared to pudding or 4 g of citric acid . The electrogastrograms remained normal and there were no differences among meals and no relation with the gastric emptying results . CONCLUSIONS: The increased intragastric urea hydrolysis associated with citric acid test meals cannot be attributed to delayed gastric emptying . Changes in the intragastric distribution of urea or a direct effect of citric acid on the bacteria (e.g . via the cytoplasmic protein, UreI) are more likely to be responsible.

Aliment Pharmacol Ther, 2001 Nov, 15(11), 1709 - 16
Review article: the immunoregulatory cytokine interleukin-10--a therapy for Crohn's disease?
Lindsay JO, Hodgson HJ.
The gastrointestinal tract serves as a barrier between the host and the vast array of foreign antigens that are contained within its lumen . The mucosal immune system must balance two opposing functions: to mount an immune response to pathogens, whilst maintaining tolerance to antigens derived from commensal bacteria and food . This balance is regulated by both cellular interactions and the release of soluble mediators called cytokines . Diseases such as ulcerative colitis and Crohn's disease are characterized by alterations in the balance of pro-inflammatory and regulatory cytokines . Interleukin-10 is a regulatory cytokine which inhibits both antigen presentation and subsequent pro-inflammatory cytokine release . In addition, there is evidence that it promotes the formation of antigen-specific regulatory T-cell clones . The pivotal role played by interleukin-10 within the mucosal immune system is demonstrated both by the chronic ileocolitis that develops in gene-targeted interleukin-10 knock-out mice, and by its therapeutic efficacy in several animal models of colitis . However, trials of daily systemic interleukin-10 administration in patients with Crohn's disease have reported only a modest clinical response . Advances in the analysis of functional polymorphisms in the interleukin-10 gene may allow therapy to be targeted to patients who will respond . Finally, therapeutic strategies utilizing gene therapy may enhance mucosal delivery and increase therapeutic response.

Biochemistry, 2001 Nov 6, 40(44), 13378 - 89
SmtB-DNA and protein-protein interactions in the formation of the cyanobacterial metallothionein repression complex: Zn2+ does not dissociate the protein-DNA complex in vitro; Kar SR et al.; The synechococcal metallothionein locus smt consists of two divergent genes: smtA coding for the metallothionein SmtA, and smtB coding for the trans-acting regulator SmtB . The latter binds at two inverted repeats, designated S1/S2 and S3/S4, in the overlapping promoter/operator sites between the two genes . We have determined the binding stoichiometries to the entire operator/promoter DNA and to the separate S1/S2 and S3/S4 half-operator oligonucleotides using sedimentation equilibrium and sedimentation velocity measurements . The full promoter/operator DNA binds two SmtB dimers . The hydrodynamic behavior of this complex supports a compact nucleoprotein structure . Each separate S1/S2 and S3/S4 operator sequence also binds two dimers . An equal molar mixture of separate S1/S2 and S3/S4 operator sequences, in excess SmtB, forms a S1/S2-SmtB:SmtB-S3/S4 bridge complex . Combining these results with previously published binding interference data, which showed consecutive S1/S2 and S3/S4 SmtB occupancy on the operator/promoter DNA, we have developed a model for the establishment of the repression complex that appears to involve significant DNA compaction, presumably DNA bending, stabilized by SmtB-SmtB bridge interactions . DNase I footprinting titrations also showed consecutive S1/S2 and S3/S4 SmtB occupancy . The footprints expand considerably in the presence of Zn2+ . Hence, SmtB remains bound to the operator sites when Zn2+ ions are present . This result is further supported by gel retardation assay . Failure of the metal ions to dissociate SmtB from the DNA points to a hitherto unknown function of SmtB in the regulation of the smt locus.

Biochemistry, 2001 Nov 6, 40(44), 13262 - 7
Catalytic coupling of the active sites in acetyl-CoA synthase, a bifunctional CO-channeling enzyme; Maynard EL et al.; Acetogenic bacteria contain acetyl-CoA synthase (ACS), an enzyme with two distinct nickel-iron-sulfur active sites connected by a tunnel through which CO migrates . One site reduces CO2 to CO, while the other synthesizes acetyl-CoA from CO, CoA, and the methyl group of another protein (CH3-CP) . Rapid binding of CO2 and a two-electron reduction activates ACS . When CoA and CH3-CP bind ACS, CO is rerouted through the tunnel to the synthase site, and kinetic parameters at the reductase site are altered . Under these conditions, the rates of CO2 reduction and acetyl-CoA synthesis are synchronized by an ordered catalytic mechanism.

Differentiation, 2001 Aug, 68(1), 22 - 30
Identification of novel elements which regulate the cell-type specificity of Dictyostelium 7E gene expression; Seager JH et al.; Previously, we have identified the Dictyostelium 7E gene promoter and shown that it is capable of driving expression in the same temporal and cAMP responsive manner as the endogenous gene during development . Furthermore, we have mapped the corresponding transcriptional regulatory sequences within the promoter . In the present study we used the lacZ reporter gene system to examine the role of 7E promoter elements in regulating cell-type specific expression during Dictyostelium morphogenesis . In situ detection of beta-galactosidase activity revealed that expression was induced within anterior prestalk cells at approximately 18 h of development . Subsequently, we found that promoter activity was independently regulated in subpopulations of prestalk cells . Element(s) upstream of position - 532 were necessary for expression in pstA cells while more proximal elements (located downstream of position - 426) were capable of directing expression in pstO cells . Deletion of a G-rich element ('GGT' box; 5'-GGT GAT GA-3') located between positions - 159 and - 152 resulted both in a loss of expression in pstA cells and aberrant expression in the prespore zone . Furthermore, the spatial organisation of reporter gene expression directed by this construct during culmination delineated a population of cells that have not been previously defined . These data suggest that the 7E gene is independently regulated in subpopulations of prestalk cells during development.

J Cell Sci, 2001 Jul, 114(Pt 14), 2627 - 40
In vivo localisation of fission yeast cyclin-dependent kinase cdc2p and cyclin B cdc13p during mitosis and meiosis; Decottignies A et al.; We investigated the in vivo localisation of fission yeast cyclin-dependent kinase cdc2p during mitosis and meiosis . Fusion to yellow fluorescent protein (YFP) revealed that cdc2-YFP is present in the cytoplasm at all stages of the cell cycle . Nuclear cdc2-YFP fluorescence oscillates with that of cdc13-YFP cyclin . At G1/S, at least one of cdc13p, cig1p or cig2p B-type cyclins is required for the accumulation of cdc2-YFP into the nucleus . Cdc2-YFP and cdc13-YFP are highly enriched on the spindle pole body of cells in late G2 or arrested at S phase . Both accumulate on the spindle pole bodies and the spindle in prophase and metaphase independently of the microtubule-associated protein dis1p . In anaphase, the cdc2p/cdc13p complex leaves the spindle prior to sister chromatid separation, and cdc13-YFP is enriched at the nuclear periphery before fluorescence disappears . If cdc13p cannot be recognized by the anaphase-promoting complex, cdc2-YFP and cdc13-YFP remain associated with the spindle . In mating cells, cdc2-YFP enters the nucleus as soon as the cells undergo fusion . During karyogamy and meiotic prophase, cdc2-YFP is highly enriched on the centromeres . In meiosis I, association of cdc2-YFP with the spindle and the spindle pole bodies shows differences to mitotic cells, suggesting different mechanisms of spindle formation . This study suggests that changes in cdc2p localisation are important for both mitosis and meiosis regulation.

J Clin Microbiol, 2001 Nov, 39(11), 4172 - 4
High prevalence of granulocytic Ehrlichiae and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks from Bulgaria; Christova I et al.; Bulgarian Ixodes ricinus ticks were examined for Ehrlichia and Borrelia coinfection: 34 and 32% of adult ticks and at least 2 and 10% of nymphs were positive for these infections, respectively . Coinfections and dual or triple Borrelia infections were frequent, although Ehrlichia phagocytophila heterogeneity was minimal . Multiple tick-borne bacteria coexist in I . ricinus ticks in southeastern Europe.

J Clin Microbiol, 2001 Nov, 39(11), 4082 - 5
Chlamydia trachomatis serology: diagnostic value of outer membrane protein 2 compared with that of other antigens; Bas S et al.; Different immunoassays using recombinant antigens or synthetic peptides were evaluated for the serodiagnosis of Chlamydia trachomatis infections . Antigens used included cysteine-rich outer membrane protein 2 (OMP2), heat shock protein 60, the polypeptide encoded by open reading frame 3 of the plasmid (pgp3), synthetic peptides derived from species-specific epitopes in variable domain IV of the major OMP (MOMP) (Labsystems, Helsinki, Finland), and a fragment of the total lipopolysaccharide (Medac, Hamburg, Germany) . Because cross-reactions between chlamydial species have been reported, Chlamydia pneumoniae-specific antibodies were also determined by immunoassays (Labsystems) . Responses obtained with serum samples from patients with well-defined diseases (i.e., urethral or endocervical samples from which C . trachomatis DNA was amplified) were compared to those obtained with samples from healthy blood donors . The best sensitivity (79%) associated with the best specificity (82%) was obtained when immunoglobulin G (IgG) responses to both MOMP and pgp3 were considered . The highest sensitivity (89%) was obtained with anti-OMP2 IgG, but the lowest specificity (57%) was obtained with this antibody, due to probable cross-reactivity with C . pneumoniae OMP2.

FEBS Lett, 2001 Oct 19, 507(1), 109 - 13
Agonist-independent and -dependent oligomerization of dopamine D(2) receptors by fusion to fluorescent proteins; Wurch T et al.; Oligomerization of the short (D(2S)) and long (D(2L)) isoforms of the dopamine D(2) receptor was explored in transfected Cos-7 cells by their C-terminal fusion to either an enhanced cyan or enhanced yellow fluorescent protein (ECFP or EYFP) and the fluorescent fusion protein interaction was monitored by a fluorescence resonance energy transfer (FRET) assay . The pharmacological properties of the fluorescent fusion proteins, as measured by both displacement of {(3)H}nemonapride binding and agonist-mediated stimulation of {(35)S}GTPgammaS binding upon co-expression with a G(alphao)Cys(351)Ile protein, were not different from the respective wild-type D(2S) and D(2L) receptors . Co-expression of D2S:ECFP+D2S:EYFP in a 1:1 ratio and D2L:ECFP+D2L:EYFP in a 27:1 ratio resulted, respectively, in an increase of 26% and 16% in the EYFP-specific fluorescent signal . These data are consistent with a close proximity of both D(2S) and D(2L) receptor pairs of fluorescent fusion proteins in the absence of ligand . The agonist-independent D(2S) receptor oligomerization could be attenuated by co-expression with either a wild-type, non-fluorescent D(2S) or D(2L) receptor subtype, but not with a distinct beta(2)-adrenoceptor . Incubation with the agonist (-)-norpropylapomorphine dose-dependently (EC(50): 0.23+/-0.06 nM) increased the FRET signal for the co-expression of D2S:ECFP and D2S:EYFP, in support of agonist-dependent D(2S) receptor oligomerization . In conclusion, our data strongly suggest the occurrence of dopamine D(2) receptor oligomers in intact Cos-7 cells.

FEBS Lett, 2001 Oct 19, 507(1), 21 - 4
Cingulin interacts with F-actin in vitro; D'Atri F et al.; Cingulin, a M(r) 140-160 kDa protein of the cytoplasmic plaque of epithelial tight junctions (TJ), interacts in vitro with TJ proteins and myosin . Here we investigated cingulin interaction with actin, using His-tagged, full-length Xenopus laevis cingulin expressed in insect cells, and glutathione S-transferase (GST) fusion proteins of fragments of cingulin expressed in bacteria . Purified full-length cingulin co-pelleted with F-actin after high speed centrifugation, and promoted the sedimentation of F-actin under low speed centrifugation, suggesting that cingulin is an actin-cross-linking protein . The actin interaction of GST fusion proteins containing fragments of Xenopus cingulin suggested that the F-actin binding site is between residues 101 and 294.

Proc Nutr Soc, 2001 Aug, 60(3), 399 - 402
Enteral and parenteral nutrition: evidence-based approach; Jeejeebhoy KN; Nutrition support for patients in hospital has become an essential form of therapy . Total parenteral nutrition (TPN) was the preferred way of giving nutrition to hospital patients for many years but enteral nutrition (EN) is now the preferred route . EN is believed to promote gut function and prevent translocation of intestinal bacteria, thus reducing the incidence of sepsis in critically ill patients . In consequence, the use of TPN has been discouraged as a dangerous form of therapy . Critical review of the data suggests that in the human subject TPN does not cause mucosal atrophy or increase translocation of bacteria through the small intestine . However, overfeeding, which is easy with TPN, can explain the results of studies which have shown that TPN increases sepsis . Furthermore, the risks of TPN-induced complications have been exaggerated . When there is risk of malnutrition and EN is not tolerated, or there is gut failure, TPN is an equally effective and safe alternative.

Biotechniques, 2001 Oct, 31(4), 802 - 4, 806, 808, 810
Picogram cloning and direct in situ sequencing of DNA from gel pieces; Meijerink E et al.; We describe a simple and rapid procedure for cloning and sequencing of DNA fragments separated by gel electrophoresis, using novel hydrophilic gels, Clearose BG, Spreadex, and Poly(NAT), that do not melt at 95 degrees C . For cloning, a band of interest is excised precisely and incubated in an extraction buffer containing 5-10 mM MgCl2 at 70 degrees C for 15-45 min . The eluted DNA is added directly to the plasmid solution . Using a topoisomerase-based ligation system, we were able to transform bacteria with a few picograms of DNA and isolate recombinant clones . For in situ sequencing, the DNA in the gel serves as the template . No treatment before cycle sequencing is necessary for fragments up to 500 bp.

Acta Odontol Scand, 2001 Oct, 59(5), 330 - 4
Soluble CD14 in human breast milk and its role in innate immune responses; Vidal K et al.; Immune factors secreted in milk are important for health in the neonatal gut . We have detected the bacterial pattern recognition receptor, soluble CD14 (sCD14) in human breast milk at different times during lactation . The molecule occurs in a single form in milk, in contrast to human serum, in which there are two isoforms . Produced by mammary epithelial cells, milk sCD14 mediates secretion of innate immune response molecules such as interleukin-8, tumor necrosis factor-alpha, and epithelial neutrophil activator-78 by CD14-negative intestinal epithelial cells exposed to lipopolysaccharide (LPS) or bacteria . Although present at low concentrations in milk, LPS-binding protein may be implicated in the biological effects observed . Our findings support the premise that milk sCD14 acts as a 'sentinel' molecule and immune modulator in homeostasis and in the defense of the neonatal intestine . In so doing, it may prevent the immune and inflammatory conditions of the gut to which non-breastfed infants are predisposed.

Nippon Yakurigaku Zasshi, 2001 Oct, 118(4), 259 - 68
{Pharmacological study on the pathological changes of the gastric mucosa in Helicobacter pylori-infected Mongolian gerbils}; Keto Y et al.; Helicobacter pylori (H . pylori) infection has been recognized to be a causal factor of gastritis, ulcers and gastric cancer in man . Using Mongolian gerbils (M . gerbils), which are suitable for an H . pylori infection animal model, we examined 1) how H . pylori infection, indomethacin and their combination affects the healing of gastric ulcers and whether or not such factors provoke a relapse of healed acetic acid ulcers; and 2) whether or not eradication of the bacteria with drugs at specified times after infection prevents the development of mucosal changes, including gastric adenocarcinoma . 1) H . pylori infection significantly delayed ulcer healing 4 weeks following infection . Indomethacin treatment showed a tendency to delay ulcer healing . Ulcer healing in H . pylori-infected M . gerbils was significantly delayed by indomethacin . H . pylori infection resulted in a relapse of healed ulcers from 1 to 6 months after infection, with a gradual increase in size . Omeprazole markedly prevented the ulcer relapse caused by H . pylori infection . 2) Four or 8 months after H . pylori inoculation, eradication was performed by concurrent treatment with omeprazole + clarithromycin . Immediately after treatment ended in both the 5 and 9 month groups, it was verified that H . pylori were completely eradicated . Autopsy performed 18 months after H . pylori inoculation revealed gastric hyperplastic polyps with erosive lesions and ulcers that were grossly visible; and atrophic gastritis, intestinal metaplasia, carcinoids, and adenocarcinomas were histologically observed in the non-treated control group . In animals eradicated after 4 months and autopsied after 18 months, however, such mucosal changes were not observed . In contrast, intestinal metaplasia and mucosal atrophy was observed in animals eradicated after 8 months and autopsied after 18 months . It was concluded that 1) H . pylori infection delayed the healing of preexisting gastric ulcers and resulted in the relapse of healed ulcers, yet indomethacin had little or no effect on ulcer healing or relapse; and 2) early eradication of H . pylori infection with drug therapy can prevent severe gastric mucosal changes, to include adenocarcinomas, in M gerbils.

Med Clin North Am, 2001 Nov, 85(6), 1545 - 63
Risk factors for nosocomial pneumonia . Focus on prophylaxis; Fleming CA et al.; Despite an increased understanding of the pathogenesis of NP and advances in diagnosis and treatment, the risk, cost, morbidity, and mortality of NP remain unacceptably high . This article has identified strategic areas for primary and secondary prophylaxis that are simple and cost-effective . Realizing that the pathogenesis of NP requires bacterial colonization and the subsequent entry of these bacteria into the lower respiratory tree helps highlight the role of cross-infection and the importance of standard infection control procedures . Similarly the role of sedation and devices as risk factors can be reduced by minimizing the duration and intensity of sedation and length of exposure to invasive devices . Additional low-cost interventions that have been shown to be effective in preventing NP are the positioning of patients in a semirecumbent position and the appropriate use of enteral feeding, antibiotics, and selected medical devices . Prophylaxis of NP and VAP is carried out best by a multidisciplinary management team comprised of physicians (critical care, pulmonary medicine, infectious diseases, and primary care), critical care and infection control nurses, and respiratory therapists, even though this approach may result in decreased professional autonomy and freedom . This group should review the current guidelines, pathways, and standards for short-term and long-term prophylaxis of NP and VAP, then integrate them into and monitor their use for routine patient care . The risk factors and prophylaxis strategies for NP discussed in this article apply primarily to patients in acute care facilities, but also are relevant to alternative health care settings as well as the care of ill patients in ambulatory settings . The routine use of effective team policies for prophylaxis needs to be monitored by the Joint Commission for the Accreditation of Health Care or other agencies . Research to delineate the most effective and feasible strategies for prophylaxis NP has been compromised by insufficient funding and lack of adequate, randomized multicenter studies to enable generalizability of results . Effective strategies for prophylaxis have not been disseminated widely or implemented in hospitals . Successful short-term and long-term strategies for prophylaxis must be evaluated and implemented by a team of physicians, nurses, and respiratory therapists . More than 100 years ago, Sir William Osler warned health care providers, "Remember how much you don't know." The authors would add that clinicians have acquired significant knowledge about risk factors and prophylaxis of NP in the 1980s and 1990s, but prophylaxis as a theory rather than an action . If the tree has not been planted, the time is now.

Boll Chim Farm, 2001 Sep-Oct, 140(5), 287 - 96
Synthesis of some new quinoline derivatives: new routes to synthesize polysubstituted 2(1H)-quinolone derivatives; el-Taweel FM et al.; Reaction of 4-hydroxyquinolines 1a,b or 1c,d with the ylidenenitriles 2b-g and 2h,c yielded pyranoquinolines 6 and 9, respectively . 4-Anilinoquinoline 10 reacted with 2a,e to give pyridoquinolines 12 . Treating of 1a or 14 with SO2Cl2 gave 3-dichloroacetyl derivative 15 . Compound 15 reacted with KCN/H2O and NaN3 to give furoquinoline 16 and the tetrazole 18, respectively . Reaction of 1a, b, f with a mixture of phenyl isothiocyanate and glycine gave pyranoquinoline 22 . This compound reacted with chloroacetic acid to yield thiazolidinones 23 . Pyranoquinolines 27 were synthesized either from the reaction of 1c,d with enamine 24 or reaction of 1e,f with hippuric acid . Condensation of 1c,d with the enamine 25 gave azlactone 28 . The latter compound reacted with 4-aminoantipyrine and 2-aminopyridine to give the imidazole derivatives 30.

Appl Environ Microbiol, 2001 Nov, 67(11), 5240 - 6
Gene cassette PCR: sequence-independent recovery of entire genes from environmental DNA; Stokes HW et al.; The vast majority of bacteria in the environment have yet to be cultured . Consequently, a major proportion of both genetic diversity within known gene families and an unknown number of novel gene families reside in these uncultured organisms . Isolation of these genes is limited by lack of sequence information . Where such sequence data exist, PCR directed at conserved sequence motifs recovers only partial genes . Here we outline a strategy for recovering complete open reading frames from environmental DNA samples . PCR assays were designed to target the 59-base element family of recombination sites that flank gene cassettes associated with integrons . Using such assays, diverse gene cassettes could be amplified from the vast majority of environmental DNA samples tested . These gene cassettes contained complete open reading frames, the majority of which were associated with ribosome binding sites . Novel genes with clear homologies to phosphotransferase, DNA glycosylase, methyl transferase, and thiotransferase genes were identified . However, the majority of amplified gene cassettes contained open reading frames with no identifiable homologues in databases . Accumulation analysis of the gene cassettes amplified from soil samples showed no signs of saturation, and soil samples taken at 1-m intervals along transects demonstrated different amplification profiles . Taken together, the genetic novelty, steep accumulation curves, and spatial heterogeneity of genes recovered show that this method taps into a vast pool of unexploited genetic diversity . The success of this approach indicates that mobile gene cassettes and, by inference, integrons are widespread in natural environments and are likely to contribute significantly to bacterial diversity.

Appl Environ Microbiol, 2001 Nov, 67(11), 5077 - 83
Anaerobic cometabolic conversion of benzothiophene by a sulfate-reducing enrichment culture and in a tar-oil-contaminated aquifer; Annweiler E et al.; Anaerobic cometabolic conversion of benzothiophene was studied with a sulfate-reducing enrichment culture growing with naphthalene as the sole source of carbon and energy . The sulfate-reducing bacteria were not able to grow with benzothiophene as the primary substrate . Metabolite analysis was performed with culture supernatants obtained by cometabolization experiments and revealed the formation of three isomeric carboxybenzothiophenes . Two isomers were identified as 2-carboxybenzothiophene and 5-carboxybenzothiophene . In some experiments, further reduced dihydrocarboxybenzothiophene was identified . No other products of benzothiophene degradation could be determined . In isotope-labeling experiments with a {(13)C}bicarbonate-buffered culture medium, carboxybenzothiophenes which were significantly enriched in the (13)C content of the carboxyl group were formed, indicating the addition of a C(1) unit from bicarbonate to benzothiophene as the initial activation reaction . This finding was consistent with the results of earlier studies on anaerobic naphthalene degradation with the same culture, and we therefore propose that benzothiophene was cometabolically converted by the same enzyme system . Groundwater analyses of the tar-oil-contaminated aquifer from which the naphthalene-degrading enrichment culture was isolated exhibited the same carboxybenzothiophene isomers as the culture supernatants . In addition, the benzothiophene degradation products, in particular, dihydrocarboxybenzothiophene, were significantly enriched in the contaminated groundwater to concentrations almost the same as those of the parent compound, benzothiophene . The identification of identical metabolites of benzothiophene conversion in the sulfate-reducing enrichment culture and in the contaminated aquifer indicated that the same enzymatic reactions were responsible for the conversion of benzothiophene in situ.

Mol Microbiol, 2001 Oct, 42(1), 37 - 46
Population density-dependent regulation of the Bradyrhizobium japonicum nodulation genes; Loh JT et al.; The nodulation genes of Bradyrhizobium japonicum are essential for infection and establishment of a nitrogen-fixing symbiosis . Here, we demonstrate that plant-produced isoflavones induce nodulation gene expression in a population density-dependent fashion . Nodulation gene induction is highest at a low population density and significantly reduced in more dense cultures . A quorum signal molecule in the conditioned medium of B . japonicum cultures mediates this repression . Repression in response to the quorum signal results from the induction of NolA which, in turn, induces NodD2 leading to inhibition of nod gene expression . Consistent with this, nolA-lacZ and nodD2-lacZ expression increased with increasing population density . Unlike the wild type, the ability to induce nodY-lacZ expression did not decline with population density in a NolA mutant . Normally, nod gene expression is repressed in planta (i.e . within nodules) . However, expression of a nodY-GUS fusion was not repressed in a NolA mutant, suggesting that quorum-sensing control may mediate in planta repression of the nod genes . Addition of conditioned medium to cultures significantly reduced nod gene expression . Treatment of inoculant cultures with conditioned medium also reduced the ability of B . japonicum to nodulate soybean plants.

Clin Microbiol Infect, 2001 Sep, 7(9), 461 - 9
Routine diagnosis of Borrelia burgdorferi (sensu lato) infections using a real-time PCR assay; Schwaiger M et al.; OBJECTIVE: To establish a one-tube fluorogenic real-time PCR assay for routine detection of Borrelia burgdorferi (sensu lato) DNA in various clinical specimens . METHODS: A fragment of the flagellin gene sequence was amplified with the TaqMan chemistry using primers and a probe common to Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii and Borrelia valaisiana . A recombinant plasmid containing the chromosomal gene coding for the flagellin protein was used as standard . RESULTS: The specificity of the assay was documented with 48 different clinically relevant Borrelia burgdorferi strains . No cross-reaction occurred with unrelated bacteria, viruses and fungi . At an analytic sensitivity of 10 copies, excellent precision within runs and between runs was observed . The potential presence of inhibitors of the Taq DNA polymerase was monitored by spiking aliquots of each sample with a plasmid containing the target sequence . Among 56 cerebrospinal fluid samples taken from 54 patients with clinical suspicion of neuroborreliosis, one (1.8%) tested positive for Borrelia burgdorferi sensu lato DNA . Borrelia burgdorferi DNA was also detected in five (17.9%) of 28 synovial fluid specimens and in one (20%) of five synovial membrane biopsies obtained from 31 patients with arthropathies . In order to test for the absence of false-positive results, 84 samples from 83 patients without evidence of Lyme disease were investigated . None of these samples showed measurable amounts of Borrelia burgdorferi DNA . CONCLUSION: By its established features, such as speed, reliability, sensitivity, specificity, the inclusion of carryover prevention and the monitoring of inhibitors in individual test tubes, this real-time PCR assay has proved to be a potent tool for the detection of Borrelia burgdorferi DNA under routine conditions in diagnostic laboratories.

J Neurochem, 2001 Oct, 79(2), 437 - 44
Inactivation of the 7B2 inhibitory CT peptide depends on a functional furin cleavage site; Hwang JR et al.; The eukaryotic subtilisin prohormone convertase 2 (PC2) is known to require in vivo exposure to the neuroendocrine protein 7B2 in order to produce an enzymatically active species capable of proteolytic action on prohormone substrates . In the present study, we examined the role of the pentabasic site within 27-kDa 7B2 in this process . We prepared two His-tagged recombinant 7B2s by overexpression in bacteria: 7B2-Ser-Ser (SS), with an inactivating mutation in the CT peptide from Lys171-Lys172 (KK) to SS, rendering the CT peptide non-inhibitory; blockade-SS, a double mutant of both the CT peptide as well as of the pentabasic furin cleavage site . These purified proteins were used in a cell-free proPC2 activation assay . Both 7B2-SS as well as blockade-SS were able to facilitate the activation of proPC2 (as judged by efficient production of enzyme activity), suggesting that cleavage at the furin site is not required for 7B2s lacking inhibitory CT peptides . Plasmids encoding proPC2 and various 7B2s were transiently transfected into human embryonic kidney (HEK293) cells and PC2 enzymatic activity and CT forms in each overnight conditioned medium were measured . Cells transfected with proPC2 and wild-type 7B2 secreted CT peptide cleavage products, but cells transfected with proPC2 and the blockade mutant overwhelmingly secreted intact, 27-kDa, blockaded 7B2 . Medium obtained from HEK293 cells transfected with proPC2 and either wild-type 7B2, 7B2-SS, or blockade-SS exhibited PC2 activity, but medium from cells expressing the 7B2 blockade mutant did not . We conclude that cleavage at the 7B2 furin consensus site is required to produce PC2 capable of efficient proteolytic inactivation of the CT peptide.

Bioorg Med Chem Lett, 2001 Nov 19, 11(22), 2983 - 6
Visualizing antibody-catalyzed retro-aldol-retro-Michael reactions; Tanaka F et al.; We developed a visible detection system for antibody-catalyzed retro-aldol-retro-Michael reactions . Aldolase antibody 38C2 catalyzed the reaction of substrate 1 to provide 6-bromo-2-napthol that forms a visible colored azo dye with diazonium salts . This system has potential for the screening of novel catalysts.

Nature, 2001 Oct 11, 413(6856), 639 - 43
In situ class switching and differentiation to IgA-producing cells in the gut lamina propria; Fagarasan S et al.; One of the front lines of the immune defence is the gut mucosa, where immunoglobulin- (IgA) is continuously produced to react with commensal bacteria and dietary antigens . It is generally accepted that, after antigenic stimulation in the Peyer's patches, IgA+ lymphoblasts (B220+IgA+) migrate through the lymph and blood circulation, and eventually home to the lamina propria of the intestine . Mice that lack activation-induced cytidine deaminase (AID) are defective in class switch recombination (CSR) and somatic hypermutation . CSR changes the immunoglobulin heavy chain constant region (CH) gene being expressed from Cmu to other CH genes, resulting in a switch of the immunoglobulin isotype from IgM to IgG, IgE or IgA . AID-/- mice also secrete large amounts of immunoglobulin-mu (IgM) into faeces, and accumulate B220-IgM+ plasma cells as well as B220+IgM+ cells in the gut . Here we show that lamina propria B220+IgA+ cells have just completed CSR, as they still express both AID and transcripts from circular DNA that has been 'looped-out' during CSR . Lamina propria IgM+ B cells seem to be pre-committed to switching to IgA+ in vitro as well as in vivo . Culturing lamina propria IgM+ B cells together with lamina propria stromal cells enhances preferential switching and differentiation of B cells to IgA+ plasma cells . We conclude that IgA+ cells in the gut lamina propria are generated in situ from B220+IgM+ lymphocytes.

J Am Soc Nephrol, 2001 Nov, 12(11), 2442 - 9
Acceleration of neutrophil apoptosis by glucose-containing peritoneal dialysis solutions: role of caspases; Catalan MP et al.; Commercial, glucose-containing peritoneal dialysis (PD) solutions have deleterious effects on leukocytes and mesothelial cells that contribute to an impaired peritoneal defense . However, the molecular mechanisms of these deleterious effects are poorly understood . The effect of PD solutions on neutrophil viability, the molecular mechanisms of cell death, its functional consequences, and the possibilities for pharmacologic modulation have now been studied . The effect of newly available, bicarbonate-buffered PD solutions were further investigated . Lactate-buffered, glucose-containing PD solutions increased the apoptosis rate of cultured neutrophils (control media versus 4.25% glucose PD solution: 31 +/- 3% versus 52 +/- 3% apoptosis at 24 h, P < 0.001) . Bicarbonate-buffered, 4.25% glucose-containing PD solutions with low concentration of glucose degradation products did not increase the rate of apoptosis . Apoptosis induced by lactate-buffered, 4.25% glucose PD solutions was not related to hyperosmolality or acidic pH and was not reproduced by increasing the glucose concentration by the addition of glucose to a commercial, lactate-buffered fluid . Neutrophil apoptosis was associated with caspase-3 activation . Inhibition of caspase-3 by the use of the caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-fmk or the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (zVAD-fmk) prevented features of apoptosis, such as morphologic changes, internucleosomal DNA degradation, and the appearance of hypodiploid cells and increased the number of viable, trypan blue-excluding neutrophils . Furthermore, zVAD-fmk increased neutrophil phagocytosis of bacteria . However, the caspase-1 inhibitor acetyl-Tyr-Val-Ala-Asp-aldehyde did not prevent cell death . These data suggest that unidentified components in commercial, lactate-buffered, high-glucose PD fluid accelerate the rate of neutrophil apoptosis . Glucose degradation products may be such unidentified components . Acceleration of neutrophil apoptosis may contribute to the impaired local defense system of patients undergoing PD.

Environ Health Perspect, 2001 Oct, 109(10), 983 - 94
Review of harmful gastrointestinal effects of carrageenan in animal experiments; Tobacman JK; In this article I review the association between exposure to carrageenan and the occurrence of colonic ulcerations and gastrointestinal neoplasms in animal models . Although the International Agency for Research on Cancer in 1982 identified sufficient evidence for the carcinogenicity of degraded carrageenan in animals to regard it as posing a carcinogenic risk to humans, carrageenan is still used widely as a thickener, stabilizer, and texturizer in a variety of processed foods prevalent in the Western diet . I reviewed experimental data pertaining to carrageenan's effects with particular attention to the occurrence of ulcerations and neoplasms in association with exposure to carrageenan . In addition, I reviewed from established sources mechanisms for production of degraded carrageenan from undegraded or native carrageenan and data with regard to carrageenan intake . Review of these data demonstrated that exposure to undegraded as well as to degraded carrageenan was associated with the occurrence of intestinal ulcerations and neoplasms . This association may be attributed to contamination of undegraded carrageenan by components of low molecular weight, spontaneous metabolism of undegraded carrageenan by acid hydrolysis under conditions of normal digestion, or the interactions with intestinal bacteria . Although in 1972, the U.S . Food and Drug Administration considered restricting dietary carrageenan to an average molecular weight > 100,000, this resolution did not prevail, and no subsequent regulation has restricted use . Because of the acknowledged carcinogenic properties of degraded carrageenan in animal models and the cancer-promoting effects of undegraded carrageenan in experimental models, the widespread use of carrageenan in the Western diet should be reconsidered.

Med Clin (Barc), 2001 Oct 20, 117(12), 441 - 5
{Community acquired pneumonia in patients older than 60 years . Incidence of atypical agents and clinical-radiological progression}; Alvarez Gutierrez FJ et al.; BACKGROUND: Seventy five patients older than 60 years with a community acquired pneumonia followed up in an outpatient clinic, were prospectively studied in order to determine the incidence of atypical agents, clinical-radiological characteristics, progression and the differences with pneumonia in younger patients . METHOD: Clinical-radiological evaluation protocols were activated in the first visit and in two subsequent controls . Etiological diagnosis was made by means of serology (in the first visit and three weeks later) . RESULTS: Initially, 85 patients older than 60 years were included of which 75 non hospitalized were fully followed up . Also, in the comparative study, 216 outpatient clinic patients 60 years old or younger were followed up during the same period . In the first group the frequency of atypical agents was 33.3% . The most frequently isolated bacteria was Coxiella burnetii (13.3%)followed by virus and Legionella pneumophila . No case of Mycoplasma pneumoniae was diagnosed . The most frequent radiological onset was alveolar infiltrate (85%) . The comparative study between the two populations (older or younger than 60 years), found few clinical differences (dyspnea more frequent in older,feverish chill in younger) and auscultation (crackles more frequent in older) . We did not find differences remaining clinical-radiological or laboratory data . Most patients presented a favourable clinical and radiological progression . Only 2 patients needed hospital admission (2.7%) . CONCLUSIONS: In outpatient clinic patients older than 60 years with community acquired pneumonia a high number of atypical agents have been found . The clinical-radiological evolution was satisfactory for most of them . Age was not a decisive element in determining hospital admissions.

J Org Chem, 1999 Jul 23, 64(15), 5441 - 5446
Amidrazone and Amidoxime Inhibitors of Squalene Hopene Cyclase; Ganem B et al.; The cyclization of squalene catalyzed by the enzyme squalene-hopene cyclase (SHC) leads to the hopanoid family of pentacyclic triterpenes, which are widely found in bacteria as membrane constituents . SHC mediates a cascade of regio- and stereoselective cyclizations that has triggered considerable interest in understanding the enzyme's mechanism of action . This paper reports synthetic studies leading to the preparation of trienylamidrazone 8, trienylamidoxime 9, and tetraenylamidoxime 10 corresponding to a partially cyclized squalene chain . All three compounds displayed significant levels of inhibition when assayed against SHC, with 9 being more active than 8, and amidoxime 10 being the most potent . Detailed profiles of their inhibition kinetics are also presented.

J Org Chem, 1999 Mar 5, 64(5), 1508 - 1511
Synthesis of 1-Deoxy-D-xylulose and 1-Deoxy-D-xylulose-5-phosphate; Blagg BS et al.; 1-Deoxy-D-xylulose (4) and the corresponding 5-phosphate (5) are substrates for the first pathway-specific enzymes in the biosynthesis of thiamine diphosphate (vitamin B(1)), pyridoxol phosphate (vitamin B(6)), and the nonmevalonate family of isoprenoid compounds recently discovered in bacteria and plant chloroplasts . Both 4 and 5 were synthesized from commercially available (-)-2,3-O-isopropylidene-D-threitol (10) . The protected tetraol was converted to (-)-3,4-O-isopropylidene-5-triisopropylsilyl-1-deoxy-D-xylulose (14) in four steps . Treatment of 14 with acetic acid gave 4 in an overall yield of 69% . The corresponding 5-phosphate was obtained by protection the carbonyl group in 14, removal of the triisopropylsilyl moiety, and treatment of the resulting alcohol with trimethyl phosphite/TeCl(4), trimethylsilyl bromide, water, and HCl in successive steps to give 5 in 58% overall yield from 10.

J Bacteriol, 2001 Nov, 183(22), 6667 - 75
Identification and inactivation of three group 2 sigma factor genes in Anabaena sp . strain PCC 7120; Khudyakov IY et al.; Three new Anabaena sp . strain PCC 7120 genes encoding group 2 alternative sigma factors have been cloned and characterized . Insertional inactivation of sigD, sigE, and sigF genes did not affect growth on nitrate under standard laboratory conditions but did transiently impair the abilities of sigD and sigE mutant strains to establish diazotrophic growth . A sigD sigE double mutant, though proficient in growth on nitrate and still able to differentiate into distinct proheterocysts, was unable to grow diazotrophically due to extensive fragmentation of filaments upon nitrogen deprivation . This double mutant could be complemented by wild-type copies of sigD or sigE, indicating some degree of functional redundancy that can partially mask phenotypes of single gene mutants . However, the sigE gene was required for lysogenic development of the temperate cyanophage A-4L . Several other combinations of double mutations, especially sigE sigF, caused a transient defect in establishing diazotrophic growth, manifested as a strong and prolonged bleaching response to nitrogen deprivation . We found no evidence for developmental regulation of the sigma factor genes . luxAB reporter fusions with sigD, sigE, and sigF all showed slightly reduced expression after induction of heterocyst development by nitrogen stepdown . Phylogenetic analysis of cyanobacterial group 2 sigma factor sequences revealed that they fall into several subgroups . Three morphologically and physiologically distant strains, Anabaena sp . strain PCC 7120, Synechococcus sp . strain PCC 7002, and Synechocystis sp . strain PCC 6803 each contain representatives of four subgroups . Unlike unicellular strains, Anabaena sp . strain PCC 7120 has three additional group 2 sigma factors that cluster in subgroup 2.5b, which is perhaps specific for filamentous or heterocystous cyanobacteria.

Phytochemistry, 2001 Nov, 58(5), 729 - 38
Herbivore-induced allene oxide synthase transcripts and jasmonic acid in Nicotiana attenuata; Ziegler J et al.; Exogenous jasmonate treatment of Nicotiana attenuata Torr . ex Wats . plants elicits durable resistance against herbivores and attack from its specialist herbivore, Manduca sexta, results in an amplification of the transient wound-induced increase in endogenous jasmonic acid levels (JA) . To understand whether this "JA burst" is under transcriptional control, we cloned allene oxide synthase (AOS; EC 4.2.1.92), the enzyme that catalyzes the dehydration of 13(S)-hydroperoxy octadecatrienoic acid to an allene oxide, the first specific reaction in JA biosynthesis . An AOS cDNA coding for a 520 aa protein (58.6 kDa) with an isoelectric point of 8.74 was overexpressed in bacteria and determined to be a functional AOS . Southern blot analysis indicated the presence of more than one gene and AOS transcripts were detected in all organs, with the highest levels in stems, stem leaves and flowers . Attack by M . sexta larvae resulted in a sustained JA burst producing an endogenous JA amount 9-fold above control levels and 3-fold above maximum wound-induced levels, a response which could be mimicked by the addition of Manduca oral secretion and regurgitant to puncture wounds . M . sexta attack, wounding and regurgitant treatment transiently increased AOS transcript in the wounded leaf, but increases were not proportional to the JA response . Moreover, transcript accumulation lagged behind JA accumulation . Systemic wound-induced increases in AOS transcript, AOS activity or JA accumulation could not be detected . We conclude that increase in AOS transcript does not contribute to the initial increase in endogenous JA, but may contribute to sustaining the JA burst.

Braz J Med Biol Res, 2001 Nov, 34(11), 1379 - 95
Sequencing and promoter analysis of the nifENXorf3orf5fdxAnifQ operon from Azospirillum brasilense Sp7; Potrich DP et al.; A 40-kb DNA region containing the major cluster of nif genes has been isolated from the Azospirillum brasilense Sp7 genome . In this region three nif operons have been identified: nifHDKorf1Y, nifENXorf3orf5fdxAnifQ and orf2nifUSVorf4 . The operons containing nifENX and nifUSV genes are separated from the structural nifHDKorf1Y operon by about 5 kb and 10 kb, respectively . The present study shows the sequence analysis of the 6045-bp DNA region containing the nifENX genes . The deduced amino acid sequences from the open reading frames were compared to the nif gene products of other diazotrophic bacteria and indicate the presence of seven ORFs, all reading in the same direction as that of the nifHDKorf1Y operon . Consensus sigma54 and NifA-binding sites are present only in the promoter region upstream of the nifE gene . This promoter is activated by NifA protein and is approximately two-times less active than the nifH promoter, as indicated by the beta-galactosidase assays . This result suggests the differential expression of the nif genes and their respective products in Azospirillum.

Nat Immunol, 2001 Nov, 2(11), 1061 - 6
Development of chronic colitis is dependent on the cytokine MIF; de Jong YP et al.; The cytokine macrophage-migration inhibitory factor (MIF) is secreted by a number of cell types upon induction by lipopolysaccharide (LPS) . Because colitis is dependent on interplay between the mucosal immune system and intestinal bacteria, we investigated the role of MIF in experimental colitis . MIF-deficient mice failed to develop disease, but reconstitution of MIF-deficient mice with wild-type innate immune cells restored colitis . In addition, established colitis could be treated with anti-MIF immunoglobulins . Thus, murine colitis is dependent on continuous MIF production by the innate immune system . Because we found increased plasma MIF concentrations in patients with Crohn's disease, these data suggested that MIF is a new target for intervention in Crohn's disease.

Surg Laparosc Endosc Percutan Tech, 2001 Oct, 11(5), 317 - 21
Seroma in laparoscopic ventral hernioplasty; Tsimoyiannis EC et al.; SUMMARY: Seroma is a frequent complication of laparoscopic or open repair of ventral hernias using expanded polytetrafluoroethylene mesh . Aspiration of this seroma has the risk of introducing bacteria, resulting in infection and the recurrence of the hernia . Between May 1996 and December 2000, 51 patents who underwent 53 laparoscopic ventral hernioplasties (44 incisional, 5 large epigastric, and 4 large umbilical) were randomized to participate in a trial comparing the intraperitoneal onlay mesh repair with or without cauterization of the hernia sac . Group A (26 patients; 28 hernias) patients were operated on by using an expanded polytetrafluoroethylene Dual Mesh patch (Gore and Associates, Flagstaff, AZ, U.S.A.) inserted intraperitoneally and secured by full-thickness stitches and endoscopic clips to cover the hernia defect, while the sac was left intact . Group B (25 patients, 25 hernias) patients were operated on according to the same technique as those in group A, but the hernia sac was cauterized by monopolar cautery (5 cases) or harmonic scalpel (20 cases) . After surgery, clinical examination and computed tomography scans were used to confirm or test the existence of seroma and recurrence . In group A, four clinically evident seromas were found . Two of them were resolved with no intervention . In the remaining two cases, multiple aspirations were needed for 4 and 7 months, respectively, but 2 and 3 months, respectively, after resolution of the seroma, a recurrence of the hernia was observed . There was one more recurrence without seroma and three with subclinical seromas (only observed on computed tomography scans) . In group B, subclinical seroma (only observed in computed tomography scan) resolved in a few days, and one recurrence without seroma was observed . Although only a small number of patients were studied, our findings suggest that the cauterization of the hernia sac prevents seromas and reduces recurrences in laparoscopic repair of ventral hernias.

J Vet Med B Infect Dis Vet Public Health, 2001 Sep, 48(7), 537 - 46
Studies on the prevalence of Riemerella anatipestifer in the upper respiratory tract of clinically healthy ducklings and characterization of untypable strains; Ryll M et al.; A total of 44 bacterial strains obtained from 49 clinically healthy ducklings of different ages originating from four different farms were identified as members of the species Riemerella anatipestifer (RA) using conventional biochemical test methods . Numerical analysis of the whole-cell fatty acid patterns of these isolates resulted in two different clusters, one of which showed a similar pattern to that of the type strain of RA . Strains having a different fatty acid methyl esters (FAME)-profile (cluster II) were designated R . anatipestifer-like (RA-like) . Sequencing of 16S rRNA genes of RA-like field isolates revealed 99% identity to RA . The significance of these observations are discussed . The present findings document for the first time that RA seems to represent a normal part of the pharyngeal flora of healthy Pekin ducks.

Nature . 1983 Sep 22;305(5932):262.
U.S . agrigenetics: suit filed against NIH; David P; KIE: Three environmental groups have filed suit against the National Institutes of Health in an attempt to block an experiment approved in April by the NIH Recombinant DNA Advisory Committee . The experiment, designed by researchers at the University of California, Berkeley, to enhance the frost resistance of crops, would release genetically engineered organisms into the general environment for the first time . The suit, filed in a federal court in the District of Columbia, claims that releasing frost-resistant bacteria into the environment could have damaging ecological consequences, that NIH should have issued an environmental impact assessment before approving any experiment involving the release of genetically engineered organisms, and that the Recombinant DNA Advisory Committee lacks the expertise to assess environmental risks .

Environ Sci Technol, 2001 Oct 1, 35(19), 3905 - 14
Interaction of phenolic uncouplers in binary mixtures: concentration-additive and synergistic effects; Escher BI et al.; The uncoupling activities of 14 binary mixtures of substituted phenols and of 4 binary mixtures of phenols and anisols were investigated at different pH values . Experiments were performed with time-resolved spectroscopy on membrane vesicles (chromatophores) of the photosynthetic bacteria Rhodobacter sphaeroides . Phenols are known to destroy the electrochemical proton gradient in energy-transducing membranes by a protonophoric mechanism . Anisols do not have protonophoric activity but disturb membrane structure and functioning as a nonspecific baseline toxicant . It was postulated in the literature that, for certain substituted phenols, the formation of a dimer between the phenoxide and the neutral phenol may contribute significantly to the overall protonophoric activity . In 13 of 14 mixtures of substituted phenols but in none of the mixtures of phenols with anisols, such a dimer appears to be formed between two different mixture partners . An extended shuttle mechanism of uncoupling, which includes a term for the contribution of such a mixed dimer, provided a good description of all experimental data . Opposite speciation favors interaction and ortho substituents abate interaction, which adds evidence for the dimerformation via a hydrogen bond between the phenol-OH and the phenoxide . These findings are significant not only regarding the mechanism of protonophoric action but also for the risk assessment process of chemical mixtures in the environment . When assessing the effect of mixtures, concentration addition is regarded as a reference X concept to estimate effects of similarly acting compounds . The substituted phenols in this work act according to the same action mechanism of uncoupling . Nevertheless, the overall effect of four of the investigated mixtures, which exhibit stronger dimer formation as compared to the single compounds or for which the resulting dimer is intrinsically more active, exceeded the effect calculated according to concentration addition considerably . In future work, this synergistic effect observed in-vitro has to be validated in-vivo to deduce its implications for the risk assessment process.

Biol Pharm Bull, 2001 Oct, 24(10), 1108 - 12
Effect of pH on metabolism of glycyrrhizin, glycyrrhetic acid and glycyrrhetic acid monoglucuronide by collected human intestinal flora; Akao T; Collected human intestinal flora (whole bacteria) was incubated with glycyrrhizin (GL), glycyrrhetic acid (GA), glycyrrhetic acid monoglucuronide (GAMG) and a combination of the three for 10 min at 37 degrees C under pH 5.6 and 7.0 . The effect of these components on GL beta-D-glucuronidase activity, GAMG beta-D-glucuronidase activity and metabolite production in whole bacteria was examined . GL and GA were not metabolized at pH 5.6 and 7.0 by whole bacteria, while the level of GAMG changed at both pH 5.6 and 7.0 . However, preincubated whole bacteria converted GA and a combination containing GA to other metabolites removed 3alpha-hydroxyglycyrrhetic acid and 3-oxoglycyrrhetic acid . The level of GL beta-D-glucuronidase activity remaining in whole bacteria after exposure to both GA and GAMG was above its initial level at pH 5.6 and 7.0, and the level of GAMG beta-D-glucuronidase activity remaining after exposure to GL, GA and GAMG was suppressed against control at pH 5.6 and 7.0 . It is found that intestinal bacteria had similar action against GL, GA and GAMG at between pH 5.6 and 7.0.

Probl Tuberk, 2001, (6), 11 - 4
{Reservoir of tuberculosis infection}; Khruleva TS; "The reservoir of tuberculous infection" is shown to imply a wider concept than does "the size of bacteria-isolating contingents" . It includes a potential reservoir (the population that is infected but not the patients with tuberculosis) and an active one (undetected and detected patients with this disease) . To reduce the total incidence of tuberculosis in the population, continuous work as to the potential reservoir should be done, by vigorously detecting those with early-stage disease . Work associated with identification of only those who isolate abundant bacteria and with their abacillation must not be recognized effective in reducing overall morbidity and improving an epidemiological situation as it is directed only against a small portion of an infection reservoir.

Med Secoli, 1989, 1(1), 79 - 86
{History of tuberculosis: a century after the discoveries of Koch and Forlanini}; Aulizio F; A century after the discovery of the etiological agent of tubercolosis, the author remembers the steps of study of the disease . Fracastoro supposed the existence of small bodies, epidemiological studies pointed attention to the transmissibility until 1882, when Koch communicated to the Physiological Society of Berlin that bacteria had been evidenced in biological material of the lungs from tuberculosis patients . The method of coloration of Koch rapidly spread, but no therapy was found . In the same year Forlanini proposed the artificial pneumothorax as cure.

Sven Med Tidskr, 1999, 3(1), 27 - 43
Not Available
Berghult B.
In ancient Egypt during the reign of Pharaoh Djoser, circa 2650 BC, the Step Pyramid was constructed by Imhotep . He was later worshiped as the God of Medicine . One of his contemporaries was the powerful writer Hesy who is reproduced on a panel showing a rebus of a swallow, a tusk and an arrow . He is therefore looked upon as being the first depicted odontologist . The art of writing begun in Egypt in about 3100 BC and the medical texts we know from different papyri were copied with hieratic signs around 1900-1100 BC . One of the most famous is the Papyrus Ebers . It was purchased by professor Ebers on a research travel to Luxor in 1873 . Two years later a beautiful facsimile in color was published and the best translation came in 1958 in German . The text includes 870 remedies and some of them are related to teeth and oral troubles like pain in the mouth, gingivitis, periodontitis and cavities in the teeth . The most common oral pain was probably pulpitis caused by extreme attrition due to the high consumption of bread contaminated with soil and/or quern minerals . Another text is the Papyrus Edwin Smith with four surgical cases of dental interest . The "toothworms" that were presumed to bring about decayed teeth have not been identified in the medical texts . It was not until 1889 W.D . Miller presented a scientific explanation that cavities were caused by bacteria . In spite of extensive research only a few evidence of prosthetic and invasive treatments have been found and these dental artifacts have probably been made post mortem . Some of the 150 identified doctors were associated with treatments of disorders of the mouth . The stele of Seneb from Sa'is during the 26th dynasty of Psamtik, 664-525 BC, shows a young man who probably was a dental healer well known to Pharaoh and his court . Clement of Alexandria mentions circa 200 AD that the written knowledge of the old Egyptians was gathered in 42 collections of papyri . Number 37-42 contained the medical writings . The household remedies in ancient Egypt were unique and future research will most likely give us new answers about pathology and health care of that time and a better understanding of old medical concepts.

Hist Sci Med, 1996, 30(1), 87 - 90
Not Available
Theodorides J.
Malta fever (brucellosis) is an infectious disease of cattle (chiefly goats) which can infect man . Although the disease is not limited to Malta, occurring also in other mediterranean countries as well as in Asia and America, it was originally described in 1863 in Malta where the responsible bacteria (Brucella melitensis) was discovered in 1887 by D . Bruce . Ten years later Wright & Smith (1897) showed that the agglutination test originally established for typhoidic patients could be used for the diagnosis of Malta fever . Finally the conclusions of the Commission for the investigation of Mediterranean fever (1905-1907) are briefly recalled.

Can Bull Med Hist, 1998, 15(2), 251 - 76
Coughing blood: tuberculosis deaths and data on the Yakama Indian Reservation, 1911-64; Trafzer CE; Tuberculosis was the foremost killer of First Nations populations during the late nineteenth and early twentieth centuries . According to data from Death Certificates collected by the Yakama County Health Department and preserved by the Office of Indian Affairs, tuberculosis among the 14 tribes of the Yakama Nation took the lives of 619 people between 1911-64 . The model age group was those Yakama between 15-19, with people between 15-39 suffering 329 (53%) deaths . Changes in contact with bacteria, diet, housing, and seasonal rounds resulting from reservation life influenced high tubercular death rates among Yakama when compared to Whites and non-Whites in the US and the population of Washington.

Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 6857 - 60
Natural abundance solid-state carbon NMR studies of photosynthetic reaction centers with photoinduced polarization; Zysmilich MG et al.; Solid-state NMR spectra of natural abundance 13C in reaction centers from photosynthetic bacteria Rhodobacter sphaeroides R-26 was measured . When the quinone acceptors were removed and continuous visible illumination of the sample was provided, exceptionally strong nuclear spin polarization was observed in NMR lines with chemical shifts resembling those of the aromatic carbons in bacteriochlorophyll and bacteriopheophytin . The observation of spin polarized 15N nuclei in bacteriochlorophyll and bacteriopheophytin was previously demonstrated with nonspecifically 15N-labeled reaction centers . Both the carbon and the nitrogen NMR studies indicate that the polarization is developed on species that carry unpaired electrons in the early electron transfer steps, including the bacteriochlorophyll dimer donor P860 and probably the bacteriopheophytin acceptor . I . Both enhanced-absorptive and emissive polarization were seen in the carbon spectrum; most lines were absorptive but the methine carbons of the porphyrin ring (alpha, beta, gamma, ) exhibited emissive polarization . The change in the sign of the hyperfine coupling at these sites indicates the existence of nodes in the spin density distribution on the tetrapyrrole cofactors flanking each methine carbon bridge.

Proc Natl Acad Sci U S A, 1995 May 23, 92(11), 4798 - 802
A multimer model for P680, the primary electron donor of photosystem II; Durrant JR et al.; We consider a model of the photosystem II (PS II) reaction center in which its spectral properties result from weak (approximately 100 cm-1) excitonic interactions between the majority of reaction center chlorins . Such a model is consistent with a structure similar to that of the reaction center of purple bacteria but with a reduced coupling of the chlorophyll special pair . We find that this model is consistent with many experimental studies of PS II . The similarity in magnitude of the exciton coupling and energetic disorder in PS II results in the exciton states being structurally highly heterogeneous . This model suggests that P680, the primary electron donor of PS II, should not be considered a dimer but a multimer of several weakly coupled pigments, including the pheophytin electron acceptor . We thus conclude that even if the reaction center of PS II is structurally similar to that of purple bacteria, its spectroscopy and primary photochemistry may be very different.

Proc Natl Acad Sci U S A, 1995 Jan 17, 92(2), 641 - 5
Chemoattraction of male gametes by a pheromone produced by female gametes of Chlamydomonas; Starr RC et al.; In isogamous species of Chlamydomonas, such as Chlamydomonas reinhardtii and Chlamydomonas eugametos, the sexual process involves the use of flagella agglutinins by which the gametes of compatible strains adhere through chance encounter and ultimately pair and fuse to form zygotes . In a newly described heterogamous species, Chlamydomonas allensworthii, the sexual process is initiated by the chemoattraction of small sperm to a sexually competent female gamete, which continues to secrete the pheromone until it has fused with one of the sperm so attracted . From bacteria-free female strains of C . allensworthii, the chemoat-tractant has been isolated and identified as a pentosylated hydroquinone (Mr = 532) whose spectral, chemical, and physical properties are in accord with the structure of a 2,3-dimethyl-5-(triprenylcarboxymethyl)-1,4-benzohydroquinone-1-(beta-xyloside) . A rapid bioassay of the pheromone uses DEAE-Toyopearl 650M beads to which the pheromone adsorbs . When such activated beads are placed in a suspension of sperm, they act as surrogate females and attract the small motile sperm . The purified pheromone shows activity at a concentration as low as 1 pM.

Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10460 - 4
Masculinization of female isopods (Crustacea) correlated with non-Mendelian inheritance of cytoplasmic viruses; Juchault P et al.; When in genetic females external male characters differentiate, the phenomenon is called "male pseudohermaphroditism." This male differentiation occurs in terrestrial isopods (Crustacea, suborder Oniscoidea) and sometimes involves only some epithelial areas (gynandromorphous mosaics) . It is not induced by male hormones or by abnormal ovary function . This intersexuality is transmitted maternally (by the intersex females) or paternally (by the brothers of intersex females) to between 30% and 60% of their offspring . Although it occurs at 20 degrees C, the male differentiation disappears when breeding takes place at 27 degrees C . Male characters differentiate in normal females--even in other Oniscoidea species (Porcellio dilatatus, Porcellio laevis, Armadillidium vulgare)--after injection of a 0.22-micron filtered tissue extract . Since an inhibitor of bacterial protein synthesis (gentamycin) does not inhibit this masculinizing effect, we infer that neither organelles nor bacteria are involved . Intersexuality is always correlated with the presence of cytoplasmic viral particles in both intersex-female and transmitter-male tissues . Striking similarities to the Drosophila S virus are noted . A reovirus-like Oniscoidea masculinizing virus, which probably acts only on the epithelial areas sensitive to the male hormones, is most likely the causative agent of this intersexuality . Here we report the conversion of secondary sexual characters putatively caused by a virus.

Proc Natl Acad Sci U S A, 2001 Nov 6, 98(23), 13454 - 9 Epub 2001 Oct 23.
Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease resistance response; Delledonne M et al.; Nitric oxide (NO) and reactive oxygen intermediates (ROIs) play key roles in the activation of disease resistance mechanisms both in animals and plants . In animals NO cooperates with ROIs to kill tumor cells and for macrophage killing of bacteria . Such cytotoxic events occur because unregulated NO levels drive a diffusion-limited reaction with O(2)(-) to generate peroxynitrite (ONOO(-)), a mediator of cellular injury in many biological systems . Here we show that in soybean cells unregulated NO production at the onset of a pathogen-induced hypersensitive response (HR) is not sufficient to activate hypersensitive cell death . The HR is triggered only by balanced production of NO and ROIs . Moreover, hypersensitive cell death is activated after interaction of NO not with O(2)- but with H(2)O(2) generated from O(2)(-) by superoxide dismutase . Increasing the level of O(2)(-) reduces NO-mediated toxicity, and ONOO(-) is not a mediator of hypersensitive cell death . During the HR, superoxide dismutase accelerates O(2)(-) dismutation to H(2)O(2) to minimize the loss of NO by reaction with O(2)(-) and to trigger hypersensitive cell death through NO/H(2)O(2) cooperation . However, O(2)(-) rather than H(2)O(2) is the primary ROI signal for pathogen induction of glutathione S-transferase, and the rates of production and dismutation of O(2)(-) generated during the oxidative burst play a crucial role in the modulation and integration of NO/H(2)O(2) signaling in the HR . Thus although plants and animals use a similar repertoire of signals in disease resistance, ROIs and NO are deployed in strikingly different ways to trigger host cell death.

J Biol Chem, 2001 Dec 28, 276(52), 48709 - 15 Epub 2001 Oct 17.
Carboxyl-terminal domain III of the delta' subunit of DNA polymerase III holoenzyme binds DnaX and supports cooperative DnaX complex assembly; Song MS et al.; The delta' subunit of the DNA polymerase-III holoenzyme is a key component of the DnaX complex; it is required for loading the beta(2) processivity factor onto a primed template . The x-ray crystal structure of delta' indicates a three domain C-shaped structure (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J . (1997) Cell 91, 335-345) . In this study, we localized the DnaX-binding domain of delta' to its carboxyl-terminal domain III by quantifying protein-protein interactions using a series of delta' fusion proteins lacking specific domains . The fusion protein corresponding to domain III of delta' bound to DnaX with an affinity approaching that of full-length delta' . In contrast, a construct bearing delta' domains I-II did not bind DnaX at detectable levels . The presence of delta and chi psi strengthened the interaction of DnaX with full-length delta' and delta' domain III . Thus, domain III of delta' not only contains the DnaX-binding site, but also contains the elements required for positive cooperative assembly of the DnaX complex . A domain III-specific anti-delta' monoclonal antibody interfered with DnaX complex formation and abolished the replication activity of DNA polymerase III holoenzyme.

Biophys J, 2001 Nov, 81(5), 2639 - 46
Single-molecule imaging of l-type Ca(2+) channels in live cells; Harms GS et al.; L-type Ca(2+) channels are an important means by which a cell regulates the Ca(2+) influx into the cytosol on electrical stimulation . Their structure and dynamics in the plasma membrane, including their molecular mobility and aggregation, is of key interest for the in-depth understanding of their function . Construction of a fluorescent variant by fusion of the yellow-fluorescent protein to the ion channel and expression in a human cell line allowed us to address its dynamic embedding in the membrane at the level of individual channels in vivo . We report on the observation of individual fluorescence-labeled human cardiac L-type Ca(2+) channels using wide-field fluorescence microscopy in living cells . Our fluorescence and electrophysiological data indicate that L-type Ca(2+) channels tend to form larger aggregates which are mobile in the plasma membrane.

Pediatr Nephrol, 2001 Oct, 16(10), 823 - 39
Pathogenesis of Shiga toxin-induced hemolytic uremic syndrome; Ray PE et al.; The term hemolytic uremic syndrome (HUS) was first introduced to describe a heterogeneous group of diseases characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure . Substantial progress has been made in our understanding of the etiology and pathogenesis of HUS . This article reviews some of the classic and new concepts related to the pathogenesis of Shiga toxin (Stx)-HUS and discusses their clinical relevance for the diagnosis and treatment of this syndrome . Infection with Stx-producing bacteria can induce HUS after a prodromal illness with or without diarrhea . Stx-induced renal endothelial injury is the primary pathogenic event . However, Stx also damages mesangial cells, as well as glomerular and renal tubular epithelial cells . Young children are at greatest risk for Stx-HUS because they express high levels of Stx receptors in renal glomeruli . Older children and adults express lower levels of glomerular Stx receptors and may develop Stx-HUS whenever the combined effects of lipopolysaccharide and cytokines upregulate the expression of Stx receptors and sensitize glomerular endothelial cells to Stx-induced injury, activate the coagulation-fibrinolytic system, and induce endothelial injury . Chemokine receptors and cytokines released by inflammatory cells (i.e., monocyte chemoattractant protein-1, interleukin-6, interleukin-8,) or injured endothelial cells (i.e., basic fibrobast growth factor) may play roles in this process . Measurement of the activity of a von Willebrand factor protease in plasma may help distinguish patients with thrombotic thrombocytopenic purpura from those with Stx-HUS.

Philos Trans R Soc Lond B Biol Sci, 1999 Nov 29, 354(1391), 1791 - 802
Termite assemblages, forest disturbance and greenhouse gas fluxes in Sabah, East Malaysia; Eggleton P et al.; A synthesis is presented of sampling work conducted under a UK government-funded Darwin Initiative grant undertaken predominantly within the Danum Valley Conservation Area (DVCA), Sabah, East Malaysia . The project concerned the assemblage structure, gas physiology and landscape gas fluxes of termites in pristine and two ages of secondary, dipterocarp forest . The DVCA termite fauna is typical of the Sunda region, dominated by Termes-group soil-feeders and Nasutitermitinae . Selective logging appears to have relatively little effect on termite assemblages, although soil-feeding termites may be moderately affected by this level of disturbance . Species composition changes, but to a small extent when considered against the background level of compositional differences within the Sunda region . Physiologically the assemblage is very like others that have been studied, although there are some species that do not fit on the expected body size-metabolic rate curve . As elsewhere, soil-feeders and soil-wood interface-feeders tend to produce more methane . As with the termite assemblage characteristics, gross gas and energy fluxes do not differ significantly between logged and unlogged sites . Although gross methane fluxes are high, all the soils at DVCA were methane sinks, suggesting that methane oxidation by methanotrophic bacteria was a more important process than methane production by gut archaea . This implies that methane production by termites in South-East Asia is not contributing significantly to the observed increase in levels of methane production worldwide . Biomass density, species richness, clade complement and energy flow were much lower at DVCA than at a directly comparable site in southern Cameroon . This is probably due to the different biogeographical histories of the areas.

Ear Nose Throat J, 2001 Oct, 80(10), 738 - 40, 742-3
Midfacial osteomyelitis in a chronic cocaine abuser: a case report; Talbott JF et al.; We describe the case of a 56-year-old man who was admitted for treatment of a progressive destruction of his hard palate, septum, nasal cartilage, and soft palate that had been caused by chronic cocaine inhalation . Biopsy of the bony septum revealed acute osteomyelitis and an extensive overgrowth of bacteria and Actinomyces-like organisms . There was no evidence of granuloma or neoplasm . The patient received intravenous ampicillin/sulbactam for 6 weeks, followed by lifetime oral amoxicillin . When there was no further evidence that destruction was progressing, the patient underwent nasal reconstruction with a cranial bone graft . The surgery was completed with no complications . To our knowledge, this is the first reported case of midfacial osteomyelitis associated with chronic cocaine abuse . The severity of this patient's complications, coupled with the success of his reconstructive surgery, makes this case particularly interesting . We believe that it is important for physicians to understand that septal perforation in a cocaine abuser should not be underestimated because it could result in a secondary bone infection . Nasoseptal destruction secondary to intranasal cocaine abuse is a result of cocaine's vasoconstrictive properties, and a decrease in the oxygen tension of intranasal tissue can facilitate the growth of anaerobic pathogens.

Prikl Biokhim Mikrobiol, 2001 Sep-Oct, 37(5), 517 - 32
{Pathogen-induced plant proteins}; Tarchevskii IA; Pathogen-induced plant proteins are classified by their functional characteristics: (a) involvement in plant cell signaling; (b) inhibition of enzymes excreted by the pathogens; (c) stabilization of plant cell walls or ability to trigger apoptosis; (d) enzymatic activity producing lysis of cell walls of pathogenic fungi and bacteria; (e) enzymatic activity in metabolic pathways of phenylpropane and terpene phytoalexins; and (f) ability to affect the pathogens directly, by disturbing the function of their cell membranes or by inactivating their ribosomes . Examples of transgenic plants with increased immunity against pathogens are also provided.

Curr Opin Biotechnol, 2001 Oct, 12(5), 516 - 22
Genomics: food and nutrition; Roberts MA et al.; Nutrition is traditionally a multidisciplinary field applying principles of molecular biochemistry and statistical epidemiology to integrative metabolism and population health . Genomics, with its global perspective, is now reinventing the future of human metabolic health . Creative experimental designs are addressing metabolic questions in nutrition ranging from energy regulation to aging, and from mechanisms of absorption to the interspecies molecular crosstalk of bacteria and human cells within the intestine.

Arch Immunol Ther Exp (Warsz), 2001, 49 Suppl 1, S33 - 9
The social life of NK cells; Martin-Fontecha A et al.; Natural killer (NK) cells represent a distinct population of lymphocytes originally identified by its ability to kill transformed cell lines in vitro . It is now clear that these cells also play an important role in the innate immune response against a variety of pathogens, such as virus, bacteria and parasites . In the past few years, different protocols have been developed to activate NK cells ex vivo, allowing a detailed molecular analysis of the interaction of these cells with their cellular targets . NK activity is regulated by signals generated by both inhibitory and stimulatory receptors expressed by target cells . Indeed, recent results indicate that, while major histocompatibility complex class I molecules expressed on target cells inhibit NK lytic activity by engaging surface inhibitory receptors, costimulatory molecules, such as B7-1, B7-2 and CD40, are able to actively trigger NK activity . This review discusses the most recent findings on the role of costimulation on NK activation and forsees the possible consequences of the interaction between NK cells and dendritic cells on the development of an adaptive immune response.

J Biol Chem, 2002 Feb 1, 277(5), 3552 - 9 Epub 2001 Oct 15.
Recruitment of activated G protein-coupled receptors to pre-existing clathrin-coated pits in living cells; Scott MG et al.; The process of clathrin-mediated endocytosis tightly regulates signaling of the superfamily of seven-transmembrane G protein-coupled receptors (GPCRs) . A fundamental question in the cell biology of membrane receptor endocytosis is whether activated receptors can initiate the formation of clathrin-coated pits (CPs) or whether they are simply mobilized to pre-existing CPs . Here, using various approaches, including a dynamic assay to monitor the distribution of CPs and GPCR-beta-arrestin complexes in live HeLa cells, we demonstrate for the first time that activated GPCRs do not initiate the de novo formation of CPs but instead are targeted to pre-existing CPs.

Appl Microbiol Biotechnol, 2001 Sep, 56(5-6), 700 - 6
Thioglucosidase activity from Sphingobacterium sp . strain OTG1; Meulenbeld GH et al.; Screening for novel thioglucoside hydrolase activity resulted in the isolation of Sphingobacterium sp . strain OTG1 from enrichment cultures containing octylthioglucoside (OTG) . OTG was hydrolysed into octanethiol and glucose by cell free extracts . Besides thioglucoside hydrolysis, several other glucoside hydrolase activities were detected in the Sphingobacterium sp . strain OTG1 cell free extract . By adding beta-glucosidase inhibitors it was possible to discriminate between these different activities . Ascorbic acid and D-gluconic acid lactone inhibited the hydrolysis of p-nitrophenyl beta-glucoside, but did not affect octyl- and octylthioglucoside hydrolase activity . Besides OTG, various other thioglucosides were hydrolysed by the novel thioglucosidase, with almost the same activities regardless of the nature of the aglycone, including the myrosinase model substrate sinigrin (a glucosinolate) . Sinigrin could also be used as a growth substrate by Sphingobacterium sp . strain OTG1, although at concentrations exceeding 0.15 mM degradation was not complete.

Bioresour Technol, 2001 Dec, 80(3), 205 - 10
Cellulolytic activity in leachate during leach-bed anaerobic digestion of municipal solid waste; Lai TE et al.; The degradation of municipal solid waste (MSW) under mesophilic conditions can be enhanced by exchanging leachate between fresh waste and stabilised waste . The optimum point in time when leachate from an anaerobically digesting waste bed can be used to initiate degradation of another waste bed might occur when the leachate of the digesting waste bed is highly active with cellulolytic and methanogenic bacteria . In this study, the cellulolytic activity of the leachate was measured using the cellulose-azure assay . As products of hydrolysis are soluble compounds, the rate of generation of these compounds was estimated based on a soluble chemical oxygen demand (SCOD) balance around the fresh waste bed . It was found that once the readily soluble material present in MSW was washed out there was very little generation of SCOD without the production of methane, indicating that flushing leachate from a stabilised waste bed resulted in a balanced inoculation of the fresh waste bed . With the onset of sustained methanogenesis, the rate of SCOD generation equalled the SCOD released from the digester as methane . The experimental findings also showed that cellulolytic activities of the leachate samples closely followed the trend of SCOD generation . reserved.

Zhonghua Yi Xue Za Zhi, 1999 Mar, 79(3), 211 - 3
{Pathogenicity in mice infected with three different strains H . pylori}; Gong G et al.; OBJECTIVE: To observe the differences of colonization and histopathology between CagA+ strains and lacking CagA by three different strains infecting C57BL/C mice . METHODS: CYP3401 wild type strain (CagA+), CYP3401(the isogenic CagA) knockout mutant CagA-, Sydney strain (CagA+) were used . The mice were administered orally with 0.5 ml, 10(9) CFU/ml of the above bacterial suspensions, respectively . Serum IgG antibodies were measured by ELISA . All the mice were sacrificed at 8 weeks after inoculation and their stomaches were removed for histopathologic study . RESULTS: The colonization of the bacteria was evident in Sydney strain and mild type strain, but characterized by wild to moderate infiltration by nutrophils and momocytes histologically . Only a small number of organisms were observed in the CagA- strains group . The levels of serum IgG antibodies were the highest in Sydney and wild strains group as compared with the control group (P < 0.01) . There was no significant difference between the CagA-group and the control group . CONCLUSION: A strong correlation was observed between CagA+, bacterial colonization, gastric mucosa inflammation, and serum IgG antibodies . The study of C57BL/C mice helps understand the pathogenicity with CagA+ strains in human body and study H . pylori infection and associated diseases.

Zhonghua Yi Xue Za Zhi, 1999 Mar, 79(3), 181 - 4
{A single strand comformation polymorphism of vacuolating cytotoxin gene in H . pylori}; Peng H et al.; OBJECTIVE: To use PCR/SSCP analysis of the vacuolating cytotoxin gene (vacA) of H . pylori for differentiation of various strains of H . pylori . METHODS: PCR was performed using the primers amplifing vacA gene of the bacteria embeded in the gastric mucosa of 159 patients with various gastric duodenal diseases . The products of PCR were further processed for SSCP analysis and southern blot hybridization . In the meantime, vacA genes of three different SSCP-patterns from three patients with duodenal ulcers were sequenced . RESULTS: The rate of detection of H . pylori with the method was 100% . vacA1 and vacA2, the two subtypes of vacA, were 76.5% (114/149) and 23.5%(35/149), respectively . Eight different SSCP-patterns were distributed in various gastroduodenal diseases, and that 80% of duodenal ulcers was predominated with B pattern . Sequencing of DNA indicated a diversity of vacA gene structure . CONCLUSION: PCR/SSCP can be used in the differentiation of different strains of H . pylori in epidemology, and in the follow up study after H . pylori eradication, especially in the differentiation between H . pylori recrudescence and reinfection.

Am J Physiol Cell Physiol, 2001 Nov, 281(5), C1734 - 42
Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists; Galietta LV et al.; Drug discovery by high-throughput screening is a promising approach to develop new therapies for the most common lethal genetic disease, cystic fibrosis . Because disease-causing mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) protein produce epithelial cells with reduced or absent Cl(-) permeability, the goal of screening is to identify compounds that restore cell Cl(-) transport . We have developed a rapid, quantitative screening procedure for analysis of CFTR-mediated halide transport in cells with the use of a conventional fluorescence plate reader . Doubly transfected cell lines were generated that express wild-type or mutant CFTR together with a yellow fluorescent protein (YFP)-based halide sensor . CFTR function was assayed from the time course of cell fluorescence in response to extracellular addition of 100 mM I(-) followed by forskolin, resulting in decreased YFP fluorescence due to CFTR-mediated I(-) entry . Cell lines were chosen, and conditions were optimized to minimize basal halide transport to maximize assay sensitivity . In cells cultured on 96-well plastic dishes, the assay gave reproducible halide permeabilities from well to well and could reliably detect a 2% activation of CFTR-dependent halide transport produced by low concentrations of forskolin . Applications of the assay are shown, including comparative dose-dependent CFTR activation by genistein, apigenin, 8-cyclopentyl-1,3-dipropylxanthine, IBMX, 8-methoxypsoralen, and milrinone as well as activation of alternative Cl(-) channels . The fluorescence assay and cell lines should facilitate the screening of novel CFTR activators and the characterization of alternative Cl(-) channels and transporters.

Antimicrob Agents Chemother, 2001 Nov, 45(11), 3234 - 7
In vitro and in vivo effects of quinupristin-dalfopristin against Pneumocystis carinii; Walzer PD et al.; Quinupristin-dalfopristin (Q-D), which is active against bacteria and Toxoplasma gondii, was examined for its activity against Pneumocystis carinii . After 72 h of incubation with rat P . carinii in an ATP cytotoxicity assay, the 50% inhibitory concentration of Q-D was 10.6 microg/ml, a level that can be achieved in serum with high-dose administration . Q-D administered intraperitoneally at doses of 50 to 200 mg per kg of body weight per day in the treatment and 100 mg/kg/day three times per week in the prophylaxis of pneumocystosis in immunosuppressed mice reduced the organism burden up to 15- and 302-fold, respectively . We conclude that Q-D has activity against P . carinii in vitro and in vivo.

Med Microbiol Immunol (Berl), 2001 Sep, 189(4), 185 - 92
Contrasting responses of human gingival and colonic epithelial cells to lipopolysaccharides, lipoteichoic acids and peptidoglycans in the presence of soluble CD14; Uehara A et al.; Gingival epithelial cells may form the first barriers of defense against oral bacteria in periodontal tissues . We stimulated human gingival epithelial cells (keratinocytes) in primary culture, the oral epithelial cell line KB and the colonic epithelial cell line SW620 with various bacterial cell-surface components in the presence or absence of soluble CD14 (sCD14) . The SW620 produced interlukin-8 (IL-8) in an sCD14-dependent manner in response to lipopolysaccharide, lipoteichoic acid and peptidoglycan . However, the primary gingival epithelial cells and KB cells did not show enhanced production of IL-8 upon stimulation with these components even in the presence of serum . These human epithelial cells were devoid of membrane CD14, as determined by flow cytometry, and CD14 mRNA expression, as determined by reverse transcriptase-PCR . In contrast, gingival epithelial cells and KB cells expressed the mRNA expression for Toll-like receptor (TLR) 2, TLR4, MD2 and MyD88 to the similar extent to those observed in SW620 cells.

Med Microbiol Immunol (Berl), 2001 Sep, 189(4), 177 - 83
Natural antibodies to nematode biotinyl-enzymes in human sera; Lorenzo S et al.; Biotinyl-enzymes are conservative molecules present in helminths, as well as in other animals, bacteria and plants . They have recently been found to be antigenic in mice, and a potential source of cross-reactivity among helminths . This study investigated the presence in human sera of antibodies reactive with biotinyl-enzymes from the nematodes Anisakis simplex, Toxocara canis and Ascaris suum . Biotinyl-enzymes from all these nematodes were recognized by IgG1 antibodies in sera from healthy subjects and from Anisakis-free patients infected with other parasites . Interestingly, IgE antibodies reactive with Anisakis simplex biotinyl-enzymes were present in about one third of the sera from Anisakis-free patients infected with other parasites . Our results also demonstrate that the anti-BE IgG1 and IgE antibodies present in the sera of Anisakis-free subjects are cross-reactive among helminths . We conclude that biotinyl-enzymes from nematodes are recognized by natural human antibodies, although Anisakis biotinyl-enzymes do not seem to be the cause of sensitization . Since sera from the Anisakis-free population also present these antibodies, as-yet unidentified factors (dietary components, intestinal inflammation and/or the presence of parasites) may contribute to the induction of anti-BE antibody background.

Drugs Aging, 2001, 18(9), 639 - 64
Specific immunotherapy of cancer in elderly patients; Matzku S et al.; The concept of immunotherapy of cancer is more than a century old, but only recently have molecularly defined therapeutic approaches been developed . In this review, we focus on the most promising approach, active therapeutic vaccination . The identification of tumour antigens can now be accelerated by methods allowing the amplification of gene products selectively or preferentially transcribed in the tumour . However, determining the potential immunogenicity of such gene products remains a demanding task, since major histocompatibility complex (MHC) restriction of T cells implies that for any newly defined antigen, immunogenicity will have to be defined for any individual MHC haplotype . Tumour-derived peptides eluted from MHC molecules of tumour tissue are also a promising source of antigen . Tumour antigens are mostly of weak immunogenicity, because the vast majority are tumour-associated differentiation antigens already 'seen' by the patient's immune system . Effective therapeutic vaccination will thus require adjuvant support, possibly by new approaches to immunomodulation such as bispecific antibodies or antibody-cytokine fusion proteins . Tumour-specific antigens, which could be a more potent target for immunotherapy, mostly arise by point mutations and have the disadvantage of being not only tumour-specific, but also individual-specific . Therapeutic vaccination will probably focus on defined antigens offered as protein, peptide or nucleic acid . Irrespective of the form in which the antigen is applied, emphasis will be given to the activation of dendritic cells as professional antigen presenters . Dendritic cells may be loaded in vitro with antigen, or, alternatively, initiation of an immune response may be approached in vivo by vaccination with RNA or DNA, given as such or packed into attenuated bacteria . The importance of activation of T helper cells has only recently been taken into account in cancer vaccination . Activation of cytotoxic T cells is facilitated by the provision of T helper cell-derived cytokines . T helper cell-dependent recruitment of elements of non-adaptive defence, such as leucocytes, natural killer cells and monocytes, is of particular importance when the tumour has lost MHC class I expression . Barriers to successful therapeutic vaccination include: (i) the escape mechanisms developed by tumour cells in response to immune attack; (ii) tolerance or anergy of the evoked immune response; (iii) the theoretical possibility of provoking an autoimmune reaction by vaccination against tumour-associated antigens; and (iv) the advanced age of many patients, implying reduced responsiveness of the senescent immune system.

Orig Life Evol Biosph, 2001 Aug-Oct, 31(4-5), 403 - 34
The evolution of acetyl-CoA synthase; Lindahl PA et al.; Acetyl-coenzyme A synthases (ACS) are Ni-Fe-S containing enzymes found in archaea and bacteria . They are divisible into 4 classes . Class I ACS's catalyze the synthesis of acetyl-CoA from CO2 + 2e-, CoA, and a methyl group, and contain 5 types of subunits (alpha, beta, gamma, delta, and epsilon) . Class II enzymes catalyze essentially the reverse reaction and have similar subunit composition . Class III ACS's catalyze the same reaction as Class I enzymes, but use pyruvate as a source of CO2 and 2e-, and are composed of 2 autonomous proteins, an alpha 2 beta 2 tetramer and a gamma delta heterodimer . Class IV enzymes catabolize CO to CO2 and are alpha-subunit monomers . Phylogenetic analyses were performed on all five subunits . ACS alpha sequences divided into 2 major groups, including Class I/II sequences and Class III/IV-like sequences . Conserved residues that may function as ligands to the B- and C-clusters were identified . Other residues exclusively conserved in Class I/II sequences may be ligands to additional metal centers in Class I and II enzymes . ACS beta sequences also separated into two groups, but they were less divergent than the alpha's, and the separation was not as distinct . Class III-like beta sequences contained approximately 300 residues at their N-termini absent in Class I/II sequences . Conserved residues identified in beta sequences may function as ligands to active site residues used for acetyl-CoA synthesis . ACS gamma-sequences separated into 3 groups (Classes I, II, and III), while delta-sequences separated into 2 groups (Class I/II and III) . These groups are less divergent than those of alpha sequences . ACS epsilon-sequence topology showed greater divergence and less consistency vis-a-vis the other subunits, possibly reflecting reduced evolutionary constraints due to the absence of metal centers . The alpha subunit phylogeny may best reflect the functional diversity of ACS enzymes . Scenarios of how ACS and ACS-containing organisms may have evolved are discussed.

J Ind Microbiol Biotechnol, 2001 Jul, 27(1), 1 - 4
The polar polysaccharide capsule of Hyphomonas adhaerens MHS-3 has a strong affinity for gold; Quintero EJ et al.; Select groups of bacteria, including prothescate species, have an unusual capacity to sequester gold and bioconcentrate it to very high levels . Hyphomonas adhaerens MHS-3 (MHS-3) is one such species, as demonstrated by Energy Dispersive Spectroscopy . Transmission electron microscopy revealed that the binding site was specific on the polar polysaccharide capsule . A capsuleless mutant and periodate-treated wild type did not sequester gold . The gold may interact with the same sites in the capsule that naturally adhere MHS-3 to surfaces in the marine environment.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2001 Oct, 92(4), 384 - 9
Substance P and mast cells: preliminary histologic analysis of the human temporomandibular joint; Henry CH et al.; PURPOSE: Neuropeptide-containing nerves can serve as a mechanism for nervous system regulation of host defense responses . Because bacteria associated with reactive arthritis have been identified in the temporomandibular joint (TMJ), this study investigates whether the presence of substance P (SP) neuropeptide-containing nerves and mast cells can be identified in the TMJ . MATERIAL AND METHODS: Posterior bilaminar tissue removed during TMJ surgery from 9 women was evaluated for the presence of neuropeptide-containing nerves by staining with a monoclonal antibody to SP . Staining of the TMJ tissue sections with 0.5% toluidine blue was performed to identify the presence of mast cells . RESULTS: SP-containing nerves and mast cells were identified within the posterior bilaminar tissue associated with the vasculature . CONCLUSIONS: The presence of neuropeptide nerves and mast cells within the TMJ has been shown . Mast cell degranulation products and SP release can contribute to TMJ inflammation.

Infect Immun, 2001 Nov, 69(11), 6738 - 48
Characterization of a Brucella species 25-kilobase DNA fragment deleted from Brucella abortus reveals a large gene cluster related to the synthesis of a polysaccharide; Vizcaino N et al.; In the present study we completed the nucleotide sequence of a Brucella melitensis 16M DNA fragment deleted from B . abortus that accounts for 25,064 bp and show that the other Brucella spp . contain the entire 25-kb DNA fragment . Two short direct repeats of four nucleotides, detected in the B . melitensis 16M DNA flanking both sides of the fragment deleted from B . abortus, might have been involved in the deletion formation by a strand slippage mechanism during replication . In addition to omp31, coding for an immunogenic protein located in the Brucella outer membrane, 22 hypothetical genes were identified . Most of the proteins that would be encoded by these genes show significant homology with proteins involved in the biosynthesis of polysaccharides from other bacteria, suggesting that they might be involved in the synthesis of a Brucella polysaccharide that would be a heteropolymer synthesized by a Wzy-dependent pathway . This polysaccharide would not be synthesized in B . abortus and would be a polysaccharide not identified until present in the genus Brucella, since all of the known polysaccharides are synthesized in all smooth Brucella species . Discovery of a novel polysaccharide not synthesized in B . abortus might be interesting for a better understanding of the pathogenicity and host preference differences observed between the Brucella species . However, the possibility that the genes detected in the DNA fragment deleted in B . abortus no longer lead to the synthesis of a polysaccharide must not be excluded . They might be a remnant of the common ancestor of the alpha-2 subdivision of the class Proteobacteria, with some of its members synthesizing extracellular polysaccharides and, as Brucella spp., living in association with eukaryotic cells.

Am J Physiol Lung Cell Mol Physiol, 2001 Nov, 281(5), L1095 - 105
Apoptosis by pan-caspase inhibitors in lipopolysaccharide-activated macrophages; Kim SO et al.; Although apoptosis has been observed in macrophages during the course of infections, the mechanism of apoptosis in activated macrophages is not fully understood . This study shows that pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD) or t-butyloxycarbonyl-Asp-fluoromethylketone (Boc-D) caused the death of lipopolysaccharide (LPS)-activated macrophages and RAW 264.7 cells with apoptotic features . The apoptosis was also observed in lipoprotein-treated bacteria but not in CpG oligonucleotide- or flagellin-treated macrophages, indicating a difference of cellular responses downstream of different Toll-like receptors . Consistent with the induction of cell death by pan-caspase inhibitors, no activation of known caspases was detected in LPS-ZVAD-treated cells, suggesting an involvement of unknown proapoptotic caspases in the cell death . ZVAD inhibited the activation of extracellular signal-regulated kinase (ERK) and p38 but not of nuclear factor (NF)-kappa B induced by LPS, suggesting that the ZVAD-sensitive molecule lies upstream of the ERK and p38 pathways but downstream of the divergent site of NF-kappa B and mitogen-activated protein kinases . Our results demonstrate that apoptosis of macrophages induced by LPS+ZVAD is independent from the known proapoptotic caspases and suggest that activity of an unidentified ZVAD-sensitive molecule(s) is involved in the survival of LPS-activated macrophages.

Am J Physiol Lung Cell Mol Physiol, 2001 Nov, 281(5), L1037 - 50
Transcriptional mechanisms of acute lung injury; Fan J et al.; Acute lung injury occurs as a result of a cascade of cellular events initiated by either infectious or noninfectious inflammatory stimuli . An elevated level of proinflammatory mediators combined with a decreased expression of anti-inflammatory molecules is a critical component of lung inflammation . Expression of proinflammatory genes is regulated by transcriptional mechanisms . Nuclear factor-kappa B (NF-kappa B) is one critical transcription factor required for maximal expression of many cytokines involved in the pathogenesis of acute lung injury . Activation and regulation of NF-kappa B are tightly controlled by a complicated signaling cascade . In acute lung injury caused by infection of bacteria, Toll-like receptors play a central role in initiating the innate immune system and activating NF-kappa B . Anti-inflammatory cytokines such as interleukin-10 and interleukin-13 have been shown to suppress inflammatory processes through inhibiting NF-kappa B activation . NF-kappa B can interact with other transcription factors, and these interactions thereby lead to greater transcriptional selectivity . Modification of transcription is likely to be a logical therapeutic target for acute lung injury.

J Theor Biol, 2001 Oct 21, 212(4), 425 - 56
A biokinetic model to describe consequences of inhibition/stimulation in DNA-proofreading and repair-1 . Development of the model; Haschke H; A biokinetic model is described which deals with the mathematical consequences of the inhibition or stimulation of DNA proofreading . It demonstrates the development of the number of DNA mismatch-dependent cells (e.g . cells with a malignant phenotype), where such mismatches arise by the in situ interaction of various substances with nucleotides of the DNA . The model can test for consequences by a logic gating on an "if-then" type of analysis in relation to the separate and consecutive processes of proofreading and repair . In particular, the consequences are considered in cases where either (i) the efficacy of proofreading and repair are reduced/prevented (inhibited) or (ii) are increased by some form of stimulation . On the chosen kinetic parameters, the model is accessible to manipulation as new data arising from further investigations become available and are introduced . The model is based on recently published data which show that an increased "mutant fraction" (see note on terms) arises in DNA replication when intracellular nucleotide pools show "asymmetries" (see note on terms) . Extraordinarily high mutant fractions can be predicted/have been recorded in the presence of proofreading inhibitors . The model expresses data in mathematical terms of the competition between the development of mismatch-dependent cells and those with authentic genetic information . (Feedback and metastasis-effects and those of wild-type replicates are included.) A computerized (numerical) integration of the corresponding set of differential equations is offered . (A diskette with the program CANCER.xls is available upon request.)

J Heart Lung Transplant, 2001 Oct, 20(10), 1064 - 74
Bronchoalveolar lavage macrophage and lymphocyte phenotypes in lung transplant recipients; Ward C et al.; Recent publications have demonstrated potentially pathologic changes in bronchoalveolar lavage (BAL) from clinically stable lung transplant recipients (SLTRs), but there are few available data on alveolar macrophages (AMs) . We formulated the hypothesis that changes in BAL AM and lymphocyte phenotypes would be apparent even in SLTRs.A cross-sectional study using a standardized 3 x 60 ml BAL, investigating lymphocyte and AM phenotypes in 19 SLTRs, 5 subjects with bronchiolitis obliterans syndrome (BOS) and 18 normal control volunteers . BAL lymphocyte and AM markers were assessed using flow cytometry.We confirmed a significant elevation of neutrophils in all lung transplant recipients with a more marked elevation in the BOS subjects . Flow-cytometric analysis showed increased numbers of natural killer (NK; CD56/CD16-positive) cells, increased CD11b- and CD11c-positive CD3 lymphocytes, increased CD8-positive lymphocytes and increased HLA-DR expression in CD8 cells from the lung transplant recipients, when compared with normals (p <.005) . In contrast, the expression of a number of AM surface markers, associated with a range of host defense functions against bacteria, fungi and viruses (CD11a, CD11b, CD11c, HLA-DR, CD14), was lower in both SLTRs and those with BOS (p <.05).These novel findings are consistent with complex lymphocyte and macrophage changes that may result from clinically silent infection, partially suppressed rejection, or both.

J Physiol Paris, 2001 Jan-Dec, 95(1-6), 469 - 75
Eradication of Helicobacter pylori has no effect on gastric acidity in duodenal ulcer patients--evaluation of 24-h pH monitoring; Racz I et al.; It is accepted that eradication of Helicobacter pylori leads to healing of chronic active gastritis facilitates ulcer healing and prevents ulcer recurrence in duodenal ulcer (DU) patients . However, it is not entirely known whether the eradication of the bacteria normalizes gastric acid secretion and abolishes dyspeptic symptoms after ulcer healing . This study was aimed to evaluate the intragastric acidity and dyspeptic complaints before, and 3 months after, eradication in 18 endoscopically proven H . pylori positive DU patients . Gastric pH was measured by 24-h continuous intraluminal recording, serum gastrin measurements and Congo-red tests were also performed . Dyspeptic complaints and antacid consumptions were recorded in diary cards, antisecretory therapy was not allowed after the cessation of eradication therapy . Endoscopy, H . pylori status and Congo-red tests were controlled at the 6th and 12th week, while pH measurements and serum gastrin tests were performed at inclusion and 3 months later . Three patients dropped out and in 14 out of the remaining subjects healing of DUs and successful eradication was achieved by the 6th and 12th week controls . The 24-h median pH and the percentage of 24-h pH readings under pH 3 were not changing significantly by the 3-month controls (from 1.9+/-0.5 to 1.8+/-0.4 and from 52.6+/-5.5% to 58.6+/-5%, respectively) . Similarly, no significant changes were observed in serum gastrin levels and dyspeptic symptom scores (from 72+/-7 pg/ml to 56.7+/-8 pg/ml and from 2.69+/-0.4 to 1.26+/-0.3, respectively) . The antacid consumption was almost stable when compared with the pre- and post-eradication periods . It was concluded that despite successful H . pylori eradication and healing of DU, intragastric acidity does not change significantly at least 3 months after the therapy . The persisting dyspeptic symptoms and the need for antacid consumption suggest that some healed ulcer patients require antisecretory therapy in the post-eradication period.

Aquat Toxicol, 2001 Nov 12, 55(3-4), 177 - 90
Environmental endocrine disruption in decapod crustacean larvae: hormone titers, cytochrome P450, and stress protein responses to heptachlor exposure; Snyder MJ et al.; A variety of enzymes and other proteins are produced by organisms in response to xenobiotic exposures . Cytochrome P450s (CYP) are one of the major phase I-type classes of detoxification enzymes found in terrestrial and aquatic organisms ranging from bacteria to vertebrates . One of the primary functions of stress proteins (HSPs) is to aid in the recovery of damaged proteins by chaperoning their refolding . These and other biomarkers of xenobiotic exposure and resulting effects have not been studied in crustacean larvae . This information is of potential importance for environmental management and risk assessment . In this work, we have given Homarus americanus larvae single 24 h exposures to the cyclodiene pesticide heptachlor, a known environmental endocrine disruptor (EDC) on different days of the 1st larval instar . We followed these larvae during the first larval stage for effects on timing of ecdysis to 2nd stage, ecdysteroid molting hormone titers, and alterations in the levels of cytochrome P450 CYP45 and HSP70 proteins . Delays in ecdysis were correlated with alterations in ecdysteroid levels . This result provides clues that this pesticide may function as an environmental endocrine disruptor in crustaceans . CYP45 and HSP70 levels were significantly elevated for several days following heptachlor exposure . The elevation in HSP70 was prolonged depending on the day of pesticide exposure and this was directly related to the increase in mortality . These results demonstrate the utility of these measurements as potential biomarkers in crustacean larval developmental toxicology and EDC effects research.

Int J Syst Evol Microbiol, 2001 Sep, 51(Pt 5), 1745 - 9
Globicatella sulfidifaciens sp . nov., isolated from purulent infections in domestic animals; Vandamme P et al.; DNA-DNA hybridization experiments and comparative 165 rDNA sequence analysis revealed that six isolates from purulent joint and lung infections in calves, from a lung lesion in a sheep, and from a joint infection of a pig represented a novel species belonging to the genus Globicatella . Whole-cell protein electrophoresis and biochemical activity testing revealed that the isolates formed a homogeneous group differing from Globicatella sanguinis, the only species of this genus described to date . These animal isolates were classified as Globicatella sulfidifaciens sp . nov . with LMG 188441 (= CCUG 44365T), isolated from the lung of a calf, as the type strain . A detailed description of its phenotypic characteristics is presented . Hydrogen sulphide production was found to be a very useful diagnostic feature.

Ann N Y Acad Sci, 2001 Sep, 941, 1 - 11
Cutaneous T cell lymphoma: the helping hand of dendritic cells; Edelson RL; Since its introduction 25 years ago, cutaneous T cell lymphoma has become the preferred designation for clonal malignancies of those CD4 thymus-derived lymphocytes ("cutaneous T cells") that preferentially migrate to skin . The varied cutaneous clinical presentations, dependent on the specific features of the dominant subclones of the malignant lymphocytes, historically led to confusing descriptive terms (mycosis fungoides, Sezary syndrome, lymphoma cutis, leukemia cutis, reticulum cell sarcoma of the skin) . Recognition that all of these clinical presentations are cancers of a single type of cell has permitted their unification under the single, clarified heading cutaneous T cell lymphoma, or CTCL . As a neoplastic amplification of the skin-homing T cells from which it is derived, CTCL's distinctive features can be explained . The triad of skin localization, remarkable avoidance of bone marrow, often even in the context of extremely high leukemic counts, and infiltration of perifollicular T cell zones of the lymph nodes and spleen reflect the migratory pathway and homing patterns of cutaneous T cells . The usually retained levels of serum immunoglobulins and the resulting capacity to defend against encapsulated bacteria, often even in advanced CTCL, are manifestations of the helper function of the malignant T cells-that is, their functional capacity to stimulate B lymphocytes to produce immunoglobulin in a polyclonal manner . In contrast, the often-extreme normal T cell deficits in advanced CTCL, equivalent to those of late-stage AIDS, probably resulting from the production of suppressive cytokines such as IL-10, cause susceptibility to a broad range of opportunistic infections, the most common direct cause of death . Pautrier microabscesses, the pathognomonic feature of epidermotropic early CTCL, hold the clues to the pathogenesis of the cancer . These intraepidermal collections of stimulated and proliferating malignant cells, adherent to the dendrites of intraepidermal dendritic antigen-presenting cells (Langerhans' cells {LCs}), indicate a dynamic communication between the two cell types . Since CTCL cells are derived from CD4 T cells, which normally receive signaling from dendritic cells (DCs) via presentation of antigenic peptides as part of class II major histocompatibility complexes to antigen-specific T cell receptors (TCRs), it seems likely that CTCL is a clonal proliferation of T cells responding to specific antigenic stimulation from LCs . This is supported by our recent finding that CTCL cells proliferate in vitro in response to TCR stimulation by autologous DCs, which have previously ingested and processed antigens from apoptotic autologous CTCL cells . In short, CTCL may be a malignancy of T cells stimulated to proliferate against its own tumor antigens . The most intriguing possibility is that a yet-unidentified transforming retrovirus, harbored by LCs, simultaneously attracts, stimulates, and transforms a single clone of antigen-specific cutaneous T cells . Longstanding disease-free remissions have been induced by transimmunization (via a photopheresis apparatus) . This treatment, introduced more than a decade ago by our group and the first and still the only FDA-approved selective anticancer immunotherapy, has been performed more than 200,000 times worldwide on advanced CTCL, as well as in reversal/prevention of heart transplant rejection and treatment of graft-versus-host disease and selected autoimmune disorders . Transimmunization induces clinically relevant suppression, and occasionally elimination, of pathogenic T cell clones . The common denominator between these diverse groups of responding patients is the presence of clonally distinctive TCRs on the disease-causing malignant or autoaggressive T cell clones . In CTCL at least one source of tumor-specific antigens is derived from the clone-specific (idiotypic) segments of the TCR protein chains . In the photopheresis apparatus, two synergistic phenomena are initiated: induction of apoptosis of the CTCL cells and mass conversion of blood monocytes to DCs . The young DCs then ingest the apoptotic CTCL cells, process and present the CTCL antigens to responding anti-CTCL cytotoxic T cells, and stimulate clinically important CTCL suppression . Now that it is better understood, transimmunization may have much broader applications in other types of cancer as well.

Int J Hematol, 2001 Aug, 74(2), 209 - 13
A clinical analysis of 52 adult patients with hemophagocytic syndrome: the prognostic significance of the underlying diseases; Takahashi N et al.; We retrospectively analyzed 52 adult patients with hemophagocytic syndrome (HPS) . The underlying diseases were heterogeneous, including malignant lymphoma (lymphoma-associated hemophagocytic syndrome {LAHS}) in 26 patients, systemic lupus erythematosus in 3 patients, viral infections in 7 patients, and bacteria} or fungal infections in 6 patients . More than 83% of patients received prednisolone as an initial treatment . Multiple-agent chemotherapies (cyclophosphamide, doxorubicin, and vincristine) were administered to 96% of LAHS patients after a histopathological diagnosis of lymphoma . HPSs were controllable and remissions were achieved except for those patients with LAHS, fulminant Epstein-Barr virus-associated HPS, and an immunosuppressive state . Twenty-one (81%) of the LAHS patients had uncontrollable HPS and died of multiple organ failure and disseminated intravascular coagulation . The median survival time of LAHS patients was 83 days . In contrast, 3 (12%) of the other HPS patients died of multiple organ failure within 44 days.The clinical manifestations and the laboratory findings of LAHS and the other HPSs were too variable to establish the prognosis based only on the findings at the onset of HPS . The prognostic factors of adult HPS were found to be the underlying diseases, notably malignant lymphoma and infections, accompanied by the immunosuppressive state.

Int J Hematol, 2001 Aug, 74(2), 134 - 43
Neutrophil functions of patients with vasculitis related to myeloperoxidase-specific anti-neutrophil antibody; Suzuki K; Neutrophils are hypothesized to cause tissue damage resulting in the development of vasculitis and glomerulonephritis, although they are known to primarily take part in host defense functions . The infiltration of inflammatory cells . notably neutrophils and macrophages, is observed in the progression of vasculitis . Neutrophils with activated status and anti-neutrophil cytoplasmic antibodies (ANCAs), especially myeloperoxidase-specific (MPO)-ANCA, have been implicated in the development of vasculitis . The target molecule of MPG-ANCA is a lysosomal enzyme MPO that usually acts to kill bacteria, viruses, and fungi and that causes damage to the tissue due to the toxicity of its product, hypochlorite (OCl-) . To elucidate the role of MPO-ANCA in the progression of vasculitis, a set of MPO-peptide fragments has been developed, and the corresponding epitope site for the specific monoclonal and/or oligoclonal antibody resulting in vasculitis has been determined . Recently some mouse models have been used for analyzing the correlation between MPO and MPO-ANCA in relation to damage of blood vessels followed by the development of vasculitis . This review focuses on the role of activated neutrophils in the development of vasculitis associated with MPO-ANCA and the target molecules of ANCA . In addition, the reactivities of ANCA and inflammatory cytokines involving leukocyte-derived chemotaxin 2 (LECT2) are also discussed.

Zhonghua Zheng Xing Shao Shang Wai Ke Za Zhi, 1999 Jul, 15(4), 298 - 300
{The time course and tissue distribution of endotoxin in rats after thermal injury}; Fang W et al.; OBJECTIVE: To observe the time course and tissue distribution of endotoxin, and to evaluate the potential role of local endotoxin in mediating multiple organ damage following burns . METHODS: Wistar rats were subjected to a 35 percent full-thickness scald injury . They were grouped according to time of sacrifice: no burn, and 12, 24, 48, 72 hours postburn . Tissue specimens from liver, kidneys, lungs and intestine were aseptically harvested to determine tissue endotoxin levels . In addition, blood samples were obtained for measurement of organ function parameters . RESULTS: It was found that endotoxin levels in liver, spleen and lungs increased markedly after thermal injury, with the highest level in liver . Meanwhile, serum GPT, GOT, CK-MB levels, and pulmonary myeloperoxidase(MPO) activities markedly elevated after scald injury, reaching a maximum between 12 and 24 hours . Intestinal diamine oxidase(DAO) activities tended to decrease at various time points postburn . In addition, there were highly positive correlations between pulmonary endotoxin levels and MPO activities, and also between renal endotoxin levels and serum Cr values . CONCLUSION: Burn injury per se can result in gut-derived endotoxin translocation which is mainly distributed to the liver, spleen and lungs . Endotoxin accumulated in local sites may play an important role in mediating multiple organ damage secondary to major burns.

Proc Natl Acad Sci U S A, 2001 Oct 23, 98(22), 12414 - 9 Epub 2001 Oct 09.
Mono(ADP-ribosyl)ation of 2'-deoxyguanosine residue in DNA by an apoptosis-inducing protein, pierisin-1, from cabbage butterfly; Takamura-Enya T et al.; Pierisin-1 is a potent apoptosis-inducing protein derived from the cabbage butterfly, Pieris rapae . It has been shown that pierisin-1 has an A small middle dotB structure-function organization like cholera or diphtheria toxin, where the "A" domain (N-terminal) exhibits ADP-ribosyltransferase activity . The present studies were designed to identify the target molecule for ADP-ribosylation by pierisin-1 in the presence of beta-{adenylate-(32)P}NAD, and we found DNA as the acceptor, but not protein as is the case with other bacteria-derived ADP-ribosylating toxins . ADP-ribosylation of tRNAs from yeast was also catalyzed by pierisin-1, but the efficiency was around 110 of that for calf thymus DNA . Pierisin-1 efficiently catalyzed the ADP-ribosylation of double-stranded DNA containing dG small middle dotdC, but not dA small middle dotdT pairs . The ADP-ribose moiety of NAD was transferred to the amino group at N(2) of 2'-deoxyguanosine to yield N(2)-(alpha-ADP-ribos-1-yl)-2'-deoxyguanosine and its beta form, which were determined by several spectral analyses including (1)H- and (13)C-NMR and mass spectrometry . The chemical structures were also ascertained by the independent synthesis of N(2)-(D-ribos-1-yl)-2'-deoxyguanosine, which is the characteristic moiety of ADP-ribosylated dG . Using the (32)P-postlabeling method, ADP-ribosylated dG could be detected in DNA from pierisin-1-treated HeLa cells, in which apoptosis was easily induced . Thus, the targets for ADP-ribosylation by pierisin-1 were concluded to be 2'-deoxyguanosine residues in DNA . This finding may open a new field regarding the biological significance of ADP-ribosylation.

Biotechnol Appl Biochem, 2001 Oct, 34(Pt 2), 99 - 102
Improvement of tagatose conversion rate by genetic evolution of thermostable galactose isomerase; Kim P et al.; To enhance the isomerization rate of galactose into tagatose, a thermostable galactose isomerase, which was isolated from bacteria growing in a hot spring, was genetically improved using the error-prone PCR method . From 500 mutant clones, a clone showing improved conversion activity was selected . The sequence of the selected clone had five amino acid changes: His(228)-->Asp, Gly(384)-->Asp, Ser(393)-->Thr, Lys(428)-->Asn and Asp(475)-->Lys . The improved galactose isomerase had an 11-fold higher reaction rate than the original.

Microbiol Immunol, 2001, 45(8), 563 - 9
Stenotrophomonas maltophilia interaction with human epithelial respiratory cells in vitro; De Vidipo LA et al.; Bacteria of Stenotrophomonas maltophilia have been isolated with increasing frequency from the airways of cystic fibrosis (CF) patients, usually following P . aeruginosa infections, but their adherence to human epithelial respiratory cells has never been investigated . In this study, various S . maltophilia strains were seen to adhere to epithelial respiratory cells in vitro, mainly along intercellular junctions . Bacteria could also enter into host cells, as determined by the gentamicin exclusion assay and transmission electron microscopy . Cells co-incubated with P . aeruginosa and S . maltophilia exhibited a significantly decreased adherence of these latter bacteria . No decrease in S . maltophilia adherence was observed when co-infection was carried out with heat-killed P . aeruginosa or when respiratory cells were first incubated with P . aeruginosa, before incubation with S . maltophilia . Our data suggest that P . aeruginosa infections do not account for the increased prevalence of S . maltophilia in CF patient airways, that thermolabile products from P . aeruginosa can control the adherence of S . maltophilia to respiratory cells and also that these two bacteria do not compete for cell receptors.

Biol Chem, 2001 Aug, 382(8), 1147 - 56
This is the end: processing, editing and repair at the tRNA 3'-terminus; Schurer H et al.; The generation of a mature tRNA 3'-end is an important step in the processing pathways leading to functional tRNA molecules . While 5'-end processing by RNase P is similar in all organisms, generation of the mature 3'-terminus seems to be more variable and complex . The first step in this reaction is the removal of 3'-trailer sequences . In bacteria, this is a multistep process performed by endo- and exonucleases . In contrast, the majority of eukaryotes generate the mature tRNA 3'-end in a single step reaction, which consists of an endonucleolytic cut at the tRNA terminus . After removal of the 3'-trailer, a terminal CCA triplet has to be added to allow charging of the tRNA with its cognate amino acid . The enzyme catalyzing this reaction is tRNA nucleotidyltransferase, homologs of which have been found in representatives of all three kingdoms . Furthermore, in metazoan mitochondria, some genes encode 3'-terminally truncated tRNAs, which are restored in an editing reaction in order to yield functional tRNAs . Interestingly, this reaction is not restricted to distinct tRNAs, but seems to act on a variety of tRNA molecules and represents therefore a more general tRNA repair mechanism than a specialized editing reaction . In this review, the current knowledge about these crucial reactions is summarized.

J Bacteriol, 2001 Nov, 183(21), 6344 - 54
Interactive control of Rhodobacter capsulatus redox-balancing systems during phototrophic metabolism; Tichi MA et al.; In nonsulfur purple bacteria, redox homeostasis is achieved by the coordinate control of various oxidation-reduction balancing mechanisms during phototrophic anaerobic respiration . In this study, the ability of Rhodobacter capsulatus to maintain a balanced intracellular oxidation-reduction potential was considered; in addition, interrelationships between the control of known redox-balancing systems, the Calvin-Benson-Bassham, dinitrogenase and dimethyl sulfoxide reductase systems, were probed in strains grown under both photoheterotrophic and photoautotrophic growth conditions . By using cbb(I) (cbb form I operon)-, cbb(II)-, nifH-, and dorC-reporter gene fusions, it was demonstrated that each redox-balancing system responds to specific metabolic circumstances under phototrophic growth conditions . In specific mutant strains of R . capsulatus, expression of both the Calvin-Benson-Bassham and dinitrogenase systems was influenced by dimethyl sulfoxide respiration . Under photoheterotrophic growth conditions, coordinate control of redox-balancing systems was further manifested in ribulose 1,5-bisphosphate carboxylase/oxygenase and phosphoribulokinase deletion strains . These findings demonstrated the existence of interactive control mechanisms that govern the diverse means by which R . capsulatus maintains redox poise during photoheterotrophic and photoautotrophic growth.

Rev Esp Cardiol, 2001 Oct, 54(10), 1135 - 40
{Inflammation and acute coronary syndromes}; Chierchia SL; A great variety of stimuli, such as free radicals, oxidized LDL or some bacteria or virus infections, can act upon the vascular surface and lead to the development of an acute inflammatory reaction . There is more and more evidence supporting the hypothesis that the mechanism responsible for the transformation of a non-complicated atherosclerotic lesion into an hemorrhagic and ulcerated lesion, with the subsequent acute and unstable clinical status, is due to the onset of an inflammatory reaction . Many studies have tried to investigate the presence of any systemic marker able to predict the prognosis of patients at risk from developing acute events, and to distinguish them from those in stable status . The increase of the levels of C-reactive-protein has been related to the development of acute coronary syndromes, though often the results obtained in the different studies have had a quite poor prognostic value when applied to the general population . The lack of direct association between the increase of the levels of C-reactive-protein and Troponin I seems to rule out the possibility that the inflammatory stimulus might be the consequence of an irreversible injury, even though there is no doubt that severe ischemia is likely to play an active role in this sense.

Biochemistry, 2001 Oct 16, 40(41), 12444 - 56
Chromophore attachment to biliproteins: specificity of PecE/PecF, a lyase-isomerase for the photoactive 3(1)-cys-alpha 84-phycoviolobilin chromophore of phycoerythrocyanin; Storf M et al.; PecE and PecF, the products of two phycoerythrocyanin lyase genes (pecE and pecF) of Mastigocladus laminosus (Fischerella), catalyze two reactions: (1) the regiospecific addition of phycocyanobilin (PCB) to Cys-alpha 84 of the phycoerythrocyanin alpha-subunit (PecA), and (2) the Delta 4-->Delta 2 isomerization of the PCB to the phycoviolobilin (PVB)-chromophore {Zhao et al . (2000) FEBS Lett . 469, 9-13} . The alpha-apoprotein (PecA) as well PecE and PecF were overexpressed from two strains of M . laminosus, with and without His-tags . The products of the spontaneous addition of PCB to PecA, and that of the reaction catalyzed by PecE/F, were characterized by their photochemistry and by absorption, fluorescence, circular dichroism of the four states obtained by irradiation with light (15-Z/E isomers of the chromophore) and/or modification of Cys-alpha 98/99 with thiol-directed reagents . The spontaneous addition leads to a 3(1)-Cys-PCB adduct, which is characteristic of allophycocyanins and phycocyanins, while the addition catalyzed by PecE and PecF leads to a 3(1)-Cys-PVB adduct which after purification was identical to alpha-PEC . The specificity and kinetics of the chromophore additions were investigated with respect to the structure of the bilin substrate: The 3-ethylidene-bilins, viz., PCB, its 18-vinyl analogue phytochromobilin, phycoerythrobilin and its dimethylester, react spontaneously to yield the conventional addition products (3-H, 3(1)-Cys), while the 3-vinyl-substituted bilins, viz., bilirubin and biliverdin, were inactive . Only phycocyanobilin and phytochromobilin are substrates to the addition-isomerization reaction catalyzed by PecE/F . The slow spontaneous addition of phycoerythrobilin is not influenced, and there is in particular no catalyzed isomerization to urobilin.

Luminescence, 2001 Sep-Oct, 16(5), 315 - 9
Effect of aqueous cigarette smoke extract on the chemiluminescence kinetics of polymorphonuclear leukocytes and on their glycolytic and phagocytic activity; Zappacosta B et al.; Water-soluble extracts of cigarette smoke are easily formed in some body compartments, such as saliva or fluid lining alveolar spaces, and can act on both cellular and extracellular compartments . In this paper we have analysed the effect of aqueous smoke extract on some metabolic and functional aspects of polymorphonuclear leukocytes . In particular, the following cellular aspects were studied: chemiluminescence, glycolysis, membrane fluidity and microscopic interaction with zymosan particles . While chemiluminescence and glycolytic activity are highly inhibited, no effect of smoke extract on membrane fluidity was observed . Moreover, the response of luminol-dependent chemiluminescence was significantly delayed, while that of lucigenin-dependent chemiluminescence was anticipated . Furthermore, the phagocytic ability of neutrophils pretreated with aqueous smoke extract was also significantly hindered . All these results might indicate that the finely tuned activity of polymorphonuclear leukocytes is somehow hampered by the aqueous extract of cigarette smoke in a way which makes these cells less effective against bacteria and more noxious towards surrounding tissues .

Trends Biochem Sci, 2001 Oct, 26(10), 624 - 31
Immunity proteins: enzyme inhibitors that avoid the active site; Kleanthous C et al.; Immunity proteins are high affinity inhibitors of colicins--SOS-induced toxins released by bacteria during times of stress . Recent work has shown that nuclease-specific immunity proteins are exosite inhibitors, binding adjacent to the enzyme active site and inhibiting colicin activity indirectly . Unusually, their binding sites comprise a near contiguous sequence that lies N-terminal to active site sequences, raising the possibility that immunity proteins bind colicins co-translationally . Exosite binding accounts for the extensive sequence diversity seen at the interfaces of colicin-immunity protein complexes, which is not only a selective advantage to colicin-producing bacteria, but also represents a powerful model system for studying specificity in protein-protein recognition.

Eur J Biochem, 2001 Oct, 268(19), 5176 - 88
Identification of coenzyme M biosynthetic 2-phosphosulfolactate phosphatase . A member of a new class of Mg(2+)-dependent acid phosphatases; Graham DE et al.; Coenzyme M (CoM; 2-mercaptoethanesulfonic acid) is the terminal methyl carrier in methanogenesis . Methanogenic archaea begin the production of this essential cofactor by sulfonating phosphoenolpyruvate to form 2-phospho-3-sulfolactate . After dephosphorylation, this precursor is oxidized, decarboxylated and then reductively thiolated to form CoM . A thermostable phosphosulfolactate phosphohydrolase (EC 3.1.3.-) catalyzing the second step in CoM biosynthesis, was identified in the hyperthermophilic euryarchaeon Methanococcus jannaschii . The predicted ORF MJ1140 in the genome of M . jannaschii encodes ComB, a Mg2+-dependent acid phosphatase that is specific for 2-hydroxycarboxylic acid phosphate esters . Recombinantly expressed purified ComB efficiently hydrolyzes rac-2-phosphosulfolactate, (S)-2-phospholactate, phosphoglycolate and both enantiomers of 2-phosphomalate . In contrast to previously studied phosphoglycolate phosphatases, ComB has a low pH optimum for activity, a narrow substrate specificity and an amino acid sequence dissimilar to any biochemically characterized protein . Like other phosphatases that function via covalent phosphoenzyme intermediates, ComB can catalyze a transphosphorylation reaction . Homologs of comB are identified in all available cyanobacterial genome sequences and in genomes from phylogenetically diverse bacteria and archaea; most of these organisms lack homologs of other CoM biosynthetic genes . The broad and disparate distribution of comB homologs suggests that the gene has been recruited frequently into new metabolic pathways.

Cell Biol Int, 2001, 25(10), 985 - 90
Detection of submicroscopic magnetite particles using reflectance mode confocal laser scanning microscopy; Green CR et al.; Light microscopy and transmission electron microscopy work at such different scales that some components of cells may be too small to detect using light microscopy but too dispersed among cells within tissues to be discovered using electron microscopy . We have used reflectance mode confocal laser scanning microscopy to detect single-domain magnetite crystals in both live and resin-embedded preparations of magnetotactic bacteria . We show that reflections from bacterial cells are uniquely associated with the magnetite, which underpins the magnetotactic response of the bacteria . En bloc viewing shows that relatively large volumes of material can be searched with sufficient resolution to enable detection of submicroscopic particles . The techniques reported here may be of interest to others wishing to detect submicroscopic objects dispersed in large volumes of tissue .

Biol Sci Space, 2000 Dec, 14(4), 353 - 62
{Psycrophilic organisms in snow and ice}; Kohshima S; Psychrophilic and psycrotrophic organisms are important in global ecology as a large proportion of our planet is cold . Two-third of sea-water covering more than 70% of Earth is cold deep sea water with temperature around 2 degrees C, and more than 90% of freshwater is in polar ice-sheets and mountain glaciers . Though biological activity in snow and ice had been believed to be extremely limited, various specialized biotic communities were recently discovered at glaciers of various part of the world . The glacier is relatively simple and closed ecosystem with special biotic community containing various psychrophilic and psycrotrophic organisms . Since psychrophilic organisms was discovered in the deep ice-core recovered from the antarctic ice-sheet and a lake beneath it, snow and ice environments in Mars and Europa are attracting a great deal of scientific attention as possible extraterrestrial habitats of life . This paper briefly reviews the results of the studies on ecology of psychrophilic organisms living in snow and ice environments and their physiological and biochemical adaptation to low temperature.

J Clin Gastroenterol, 2001 Oct, 33(4), 330 - 2
Esophageal dysmotility in an adult with chronic granulomatous disease; Golioto M et al.; Chronic granulomatous disease (CGD) is a group of hereditary disorders of impaired intracellular destruction of phagocytosed bacteria . Gastrointestinal manifestations are present, with hepatic abscess being the most common . In this case report, we present an adult with CGD with esophageal involvement, which has been described in only one other adult . The clinical history, modalities of diagnosis (including endoscopy, barium radiography, and esophageal manometry), and therapeutic strategies pertaining to the esophageal manifestations of CGD are discussed . A review of the pertinent available literature is provided.

Science, 2001 Oct 5, 294(5540), 96 - 100
Making sense of eukaryotic DNA replication origins; Gilbert DM; DNA replication is the process by which cells make one complete copy of their genetic information before cell division . In bacteria, readily identifiable DNA sequences constitute the start sites or origins of DNA replication . In eukaryotes, replication origins have been difficult to identify . In some systems, any DNA sequence can promote replication, but other systems require specific DNA sequences . Despite these disparities, the proteins that regulate replication are highly conserved from yeast to humans . The resolution may lie in a current model for once-per-cell-cycle regulation of eukaryotic replication that does not require defined origin sequences . This model implies that the specification of precise origins is a response to selective pressures that transcend those of once-per-cell-cycle replication, such as the coordination of replication with other chromosomal functions . Viewed in this context, the locations of origins may be an integral part of the functional organization of eukaryotic chromosomes.

Cancer Epidemiol Biomarkers Prev, 2001 Oct, 10(10), 1021 - 7
An association of cervical inflammation with high-grade cervical neoplasia in women infected with oncogenic human papillomavirus (HPV); Castle PE et al.; Previous reports of genital conditions, such as nonspecific genital infection/sore or vaginal discharge associated with cervical cancer (L . A . Brinton et al., J . Natl . Cancer Inst . (Bethesda), 79: 23-30, 1987; C . J . Jones et al., Cancer Res., 50: 3657-3662, 1990), suggest a possible link between either genital tract inflammation or changes in bacteria flora consistent with bacterial vaginosis (BV) and cervical cancer . To test whether changes in vaginal bacterial flora or the degree of cervical inflammation are associated with women having a human papillomavirus (HPV) infection or with women infected with oncogenic HPV having high-grade cervical lesions (high-grade squamous intraepithelial lesions or cancer), we conducted a case-control study of women <50 years old enrolled in the Costa Rican natural history study of HPV and cervical neoplasia . To test whether BV and inflammation were associated with HPV DNA positivity, Analysis 1 was restricted to women with no or mild (low-grade or equivocal) cytological abnormalities, and the degree of inflammation and Nugent score (a measure of BV) were compared between women infected (n = 220) and not infected (n = 130) with HPV . To test whether BV and inflammation were associated with high-grade lesions, Analysis 2 was restricted to women infected with oncogenic HPV, and the degree of inflammation and Nugent score were compared between women with (n = 95) and without (n = 158) high-grade cervical lesions . In Analysis 1, BV and cervical inflammation were not associated with HPV infection . In Analysis 2, BV was not associated with high-grade lesions . However, we found a marginally significant positive trend of increasing cervical inflammation associated with high-grade lesions in oncogenic HPV-infected women, (P(trend) = 0.05) . Overt cervicitis was associated with a 1.9-fold increase in risk of high-grade lesions (95% confidence interval, 0.90-4.1) . The results of this study suggest that cervical inflammation may be associated with high-grade lesions and may be a cofactor for high-grade cervical lesions in women infected with oncogenic HPV.

Curr Opin Microbiol, 2001 Oct, 4(5), 595 - 601
The replication-recombination connection: insights from genomics; Gruss A et al.; One amazing characteristic of DNA processing enzymes is their breakneck speed . However, considering the metabolic traffic on DNA, it is not surprising that accidents that interrupt the replication process and require immediate repair occur . Recombination is an example of a repair mechanism, and as a result of accidents, the arrested or broken fork becomes the recombination substrate . Repair proteins are on hand to assist at accident sites, but responses may be sensitive to conditions and depend on the organism . Genomics has been used to identify genetic clues that indicate the occurrence of accidents and to detect sites of recombination . DNA and protein features that may affect replication and recombination are examined and discussed.

Plant Mol Biol, 2001 Oct, 47(3), 379 - 88
Characterization of a second carotenoid beta-hydroxylase gene from Arabidopsis and its relationship to the LUT1 locus; Tian L et al.; Xanthophylls are oxygenated carotenoids that perform critical roles in plants . Beta-carotene hydroxylases (beta-hydroxylases) add hydroxyl groups to the beta-rings of carotenes and have been cloned from several bacteria and plants, including Arabidopsis . The lut1 mutation of Arabidopsis disrupts epsilon-ring hydroxylation and has been suggested to identify a related carotene hydroxylase that functions specifically on epsilon-ring structures . We have used library screening and genomics-based approaches to isolate a second beta-hydroxylase genomic clone and its corresponding cDNA from Arabidopsis . The encoded protein is 70% identical to the previously reported Arabidopsis beta-hydroxylase 1 . Phylogenetic analysis indicates a common origin for the two proteins, however, their different chromosomal locations, intron positions and intron sizes suggest their duplication is not recent . Although both hydroxylases are expressed in all Arabidopsis tissues analyzed, beta-hydroxylase 1 mRNA is always present at higher levels . Both cDNAs encode proteins that efficiently hydroxylate the C-3 position of beta-ring containing carotenes and are only weakly active towards epsilon-ring containing carotenes . Neither beta-hydroxylase cDNA maps to the LUT1 locus, and the genomic region encompassing the LUT1 locus does not contain a third related hydroxylase . These data indicate that the LUT1 locus encodes a protein necessary for epsilon-ring hydroxylation but unrelated to beta-hydroxylases at the level of amino acid sequence.

Gastroenterol Clin North Am, 2001 Sep, 30(3), 679 - 92, viii
Infectious causes of chronic diarrhea; Lee SD et al.; Chronic diarrhea can be seen in association with specific pathogens, usually parasites and occasionally some bacteria . This article reviews pathogens causing chronic diarrhea in immunocompetent individuals and provides a rational diagnostic approach.

J Allergy Clin Immunol, 2001 Oct, 108(4 Suppl), S91 - 4
Ensuring the pathogen safety of intravenous immunoglobulin and other human plasma-derived therapeutic proteins; Miller JL et al.; Countless patients and clinicians rely on therapeutic proteins, such as intravenous immunoglobulins (IVIGs), isolated from human blood plasma . Since plasma is predisposed to contamination by a variety of blood-borne pathogens, ascertaining and ensuring the pathogen safety of plasma-derived therapeutics is a priority among manufacturers . Even though the pathogen safety records for IVIG and other plasma proteins are excellent, the industry remains active in research programs aimed at improving the margin of safety . Industry initiatives designed to increase the safety of plasma-derived products range from donor screening and testing to implementing methods into the manufacturing processes that can inactivate or remove pathogens from product streams . In general, the industry's comprehensive strategy is designed to provide patients and caregivers with the safest plasma products possible.

J Biol Chem, 2001 Dec 7, 276(49), 45862 - 7 Epub 2001 Oct 03.
A single amidotransferase forms asparaginyl-tRNA and glutaminyl-tRNA in Chlamydia trachomatis; Raczniak G et al.; Aminoacyl-tRNA is generally formed by aminoacyl-tRNA synthetases, a family of 20 enzymes essential for accurate protein synthesis . However, most bacteria generate one of the two amide aminoacyl-tRNAs, Asn-tRNA or Gln-tRNA, by transamidation of mischarged Asp-tRNA(Asn) or Glu-tRNA(Gln) catalyzed by a heterotrimeric amidotransferase (encoded by the gatA, gatB, and gatC genes) . The Chlamydia trachomatis genome sequence reveals genes for 18 synthetases, whereas those for asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase are absent . Yet the genome harbors three gat genes in an operon-like arrangement (gatCAB) . We reasoned that Chlamydia uses the gatCAB-encoded amidotransferase to generate both Asn-tRNA and Gln-tRNA . C . trachomatis aspartyl-tRNA synthetase and glutamyl-tRNA synthetase were shown to be non-discriminating synthetases that form the misacylated tRNA(Asn) and tRNA(Gln) species . A preparation of pure heterotrimeric recombinant C . trachomatis amidotransferase converted Asp-tRNA(Asn) and Glu-tRNA(Gln) into Asn-tRNA and Gln-tRNA, respectively . The enzyme used glutamine, asparagine, or ammonia as amide donors in the presence of either ATP or GTP . These results suggest that C . trachomatis employs the dual specificity gatCAB-encoded amidotransferase and 18 aminoacyl-tRNA synthetases to create the complete set of 20 aminoacyl-tRNAs.

Cell Immunol, 2001 Jul 10, 211(1), 43 - 50
Inflammation and lymphocyte activation during mycobacterial infection in the interferon-gamma-deficient mouse; Pearl JE et al.; Interferon-gamma is a pivotal cytokine in the protective response to tuberculosis . In its absence rampant bacterial growth results in tissue destruction and death . While macrophage activation is key, this pleiotropic cytokine has other secondary but significant roles . To investigate these roles, both intravenous and aerosol infection of the IFN-gamma gene disrupted (GKO) mouse was performed . For the first time we describe the very similar growth of bacteria, during the initial phase of infection, between control and GKO mice . During this initial phase, however, very different histopathologic consequences between control and GKO mice were observed . Key observations included an early increased accumulation of granulocytes and a much more rapid and pronounced interstitial pneumonia in the GKO mice . As infection developed, GKO mice mounted an antigen-specific response; however, lymphocyte activation was much more rapid in these mice . Of interest is the fact that this increased rapidity occurred prior to significant differences in bacterial number . Taken together these data support a role for IFN-gamma in limiting both initial cellular recruitment and acquired lymphocytic responses to mycobacterial infection . This role may be key in surviving the kind of chronic stimulatory disease caused by Mycobacterium tuberculosis .

Am J Infect Control, 2001 Oct, 29(5), 301 - 5
A randomized clinical trial to compare the effects of a heat and moisture exchanger with a heated humidifying system on the occurrence rate of ventilator-associated pneumonia; Memish ZA et al.; BACKGROUND: The purpose of this study was to compare the performance of heat and moisture exchanger filters with heated humidifying systems in the mechanical ventilator circuit on the incidence of ventilator-associated pneumonia (VAP) and bacterial colonization . METHOD: Two hundred and forty-three consecutive patients who required mechanical ventilation for 48 hours or more in the adult intensive care unit were randomized to either a heat and moisture exchanger (HME) or a heated humidifying breathing circuit . RESULTS: The VAP rate among the group with HME was 11.4%; the rate among the group with heated humidifying system (HHS) was 15.8% . The difference was not statistically significant . Approximately 68% of the patients in the HME group had no pathogen isolated compared with 50% of the patients in the HHS group . This difference was statistically significant (P =.006) . However, the distribution of the pathogens among those patients who had the isolated pathogens was mostly identical in the 2 groups . CONCLUSION: Even though the study did not find HME to be significantly advantageous over the HHS, in as much as VAP rate is concerned, other advantages such as reduced nurses workload, reduced financial cost, and better safety made HME a more favorable device for use in our adult intensive care unit.

Dis Colon Rectum, 2001 Sep, 44(9), 1241 - 3
The concept and application of antisense oligonucleotides; Yacyshyn BR et al.; Since the identification of the double-stranded DNA helix by Watson and Crick in 1953, the knowledge of nucleotide structure and function has been an important potential tool in the study and therapy of disease . There is recent clinical evidence that antisense oligonucleotides may be important therapeutic compounds in the clinical therapy of a range of diseases, including infection (viruses and bacteria), oncology, and inflammation . Our laboratory-based understanding of antisense oligonucleotide activity has provided a foundation for their use in several human diseases . Potentially relevant applications include inflammatory bowel disease therapy, psoriasis, transplantation, rheumatoid arthritis, cytomegalovirus retinitis, hepatitis C, and solid tumor therapy . Here we will outline these applications as well as our ongoing clinical trials for Crohn's disease.

Nat Biotechnol, 2001 Oct, 19(10), 940 - 5
Use of isogenic human cancer cells for high-throughput screening and drug discovery; Torrance CJ et al.; Cell-based screening for novel tumor-specific drugs has been compromised by the lack of appropriate control cells . We describe a strategy for drug screening based on isogenic human cancer cell lines in which key tumorigenic genes have been deleted by targeted homologous recombination . As a test case, a yellow fluorescent protein (YFP) expression vector was introduced into the colon cancer cell line DLD-1, and a blue fluorescent protein (BFP) expression vector was introduced into an isogenic derivative in which the mutant K-Ras allele had been deleted . Co-culture of both cell lines allowed facile screening for compounds with selective toxicity toward the mutant Ras genotype . Among 30,000 compounds screened, a novel cytidine nucleoside analog was identified that displayed selective activity in vitro and inhibited tumor xenografts containing mutant Ras . The present data demonstrate a broadly applicable approach for mining therapeutic agents targeted to the specific genetic alterations responsible for cancer development.

Microbes Infect, 2001 Oct, 3(12), 997 - 1003
Protective humoral immunity induced by surface-associated material from Actinobacillus actinomycetemcomitans in mice; Herminajeng E et al.; The aim of the present study was to determine the role of antibodies specific to anti-surface-associated material from Actinobacillus actinomycetemcomitans (anti-SAM-Aa) in an infection induced by this periodontopathogen in mice . When SAM-Aa obtained by saline extraction of A . actinomycetemcomitans Y4 was separated on one-dimensional gel electrophoresis, this constituent contained antigen fragments with molecular weights ranging from 14000 to 79000 . Immunoblot analysis revealed that increased antigen dose/immunization resulted in increased numbers of antigen epitopes recognized by serum antibodies of the immunized mice . Rapid healing of the primary lesions and high levels of specific IgG antibodies after challenge with live A . actinomycetemcomitans were seen in the immunized mice, especially at the highest-dose level of 100 microg/immunization . Transfer of SAM-Aa-immunized, but not the SAM-Aa-immunized and adsorbed, serum prior to challenge with live bacteria led to rapid healing of the lesions in the recipient mice . Increased phagocytosis of A . actinomycetemcomitans by murine macrophages (RAW264.7 cells) was observed when this periodontopathogen was opsonized by the SAM-Aa-immunized, but not SAM-Aa-immunized and adsorbed, serum . These results suggest that in mice, SAM-Aa antigens may induce protective antibodies by acting, at least, as an opsonin against challenge with live A . actinomycetemcomitans.

Artif Life, 2001 Spring, 7(2), 171 - 90
Evolving control metabolisms for a robot; Ziegler J et al.; This article demonstrates a new method of programming artificial chemistries . It uses the emerging capabilities of the system's dynamics for information-processing purposes . By evolution of metabolisms that act as control programs for a small robot one achieves the adaptation of the internal metabolic pathways as well as the selection of the most relevant available exteroceptors . The underlying artificial chemistry evolves efficient information-processing pathways with most benefit for the desired task, robot navigation . The results show certain relations to such biological systems as motile bacteria.

Mol Microbiol, 2001 Sep, 41(6), 1409 - 17
Quantitative analysis of expression of two circadian clock-controlled gene clusters coding for the bidirectional hydrogenase in the cyanobacterium Synechococcus sp . PCC7942; Schmitz O et al.; Hydrogen metabolism is of central interest in cyanobacterial research because of its potential applications . The gene expression and physiological role of the cyanobacterial bidirectional NAD(P)+-reducing hydrogenase are poorly understood . Transcription rates of hoxEF and hoxUYH encoding this enzyme have been studied in Synechococcus sp . PCC7942 . PhoxU activity was about three times higher than that of PhoxE . Circadian phasing of both promoters was found to be synchronous and influenced expression levels by at least one order of magnitude . This is the first demonstration of circadian control of gene expression for any hydrogenase . For the majority of PhoxU-driven messages, transcription presumably terminates between hoxU and hoxH . Being part of a polycistronic hoxUYHW.. . operon, hoxW, encoding a protease involved in C-terminal processing of the hydrogenase large-subunit HoxH, is mainly expressed by its own promoter, PhoxW . The complex transcript formation may be a key feature for controlling bidirectional hydrogenase expression in vivo.

Phys Rev Lett . 2001 Sep 24;87(13):136101 . Epub 2001 Sep 05.
Maximal height scaling of kinetically growing surfaces; Raychaudhuri S et al.; The scaling properties of the maximal height of a growing self-affine surface with a lateral extent L are considered . In the late-time regime its value measured relative to the evolving average height scales like the roughness: h*(L) approximately L alpha . For large values its distribution obeys logP(h*(L)) approximately (-)A(h*(L)/L(alpha))(a) . In the early-time regime where the roughness grows as t(beta), we find h*(L) approximately t(beta){lnL-(beta/alpha)lnt+C}(1/b), where either b = a or b is the corresponding exponent of the velocity distribution . These properties are derived from scaling and extreme-value arguments . They are corroborated by numerical simulations and supported by exact results for surfaces in 1D with the asymptotic behavior of a Brownian path.

Phys Rev E Stat Nonlin Soft Matter Phys . 2001 Sep;64(3 Pt 1):031903 . Epub 2001 Aug 24.
Measure representation and multifractal analysis of complete genomes; Yu ZG et al.; This paper introduces the notion of measure representation of DNA sequences . Spectral analysis and multifractal analysis are then performed on the measure representations of a large number of complete genomes . The main aim of this paper is to discuss the multifractal property of the measure representation and the classification of bacteria . From the measure representations and the values of the D(q) spectra and related C(q) curves, it is concluded that these complete genomes are not random sequences . In fact, spectral analyses performed indicate that these measure representations, considered as time series, exhibit strong long-range correlation . Here the long-range correlation is for the K-strings with dictionary ordering, and it is different from the base pair correlations introduced by other people . For substrings with length K=8, the D(q) spectra of all organisms studied are multifractal-like and sufficiently smooth for the C(q) curves to be meaningful . With the decreasing value of K, the multifractality lessens . The C(q) curves of all bacteria resemble a classical phase transition at a critical point . But the "analogous" phase transitions of chromosomes of nonbacteria organisms are different . Apart from chromosome 1 of C . elegans, they exhibit the shape of double-peaked specific heat function . A classification of genomes of bacteria by assigning to each sequence a point in two-dimensional space (D(-1),D1) and in three-dimensional space (D(-1),D1,D(-2)) was given . Bacteria that are close phylogenetically are almost close in the spaces (D(-1),D1) and (D(-1),D1,D(-2)).

J Struct Biol, 2001 Aug, 135(2), 157 - 69
Gene duplication and the evolution of group II chaperonins: implications for structure and function; Archibald JM et al.; Chaperonins are multisubunit protein-folding assemblies . They are composed of two distinct structural classes, which also have a characteristic phylogenetic distribution . Group I chaperonins (called GroEL/cpn60/hsp60) are present in Bacteria and eukaryotic organelles while group II chaperonins are found in Archaea (called the thermosome or TF55) and the cytoplasm of eukaryotes (called CCT or TriC) . Gene duplication has been an important force in the evolution of group II chaperonins: Archaea possess one, two, or three homologous chaperonin subunit-encoding genes, and eight distinct CCT gene families (paralogs) have been described in eukaryotes . Phylogenetic analyses indicate that while the duplications in archaeal chaperonin genes have occurred numerous times independently in a lineage-specific fashion, the eight different CCT subunits found in eukaryotes are the products of duplications that occurred early and very likely only once in the evolution of the eukaryotic nuclear genome . Analyses of CCT sequences from diverse eukaryotic species reveal that each of the CCT subunits possesses a suite of invariant subunit-specific amino acid residues ("signatures") . When mapped onto the crystal structure of the archaeal chaperonin from Thermoplasma acidophilum, these signatures are located in the apical, intermediate, and equatorial domains . Regions that were found to be variable in length and/or amino acid sequence were localized primarily to the exterior of the molecule and, significantly, to the extreme tip of the apical domain (the "helical protrusion") . In light of recent biochemical and electron microscopic data describing specific CCT-substrate interactions, our results have implications for the evolution of subunit-specific functions in CCT .

J Struct Biol, 2001 Aug, 135(2), 126 - 38
The Chaperones of the archaeon Thermoplasma acidophilum; Ruepp A et al.; Chaperonesare an essential component of a cell's ability to respond to environmental challenges . Chaperones have been studied primarily in bacteria, but in recent years it has become apparent that some classes of chaperones either are very divergent in bacteria relative to archaea and eukaryotes or are missing entirely . In contrast, a high degree of similarity was found between the chaperonins of archaea and those of the eukaryotic cytosol, which has led to the establishment of archaeal model systems . The archaeon most extensively used for such studies is Thermoplasma acidophilum, which thrives at 59 degrees C and pH 2 . Here we review information on its chaperone complement in light of the recently determined genome sequence .

Water Sci Technol, 2001, 44(4), 229 - 36
Post-treatment of anaerobic effluents in an overland flow system; Chernicharo CA et al.; This research aimed at the investigation of an overland flow system applied to the post-treatment of anaerobic effluents . The system treated domestic sewage in Itabira City (Brazil), being composed by an anaerobic reactor and an overland flow system, the latter working as a post-treatment unit . A portion of the reactor's effluent was directed to a group of three overland flow slopes (demonstration scale), that were operated with different application rates . During Phase 1 of the research the overland flow system was fed under a permanent hydraulic regime (constant flows), having as inflow the effluent from an UASB reactor (full-scale, volume of 477 m3) . During Phase 2, the overland system was fed under a hydraulic transient pattern (variable flows with hourly variations), having as inflow the effluent from a partitioned UASB reactor (demonstration-scale, volume of 9 m3) . In general, the performance of the overland flow system as a polishing step was very good, mainly because of the low solids and organic matter concentration in the final effluent (average values of BOD from 48 to 62 mg/L; COD from 98 to 119 mg/L and SS from 17 to 57 mg/L) . Regarding nutrients and coliforms, the system also reached satisfactory efficiency levels . Based on the experience obtained with this study, it is suggested that overland flow systems, working as post-treatment step of UASB reactors, can work with application rates in the range of 0.4 to 0.5 m3/m.h, which are higher than those normally applied.

Front Biosci, 2001 Oct 01, 6, D1161 - 72
Nitric oxide: one of the more conserved and widespread signaling molecules; Torreilles J; After the discovery of the vasodilatory functions of nitric oxide (NO), many signaling mechanisms involving NO were identified through experiments on mammals . NO activates soluble guanylyl cyclase to induce the formation of cGMP, stimulates ADP-ribosylation of GAPDH to alter cell energy production, and combines with superoxide to generate peroxynitrite . It then became clear that NO was a major messenger molecule in mammals, involved in the regulation of blood vessel dilatation, immune function and neurotransmission in the brain and peripheral nervous system . The wide spectrum of physiological effects of NO in mammals prompted researchers to look for the presence of NO in vertebrates and invertebrates . Parallel findings on the presence of NO signaling in vertebrates and invertebrates were observed, and then NO was found to be a signaling molecule widely spread throughout the metazoan kingdom and whose functions were highly conserved during evolution . These features were extended to the entire animal kingdom after the discovery of NOS activity in protozoa, yeasts and bacteria . Recently, the involvement of NO and NOS in plant disease resistance to infection was documented and many close similarities were detected between NO-dependent signaling mechanisms involved in plants and those identified in animals . All of these results indicated that NO is one of the earliest and most widespread signaling molecules in living organisms . This short review was aimed at marshalling recent information that led to this conclusion.

Curr Opin Chem Biol, 2001 Oct, 5(5), 535 - 40
The non-mevalonate pathway of isoprenoids: genes, enzymes and intermediates; Rohdich F et al.; Although the mevalonate pathway had been considered for a long time as the unique source of biosynthetic isoprenoids, an alternative pathway has recently been discovered . The first intermediate, 1-deoxy-D-xylulose 5-phosphate, is assembled by condensation of glyceraldehyde 3-phosphate and pyruvate . A skeletal rearrangement coupled with a reduction step affords the branched-chain polyol, 2C-methyl-D-erythritol 4-phosphate, which is subsequently converted into a cyclic 2,4-diphosphate by the consecutive action of three enzymes via nucleotide diphosphate intermediates . The genes specifying these enzymes have been cloned from bacteria, plants and protozoa . Their expression in recombinant bacterial hosts has opened the way to the identification of several novel pathway intermediates.

Curr Opin Chem Biol, 2001 Oct, 5(5), 525 - 34
Tailoring enzymes that modify nonribosomal peptides during and after chain elongation on NRPS assembly lines; Walsh CT et al.; Nonribosomal peptide synthetases are large enzyme complexes that synthesize a variety of peptide natural products through a thiotemplated mechanism . Assembly of the peptides proceeds through amino acid loading, amide-bond formation and chain translocation, and finally thioester lysis to release the product . The final products are often heavily modified, however, through methylation, epimerization, hydroxylation, heterocyclization, oxidative cross-linking and attachment of sugars . These activities are the province of specialized enzymes (either embedded in the multidomain nonribosomal peptide synthetase structure or standalone).

J Comp Pathol, 2001 Aug-Oct, 125(2-3), 224 - 7
Abomasal ulcers in captive white-tailed deer (Odocoileus virginianus); Palmer MV et al.; Abomasal ulceration was noted in 32 of 200 white-tailed deer . Ulceration was most common in the abomasal pylorus and at the abomasal-duodenal junction . Abomasal ulceration was characterized by focal to multifocal, sharply demarcated areas of coagulation necrosis and haemorrhage extending through the mucosa, with fibrin thrombi in mucosal blood vessels of small diameter . Ulcerated areas were often covered by a mixture of mucus, debris and neutrophils . Visible bacteria were not associated with ulcerative lesions . All deer with abomasal ulceration had intercurrent disease, including bacterial pneumonia, enterocolitis, intussusception, chronic diarrhoea, capture myopathy, or experimentally induced tuberculosis . The anatomical distribution of abomasal ulcers in this population of captive white-tailed deer resembled that seen in veal calves . Copyright Harcourt Publishers Ltd.

J Comp Pathol, 2001 Aug-Oct, 125(2-3), 137 - 44
T cell-dependent inducible nitric oxide synthase production and ultrastructural morphology in BALB/c mice infected with Mycobacterium avium subspecies paratuberculosis; Thomsen BV et al.; Euthymic BALB/c and athymic nude BALB/c mice aged 3-8 days were infected intraperitoneally with Mycobacterium avium subspecies paratuberculosis (ATCC strain 19698) . After euthanasia at 5 months post-inoculation, hepatic granulomas were evaluated by morphometric analysis of digital images captured from light microscopy sections, by electron microscopy and by immunohistochemical methods . Euthymic mice differed from athymic mice in that (1) their hepatic granulomas were smaller, contained fewer bacteria, and produced more inducible nitric oxide synthase, and (2) their hepatic macrophages contained fewer bacteria, a higher percentage of degraded bacteria, and increased numbers of primary lysosomes . The study showed that macrophage activation was markedly less in the T cell-deficient athymic mice than in the euthymic mice . Copyright Harcourt Publishers Ltd

Mediators Inflamm, 2001 Aug, 10(4), 173 - 8
Effects after inhalation of (1-->3)-beta-D-glucan in healthy humans; Thorn J et al.; BACKGROUND AND AIM: This study was performed to assess the effects of an exposure to a pure (1-->3)-beta-D-glucan, a cell wall component of fungi, plants and certain bacteria . METHODS: Twenty-one healthy subjects inhaled saline or (1-->3)-beta-D-glucan suspended in saline in a random, double-blind, cross-over design . They were examined before exposure and 24 and 72h afterwards with spirometry, blood sampling and collection of induced sputum . Differential cell counts and eosinophilic cationic protein (ECP) were determined in blood and sputum, and myeloperoxidase (MPO), tumour necrosis factor-alpha (TNF-alpha), and interleukin (IL)-8 and IL-10 were determined in sputum supernatants . TNF-alpha was determined after cultivation of blood mononuclear cells . RESULTS: In sputum, inhalation of saline caused a significant increase in ECP and TNF-alpha . (1-->3)-beta-D-Glucan inhalation caused a further increase in these cytokines, although not statistically significantly different from the increase induced by inhalation of saline alone . In blood, the number of eosinophils was significantly decreased 72 h after the challenge with (1-->3)-beta-D-glucan . This effect was not found after the inhalation of saline alone . TNF-alpha production from stimulated blood mononuclear cells was significantly decreased 72 h after the (1-->3)-beta-D-glucan inhalation as compared with the increase induced by saline inhalation . CONCLUSIONS: The results suggest that (1-->3)-beta-D-glucan causes a different type of response as compared with inflammatory agents such as bacterial endotoxin that cause a neutrophil-dominated inflammatory response.

Cell Mol Life Sci, 2001 Aug, 58(9), 1167 - 78
Animal cellulases; Watanabe H et al.; Previous dogma has maintained that cellulose, ingested by xylophagous or herbivorous animals, is digested by cellulolytic symbiotes . The first evidence in conflict with this contention involved the demonstration of cellulolytic activities in symbiote-free secreting organs (e.g., the salivary glands of termites) or defaunated guts . Following these demonstrations, possible endogenous cellulase components were purified from several cellulose-digesting invertebrates, but this research did little to change the general view concerning animal cellulose digestion . Thanks to recent developments in molecular biology, the existence of cellulases of animal origin has been firmly established . To date, cellulase genes have been reported from arthropods (insects and a crayfish) and nematodes . This paper describes and discusses the presence and nature of endogenous cellulases in higher animals.

J Periodontol, 2001 Sep, 72(9), 1228 - 35
A sensitive method for detecting Porphyromonas gingivalis by polymerase chain reaction and its possible clinical application; Nozaki T et al.; BACKGROUND: It is useful for the clinical diagnosis of periodontitis to monitor the colonization of periodontopathic bacteria in periodontal pockets . In this study, we attempted to establish and possibly identify the clinical application of a sensitive method to detect Porphyromonas gingivalis (P.g.), one of the putative periodontopathic bacteria related to chronic periodontitis . METHODS: Genomic DNA extracted from cultured P.g . 381 and clinically isolated subgingival plaque samples were used as a template of polymerase chain reaction (PCR) . We designed primers to amplify the genomic DNA coding 40 kDa outer membrane protein (OMP), one of the unique proteins to all strains of P.g . The efficiency and specificity of amplification were evaluated by agarose gel electrophoresis and subsequent Southern hybridization with a digoxygenin-labeled oligonucleotide probe . RESULTS: Fewer than 100 P.g . bacterial cells in the specimen were reproducibly detected by PCR-hybridization assay . This PCR-hybridization assay was at least 100 times more sensitive than the conventional indirect immunofluorescence assay (IIF) . Furthermore, the imaging analysis showed that there is a linear correlation between the strength of the signal and the cell number of P.g . from which the template DNA was extracted semiquantitatively . It is noteworthy that the PCR assay could also be applied to detect P.g . from clinical plaque samples and that it was approximately 100 times more sensitive than a conventional IIF assay . CONCLUSION: The PCR assay established in this study can be a powerful tool to detect P.g . in periodontal pockets and monitor the colonization and/or recolonization of P.g . at the very early phase.

Microbiology, 2001 Oct, 147(Pt 10), 2757 - 67
The replicon of pSW800 from Pantoea stewartii; Wu CY et al.; A 2019 bp DNA fragment containing the replicon of pSW800 from Pantoea stewartii SW2 was cloned and characterized . This replicon contains two genes--repA and repB, which encode a 36.5 kDa replication initiation protein (RepA) and a peptide of 18 aa, respectively . These two genes overlap by 8 bases with repB situated upstream . The replicon also transcribes an antisense RNA (RNAI) that inhibits the expression of repA and repB . The ribosome-binding sequence (RBS) of repA is likely to be hidden in a stem-loop structure, inhibiting the translation of repA . Furthermore, translation of repB is likely to disrupt the stem-loop structure, which is one of the criteria allowing the translation of repA to begin . A mutagenesis study revealed that a sequence (5'-GCACGGG-3') located 111 nt upstream from repA is crucial; mutation of this sequence prevented the translation of repA . Additionally, this region and the stem-loop structure containing the RBS of repA may form an RNA pseudoknot . Results in this study demonstrate that a mechanism similar to that regulating plasmid replication in the IncB, IncIalpha and IncL/M groups also regulates pSW800 replication.

J Clin Pathol, 2001 Oct, 54(10), 774 - 7
Helicobacter heilmannii gastritis: a histological and immunohistochemical trait; Ierardi E et al.; AIM: Biopsies of the gastric antrum were reviewed over a period of 10 years to determine the prevalence of Helicobacter heilmannii in symptomatic subjects from this geographical area and to relate its presence to distinctive histopathological and immunohistochemical features . METHODS: Biopsies from 7926 symptomatic patients were reviewed . Ten serial sections were stained with haematoxylin and eosin for conventional histology . Another 10 sections were stained with the Gram method for spiral bacteria . When H heilmannii was suspected, 10 additional serial sections were stained with methylene blue to obtain homogeneous colouring . An equal number of sections from patients affected by isolated H heilmannii or H pylori gastritis were analysed by immunohistochemistry to evaluate lymphoid aggregate/mucosal lymphocyte clonality (CD20 and CD3) and tumour necrosis factor alpha (TNF-alpha) in stromal cells . RESULTS: The prevalence of H heilmannii was 0.1% (eight of 7926), whereas H pylori was present in 60.7% of patients (4813 of 7926) . In two of the eight H heilmannii positive patients both helicobacters were found . In all subjects infected by H heilmannii only, distinctive histology (lymphocyte exudation into gastric foveolae) was seen . Lymphoid aggregates, chronic mucosal inflammation with patchy activity, and the absence of epithelial mucus depletion were regular features of H heilmannii gastritis . Immunohistochemistry did not reveal different lymphocyte clonal patterns between H pylori and H heilmannii gastritis: CD20 positive cells were predominant in the centre of aggregates and mucosal infiltrates, whereas CD3 positive cells were prevalent at the periphery of follicles . Only H pylori gastritis showed a significant increase in TNF-alpha positive stromal cells . CONCLUSION: These data suggest that an unusual lymphocyte reaction, with the tendency to invade the foveolar lumen, is a distinctive histopathological aspect of H heilmannii chronic gastritis, although further studies in a larger series are necessary to confirm this fact . Nevertheless, lymphocyte clones do not differ qualitatively from those found in H pylori infection . Moreover, compared with H heilmannii, H pylori provokes a more intense release of TNF-alpha, suggesting that different inflammatory responses exist to these two organisms.

Plant J, 2001 Sep, 27(6), 551 - 60
Arabidopsis polyamine biosynthesis: absence of ornithine decarboxylase and the mechanism of arginine decarboxylase activity; Hanfrey C et al.; Unlike other eukaryotes, which can synthesize polyamines only from ornithine, plants possess an additional pathway from arginine . Occasionally non-enzymatic decarboxylation of ornithine could be detected in Arabidopsis extracts; however, we could not detect ornithine decarboxylase (ODC; EC 4 . 1.1.17) enzymatic activity or any activity inhibitory to the ODC assay . There are no intact or degraded ODC sequences in the Arabidopsis genome and no ODC expressed sequence tags . Arabidopsis is therefore the only plant and one of only two eukaryotic organisms (the other being the protozoan Trypanosoma cruzi) that have been demonstrated to lack ODC activity . As ODC is a key enzyme in polyamine biosynthesis, Arabidopsis is reliant on the additional arginine decarboxylase (ADC; EC 4.1.1.9) pathway, found only in plants and some bacteria, to synthesize putrescine . By using site-directed mutants of the Arabidopsis ADC1 and heterologous expression in yeast, we show that ADC, like ODC, is a head-to-tail homodimer with two active sites acting in trans across the interface of the dimer . Amino acids K136 and C524 of Arabidopsis ADC1 are essential for activity and participate in separate active sites . Maximal activity of Arabidopsis ADC1 in yeast requires the presence of general protease genes, and it is likely that dimer formation precedes proteolytic processing of the ADC pre-protein monomer.

J Mol Biol, 2001 Sep 28, 312(4), 583 - 9
Promoter-specific involvement of the FixJ receiver domain in transcriptional activation; Ton-Hoang B et al.; The "two-component" FixLJ system activates nitrogen fixation genes via nifA and fixK in Sinorhizobium meliloti . Like other response regulators, the FixJ protein can be decomposed into an N-terminal phosphorylatable "receiver" domain FixJN and a C-terminal transcriptional activator domain FixJC . The FixJN receiver domain was known to regulate activity of FixJC negatively at the nifA promoter . Here we show a different situation at the fixK promoter where FixJN also contributes positively to transcriptional activation . This promoter-specific effect was mapped by alanine-scanning mutagenesis to the beta2 strand of the receiver domain . This interaction with FixJN is required for the recruitment of RNA polymerase at the fixK promoter by phosphorylated FixJ . Altogether the FixJ receiver domain appears to carry at least four functions, some of which can be separated by mutation: (1) autophosphorylation; (2) inhibition of FixJC; (3) dimerization; (4) transcriptional activation at pfixK . This example illustrates the formidable functional plasticity of receiver domains .

Water Sci Technol, 2001, 44(4), 63 - 70
Bamboo as an anaerobic medium: effect of filter column height; Camargo SA et al.; Sewage was treated in anaerobic filters filled with rings of bamboo (either whole or cut), and their effectiveness in the reduction of chemical oxygen demand (COD) was monitored along the entire extension of the filter . This efficiency was determined based on the COD reduction in both the brute effluent (COD-T) and that passed through a glass filter (COD-F) . The quantity of total suspended solids (TSS) present at various heights in the filter column, measured at approximately 10-cm intervals from the false bottom to the height of the filter outflow (80 cm above) for various hydraulic detention times (HDT) . The performance of the system resulted in little variation up to 5 hours of HDT, with the percentage of reduction of COD-T and COD-F being situated in the range of 60-80% and 40-80%, respectively . The first 40 cm of the filter proved to be relatively effective, and no significant differences in performance between whole and cut rings of bamboo were observed . A shock in pH occurring after 562 days of operation provoked an immediate and marked decrease in the performance of the reactors, especially that operating with a HDT of only 2 hours . This latter filter required more time to return to normal operational conditions than did the other reactors operating with longer HDT.

Water Sci Technol, 2001, 44(4), 33 - 40
Anaerobic treatment of domestic sewage at low temperature; Elmitwalli T et al.; The results of research concerning the feasibility of anaerobic treatment of domestic sewage at low temperature are summarized in this article . The batch tests demonstrated a high biodegradability of domestic sewage at 20 degrees C (74%) . Both batch and continuous experiments for the treatment of domestic sewage showed that the removal of SS prior to anaerobic treatment of domestic sewage not only provides a stable reactor performance but also improves the removal of both colloidal (CODcol) and dissolved COD (CODdis) . The results of the pre-treatment of domestic sewage in an anaerobic filter (AF) and an anaerobic hybrid (AH) reactor showed that the AF reactor is an efficient process for the removal of suspended COD (CODss), viz . 82%, at an HRT of 4 h and 13 degrees C . The novel AF reactor consists of vertical sheets of reticulated polyurethane foam with knobs, where the biomass was only in attached form . For the treatment of pre-settled sewage at 13 degrees C, the AH reactor, with granular sludge, showed a higher total COD (CODt) removal than the UASB reactor as a result of higher CODcol removal . Therefore, the performance of a two-step system, AF + AH (with granular sludge) reactor, was investigated with different HRTs at 13 degrees C . For optimization of CODss and CODdis an HRT of 4 + 4 h is needed, while for optimization of CODcol removal an HRT of 4 + 8 h is required . A CODt removal of 71% was achieved with 60% conversion to methane from the removed CODt when the AF + AH system was operated at an HRT of 4 + 8 h at 13 degrees C.

Water Sci Technol, 2001, 44(4), 15 - 22
Simultaneous organic nitrogen and sulfate removal in an anaerobic GAC fluidised bed reactor; Fdz-Polanco F et al.; A granular activated carbon (GAC) anaerobic fluidised bed reactor treating vinasse from an ethanol distillery of sugar beet molasses was operated for 250 days under three different organic loading rates . The reactor showed good performance in terms of organic matter removal and methane production but an anomalous behaviour in terms of unusual high concentrations of molecular nitrogen and low concentration of hydrogen sulphide in the biogas . The analysis of the different nitrogenous and sulphur compounds and the mass balances of these species in the liquid and gas phases clearly indicated an uncommon evolution of nitrogen and sulphur in the reactor . Up to 55% of the TKN and up to 80% of the sulphur disappear in the liquid phase . This is the opposite to any previously reported results in the bibliography . The new postulated anaerobic process of ammonia and sulphate removal seems to follow the mechanism: SO4 = +2 NH4+-->S + N2 + 4H2O (delta G degree = -47.8 kJ/mol).

Water Sci Technol, 2001, 44(4), 109 - 16
Study of thermal hydrolysis as a pretreatment to mesophilic anaerobic digestion of pig slurry; Bonmati A et al.; Feasibility of anaerobic digestion of pig slurry is dependent, among other factors, on the biogas production rate, which is low compared with other organic wastes, and on the profitable uses of surplus thermal energy produced, a limiting factor in warm geographical areas . The objectives of this work are determining whether low temperature thermal pretreatment (< 90 degrees C) improves pig slurry anaerobic digestion, and determining whether organic matter degradation during the thermal pretreatment is due to thermal phenomena (80 degrees C) or to enzymatic ones (60 degrees C) . The thermal degradation tests showed that hydrolysis occurring during the thermal pretreatment is due to thermal phenomena . The increase in soluble substances were significantly larger at 80 degrees C than at 60 degrees C (both during 3 h) . Two types of slurry were used in the batch anaerobic digestion tests . The effect of thermal pretreatment differed with the type of slurry: it was positive with almost non-degraded slurries containing low NH4(+)-N concentration, and negative (inhibition of the anaerobic digestion process) when using degraded slurries with high NH4(+)-N content.

Water Sci Technol, 2001, 44(4), 1 - 6
Use of activated carbon and natural zeolite as support materials, in an anaerobic fluidised bed reactor, for vinasse treatment; Fernandez N et al.; In Cuba, the alcohol distillation process from cane sugar molasses, produces a final waste (vinasse), with an enormous polluting potential and a high sulfate content . Applying the anaerobic technology, most of the biodegradable organic matter can turn into biogas, rich in methane but with concentrations of sulfide above 1% . The present work develops two experiences with anaerobic fluidized bed reactors (AFBR) using both Cuban raw material, activated carbon and natural zeolite, as support media, with the purpose of obtaining high organic matter removal rates and keeping sulfide and ammonium concentrations in the permissible ranges . The reactors were operated during 120 days, achieving an organic loading rate of 10 kg COD/m3 day, with COD removal above 70%, and a methane production of 2 L/d . The activated carbon and natural zeolite used support materials in anaerobic fluidized bed reactors, and showed good results of distillery waste removal.

Int Arch Allergy Immunol, 2001 Aug, 125(4), 273 - 9
Chemoattractant receptors expressed on type 2 T cells and their role in disease; Cosmi L et al.; The existence of two functionally distinguished populations among T cells has been established in both mice and humans . Type 1 T helper (Th1) cells are involved in the defense against intracellular bacteria and many viruses, while type 2 Th cells (Th2) are the major actors in the response against parasites and play a central role in allergic inflammation . More recently, several data have suggested that some chemokine receptors are tightly regulated on T cells, and in accordance with this selective expression, Th1 and Th2 cells can be differentially recruited by specific chemokines to the inflammatory sites . Among Th2-associated chemokine receptors, CCR3, CCR4 and CCR8 have been described to play a central role in allergic inflammation . However, CCR3 is mainly expressed on basophils, eosinophils and mast cells, but it is poorly expressed by Th2 cells, and CCR4 is also expressed by Th subsets different from Th2 cells . So far, the chemoattractant receptors which among T cells appear to be selectively expressed by Th2 cells or their subsets are CCR8 and CRTH2 . The ligand for CRTH2 is not a chemokine, but is prostaglandin (PG)D2, which is able to attract basophils, eosinophils, Th2 cells and type 2 cytotoxic (Tc2) CD8+ T lymphocytes . The selective expression of CRTH2 on Th2 and Tc2 cells may be useful to develop new therapeutic strategies against allergic diseases and against other immune disorders . Additional studies, however, are required to understand its effective importance in the induction and maintenance of Th2- or Tc2-mediated response and inflammation .

J Clin Microbiol, 2001 Oct, 39(10), 3666 - 71
Borrelia burgdorferi sensu lato and Ehrlichia spp . in Ixodes ticks from southern Norway; Jenkins A et al.; We report the results of a study of the prevalence of Ehrlichia and Borrelia species in 341 questing Ixodes ricinus ticks from two locations in southern Norway . The prevalences of Borrelia burgdorferi sensu lato and Ehrlichia spp . were, respectively, 16 and 11.5% at site 1 and 17 and 6% at site 2 . Prevalence and species composition of Borrelia and Ehrlichia varied with location and date of collection . The dominant Borrelia species at both sites was Borrelia afzelii, followed by Borrelia burgdorferi sensu stricto . Borrelia garinii was found in only a single tick . The dominant member of the Ehrlichia group was a recently described Ehrlichia-like organism related to the monocytic ehrlichiae . Variants of Ehrlichia phagocytophila and the agent of human granulocytic ehrlichiosis were also found . The highest prevalences for B . afzelii, B . burgdorferi sensu stricto, and the Ehrlichia-like organism were observed in May . B . afzelii was most prevalent in females, less prevalent in nymphs, and least prevalent in males, while the prevalence of Ehrlichia was highest in nymphs, lower in females, and least in males . Double infections with B . afzelii and B . burgdorferi sensu stricto and with B . afzelii and the Ehrlichia-like organism were significantly overrepresented . Tick densities were highest in May, when densities of more than 200 ticks/100 m2 were observed, and declined during the summer months to densities as low as 20 ticks/100 m2 . We conclude that estimates of the prevalence of tick-borne bacteria are sensitive to the choice of date and site for collection of ticks . This is the first study of tick-borne Borrelia and Ehrlichia in Norway and the lowest reported B . garinii prevalence in Northern Europe . The prevalence of the Ehrlichia-like organism is described for the first time in questing ticks.

Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11175 - 80
Definition of EGF-like, closely interacting modules that bear activation epitopes in integrin beta subunits; Takagi J et al.; Integrin beta subunits contain four cysteine-rich repeats in a long extracellular stalk that connects the headpiece to the membrane . Most mAbs to integrin activation epitopes map to these repeats, and they are important in propagating conformational signals from the membrane/cytosol to the ligand-binding headpiece . Sequence analysis of a protein containing only 10 integrin-like, cysteine-rich repeats suggests that these repeats start one cysteine earlier than previously reported . By using the new repeat boundaries, statistically significant sequence homology to epidermal growth factor-like domains is found, and a disulfide bond connectivity of the eight cysteines is predicted that differs in three of four disulfides from a previous prediction of epidermal growth factor-like modules {Berg, R . W., Leung, E., Gough, S., Morris, C., Yao, W.-P., Wang, S.-x., Ni, J . & Krissansen, G . W . (1999) Genomics 56, 169-178} . N-terminally truncated beta2 integrin stalk fragments were well expressed and secreted from 293 T cells when they began at repeat boundaries but not when they began one cysteine earlier or later . Furthermore, peptides that correspond to module 3 or modules 2 + 3 were expressed in bacteria and refolded . The module 2 + 3 fragment was as reactive with three mAbs to activation epitopes as a beta2 fragment expressed in eukaryotic cells, indicating a native fold . Only one residue intervenes between the last cysteine of one module and the first cysteine of the next . This arrangement is consistent with a tight intermodule connection, a prerequisite for signal propagation from the membrane to the ligand binding headpiece.

Clin Lab Med, 2001 Sep, 21(3), 435 - 73
Clinical recognition and management of patients exposed to biological warfare agents; Franz DR et al.; Concern regarding the use of biological agents (bacteria, viruses, or toxins) as tools of warfare or terrorism has led to measures to deter their use or, failing that, to deal with the consequences . Unlike chemical agents, which typically lead to severe disease syndromes within minutes at the site of exposure, diseases resulting from biological agents have incubation periods of days . Rather than a paramedic, it will likely be a physician who is first faced with evidence of the results of a biological attack . Provided here is an updated primer on 11 classic BW and potential terrorist agents to increase the likelihood of their being considered in a differential diagnosis . Although the resultant diseases are rarely seen in many countries today, accepted diagnostic and epidemiologic principles apply; if the cause is identified quickly, appropriate therapy can be initiated and the impact of a terrorist attack greatly reducedPublication Types:

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