Bacteria

Med Microbiol Immunol (Berl), 2002 May, 191(1), 5 - 10
Asialo-GM1 and asialo-GM2 are putative adhesion molecules for Moraxella catarrhalis; Ahmed K et al.; Moraxella catarrhalis is an important pathogen of respiratory and middle ear infections . We previously reported that the attachment of M . catarrhalis to pharyngeal epithelial cells is mediated by ganglioside M2 (GM2) . Several sets of adhesins or receptors are involved in such attachment process . In this study, we used the same strains and similar bacterial culture conditions as those in our previous study, and demonstrated by thin layer chromatography that M . catarrhalis can also bind to asialo-GM1 (Gg4Cer) and asialo-GM2 (Gg3Cer) . GalNAcbeta1-->4Galbeta1 is a common sequence in both Gg4Cer and Gg3Cer, and in many respiratory bacteria, this sequence acts as a receptor for attachment to host cells . Treatment of human pharyngeal epithelial cells with anti-GM2 and anti-Gg4Cer antibodies significantly decreased attachment of M . catarrhalis to these cells; however, treatment with anti-Gg3Cer antibody did not decrease M . catarrhalis attachment . Immunofluorescence microscopy revealed that human pharyngeal epithelial cells are positive for GM2 and Gg4Cer, but not for Gg3Cer . Our results indicate that Gg4Cer on human pharyngeal epithelial cells, and Gg3Cer,possibly on other cells, could serve as molecules for attachment of M . catarrhalis.

J Forensic Sci, 2002 Jul, 47(4), 757 - 72
Characterization and validation studies of powerPlex 2.1, a nine-locus short tandem repeat (STR) multiplex system and penta D monoplex; Levedakou EN et al.; In order to increase the power of discrimination for human identification purposes, a nine-locus short tandem repeat (STR) multiplex, the GenePrint PowerPlex 2.1 system (PowerPlex 2.1) developed by Promega Corporation and a separate pentanucleotide-repeat locus, Penta D, were tested . This megaplex system includes the highly polymorphic loci FGA, TPOX, D8S1179, vWA, Penta E, D18S51, D21S11, TH01, and D3S1358 and may be used in combination with the eight-locus STR multiplex, the GenePrint PowerPlex 1.1 system (PowerPlex 1.1) that has been previously developed . Three of the loci, TPOX, TH01 and vWA, have been included in both systems for quality control purposes . As with PowerPlex 1.1, PowerPlex 2.1 is also based on a two-color detection of fluorescent-labeled DNA products amplified by polymerase chain reaction (PCR) and provides a valuable tool for accurate and rapid allele determination . The primer sequences used in the PowerPlex 2.1/Penta D system are also presented in this report . To meet the "Quality Assurance Standards for Forensic DNA Testing Laboratories" (FBI), we tested the efficiency and reproducibility of the PowerPlex 2.1/PentaD system by several validation studies that were conducted as a joint project among seven laboratories . Validation tests included concordance studies, sensitivity, and species specificity determination, as well as performance in forensic and environmentally impacted samples . The results produced from these tests demonstrated the consistency and reliability of the PowerPlex 2.1/Penta D system.

Kansenshogaku Zasshi, 2002 Jun, 76(6), 425 - 31
{Pathogenicities of Mycobacterium intracellulare and M . avium strains to the mice which were isolated from non-tuberculous mycobactriosis patients}; Goto Y et al.; The virulence of 5 strains of M . intracellulare and 6 strains of M . avium to mice were examined . The former bacteria were obtained from the patients with mycobacterial pulmonary disease and the latter were from AIDS patients respectively . C57BL/6 (NRAMP-1 susceptible) and its NRAMP-1 congenic mice (resistant) were used to evaluate the virulence of these bacteria . Three of the 5 strains of M . intracelllulare showed a relatively high virulence . They grew in the liver, spleen and lungs of susceptible mice and even in the lungs of resistant mice . On the other hand, none of 6 strains of M . avium could grow in either susceptible or resistant mice . In conclusion, our mouse model may be a useful tool for evaluating the virulence of bacteria isolated from mycobacteriosis patients.

Nucleic Acids Res, 2002 Jul 15, 30(14), 3067 - 77
A genetic screen identifies novel non-compatible loxP sites; Langer SJ et al.; The ability of the Cre/lox system to make precise genomic modifications is a tremendous accomplishment . However, recombination between cis-linked heterospecific lox sites limits the use of Cre- mediated exchange of DNA to systems where genetic selection can be applied . To circumvent this problem we carried out a genetic screen designed to identify novel mutant spacer-containing lox sites displaying enhanced incompatibility with the canonical loxP site . One of the mutant sites recovered appears to be completely stable in HEK293 cells constitutively expressing Cre recombinase and supports recombinase-mediated cassette exchange (RMCE) in bacteria and mammalian cell culture . By preventing undesirable recombination, these novel lox sites could improve the efficiency of in vivo gene transfer.

FEBS Lett, 2002 Jul 31, 524(1-3), 127 - 33
Transient interaction of cpSRP54 with elongating nascent chains of the chloroplast-encoded D1 protein; 'cpSRP54 caught in the act'; Nilsson R et al.; The signal recognition particle (SRP) in bacteria and endoplasmic reticulum is involved in co-translational targeting . Plastids contain cpSRP54 and cpSRP43, unique to plants, but lack a SRP RNA molecule . A role for cpSRP in biogenesis of plastid-encoded membrane proteins has not been firmly established yet . In this study, a transient interaction between cpSRP54 and elongating D1 protein was observed using a homologous chloroplast translation system . Using the novel approach of cross-linking at different time points during elongation of full-length D1 protein, we showed that cpSRP54 interacts strongly with the elongating nascent chain forming two distinct cross-linked products . However, this interaction did not lead to an elongation arrest and cpSRP54 was released from the nascent chains, once they were longer than approximately 14 kDa . Detailed mutant analysis showed that the cpSRP54 interaction occurred via the first transmembrane domain, which could be replaced by other hydrophobic domains of more than 10 amino acids.

Am J Trop Med Hyg, 2002 Jan, 66(1), 108 - 11
Field prevalence of Wolbachia in the mosquito vector Aedes albopictus; Kitrayapong P et al.; The endosymbiotic bacteria in the genus Wolbachia have been proposed as a potential candidate to deliver pathogen-blocking genes into natural populations of medically important insects . The successful application of Wolbachia in insect vector control depends on the ability of the agent to successfully invade and maintain itself at high frequency under field conditions . Here, we evaluated the prevalence of Wolbachia infections in a field population of the Wolbachia-superinfected mosquito Aedes albopictus . A field prevalence of 100% (n = 1,016) was found in a single population in eastern Thailand via polymerase chain reaction (PCR) testing of Wolbachia both from individual parent females and their corresponding F1 offspring . This is the first report of accurate Wolbachia prevalence in a field population of an insect disease vector . The prevalence of superinfection was estimated to be 99.41% . All single-infected individual mosquitoes (n = 6) were found to harbor group A Wolbachia . For this particular population, none was found to be single-infected with group B Wolbachia . Our results also show that PCR testing of field materials alone without checking F1 offspring overestimated the natural prevalence of single infection . Thus, the confirmation of infection status by means of F1 offspring was critical to the accurate estimates of Wolbachia prevalence under field conditions.

Am J Trop Med Hyg, 2002 Jan, 66(1), 103 - 7
Maternal transmission efficiency of Wolbachia superinfections in Aedes albopictus populations in Thailand; Kittayapong P et al.; We examined the transmission efficiency of 2 strains of Wolbachia bacteria that cause cytoplasmic incompatibility in field populations of Aedes albopictus by polymerase chain reaction assay . We found mainland and island populations throughout Thailand to be superinfected with group A and B bacteria . Of 320 Wolbachia-positive adult mosquitoes, 97.5% were infected with both groups . Single infected individuals of each Wolbachia group were encountered in nearly equal numbers . We screened 550 offspring from 80 field-collected mothers and found the transmission efficiency of group A Wolbachia to be 96.7% and that of group B Wolbachia to be 99.6% . Mothers that did not transmit both Wolbachia infections to all of their offspring were significantly larger in size than those with perfect transmission fidelity . We discuss our findings in relation to the prospects of the use of Wolbachia as a gene-driving mechanism.

Arch Pharm Res, 2002 Jun, 25(3), 258 - 69
Synthesis of new 2-thiouracil-5-sulfonamide derivatives with biological activity; Fathalla A et al.; 2-Thiouracil-5-sulfonylchloride 1 reacted with a series of aromatic and heterocyclic amines to give 2a-j . The same compound 1 was reacted with a series of sulphonamides giving different sulphonamides of type 3a-e . On the other hand compound 1 was allowed to react with p-aminoacetophenone givining compound 4 which in turn was allowed to react with derivatives of alkyl thiosemicarbazides to give thiosemicarbazones of type 5a-e, also compound 4 was monobrominated to give compound 6 which in turn was reacted thiosemicarbazones of some aldehydes to give the corresponding thiazole derivatives 7a-f . In the same time compound 4 was reacted with a series of aromatic and heterocyclic aldehydes givining chalcones 8a-g (Claisen-Schemidt reaction) . Also compound 4 was allowed to react with a series of aromatic and heterocyclic aldehydes, ethyl cyano acetate and/or malononitrile, and ammonium acetate giving pyridine derivatives 9a-d and 10a-e respectively . The biological effects of some of the new synthesized compounds was also investigated.

Pharm Res, 2002 Jun, 19(6), 825 - 31
Transport and utilization of arginine and arginine-containing peptides by rat alveolar macrophages; Yang XD et al.; PURPOSE: To demonstrate that rat alveolar macrophages (AM) exhibited the PepT1-like transporter for the uptake of arginine (Arg)-containing small peptides and utilized these peptides as direct substrates for nitric oxide (NO) production . NO is an important mediator that, on one hand, protects the lung from bacteria infection and, on the other hand, augments inflammatory lung injury . METHOD: The uptake of small peptides by rat AM was evaluated using fluorescein isothiocyanate (FITC)-labeled (*) peptides (Arg-Lys*, Gly-Sar-Lys*, and beta-Ala-Lys*), high-performance liquid chromatography (HPLC) analysis of potential peptide degradation, and known inhibitors of Arg and PepT1 transport . NO production by AM through Arg and Arg-containing peptides was studied with and without inhibition by transport inhibitors . The presence of PepT1-like transporter on AM was evaluated using anti-PepT1 antisera and Western blot analysis . The substrate specificity of Arg-Gly and Arg-Gly-Asp was determined using purified inducible NO synthase (iNOS) . The availability of Arg-containing peptides in the lung was determined by HPLC analysis of bronchoalveolar lavage (BAL) fluid . RESULTS: The FITC-labeled peptides were internalized by AM without degradation . The uptake of Arg-Lys*, beta-Ala-Lys*, and Gly-Sar-Lys* was blocked (approximately 50%) by cephradine (an inhibitor of PepT1 for peptide transport) but not by Lys (an inhibitor on cationic amino acid transporter 2B for Arg transport) . The NO production by AM through Arg-containing peptides was significantly blocked only by PepT1 inhibitors and by an anti-PepT1 antibody in a dose-dependent manner . These inhibitors had no effect on the AM production of NO using Arg as a substrate . Arg-Gly and Arg-Gly-Asp were found to be direct substrates for iNOS with similar Km and Vmax values to those of Arg . But, the production of NO by AM using these peptides as substrates was 2-fold higher than using Arg as a substrate . Both Arg-Gly and Arg-Gly-Asp were found in the BAL fluid . The presence of a PepT1-like transporter on AM was confirmed by Western blotting . CONCLUSION: This study shows that AM exhibit PepT1-like transporter for small peptide uptake . Arginine-containing peptides, through the PepT1 transporter system, can serve as direct substrates of iNOS for the production of NO by AM.

J Immunol Methods, 2002 Aug 1, 266(1-2), 197 - 207
Isolation and characterization of rabbit single chain antibodies to human Reg Ialpha protein; Chi XS et al.; To investigate the role of Reg Ialpha in human inflammatory bowel disease (IBD), we made two phage-displayed single chain variable fragment (scFv) libraries from rabbits immunized with recombinant or native human Reg Ialpha . After one to three rounds of panning, we were able to isolate phage-displaying scFvs, which bound to human Reg Ialpha . Anti-Reg Ialpha scFvs from both libraries showed similar immunoreactivity to different processed forms of the protein . Despite several DNA fingerprint patterns among these clones, their deduced amino acid sequences are highly homologous with 100% identity in the complementarity-determining regions (CDRs) of the variable segment of heavy chain (VH) region and a small variation in the CDR1 of the variable segment of light chain (VL) region . We also expressed and purified soluble myc-tagged or glutathione S-transferase (GST) fusion scFv proteins from bacteria . Immunohistochemical studies using one of our anti-Reg Ialpha scFv antibodies showed prominent staining in the metaplastic Paneth cell population and light staining in the lamina propria . This scFv antibody is now being used for studies of the role of Reg Ialpha in human IBD.

Z Naturforsch {C}, 2002 May-Jun, 57(5-6), 516 - 21
Discriminatory power of RAPD, PCR-RFLP and southern blot analyses of ureCD or ureA gene probes on Helicobacter pylori isolates; Smith S et al.; The genetic diversity of 33 Nigerian Helicobacter pylori isolates were studied using RAPD, PCR-RFLP and Southern blot analysis of ureA or ureCD gene probes . RAPD was able to distinguish the following number of isolates using the primers 3880: 5'-AAGAGCCCGT-3' (28), 3881 :5'-AACGCGCAAC-3' (33) and OPH8 :5'-GAAACACCCC-3' (25) . Southern blot analysis using the ureCD probe was also able to distinguish the 12 isolates tested into ten different patterns . The PCR-RFLP technique distinguished all 33 isolates into six types . In conclusion, considering typeability, discriminatory power, and convenience, RAPD with the 3881 primer was considered the most useful technique.

Biol Pharm Bull, 2002 Jul, 25(7), 861 - 6
Prevention of growth and metastasis of murine melanoma through enhanced natural-killer cytotoxicity by fatty acid-conjugate of protopanaxatriol; Hasegawa H et al.; Ginsenosides, the glycosides of Panax ginseng, are metabolized (deglycosylated) by intestinal bacteria after oral administration . 20(S)-Protopanaxatriol (M4) is the main bacterial metabolite of protopanaxatriol-type ginsenosides and mediates their antitumor effects . To clarify the mechanism of the M4-mediated antitumor effect, the antitumor activity and metabolism of M4 was examined, using the C57BL/6 mice implanted with B16-BL6 melanoma . The chronic oral administration of M4 inhibited the growth of B16-BL6 melanoma at the implanted site . Analyses using TLC, HPLC, MS and NMR suggest that orally administered M4 was absorbed from the small intestine into the mesenteric lymphatics followed by the rapid esterification of M4 with fatty acids and its accumulation in the tissues including the liver and lung . The administration of M4 prior to the intravenous injection of B16-BL6 cells abrogated the enhanced lung metastasis in the mice pretreated with 2-chloroadenosine more effectively than in those pretreated with anti-asialo GM1 . The esterified M4 (EM4) did not directly affect tumor growth in vitro, whereas it stimulated splenic NK cells to become cytotoxic to tumor cells . These results indicate that the antitumor activity of M4 is based on the NK cell-mediated tumor lysis enhanced by EM4.

Salud Publica Mex, 2002 May-Jun, 44(3), 201 - 6
{Acute respiratory infections in children attending a child day care center}; Nandi-Lozano E et al.; OBJECTIVE: To assess the incidence of acute respiratory infections and bacterial colonization in children attending a daycare center . MATERIAL AND METHODS: A cohort study was conducted from April to Octuber 1999, among 85 children aged under four years, who attended the daycare center at Hospital Infantil de Mexico (Mexico City's Children's Hospital) "Federico Gomez" . Acute respiratory infection incidence rates and quarterly point prevalence figures of nasopharyngeal colonization were obtained . Data were analyzed using descriptive statistics . RESULTS: A total of 85 children were studied (40 girls and 45 boys) during 9,090 children-days of follow-up . Three children had a history of atopia (3.5%), six a history of asthma (7%), and 39 (46%) were exposed to passive smoking . There were 258 events of respiratory tract infection for an incidence rate of 10.3 infections per person-year (95% CI 8.7-12.0) . The main clinical syndromes were pharyngitis (95%), acute otitis media (3.5%), and bronchiolitis (1%) . The incidence rates of otitis and bronchiolitis were 0.36 and 0.12 per child-year of observation, respectively . The prevalence figures of nasopharyngeal colonization for the three main bacteria were: S . pneumoniae 20.4%; nontypable H . influenzae 13%; and Moraxella catarrhalis 8% . CONCLUSIONS: Study results show a high prevalence of colonization due to invasive strains, as well as a two-fold incidence rate of acute respiratory infection, higher than those reported in community surveys . These results add to the description of this poorly documented infectious disease in Mexico . The English version of this paper is available at: http://www.insp.mx/salud/index.html.

J Prosthet Dent, 2002 Jun, 87(6), 674 - 8
Endodontic failure caused by inadequate restorative procedures: review and treatment recommendations; Heling I et al.; A review of the literature was performed to determine whether prompt placement of coronal restorations, including sealing and placement of posts and cores, can positively influence the long-term prognosis of teeth after root canal therapy . Both hand and MEDLINE searches were employed to identify peer-reviewed articles on radicular apical integrity after coronal restorations, especially where root canal space was used for post and core fabrication . A total of 41 articles published between 1969 and 1999 (the majority from the 1990s) were reviewed . The literature suggests that the prognosis of root canal-treated teeth can be improved by sealing the canal and minimizing the leakage of oral fluids and bacteria into the periradicular areas as soon as possible after the completion of root canal therapy.

Chem Pharm Bull (Tokyo), 2002 Jul, 50(7), 892 - 5
An in vitro and in vivo investigation into the suitability of bacterially triggered delivery system for colon targeting; Raghavan CV et al.; The colon specific drug delivery systems based on polysaccharides; locust bean gum and chitosan in the ratio of 2 : 3, 3 : 2 and 4 : 1 were evaluated using in vitro and in vivo methods . The in vitro studies in pH 6.8 phosphate buffer containing 2% w/v rat caecal contents showed that the cumulative percentage release of mesalazine after 26 h were 31.25+/-0.56, 46.25+/-0.96, 97.5+/-0.26 (mean+/-S.D.), respectively . The in vivo studies conducted in nine healthy male human volunteers for the various formulations revealed that, the drug release was initiated only after 5 h (i.e.) transit time of small intestine and the bioavailability (AUC(0-->t*)) of the drug was found to be 85.24+/-0.10, 196.08+/-0.12, 498.62+/-0.10 microg x h/ml 26 (mean+/-S.D.), respectively . These studies on the polysaccharides demonstrated that the combination of locust bean gum and chitosan as a coating material proved capable of protecting the core tablet containing mesalazine during the condition mimicking mouth to colon transit . In particular, the formulation containing locust bean gum and chitosan in the ratio of 4 : 1 held a better dissolution profile, higher bioavailability and hence a potential carrier for drug targeting to colon.

Zoolog Sci, 2002 Jun, 19(6), 695 - 701
A comparative study of body wall structures of a pogonophore and an annelid from a phylogenetic viewpoint; Matsuno A et al.; Pogonophores are tube worms that live in reducing deep-sea waters where sunlight does not penetrate . They are highly adapted for their special habitat in lacking guts and possessing endosymbiotic chemosynthetic bacteria . Because of these peculiar characteristics, it is not yet clear whether they should be classified as annelids or not . Electron-microscopic observations of sections of a Japanese pogonophore (Oligobrachia mashikoi) show that the body wall has circular and longitudinal muscular systems . These muscular systems, however, differ from the annelid (Branchiura sowerbyi) in these ways: (1) The outer circular muscle of the pogonophore was constructed of smooth muscle cells . In contrast, that of the annelid was composed of obliquely-striated muscle cells, even though the cells were small and bore undeveloped characteristics . (2) The inner longitudinal muscle of the pogonophore was constructed of undeveloped obliquely-striated muscle cells, whereas that of the annelid was composed of well-developed ones . These observations suggest that this pogonophore can not be classified as an annelid, although many previous studies have placed pogonophores in that phylum.

J Biol Chem, 2002 Sep 27, 277(39), 36351 - 6 Epub 2002 Jul 18.
NMR structure of PW2 bound to SDS micelles . A tryptophan-rich anticoccidial peptide selected from phage display libraries; Tinoco LW et al.; PW2 (HPLKQYWWRPSI) was selected from phage display libraries through an alternative panning method using living sporozoites of Eimeria acervulina as target . Synthetic PW2 shows anticoccidial activity against E . acervulina and Eimeria tenella with very low hemolytic activity . It also displays antifungal activity but no activity against bacteria . We present the solution structure of the PW2 bound to SDS micelles . In the absence of an interface, PW2 is in random coil conformation . In micelles, structural calculation shows that Trp-7 forms the hydrophobic core that is important for the peptide folding . Lys-4, Tyr-6, Trp-8, and Arg-9 are in the same surface, possibly facing the micelle interface . This possibility was supported by the fact that chemical shift differences for these residues were more pronounced when compared with PW2 in water and in SDS . PW2 gains structure upon binding to SDS micelles . Lys-4, Tyr-6, Trp-8, and Arg-9 were found to bind to the micelle . Trp-7, Trp-8, and Arg-9 composed the WW+ consensus found in the sequence of the peptides selected with the phage display technique against E . acervulina sporozoites . This suggested that Trp-7, Trp-8, and Arg-9 are probably key residues not only for the peptide interaction with SDS micelles but also for the interaction with E . acervulina sporozoites surface.

Comp Biochem Physiol B Biochem Mol Biol, 2002 Aug, 132(4), 739 - 50
Proline biosynthesis genes and their regulation under salinity stress in the euryhaline copepod Tigriopus californicus; Willett CS et al.; Diverse organisms regulate concentrations of intracellular organic osmolytes in response to changes in environmental salinity or desiccation . In marine crustaceans, accumulation of high concentrations of proline is a dominant component of response to hyperosmotic stress . In the euryhaline copepod Tigriopus californicus, synthesis of proline from its metabolic precursor glutamate is tightly regulated by changes in environmental salinity . Here, for the first time in a marine invertebrate, the genes responsible for this pathway have been cloned and characterized . The two proteins display the sequence features of homologous enzymes identified from other eukaryotes . One of the cloned genes, delta1-pyrroline-5-carboxylase reductase (P5CR), is demonstrated to have the reductase enzyme activity when expressed in proline-auxotroph bacteria, while the second, delta1-pyrroline-5-carboxylase synthase (P5CS), does not rescue proline-auxotroph bacteria . In contrast to results from higher plants, neither levels of P5CS nor P5CR mRNAs increase in response to salinity stress in T . californicus . Hence, regulation of proline synthesis during osmotic stress in T . californicus is likely mediated by some form of post-transcriptional regulation of either P5CS or P5CR . Understanding the regulation this pathway may elucidate the mechanisms limiting the salinity ranges of marine taxa .

Microb Pathog, 2002 Jul, 33(1), 7 - 15
Previous infection with the nematode Nippostrongylus brasiliensis alters the immune specific response against Chlamydophila abortus infection; Buendia A et al.; An experimental mouse model to analyze the interaction between the immune responses elicited following infection with Nippostrongylus brasiliensis and Chlamydophila abortus has been established . Mice infected with C . abortus 7 days after N . brasiliensis showed an increased bacterial multiplication in spleen and liver compared to bacteria-alone infected mice . However the morbidity of these mice, expressed as weight loss, was significantly lower . Analysis of the immune responses elicited showed that spleen from co-infected mice had reduced IFN-gamma production in response to C . abortus antigen . The bias towards a type 2 response in co-infected mice was confirmed by an increase in the production of IL-4 and in the lower ratio IgG2a/IgG1 . In pregnant mice co-infection caused a delay in the time of abortion and an increased systemic susceptibility to C . abortus infection.

Vaccine, 2002 Jul 26, 20(23-24), 2981 - 8
Serum-derived IgG1-mediated immune exclusion as a mechanism of protection against H . pylori infection; Keenan J et al.; The induction of protective immunity against Helicobacter challenge in a murine model was found to correlate with the magnitude of IgG (serum and gastric lavage) responsiveness to intra-nasal (i.n.) immunisation . IgG1-secreting hybridoma backpacks in Helicobacter pylori (H . pylori)-infected mice revealed serum transudation into the stomach . A Lpp20-specific monoclonal antibody was associated with significantly reduced H . pylori colonisation . Histology revealed aggregates of the remaining H . pylori in these mice, suggesting a role for IgG1-mediated immune exclusion of the bacteria . In vitro immunogold electron microscopy supported this hypothesis, but also suggested that a threshold of H . pylori-specific antibody needs to be maintained if immune exclusion by the host is to overcome immune evasion by the bacteria.

Toxicology, 2002 Aug 1, 177(1), 11 - 22
Tyrosyl radical production by myeloperoxidase: a phagocyte pathway for lipid peroxidation and dityrosine cross-linking of proteins; Heinecke JW; To kill invading bacteria, viruses, and fungi, phagocytes secrete hydrogen peroxide (H(2)O(2)) and the heme enzyme myeloperoxidase . We have explored the possibility that myeloperoxidase might use H(2)O(2) to convert L-tyrosine to tyrosyl radical . Activated human neutrophils and monocytes used the system to oxidize free L-tyrosine to o,o'-dityrosine, a stable product of tyrosyl radical . Protein-bound tyrosyl residues exposed to myeloperoxidase, H(2)O(2), and L-tyrosine were also oxidized to o,o'-dityrosine . The cross-linking reaction required free L-tyrosine, suggesting that myeloperoxidase converts the amino acid to a diffusible radical catalyst that promotes protein oxidation . We used electron paramagnetic resonance to provide direct evidence that the oxidizing intermediate is free tyrosyl radical . Myeloperoxidase-generated tyrosyl radical also initiates lipid peroxidation, suggesting that activated phagocytes might also be able to oxidize lipids in host tissues . Moreover, myeloperoxidase is present and active in human atherosclerotic tissue, and levels of protein-bound dityrosine are elevated in such lesions . Our recent studies indicate that activated neutrophils use oxidants generated by the phagocyte NADPH oxidase to produce protein-bound dityrosine during acute inflammation . Collectively, these findings suggest that generation of tyrosyl radical by myeloperoxidase allows activated phagocytes to damage both proteins and lipids . Elevated levels of o,o'-dityrosine have been detected in inflammatory lung disease, neurodegenerative disorders, and aging . Thus, oxidation of tyrosine to tyrosyl radical might play a role in the pathogenesis of many diseases.

Free Radic Biol Med, 2002 Aug 1, 33(3), 323 - 36
Molecular and cellular mechanisms involved in Helicobacter pylori-induced inflammation and oxidative stress; Naito Y et al.; Helicobacter pylori (H . pylori)-infection leads to different clinical and pathological outcomes in humans, including chronic gastritis, peptic ulcer disease, and gastric neoplasia . The key determinants of these outcomes are the severity and distribution of the H . pylori-induced inflammation . Antral-type gastritis is associated with excessive acid secretion and a high risk of duodenal ulcer . In contrast, gastritis that involves the acid-secreting corpus region leads to hypochlorhydria, progressive gastric atrophy, and an increased risk of gastric cancer . The key pathophysiological event in H . pylori infection is initiation and continuance of an inflammatory response . Bacteria or their products trigger this inflammatory process and the main mediators are cytokines . Identification of both host- and bacterial-factors that mediate is an intense area of interest in current researches . Recent data indicates that the cag pathogenicity island plays a crucial role in H . pylori-induced gastric inflammation via the activation of gene transcription . It has been demonstrated that oxidative and nitrosative stress associated with inflammation plays an important role in gastric carcinogenesis as a mediator of carcinogenic compound formation, DNA damage, and cell proliferation . Genetic information regulating such stress would be one of the host factors determining the outcome--particularly when the outcome is gastric cancer--of H . pylori infection, and the compound that attenuates such stress may be a candidate for use in chemoprevention . This review highlights recent advances in understanding of the mechanisms underlying chronic inflammation following infection with H . pylori.

J Org Chem, 2002 Jul 26, 67(15), 5416 - 8
Synthesis of 4-diphosphocytidyl-2-C-methyl-D-erythritol and 2-C-methyl-D-erythritol-4-phosphate; Koppisch AT et al.; 2-C-Methyl-D-erythritol 4-phosphate (MEP, 2) and 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDPME, 3) are metabolites in the MEP pathway for biosynthesis of isoprenoid compounds in bacteria, plant chloroplasts, and algae . The free phosphoacid of 2 was prepared from benzyloxyacetone in five steps with an overall yield of 27% and an enantiomeric ratio (er) of 75:25 . Following titration to the corresponding tributylammonium salt, 2 was coupled to cytidine 5'-monophosphate using a protocol originally developed for synthesis of base-sensitive nucleoside diphosphate sugars to give 3 in 40% yield, following purification by size exclusion chromatography.

J Mol Microbiol Biotechnol, 2002 Jul, 4(4), 405 - 15
Bioinformatic analyses of the tRNA: (guanine 26, N2,N2)-dimethyltransferase (Trm1) family; Bujnicki JM et al.; Functional analyses of the tRNA:(guanine 26, N2,N2)-dimethyltransferase (Trm1) have been hampered by a lack of structural information about the enzyme and by low sequence similarity to better studied methyltransferases . Here we used computational methods to detect novel homologs of Trm1, infer the evolutionary relationships of the family, and predict the structure of the Trm1 methyltransferase . The N-terminal region of the protein is predicted to form an S-adenosylmethionine-binding domain, which harbors the active site . The C-terminal region is rich in predicted alpha-helices and, in analogy to other nucleic acid methyltransferases, may constitute the target recognition domain of the enzyme . Interposing these two domains, most Trm1 homologs possess a highly variable inserted sequence that is delimited by a Cys4 cluster, likely forming a Zn-finger structure . The residues of Trm1 predicted to participate in cofactor binding, target recognition, and catalysis, were mapped onto a preliminary structural model, providing a platform for designing new experiments to better understand the molecular functions of this protein family . In addition, identification of novel, atypical Trm1 homologs suggests candidates for cloning and biochemical characterization.

Ann Dermatol Venereol, 2002 May, 129(5 Pt 1), 728 - 31
{Mycobacterial bovis BCG cutaneous infections following mesotherapy: 2 cases}; Marco-Bonnet J et al.; INTRODUCTION: Infectious complications following mesotherapy are usually due to ordinary bacteria or atypical mycobacteria . We report two new cases of mycobacterial bovis BCG infections following mesotherapy . To our knowledge only one case has already been reported.CASES REPORTS: A 52 year-old woman developed vaccinal MERIEUX BCG cutaneous abscesses following mesotherapy . Identification was made by a novel class of repeated sequences: Mycobacterial interspersed repetitive units . Despite prolonged anti-tuberculous therapy, complete remission was not obtained and surgical excision was performed . The second case was a 49 year-old man who developed a mycobacterial bovis BCG cutaneous abscess (Connaught) after mesotherapy, the regression of which was obtained with anti-tuberculous therapy.DISCUSSION: The severity of these two mycobacterial infections following mesotherapy illustrate the potential risks of mesotherapy . Identification is possible by molecular biology techniques (PCR and sequencing) . The origin of this infection is unclear and therapeutic decision is difficult . Some authors recommend anti-tuberculous therapy but surgical excision may be necessary as in our cases.

Anal Biochem, 2002 Jul 15, 306(2), 171 - 80
23Na NMR study of Fibrobacter succinogenes S85: comparison of three chemical shift reagents and calculation of sodium concentration using ionophores; Delort AM et al.; In order to measure intracellular sodium concentrations in resting cells of Fibrobacter succinogenes S85 by (23)Na NMR spectrometry, two methodological aspects were studied . First, three different shift reagents (Dy(PPP(i))(7-)(2), Tm(DOTP)(5-), and Dy(TTHA)(3-)) were tested for their ability to separate internal and external (23)Na NMR resonances . Their toxicity toward F . succinogenes cells was evaluated by in vivo(13)C NMR experiments . Tm(DOTP)(5-) was found to be the most efficient shift reagent while being nontoxic . Second, a new methodology was developed to calculate intracellular sodium concentration in F . succinogenes by using ionophores . This approach avoided the problem of intracellular volume measurement and that of sodium visibility determination.

Mol Microbiol, 2002 Jul, 45(2), 365 - 74
Role of the extracytoplasmic-function sigma factor sigma(H) in Mycobacterium tuberculosis global gene expression; Manganelli R et al.; Like other bacterial species, Mycobacterium tuberculosis has multiple sigma (sigma) factors encoded in its genome . In previously published work, we and others have shown that mutations in some of these transcriptional activators render M . tuberculosis sensitive to various environmental stresses and, in some cases, cause attenuated virulence phenotypes . In this paper, we characterize a M . tuberculosis mutant lacking the ECF sigma factor sigma(H) . This mutant was more sensitive than the wild type to heat shock and to various oxidative stresses, but did not show decreased ability to grow inside macrophages . Using quantitative reverse transcription-PCR and microarray technology, we have started to define the sigma(H) regulon and its involvement in the global regulation of the response to heat shock and the thiol-specific oxidizing agent diamide . We identified 48 genes whose expression increased after exposure of M . tuberculosis to diamide; out of these, 39 were not induced in the sigH mutant, showing their direct or indirect dependence on sigma(H) . Some of these genes encode proteins whose predicted function is related to thiol metabolism, such as thioredoxin, thioredoxin reductase and enzymes involved in cysteine and molybdopterine biosynthesis . Other genes under sigma(H) control encode transcriptional regulators such as sigB, sigE, and sigH itself.

Gastroenterol Clin North Am, 2002 Mar, 31(1), 63 - 76
The genetics of inflammatory bowel disease; Duerr RH; The complex genetics of IBD is characterized by more than one susceptibility locus, genetic heterogeneity, incomplete penetrance, and probable gene-gene and gene-environment interactions . Functional candidate gene association studies during the past few decades have revealed only modest associations between IBD and genetic variants in the HLA genes and a limited number of other genes that are involved in immune regulation and the inflammatory response . Important advances in IBD genetics research have come about from systematic genome searches for IBD loci . The identification of Crohn's disease-associated NOD2 genetic variants that appear to alter the innate immune response to bacteria is a seminal finding that perhaps is the greatest advance toward understanding the pathogenesis of IBD in decades . The future discovery of other IBD genetic risk factors, facilitated by the completion of the human genome sequencing and annotation, may allow the development of better therapies, possibly including preventive therapies, for patients with Crohn's disease and ulcerative colitis.

Z Gastroenterol, 2002 Jul, 40(7), 525 - 9
Role of the intestinal epithelium in orchestrating innate and adaptive mucosal immunity; Maaser C et al.; The mucosa that lines the human colon and small intestine is a site of chronic regulated "physiologic" inflammation . This contrasts markedly with other mucosal sites in that if the numbers of T and B cells, eosinophils, mast cells, macrophages, and dendritic cells that are present in the human intestinal tract were to be present in other sites, those sites would be considered to be sites of chronic pathological inflammation . This review examines the role of the intestinal epithelium in the development of "physiologic" intestinal mucosal inflammation and focuses on its role in signalling and mediating host innate and adaptive mucosal immune responses.

Proc Natl Acad Sci U S A, 2002 Aug 6, 99(16), 10417 - 22 Epub 2002 Jul 16.
The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans; Morley JF et al.; Studies of the mutant gene in Huntington's disease, and for eight related neurodegenerative disorders, have identified polyglutamine (polyQ) expansions as a basis for cellular toxicity . This finding has led to a disease hypothesis that protein aggregation and cellular dysfunction can occur at a threshold of approximately 40 glutamine residues . Here, we test this hypothesis by expression of fluorescently tagged polyQ proteins (Q29, Q33, Q35, Q40, and Q44) in the body wall muscle cells of Caenorhabditis elegans and show that young adults exhibit a sharp boundary at 35-40 glutamines associated with the appearance of protein aggregates and loss of motility . Surprisingly, genetically identical animals expressing near-threshold polyQ repeats exhibited a high degree of variation in the appearance of protein aggregates and cellular toxicity that was dependent on repeat length and exacerbated during aging . The role of genetically determined aging pathways in the progression of age-dependent polyQ-mediated aggregation and cellular toxicity was tested by expressing Q82 in the background of age-1 mutant animals that exhibit an extended lifespan . We observed a dramatic delay of polyQ toxicity and appearance of protein aggregates . These data provide experimental support for the threshold hypothesis of polyQ-mediated toxicity in an experimental organism and emphasize the importance of the threshold as a point at which genetic modifiers and aging influence biochemical environment and protein homeostasis in the cell.

Oper Dent, 2002 Jul-Aug, 27(4), 330 - 42
Histopathologic study on pulp response to single-bottle and self-etching adhesive systems; Medina VO 3rd et al.; This study compared the pulp response to seven adhesive resins (three single-bottle and four self-etching primers) and their companion resin composite systems with a commercial calcium hydroxide material when applied to exposed monkey pulps . The control group was capped with Dycal (DY), while the experimental groups were capped with one of the following adhesive resin systems: AQ Bond (AQ), Single Bond (SB), Imperva Fluorobond (IF), One Step (OS), Prime&Bond NT (PBNT), Perme Bond F (PBF) and One-up Bond F (OBF) . Histopathologic evaluation of pulp tissue disorganization, inflammatory cell infiltration, reparative dentin formation and bacterial penetration at the 3rd, 30th and 90th post-operative days was done using light microscopy . Data were analyzed using the Kruskal-Wallis test followed by the Least Significant Difference Test to determine differences between the control group and the experimental groups at each observation period . The correlation of inflammatory cell infiltration and bacterial presence was investigated by the Kendall correlation analysis . All tests were performed at a 95% level of confidence . The pulpal responses of groups DY, SB, OS, PBF and OBF were generally characterized by none-to-mild pulp tissue disorganization and inflammatory cell infiltration . Also, initiation of reparative dentin formation was found earlier in Group DY, resulting in more complete dentin bridges at the 30- and 90-day observation periods . Groups AQ, IF and PBNT had significantly more inflammatory cell infiltration and a lower incidence of reparative dentin formation than Group DY . A significant correlation was detected between inflammatory cell infiltration and the presence of bacteria . It is concluded that the pulp response to SB, OS, PBF and OBF is not significantly different from the calcium hydroxide preparation . However, calcium hydroxide capping resulted in a higher incidence and faster rate of reparative dentin formation.

Chem Commun (Camb), 2002 Feb 21, (4), 318 - 9
Redox control of the P450cam catalytic cycle: effects of Y96F active site mutation and binding of a non-natural substrate; Reipa V et al.; Spectroelectrochemistry measurements are used to demonstrate that active site mutation and binding of an non-natural substrate to P450cam (CYP101) reduces the shift in the redox potential caused by substrate-binding, and thereby results in slower catalytic turnover rate relative to wild-type enzyme with the natural camphor substrate.

Int J Lepr Other Mycobact Dis, 2002 Mar, 70(1), 25 - 33
Light and ultrastructural study of sciatic nerve lesions induced using intraneural injection of viable Mycobacterium leprae in normal and immunosuppressed Swiss white mice; Shetty VP et al.; Freshly harvested M . leprae were microinjected into the sciatic nerves of nonimmunosuppressed (non-TR) and immunosuppressed (TR) mice using the technique described by Wisniewski and Bloom . The lesions thus induced, on bypassing the blood-nerve barrier, were biopsied at regular intervals beginning 24 hr and followed up to one year . The fate of M . leprae and the ensuing inflammation and nerve damage were studied using light and electron microscopy . The lesions in both non-TR and TR mice at 24 hr showed an influx of polymorphonuclear leukocytes and an increase in mast cells . The influx and peaking of lymphocytes were delayed by two weeks and 6 weeks, respectively, in TR mice, but the density of lymphocytes at the peak intervals was comparable in both . The plasma cells denoting the humoral response were seen in both, but there was a delay of 3 weeks in non-TR mice . The lesions in non-TR mice showed differentiation of macrophages into epithelioid cells and the formation of giant cells depicting borderline tuberculoid leprosy (BT), Whereas in TR mice, the macrophages showed foamy cytoplasmic changes depicting borderline lepromatous leprosy (BL) . Other significant observations common to both non-TR and TR mice were: a) The lesions remained highly localized and showed signs of regression at the 6th and the 12th month intervals . b) The characteristic segmental demyelination and some attempt at remyelination were seen at the site . c) The influx of lymphocytes concorded well with demyelination . d) Bacteria were only seen in the macrophages and never in the Schwann cells or endothelial cells . e) Bacteria persisted in the macrophages, but appeared progressively degenerate at the 6th and 12th post-inoculation months, suggesting loss of viability . The study shows that there was a very effective containment of the infection and that the Schwann cells were resistant to M . leprae infection in the neural milieu . Nerve damage and Schwann cell bacillation do not go hand-in-hand.

Proc Natl Acad Sci U S A, 2002 Jul 23, 99(15), 10173 - 8 Epub 2002 Jul 15.
Chaotic mixing deep in the lung; Tsuda A et al.; Our current understanding of the transport and deposition of aerosols (viruses, bacteria, air pollutants, aerosolized drugs) deep in the lung has been grounded in dispersive theories based on untested assumptions about the nature of acinar airflow fields . Traditionally, these have been taken to be simple and kinematically reversible . In this article, we apply the recently discovered fluid mechanical phenomenon of irreversible low-Reynolds number flow to the lung . We demonstrate, through flow visualization studies in rhythmically ventilated rat lungs, that such a foundation is false, and that chaotic mixing may be key to aerosol transport . We found substantial alveolar flow irreversibility with stretched and folded fractal patterns, which lead to a sudden increase in mixing . These findings support our theory that chaotic alveolar flow--characterized by stagnation saddle points associated with alveolar vortices--governs gas kinematics in the lung periphery, and hence the transport, mixing, and ultimately the deposition of fine aerosols . This mechanism calls for a rethinking of the relationship of exposure and deposition of fine inhaled particles.

Atherosclerosis, 2002 Sep, 164(1), 153 - 60
Positive Chlamydia pneumoniae serology is associated with elevated levels of tumor necrosis factor alpha in patients with coronary heart disease; Schumacher A et al.; Infectious agents are possible stimulators of inflammation in atherogenesis . The aim of this study was to investigate if Chlamydia pneumoniae and Helicobacter pylori were associated with elevated levels of tumor necrosis factor alpha (TNFalpha) and interleukin-6 in coronary heart disease (CHD) patients (n=193) and age- and sex-matched controls (n=193) as markers of increased inflammatory activity . C reactive protein (CRP) and fibrinogen were also included . Serologic status towards the two bacteria was measured and levels of the inflammatory markers were compared between seropositives and seronegatives, each study group being evaluated separately . In CHD patients Chlamydia lipopolysaccharide (LPS) IgA seropositivity predicted elevated TNFalpha levels (P=0.009), still statistically significant after adjustment for traditional cardiovascular risk factors (P=0.005) . Chlamydia LPS IgG seropositivity independently predicted fibrinogen levels in CHD patients (P=0.028), while no association between serology and inflammatory markers were observed among controls . H . pylori seropositivity alone was not associated with any increase in the inflammatory markers in any of the two groups . However, in CHD patients seropositivity to both agents predicted higher levels of TNFalpha (P=0.041), CRP (P=0.037) and fibrinogen (P=0.001) compared to double seronegativity . We conclude that C . pneumoniae LPS seropositivity may contribute to increased vascular inflammation in CHD patients, possibly even more pronounced when present in combination with H . pylori seropositivity.

Vet Microbiol, 2002 Aug 2, 88(1), 59 - 74
Characterization of the in vitro adhesion of Actinobacillus pleuropneumoniae to swine alveolar epithelial cells; Van Overbeke I et al.; Actinobacillus pleuropneumoniae biovar 1 serotypes 2, 5a, 9 and 10 strains were tested for their ability to adhere to alveolar epithelial cells in culture . For the serotypes 5a, 9 and 10 strains, optimal adherence was observed after growth of bacterial cells in a NAD-restricted medium (0.001% NAD) . This condition was also associated with the expression of a 55 kDa outer membrane protein (OMP) and of fimbriae . For the serotype 2 strain, adherence and expression of fimbriae and a 55 kDa OMP was less influenced by the growth conditions . The N-terminal amino acid sequence of the 55 kDa OMP had no homology with any known sequence, suggesting that it is an as yet unknown protein . Adherence capabilities were significantly reduced following treatment of the bacteria with proteolytic enzymes or heat . These findings suggest that proteins are involved in adhesion . The hydrophobic bond-breaking agent tetramethylurea was unable to inhibit the adherence of A . pleuropneumoniae to alveolar epithelial cells . Treatment of the bacteria with sodium metaperiodate resulted in lower adhesion scores for the serotypes 2 and 9 strains but the inhibition of adhesion was clearly lower than after treatment with proteolytic enzymes . This indicates that, besides proteins, carbohydrates might also be involved in adhesion of A . pleuropneumoniae to alveolar epithelial cells . The finding that inhibition of adhesion was very high when bacteria were treated with a combination of sodium metaperiodate and pronase also suggests that more than one adhesin is involved.

Gene, 2002 Jun 12, 292(1-2), 245 - 59
Structure, expression, and functional characterization of the mouse CLP-1 gene; Huang F et al.; Mouse CLP-1, a potential cardiac transcriptional regulatory factor, is encoded by a single copy gene lacking introns that is expressed into two mRNAs via alternative polyadenylation . Both mRNAs encode the same 41 kDa protein, a novel protein that is 85.3% homologous with a human homologue called HIS1 . Mouse CLP-1 is widely expressed in a number of tissues as well as in early development and is localized to the nucleus . The CLP-1 gene promoter is active in different cell types and sequence analysis shows a number of potential binding sites for cardiogenic transcription factors such as Nkx2.5 and GATA-4, indicating a potential role in development . CLP-1 appears to "squelch" the cardiac MLC-2v promoter in a concentration-dependent manner in cardiac but not other cell types, suggesting that CLP-1 may be interacting with a cardiac-specific factor to regulate cardiac MLC-2v expression . The overall expression pattern of CLP-1 is similar to that of LCR-F1 and Oct-1, two widely expressed transcription factors that also play specific roles in the transcription of cell-specific genes . CLP-1 may be a transcriptional mediator capable of interacting with and potentiating cell-specific transcription factors.

Biochemistry, 2002 Jul 23, 41(29), 9237 - 47
New insights into the heme cavity structure of catalase-peroxidase: a spectroscopic approach to the recombinant synechocystis enzyme and selected distal cavity mutants; Heering HA et al.; Catalase-peroxidases (KatGs) are heme peroxidases with homology to yeast cytochrome cperoxidase (CCP) and plant ascorbate peroxidases (APXs) . KatGs exhibit a peroxidase activity of broad specificity and a high catalase activity, which strongly depends on the presence of a distal Trp as part of the conserved amino acid triad Arg-Trp-His . By contrast, both CCP and APX do not have a substantial catalase activity despite the presence of the same triad . Thus, to elucidate structure-function relationships of catalase-peroxidases (for which no crystal structure is available at the moment), we performed UV-Vis and resonance Raman studies of recombinant wild-type KatG from the cyanobacterium SynechocystisPCC 6803 and the distal side variants (His123-->Gln, Glu; Arg119-->Ala, Asn; Trp122-->Phe, Ala) . The distal cavity of KatG is very similar to that of the other class I peroxidases . A H-bond network involving water molecules and the distal Trp, Arg, and His is present, which connects the distal and proximal sides of the heme pocket . However, distal mutation not only affects the heme Fe coordination state and perturbs the proximal Fe-Im bond, as previously observed for other peroxidases, but also alters the stability of the heme architecture . The charge of the distal residues appears particularly important for maintaining the heme architecture . Moreover, the Trp plays a significant role in the distal H-bonding, much more pronounced than in CCP . The relevance of these findings for the catalase activity of KatG is discussed in light of the complete loss of catalase activity in the distal Trp mutants.

Infect Immun, 2002 Aug, 70(8), 4581 - 90
Helicobacter pylori-induced activation of human endothelial cells; Innocenti M et al.; Helicobacter pylori infection causes active chronic inflammation with a continuous recruitment of neutrophils to the inflamed gastric mucosa . To evaluate the role of endothelial cells in this process, we have examined adhesion molecule expression and chemokine and cytokine production from human umbilical vein endothelial cells stimulated with well-characterized H . pylori strains as well as purified proteins . Our results indicate that endothelial cells actively contribute to neutrophil recruitment, since stimulation with H . pylori bacteria induced upregulation of the adhesion molecules VCAM-1, ICAM-1, and E-selectin as well as the chemokines interleukin 8 (IL-8) and growth-related oncogene alpha (GRO-alpha) and the cytokine IL-6 . However, there were large variations in the ability of the different H . pylori strains to stimulate endothelial cells . These interstrain variations were seen irrespective of whether the strains had been isolated from patients with duodenal ulcer disease or asymptomatic carriers and were not solely related to the expression of known virulence factors, such as the cytotoxin-associated gene pathogenicity island, vacuolating toxin A, and Lewis blood group antigens . In addition, one or several unidentified proteins which act via NF-kappaB activation seem to induce endothelial cell activation . In conclusion, human endothelial cells produce neutrophil-recruiting factors and show increased adhesion molecule expression after stimulation with certain H . pylori strains . These effects probably contribute to the continuous recruitment of neutrophils to H . pylori-infected gastric mucosa and may also contribute to tissue damage and ulcer formation.

Infect Immun, 2002 Aug, 70(8), 4501 - 9
Dissemination of Mycobacterium tuberculosis is influenced by host factors and precedes the initiation of T-cell immunity; Chackerian AA et al.; We report that dissemination of Mycobacterium tuberculosis in the mouse is under host control and precedes the initiation of T-cell immunity . Nine to eleven days after aerosol inoculation, M . tuberculosis disseminates to the pulmonary lymph nodes (LN), where M . tuberculosis-specific T cells are detected 2 to 3 days thereafter . This indicates that the initial spread of bacteria occurs via lymphatic drainage and that the acquired T-cell immune response is generated in the draining LN . Dissemination to peripheral sites, such as the spleen and the liver, occurs 11 to 14 days postinfection and is followed by the appearance of M . tuberculosis-specific T cells in the lung and the spleen . In all cases studied, dissemination to the LN or the spleen preceded activation of M . tuberculosis-specific T cells in that organ . Interestingly, bacteria disseminate earlier from the lungs of resistant C57BL/6 mice than from the lungs of susceptible C3H mice, and consequently, C57BL/6 mice generate an immune response to M . tuberculosis sooner than C3H mice generate an immune response . Thus, instead of spreading infection, early dissemination of M . tuberculosis may aid in the initiation of an appropriate and timely immune response . We hypothesize that this early initiation of immunity following inoculation with M . tuberculosis may contribute to the superior resistance of C57BL/6 mice.

J Food Prot, 2002 Jul, 65(7), 1200 - 6
A review of aerobic and psychrotrophic plate count procedures for fresh meat and poultry products; Jay JM; This is a review of reports that employed aerobic plate counts on fresh meat and poultry products since 1985; it lists synopses of 100 applications . A total of 15 different plating media were used, with 48 (48%) being either plate count agar (PCA) or tryptone glucose yeast extract agar . The temperature-time relations ranged from a low temperature of 20 degrees C for 120 h to 37 degrees C for 24 h . Some 29 different temperature-time combinations were used among the total of 109, with 21 (19.3%) being 35 degrees C/48 h, followed by 12 (11.0%) at 32 degrees C/48 h, 11 (10.1%) at 25 degrees C/48 h, and 9 (8.3%) at 25 degrees C/72 h . Fifty-four (49.5%) plate count applications employed incubation temperatures of 30 degrees C and below . From the 26 reports that employed psychrotrophic counts, 16 (61.5%) used PCA; 18 different temperature-time combinations were used, with 7 degrees C/10 d employed by only four . Twenty-one (80.8%) employed an incubation temperature at or <10 degrees C, and five employed an incubation temperature >10 degrees C . There is a serious need for some consensus on methodologies for aerobic and psychrotrophic counts on fresh meat and poultry products.

Equine Vet J, 2002 Jul, 34(4), 405 - 10
Intradermal skin testing in Icelandic horses in Austria; Kolm-Stark G et al.; Icelandic horses in Austria are commonly affected by an allergic inflammatory skin disease recurring during the summer seasons, which shares characteristic features with Culicoides hypersensitivity . However, the causative agents have not yet been identified . Therefore, intradermal skin testing (IDST) with a standardised extract of Culicoides variipennis and 21 other allergens relevant within Austria was performed in 81 Icelandic horses . All horses included into the study were treated regularly with ivermectin and had no history of administration of anti-inflammatory drugs . Forty-three of these horses were affected by summer seasonal recurrent dermatitis (SSRD) . No history or signs of any other disease were evident in any horse . Pruritic dermatitis due to ectoparasites, bacteria and dermatophytes were ruled out by means of fungal culture, skin scraping and biopsy . Culicoides variipennis antigens evoked a positive cutaneous reaction in 1 of 38 normal and 3 of 43 SSRD horses at the proposed dilution of 1:50,000 or 1:25,000, and in 24 of 38 normal and 13 of 43 SSRD horses at a dilution of 1:10,000 . Furthermore, no significant differences in onset or intensity of skin reactions to the 21 other allergens, including pollens, moulds, mites and insects, except deerfly and horsefly, were obvious between the 2 groups . Efficiency (percentage of correct results) for the used antigens in the skin test was 0.47-0.60 . Maximal sensitivity was 0.51 . Altogether, 38 of 43 SSRD horses and 28 of 38 normal horses were positive 4 h after allergen administration . The divergence between IDST results and manifestation of clinical signs found in this study underlines the difficulties associated with establishing a skin test protocol in horses within a geographic area . Whether the outcome of this study would have been influenced significantly by using Culicoides spp . present in Austria has to be clarified in future research.

Med Device Technol, 2002 Jun, 13(5), 20 - 3
Trends in medical filtration; Hogan B; Advances in materials, mould tooling and control systems are offering the industry greater design choices in filtration as well as the potential to reduce manufacturing costs . This article describes what is possible.

J Med Virol, 2002 Aug, 67(4), 603 - 7
Foodborne outbreak caused by a Norwalk-like virus in India; Girish R et al.; An outbreak of acute gastroenteritis occurred in the nurses' hostel of a civil hospital in Delhi, after a farewell party involving 130 nurses and some of the housekeeping staff . All affected persons had eaten salad sandwiches at the party . Stool samples were collected from six of these patients on the second day of infection . All six samples, when tested for the presence of common bacteria, parasites, and rotavirus, were found to be negative . The clinical features of this outbreak matched the criteria set for outbreaks caused by Norwalk-like viruses (NLVs) . Reverse transcription-polymerase chain reaction (RT-PCR) was carried out on these six samples, using primers from the RNA-dependent RNA polymerase (RdRp) gene of NLVs . Immunoelectron microscopy was carried out on two of the samples, using convalescent phase serum . All six samples were positive for genogroup (GG) II NLVs by RT-nested PCR . Aggregates of 32-nm viral particles were visualized by immunoelectron microscopy in one of the two samples . Sequencing of the RdRp gene was done on amplicons from three samples; phylogenetic analysis placed the isolates NDV/1999 in a Toronto virus cluster of GG II NLVs . This is the first report of a food-borne outbreak attributable to NLVs from India .

Biotechnol Bioeng, 2002 Aug 5, 79(3), 314 - 22
Conversion and toxicity characteristics of formaldehyde in acetoclastic methanogenic sludge; Gonzalez-Gil G et al.; An unadapted mixed methanogenic sludge transformed formaldehyde into methanol and formate . The methanol to formate ratio obtained was 1:1 . Formaldehyde conversion proceeded without any lag phase, suggesting the constitutive character of the formaldehyde conversion enzymes involved . Because the rate of formaldehyde conversion declined at increased formaldehyde additions, we hypothesized that some enzymes and/or cofactors might become denatured as a result of the excess of formaldehyde . Furthermore, formaldehyde was found to be toxic to acetoclastic methanogenesis in a dual character . Formaldehyde toxicity was partly reversible because once the formaldehyde concentration was extremely low or virtually removed from the system, the methane production rate was partially recovered . Because the degree of this recovery was not complete, we conclude that formaldehyde toxicity was partly irreversible as well . The irreversible toxicity likely can be attributed to biomass formaldehyde-related decay . Independent of the mode of formaldehyde addition (i.e., slug or continuous), the irreversible toxicity was dependent on the total amount of formaldehyde added to the system . This finding suggests that to treat formaldehyde-containing waste streams, a balance between formaldehyde-related decay and biomass growth should be attained .

Arch Microbiol, 2002 Aug, 178(2), 85 - 93 Epub 2002 Jun 14.
Genetics and control of CO(2) assimilation in the chemoautotroph Ralstonia eutropha; Bowien B et al.; The nutritional versatility of facultative autotrophs requires efficient overall control of their metabolism . Most of these organisms are Proteobacteria that assimilate CO(2) via the highly energy-demanding Calvin-Benson-Bassham reductive pentose-phosphate cycle . The enzymes of the cycle are encoded by cbb genes organized in cbb operons differing in size and composition, although conserved features are apparent . Transcription of the operons, which may form regulons, is strictly controlled, being induced during autotrophic but repressed to varying extents during heterotrophic growth of the bacteria . The chemoautotroph Ralstonia eutropha is one of the organisms studied extensively for the mechanisms involved in the expression of cbb gene systems . CbbR is a LysR-type transcriptional regulator and the key activator protein of cbb operons . The cbbR gene is typically located adjacent and in divergent orientation to its cognate operon . The activating function of CbbR seems to be modulated by metabolites signaling the nutritional state of the cell to the cbb system . Phosphoenolpyruvate is such a signal metabolite acting as a negative effector of R . eutropha CbbR, whereas NADPH has been proposed to be a coactivator of the protein in two other chemoautotrophs, Xanthobacter flavus and Hydrogenophilus thermoluteolus . There is evidence for the participation of additional regulators in cbb control . In the photoautotrophs Rhodobacter capsulatus and Rhodobacter sphaeroides, response regulator RegA of the global two-component signal transduction system RegBA serves this function . It is conceivable that specific variants of cbb control systems have evolved to ensure their optimal integration into regulatory networks operating in the diverse autotrophs characterized by different metabolic capabilities.

Plant Physiol, 2002 Jul, 129(3), 1216 - 21
Effect of short-term N(2) deficiency on expression of the ureide pathway in cowpea root nodules; Smith PM et al.; Root systems of 28-d-old cowpea (Vigna unguiculata L . Walp cv Vita 3: Bradyrhizobium sp . strain CB756) plants bearing nitrogen-fixing nodules in sand culture were exposed to an atmosphere of Ar:O(2) (80:20, v/v) for 48 h and then returned to air . Root systems of control plants were maintained in air throughout . Nodules were harvested at the same times in control and Ar:O(2)-treated root systems . Activities of two enzymes of de novo purine synthesis, glycinamide ribonucleotide transformylase (GART; EC 2.1.2.2), aminoimidazole ribonucleotide synthetase (AIRS; EC 6.3.3.1), uricase (EC 1.7.3.3), and phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) were measured together with the protein level of each using immune-specific polyclonal antibodies . AIRS activity and protein both declined to very low levels within 6 h in Ar:O(2) together with a decline in transcript level of pur5, the encoding gene . GART activity, protein, and transcript (pur3) levels were relatively stable . Uricase activity declined in Ar:O(2) as rapidly as AIRS activity but the protein was stable . PEPC activity showed evidence of increased sensitivity to inhibition by malate but the protein level was stable . The data indicate that the flux of fixed N from bacteroids (N(2)-fixing nodule bacteria) is in some way associated with transcriptional control over pur5 and possibly also catabolism of AIRS protein . In contrast, there is limited posttranslational control over GART and PEPC and close posttranslational control over uricase activity . The significance of these different levels of regulation is discussed in relation to the overall control of enhanced expression of plant enzymes in the cowpea symbiosis.

Trends Mol Med, 2002 Jul, 8(7), 355 - 8
Blood-bank testing for infectious diseases: how safe is blood transfusion?
Strong DM, Katz L.
Remarkable progress has been made in transfusion safety from infection over the past three decades . Donor deferrals for at-risk behaviors, the introduction of more-sensitive viral-screening assays and the recent introduction of nucleic-acid amplification technology have nearly eliminated transmission of HIV and hepatitis C virus (HCV) by blood transfusion in North America . Nevertheless, risks of other infectious agents for which such robust screening tools have not been developed, such as bacteria and parasites, still remain . As a result of these successes, the non-infectious risks such as misidentification of patients and inadequate and inappropriate transfusion have become the primary sources of transfusion risk.

J Periodontal Res, 2002 Jun, 37(3), 210 - 4
Treatment with SRL172 (heat-killed Mycobacterium vaccae) inhibits progression of established experimental periodontal disease in Wistar rats; Brelvik T et al.; Periodontal disease is accompanied by a change in the periodontal bacterial flora, and an increase in Th2 cytokine expression . Therefore, preparations that can down-regulate Th2 responses and increase Th1 responses to bacteria may have therapeutic effects . SRL172, a preparation of heat-killed Mycobacterium vaccae (NCTC11659), has been shown to have these properties in man and animals and, in a previous study, a single subcutaneous injection of this material 13 days before application of ligatures to the right second molars of Wistar rats strikingly reduced subsequent destruction of the tooth-supporting tissues . The same material has therefore been tested in a therapeutic protocol, in rats with ongoing periodontal disease . A silk ligature was placed round the maxillary right second molar in the gingival sulcus on day 0 . This was removed after two weeks, and the animals immediately received 0.1 mg SRL172 s.c . or saline . One week later (i.e . at three weeks), a boost of 1.0 mg SRL172 or saline was given . Animals were sacrificed at eight weeks (i.e . five weeks after the booster dose) . Treatment of ongoing periodontal disease with SRL172 significantly reduced fibre loss (p = 0.046 by histometry) and bone loss (p = 0.0008 by radiography) on the ligatured side, and reduced fibre loss (p = 0.0086) on the control side . Thus SRL172 is an effective treatment in this model . As SRL172 has undergone extensive safety testing in man, these results justify clinical studies in periodontal disease, and such studies are now planned.

J Periodontal Res, 2002 Jun, 37(3), 177 - 83
Upregulation of co-stimulatory molecule expression and dendritic cell marker (CD83) on B cells in periodontal disease; Mahanonda R et al.; T cells and their cytokines are well known for their important role in the pathogenesis of periodontitis . To date, the role of antigen presenting cells (APCs), which are known to be critical in the regulation of T cell response, has been poorly investigated in periodontitis . In this study, we analyzed the expression of co-stimulatory molecules (CD80 and CD86) and CD83, which is a marker of mature dendritic cells, on gingival cells that were isolated from severe periodontitis tissues, with the use of flow cytometry . Significant upregulation of CD86 and CD83 expression was detected in periodontitis lesions, and most of this occurred on B cells . In vitro peripheral blood mononuclear cell cultures showed that stimulation with different periodontopathic bacteria, that included Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Actinomyces viscosus, upregulated both CD86 and CD83 expression on B cells . Therefore, the presence of plaque bacteria may be responsible for the enhanced expression seen in vivo on gingival B cells . APC function by bacterial-activated B cells was further investigated using allogeneic mixed leukocyte reactions . After 24 h culture with either A . actinomycetemcomitans or P . gingivalis, these activated B cells performed as potent APCs in mixed leukocyte reactions, and they stimulated T cells to produce high levels of gamma interferon and minimal interleukin-5 . In conclusion, periodontopathic bacterial-induced B cell activation with upregulation of CD86 and CD83 may be associated with enhanced APC function . The results of this study suggest, therefore, that infiltrated gingival B cells have a possible role as APCs in the regulation and maintenance of local T cell response in periodontitis.

Ground Water, 2002 Jul-Aug, 40(4), 416 - 24
Localized thermal anomalies in haloclines of coastal Yucatan sinkholes; Stoessell RK et al.; A temperature spike is reported in the haloclines of three Yucatan sinkholes along a 1 km NW-SE transect from 5 to 4 km inland from the Caribbean coast . The temperature spike decreases in magnitude from 3.5 degrees C to 0.2 degrees C, approaching the coast . The anomaly does not vary diurnally and does not extend down into the underlying sea water . These conditions are inconsistent with explanations such as radiation absorption within the halocline, in situ microbially mediated sulfate reduction within the halocline and the underlying sea water, and sulfide oxidation by photosynthetic purple and green bacteria within the halocline . One explanation consistent with the shape and halocline location of the temperature spike involves a localized sea water convection cell operating near the coast . Cold sea water from the Caribbean Sea enters the coastal limestone at depths of a few hundred meters and heats up because of the geothermal gradient, buoyantly rising in vertical fractures within the unconfined aquifer . Blocked by the less dense fresh water, the movement stops in the halocline where the warm sea water mixes with brackish water . The convection cycle would be completed by the coastward movement of cooling brackish water . The observed temperature anomalies could possibly be snapshots of this warm layer moving coastward.

Expert Rev Anticancer Ther, 2002 Jun, 2(3), 309 - 21
Gene therapy for prostate cancer; Gdor Y et al.; Prostate cancer is the most common noncutaneous cancer in man . When confined to the prostate it can be cured by radical prostatectomy or irradiation therapy . However, there are no curative therapies for locally advanced, recurrent or metastatic disease . Prostate cancer gene therapy has recently transition from preclinical studies to clinical trials with the goal of developing novel treatments for prostate cancer . The greatest challenge in treating advanced prostate cancer is therapeutic access to and the elimination of metastases . This review details two aspects of prostate cancer gene therapy, the types of delivery systems under development and specific categories of therapeutic genes available with an emphasis on the mechanism of action of specific gene therapy strategies.

Proteomics, 2002 Jun, 2(6), 775 - 83
Immunogenic proteins of Helicobacter pullorum, Helicobacter bilis and Helicobacter hepaticus identified by two-dimensional gel electrophoresis and immunoblotting; Kornilovs'ka I et al.; The ecological niches occupied by various species of Helicobacter are not yet known and the full spectrum of diseases associated with Helicobacter infections are not yet defined . Since these fastidious microaerofilic bacteria require special growth conditions new and improved molecular and serologic diagnostic methods have been developed to increase our understanding of their pathogenesis and virulence characteristics . Immunogenic cell surface proteins of Helicobacter pullorum, Helicobacter bilis, and Helicobacter hepaticus were characterised by proteomic techniques using two-dimensional electrophoresis and immunoblotting with antisera from immunised rabbits . Cross-reactivity between the three Helicobacter species were analysed after a four-step cross-absorption experiment . For H . pullorum, H . bilis and H . hepaticus 21, 13 and 27 specific immunogenic proteins, respectively, were identified . These proteins could be of important sero-diagnostic value for analyses of sera from humans, laboratory animals and for the veterinarian field.

Environ Toxicol, 2002, 17(3), 149 - 59
Biomarker study of a municipal effluent dispersion plume in two species of freshwater mussels; Gagne F et al.; The toxicological effects of a primary-treated municipal effluent plume were investigated in two species of freshwater mussels, Elliptio complanata and Dreissena polymorpha, exposed for 62 days at sites upstream and downstream of an effluent outfall in the St . Lawrence River (Quebec, Canada) . Levels of metallothioneins (MT), cytochrome P4501A1 activity, DNA damage, total lipids, relative levels of vitellins, and phagocytic activity (in E . complanata hemocytes) were determined after the exposure period . A parallel analysis measured heavy metals and coprostanol in mussel tissues . The results show that significant levels of coprostanol and some metals (specifically, Cu, Hg, Sb, Se, and Zn) had accumulated in mussels caged 5 km downstream of the effluent plume . Mixed-function oxidase activity, MT in gills, total lipids, DNA damage (in D . polymorpha only), and total hemolymph bacteria (in E . complanata only) had increased in these mussels, while levels of total cadmium (Cd), MT in digestive glands or whole soft tissues, phagocytic activity, and DNA damage in the digestive gland (in E . complanata only) were diminished . The exposure of mussels to surface waters contaminated by a municipal effluent led to many stress responses, depending on both the tissues and the species being examined .

Microsc Res Tech, 2002 Jun 15, 57(6), 491 - 7
Are thrombocytes and platelets true phagocytes?
Meseguer J, Esteban MA, Rodriguez A.
Thrombocytes and platelets, beyond their primary function in hemostasis, seem to play an active role in inflammation . As regards their phagocytic ability, the results to date are confusing, incomplete, and somewhat contradictory . Whereas the interaction of avian thrombocytes or mammalian platelets with bacteria both in vitro and in vivo has received wide attention, almost no information exists on the topic in "lower" vertebrates . The aim of this work is to review the available information on the phagocytic properties of thrombocytes and platelets . Particular attention is payed to the ontogeny of these cells, the soluble factors involved in the inflammatory process derived from them, and their interaction with particulate material, mainly with bacteria .

J Cell Biochem, 2002, 86(2), 277 - 89
Sox9 transactivation and testicular expression of a novel human gene, KIAA0800; Zhao LJ et al.; The Sry and Sox9 sex-determination factors initiate and promote testis differentiation by gene transactivation through similar promoter elements . However, knowledge is limited concerning what genes are regulated by Sry/Sox9 in the testis . Identification and characterization of Sry/Sox9-regulated genes are critical for understanding sexual differentiation . We now demonstrate that a novel human gene, KIAA0800, is preferentially expressed in the testis and is transactivated by Sox9 . The KIAA0800 promoter is repressed by an upstream element involving a polyT track and two Alu repeats . Two specific Sox9-bindings sites have been identified in the KIAA0800 promoter by using DNaseI footprinting assays and gel electrophoretic mobility shift assays . Sox9 transactivation of the KIAA0800 promoter appears to be exerted mainly through the relief of promoter repression . Genes homologous to the human KIAA0800 exist in organisms with differentiated sex tissues including mouse, Drosophila, and C . elegans, but not in unicellular organisms, including yeast and bacteria . Further, our recent sequence analysis shows that KIAA0800 protein is 97% identical between human and mouse . Thus, KIAA0800 is a novel Sox9-activated gene that is evolutionarily conserved and potentially involved in sexual differentiation .

Eur J Clin Microbiol Infect Dis, 2002 Jun, 21(6), 455 - 60 Epub 2002 Jun 14.
Evaluation of a new commercial assay for diagnosis of pulmonary and nonpulmonary tuberculosis; Johansen IS et al.; A new commercial assay for the diagnosis of tuberculosis, the BDProbeTec ET Direct Detection assay (Becton Dickinson, USA), was evaluated using 351 respiratory and 372 nonrespiratory specimens . The results were compared to detection of Mycobacterium tuberculosis complex (MTC) by conventional culture . Among the 351 respiratory specimens, MTC bacteria were identified in 150, of which 85 were positive by both microscopy and the assay . Sixty-five specimens culture positive for MTC were microscopy negative; of these, 39 were positive in the assay . All 26 specimens culture positive for nontuberculous mycobacteria (NTM) were negative by the assay . Of 175 specimens culture negative for MTC, 3 were falsely positive by the assay and 1 yielded inhibition . The overall sensitivity and specificity values were 82.7% and 98.5%, respectively . The sensitivity for microscopy-positive and -negative respiratory specimens was 100% and 60%, respectively . After correction for discrepancies, the specificity was 99% compared with notification data . The BDProbeTec ET assay detected 66 of 67 microscopy-positive and 50 of 125 microscopy-negative nonrespiratory specimens . The result for one specimen was inconclusive . All nine specimens containing NTM were negative by the assay . Of 171 specimens culture negative for MTC, 6 were falsely positive by the assay . The overall sensitivity and specificity values obtained with nonrespiratory specimens were 60.7% and 96.7%, respectively . After examining discrepancies by reviewing the patients' histories, the specificity was 98.9% . The sensitivity was 98.5% in microscopy-positive specimens and 40.3% in microscopy-negative specimens . The overall inhibition rate was 0.3% . The BDProbeTec ET assay is a fast, effective, and user-friendly system that can be used for rapid detection of MTC bacteria in respiratory and microscopy-positive nonrespiratory specimens as an important supplement to smear and culture.

Mol Genet Genomics, 2002 Jun, 267(4), 526 - 35 Epub 2002 May 29.
Identification and genomic organization of gene loci negatively controlled by the virulence regulatory BvgAS two-component system in Bordetella bronchiseptica; Schneider B et al.; The two-component system BvgAS positively controls transcription of the virulence genes of Bordetella pertussis and B . bronchiseptica, which include several genes for toxins and adhesins . On the other hand, the BvgAS system negatively controls the expression of a poorly characterized set of genes, the so-called virulence repressed ( vrg) genes . To investigate the function of this group of genes and their relationship to virulence, we identified several novel vrg genes of B . bronchiseptica via the generation of transcriptional fusions with gfp (the ORF encoding Green Fluorescent Protein) by transposon mutagenesis . Expression of all of the vrg genes was enhanced under phenotype-modulating growth conditions and in phase variants, demonstrating that their transcription is indeed controlled by the BvgAS system . In addition, transcription of most of these new vrg genes was found to be affected by the growth phase of the bacteria, with maximal expression being observed in the late logarithmic or stationary phase . The majority of these genes encode putative metabolic functions involved in redox reactions and amino acid transport . Interestingly, several vrg genes of B . bronchiseptica are not expressed or have been lost in B . pertussis, indicating that, possibly as a consequence of its adaptation to a single host organism, many vrg genes of B . pertussis are gradually decaying.

Appl Microbiol Biotechnol, 2002 Jul, 59(2-3), 231 - 8 Epub 2002 May 16.
Introduction of the carbohydrate-activated promoter P(malK) for recombinant protein production; Bostrom M et al.; A production protocol for the use of the malK promoter was established . The protocol includes two phases: an initial fed-batch phase on glucose to reach a high cell density and a fed-batch phase on maltose for production of the desired recombinant protein . It is suggested that this cultivation scheme could be used for all promoters that are catabolite repressed by glucose and where growth and production need to be separated . The specific feature of this system is shown by its ability to control the rate of synthesis of the product protein, ss-galactosidase . In the production phase with a constant feed or an exponential feeding of 0.1 h(-1) it took 4 h longer to reach the maximum specific production rate than with the higher dilution rates of 0.25 h(-1) and 0.4 h(-1), respectively . In the above experiments a dilution rate of 0.3 h(-1) in the growth phase was used . The volumetric production of this system could furthermore be extended to 40 h . All protocol procedures so far tested resulted in the same maximum production rate, but reached in different lengths of time . It is argued that this system is particularly well suited for the production of proteins that have a complex structure and/or need to be produced in a soluble form or to be exported to the periplasm.

FEMS Immunol Med Microbiol, 2002 Jul 12, 33(3), 191 - 200
Major histocompatibility complex class I and II expression on macrophages containing a virulent strain of Brucella abortus measured using green fluorescent protein-expressing brucellae and flow cytometry; Murphy E et al.; Immune responses appropriate for control of an intracellular pathogen are generated in mice infected with Brucella abortus, shown by the ability of T cells to adoptively transfer resistance to naive mice . The infection nevertheless persists for months . It was hypothesized that one factor in maintaining the infection despite the presence of immune T cells was suboptimal expression of major histocompatibility complex (MHC) molecules on macrophages containing brucellae . This would allow B . abortus to elude detection by the host's immune system . To test this, B . abortus organisms expressing green fluorescent protein (GFP-Brucella) were constructed and three-color flow cytometry used to evaluate MHC expression on macrophages following in vitro or in vivo infection . When infected in vitro, the levels of MHC class I and class II expression on J774 macrophages containing GFP-Brucella were the same or higher than on macrophages without GFP-Brucella in the same cultures . Similarly, the MHC expression was higher on GFP(+) peritoneal exudate cells following infection or phagocytosis of heat-killed GFP-Brucella than it was on uninfected peritoneal exudate cells . Following in vivo infection of mice the level of MHC class I and II expression on GFP(+) cells in their spleens (the main site of infection) also tended to be as high as or higher than that on the GFP-negative cells . The only in vivo GFP(+) cells that showed a decreased MHC expression was a population of splenic Mac1(+) cells recovered from interferon-gamma gene-disrupted mice at the time of their death due to an overwhelming number of bacteria per spleen . Overall, it was concluded that decreased MHC expression is not a general principle associated with brucella infection of macrophages and thus not likely to contribute to maintenance of the chronic infection.

Crit Rev Microbiol, 2002, 28(2), 79 - 122
Ribonucleases from T2 family; Deshpande RA et al.; Ribonucleases are ubiquitous in distribution . Ribonucleases that hydrolyse RNA to 3' mononucleotides via 2', 3' cyclic nucleotides are classified into three groups, RNase A, RNase T1, and RNase T2 families . Apart from salvage of cellular or extracellular RNAs, RNases participate in vital cellular functions such as DNA replication, transcription and RNA processing, splicing and editing, and control of translation by determining the turnover of RNA . T2 family RNases have been implicated in nutrition, phosphate remobilization, self-incompatibility, senescence, and defense against pathogens . They are important analytical enzymes and have been exploited for the structural determination of RNAs . Although considerable information is available on RNase A and T1 family RNases, less information is available on RNases from T2 family except RNase Rh from Rhizopus niveus and RNase LE from tomato . However, during the last few years, the primary structure, active site nature based on sequence homology, and probable mechanism of action have been postulated for some of these enzymes . RNases of T2 family, their occurrence, purification, characteristics, biological role, and applications have been reviewed.

Sci Total Environ, 2002 Jul 3, 293(1-3), 219 - 31
Bioaerosol exposure during refuse collection: results of field studies in the real-life situation; Neumann HD et al.; To determine the bioaerosol exposure of refuse collectors, field measurements were performed under real working conditions within the framework of a research project . Influencing variables such as different types of refuse, community structure, collection interval and season were taken into account . Overall, 1612 samples were taken in towns of Westfalia, Germany . With workplace levels on a scale of 10(3) to less than 10(4) CFU/m3 for the loader, the results show a surprisingly low total fungi concentration in comparison with earlier studies . Total bacteria concentrations, in contrast, were largely on a scale of 10(4) CFU/m3, with 10(5) CFU/m3 being registered sporadically, especially in apartment-block districts . Endotoxin levels were high especially in the summer months, occasionally reaching values of more than 50 EU/m3, whereas they were normally below 10 EU/m3 in autumn and winter . Inside the cab, the exposure level for the entire spectrum was at least one power of ten lower . The factors believed to account primarily for the low total fungi concentration were workplace hygiene, the prevailing 1-week collection interval, and the low in-process exposure time resulting from the effective deployment of automatic lifting devices . In contrast, the type of refuse was not found to have a significant influence.

Curr Gene Ther, 2001 May, 1(1), 53 - 100
Genetic vaccination for the active immunotherapy of cancer; Bronte V; Molecular biology techniques have given novel impetus to the immunotherapy of cancer because they have catalyzed the identification of several potential tumor antigens, and permitted the generation of vectors for the delivery of genetic material encoding these antigens . Vaccines can be defined "genetic" when the antigen they enclose is present as DNA or RNA . Microrganisms used as vectors can deliver the genetic information, but naked nucleic acids have also been shown to be effective immunogens thanks to built-in adjuvants that activate professional antigen presenting cells . Although gene-based cancer vaccines have been tested in mouse models and selected for pilot clinical trials, enthusiasm has somewhat waned due to an apparently major drawback of cancer vaccination: tumor antigens are weak, and therefore fail to stimulate a sterilizing immune response in tumor-bearing patients . Mouse studies, however, have shown that cancer vaccines are extremely efficacious in establishing a state of active immunosurvellance against tumor growth . This review reconsiders the findings emerging from preclinical studies in the context of our current knowledge of the cellular and molecular bases of the immune responses to vaccines, in an attempt to approach critically the use of genetic vaccination for the treatment of cancer.

Water Res, 2002 May, 36(9), 2225 - 32
Treatment of domestic sewage in a two-step anaerobic filter/anaerobic hybrid system at low temperature; Elmitwalli TA et al.; The treatment of domestic sewage at low temperature of 13 degrees C was investigated in a two-step system consisting of an anaerobic filter (AF) +an anaerobic hybrid (AH) reactor operated at different hydraulic retention times (HRTs) . The AF reactor was efficient in the removal of suspended COD, viz . 81%, 58% and 57% at an HRT of, respectively, 4, 2 and 3 h . For optimisation of the removal of suspended COD and dissolved COD, an HRT of 4 + 4 h is required for the AF + AH system . For additional optimisation of colloidal COD removal, the AH reactor needs an HRT of 8 h . The AF + AH system operated at an HRT of 4 + 8 h at 13 degrees C provided a high removal efficiency for all COD fractions . The achieved total COD removal was as high as 71% which is similar to values found in tropical areas . Moreover, 60% of the removed COD was converted to methane.

Br J Cancer, 2002 Jul 15, 87(2), 187 - 93
Establishment and characterisation of six human biliary tract cancer cell lines; Ku JL et al.; Human cell lines established from biliary tract cancers are rare, and only five have been reported previously . We report the characterisation of six new six biliary tract cancer cell lines (designated SNU-245, SNU-308, SNU-478, SNU-869, SNU-1079 and SNU-1196) established from primary tumour samples of Korean patients . The cell lines were isolated from two extrahepatic bile duct cancers (one adenocarcinoma of common bile duct, one hilar bile duct cancer), two adenocarcinomas of ampulla of Vater, one intrahepatic bile duct cancer (cholangiocarcinoma), and one adenocarcinoma of the gall bladder . The cell phenotypes, including the histopathology of the primary tumours and in vitro growth characteristics, were determined . We also performed molecular characterisation, including DNA fingerprinting analysis and abnormalities of K-ras, p15, p16, p53, hMLH1, hMSH2, DPC4, beta-catenin, E-cadherin, hOGG1, STK11, and TGF-betaRII genes by PCR-SSCP and sequencing analysis . In addition, we compared the genetic alterations in tumour cell lines and their corresponding tumour tissues . All lines grew as adherent cells . Population doubling times varied from 48-72 h . The culture success rate was 20% (six out of 30 attempts) . All cell lines showed (i) relatively high viability; (ii) absence of mycoplasma or bacteria contamination; and (iii) genetic heterogeneity by DNA fingerprinting analysis . Among the lines, three lines had p53 mutations; and homozygous deletions in both p16 and p15 genes were found three and three lines, respectively; one line had a heterozygous missense mutation in hMLH1; E-cadherin gene was hypermethylated in two lines . Since the establishment of biliary tract cancer cell lines has been rarely reported in the literature, these newly established and well characterised biliary tract cancer cell lines would be very useful for studying the biology of biliary tract cancers, particularly those related to hypermethylation of E-cadherin gene in biliary tract cancer.

J Mol Evol, 2002 Aug, 55(2), 153 - 60
Updating carbamoylphosphate synthase (CPS) phylogenies: occurrence and phylogenetic identity of archaeal CPS genes; Cammarano P et al.; Among Bacteria the carA and carB genes encoding the small (CarA) and large (CarB) subunits of carbamoylphosphate synthase (CPS) have been lost in certain symbionts (Haemophylus influenzae) and in most obligate intracellular parasites (Chlamydiae, Spirochaetes, Mycoplasmatales, Rickettsiae) having genome sizes in the 0.7- to 1.1-Mb range . Compared to Bacteria, Archaea exhibit a more varied pattern of CPS gene losses and an unusual propensity to incorporate CPS genes derived from both Bacteria and other Archaea . Schematically they fall into three groups . Group 1 taxa (the crenarchaeon Aeropyrum pernix and the euryarchaea Pyrococcus horikoshi and Pyrococcus abyssii) lack CPS genes altogether . Group 2 taxa (comprising Halobacteriales, Thermoplasmales, Methanococcales, Methanomicrobiales, Archaeoglobales) harbor CPS genes whose encoded CarB and CarA subunit proteins are ostensibly bacterial in origin; that is, they are intermixed with bacterial homologues on a phylogeny of concatenated CarA and CarB sequences and are not distinguishable from bacterial sequences after searching for domain-specific amino acid residue positions . Group 3 taxa (the crenarchaea Pyrobaculum aerophilum, Sulfolobus solfataricus, and Sulfolobus tokodaii and the euryarchaeon Pyrococcus furiosus) harbor CPS genes whose encoded proteins appear to be archaeal: consistent with an archaeal origin, the CarA and CarB sequences in this group possess both unique signatures and signatures affiliating them to Eukarya . Based on the topology of the clade comprising the four Group 3 taxa, we argue that CPS genes of P . furiosus (a euryarchaeon) and those of the crenarchaea P . aerophilum, S . solfataricus, and S . tokodaii are of a single type, resulting from the two genes being laterally transferred from a crenarchaeon to P . furiosus.

J Mol Evol, 2002 Aug, 55(2), 127 - 37
Comparative molecular evolution of primary (Buchnera) and secondary symbionts of aphids based on two protein-coding genes; Moya A et al.; A+T content, phylogenetic relationships, codon usage, evolutionary rates, and ratio of synonymous versus non-synonymous substitutions have been studied in partial sequences of the atpD and aroQ/pheA genes of primary ( Buchnera) and secondary symbionts of aphids and a set of selected non-symbiotic bacteria, belonging to the five subdivisions of the Proteobacteria . Compared to the homologous genes of the last group, both genes belonging to Buchnera behave in a similar way, showing a higher A+T content, forming a monophyletic group, a loss in codon bias, especially in third base position, an evolutionary acceleration and an increase in the number of non-synonymous substitutions, confirming previous results reported elsewhere for other genes . When available, these properties have been partly observed with the secondary symbionts, but with values that are intermediate between Buchnera and free living Proteobacteria . They show high A+T content, but not as high as Buchnera, a non-solved phylogenetic position between Buchnera, and the other gamma-Proteobacteria, a loss in codon bias, again not as high as in Buchnera and a significant evolutionary acceleration in the case of the three atpD genes, but not when considering aroQ/pheA genes . These results give support to the hypothesis that they are symbionts at different stages of the symbiotic accommodation to the host.

Free Radic Biol Med, 2002 Jul 15, 33(2), 220 - 35
The metabolic fate of dietary polyphenols in humans; Rechner AR et al.; Dietary polyphenols are widely considered to contribute to health benefits in humans . However, little is yet known concerning their bioactive forms in vivo and the mechanisms by which they may contribute toward disease prevention . Although many studies are focusing on the bioavailability of polyphenols through studying their uptake and the excretion of their conjugated forms, few are emphasizing the occurrence of metabolites in vivo formed via degradation by the enzymes of colonic bacteria and subsequent absorption . The purpose of this research was to investigate the relationship between biomarkers of the colonic biotransformation of ingested dietary polyphenols and the absorbed conjugated polyphenols . The results show that the majority of the in vivo forms derive from cleavage products of the action of colonic bacterial enzymes and subsequent metabolism in the liver . Those include the glucuronides of 3-hydroxyphenylacetic, homovanillic, vanillic and isoferulic acid as well as 3-(3-methoxy-4-hydroxyphenyl)-propionic, 3-(3-hydroxyphenyl)-propionic acid, and 3-hydroxyhippuric acid . In contrast, intact conjugated polyphenols themselves, such as the glucuronides of quercetin, naringenin and ferulic, p-coumaric, and sinapic acid were detected at much lower levels . The results suggest that consideration should be given to the cleavage products as having a putative role as physiologically relevant bioactive components in vivo.

Microbes Infect, 2002 Jul, 4(9), 903 - 14
Toll receptors, CD14, and macrophage activation and deactivation by LPS; Dobrovolskaia MA et al.; This review will focus on the molecular mechanisms of macrophage activation and desensitization by bacterial lipopolysaccharide (LPS) . The most recent advances in the understanding of the function of the LPS receptor complex and its role in the development of the septic shock syndrome and endotoxin tolerance will be discussed.

Microbes Infect, 2002 Jul, 4(9), 887 - 95
Genetic approaches to the study of Toll-like receptor function; Takeuchi O et al.; The Toll-like receptor (TLR) family plays a role in sensing invading pathogens . Ten members have been reported to date, seven of which were found to recognize discrete bacterial components . Mouse models lacking each TLR and its signaling molecule are useful tools for the analysis of the innate immune system.

Gastroenterology, 2002 Jul, 123(1), 187 - 95
Mechanisms of acid resistance due to the urease system of Helicobacter pylori; Scott DR et al.; BACKGROUND & AIMS: Helicobacter pylori, a neutralophile, uses acid neutralization by urease to combat gastric acidity, allowing gastric colonization . Both acute and chronic acid resistance mechanisms are present . Acute mechanisms of acid adaptation could be due to surface urease, increased inner-membrane urea permeability via UreI, or both . Slower mechanisms may involve increased nickel insertion into apoenzyme, posttranscriptional regulation, or increased enzyme synthesis . The aim of this study was to further define regulation of urease under acidic conditions . METHODS: Surface-bound urease was analyzed by measurement of free and bound urease after centrifugation through a step gradient and by quantitative urease immunostaining of intact and fixed bacteria . Changes in urease synthesis or assembly were determined by incubation of the organisms at pH 5.5 or 7.0 in the absence and presence of chloramphenicol, urea, or nickel chelator and in ureI-positive and -negative organisms . RESULTS: The amount of surface urease was below detection limits with either centrifugation washing or immunostaining . Total bacterial urease activity was increased 3-5-fold by incubation at pH 5.5 in the presence of chloramphenicol but not in nickel-free medium or in ureI knockout organisms . There was also a 3-fold increase in survival of acid shock in acid-adapted organisms . CONCLUSIONS: Surface-bound urease is too low to contribute to acid resistance . Acidic medium pH induces UreI-dependent nickel incorporation into apoenzyme . This augmentation of urease activity increases survival in acid and is part of the gastric colonization strategy of the organism.

Cell Microbiol, 2002 Jul, 4(7), 447 - 60
Identification of MEK- and phosphoinositide 3-kinase-dependent signalling as essential events during Chlamydia pneumoniae invasion of HEp2 cells; Coombes BK et al.; The ability of Chlamydia pneumoniae to survive and cause disease is predicated on efficient invasion of cellular hosts . While it is recognized that chlamydial determinants are important for mediating attachment and uptake into non-phagocytic cells, little is known about the bacterial ligands and cellular receptors that facilitate invasion or host cell signal transduction pathways implicated in this process . We used transmission and scanning electron microscopy to demonstrate that attachment of bacteria to host cells induced the appearance of microvilli on host cell membranes . Invasion occurred 30-120 min after cell contact with the subsequent loss of membrane microvilli . Using an epithelial cell infection model, C . pneumoniae invasion caused a rapid and sustained increase in MEK-dependent phosphorylation and activation of ERK1/2, followed by PI 3-kinase-dependent phosphorylation and activation of Akt . Tyrosine phosphorylation of focal adhesion kinase (FAK) preceded its appearance in a complex with the p85 subunit of PI 3-kinase during chlamydial invasion and isoform-specific tyrosine phosphorylation of the docking protein Shc also occurred at the time of attachment and entry of bacteria . Chlamydia entry but not attachment could be abrogated with specific inhibitors of MEK, PI 3-kinase and actin polymerization, demonstrating the importance of these signalling pathways and an intact actin cytoskeleton for C . pneumoniae invasion . These results suggest that activation of specific cell signalling pathways is an essential strategy used by C . pneumoniae to invade epithelial cells.

Cell Microbiol, 2002 Jul, 4(7), 411 - 24
Characterization of a secreted Chlamydia protease; Shaw AC et al.; Chlamydiae are obligate intracellular bacteria that are important human pathogens . The Chlamydia genomes contain orthologues to secretion apparatus proteins from other intracellular bacteria, but only a few secreted proteins have been identified . Most likely, effector proteins are secreted in order to promote infection . Effector proteins cannot be identified by motif or similarity searches . As a new strategy for identification of secreted proteins we have compared 2D-PAGE profiles of {35S}-labelled Chlamydia proteins from whole lysates of infected cells to 2D-PAGE profiles of proteins from purified Chlamydia . Several secretion candidates from Chlamydia trachomatis D and Chlamydia pneumoniae were detected by this method . Two protein spots were identified among the candidates . These represent fragments of the 'chlamydial protease- or proteasome-like activity factor' (CPAF) and were clearly present in 2D-PAGE profiles of whole lysates of infected cells but absent from purified Chlamydia . CPAF was recently identified by Zhong and colleagues as a secreted protease which cleaves host cell transcription factors essential for MHC class I and II antigen presentation . The identification of CPAF in this paper verifies the applicability of the described method for the identification of secreted proteins . We extend the findings by Zhong et al . by proteome studies of expression and turnover of C . trachomatis CPAF showing that the degradation of C . trachomatis D CPAF in the host cell is very limited . Furthermore, we show that two fragments of CPAF exist in C . pneumoniae as well as in C . trachomatis.

Biochemistry, 2002 Jul 16, 41(28), 8899 - 906
DNA adducts formed from 4-hydroxytamoxifen are more mutagenic than those formed by alpha-acetoxytamoxifen in a shuttle vector target gene replicated in human Ad293 cells; McLuckie KI et al.; The drug tamoxifen, used to treat breast cancer, causes liver cancer in rats and endometrial cancer in women . Tamoxifen forms liver DNA adducts in both short- and long-term dosing of rodents, and DNA adducts have also been reported in tissues of women undergoing tamoxifen therapy . It is not known if the induction of endometrial cancer in women is through these DNA adducts or through the estrogenic nature of the drug . In this study, we have investigated the mutagenicity of two model reactive intermediates of tamoxifen, alpha-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide (4-OHtamQM) . These form the same DNA adducts as those found in tamoxifen-treated rats . The two compounds were used to treat the pSP189 plasmid containing the supF gene, which was replicated in Ad293 cells before being screened in indicator bacteria . Plasmid reacted with 4-OHtamQM was more likely to be mutated (2-7-fold increase) than that reacted with alpha-acetoxytamoxifen, despite having a lower level of DNA damage (12-20-fold less), as assayed by (32)P-postlabeling . The two compounds induced statistically different mutation spectra in the supF gene . The majority of mutations in alpha-acetoxytamoxifen-treated plasmid were GC -->TA transversions while GC-->AT transitions were formed in 4-OHtamQM-treated plasmid . 4-OHTamQM-treated DNA induced a larger proportion of multiple mutations and large deletions compared to alpha-acetoxytamoxifen . Sites of mutational hotspots were observed for both compounds . In conclusion, the quantitatively minor DNA adduct of tamoxifen (dG-N(2)-4-hydroxytamoxifen) is more mutagenic than the major tamoxifen DNA adduct (dG-N(2)-tamoxifen).

Biol Sci Space, 2001 Oct, 15 Suppl, S203 - 10
Biological effects of space radiation; Ohnishi T et al.; To determine the effects of space radiation on human health for long-term stays in space, we performed 21 space experiments on radiation biology . Two main characteristics of space are microgravity and space radiation that consists of low dose, chronic exposure at low dose-rates, and heavy particles . Through space experiments, we demonstrated the formation of DNA strand breaks, induced mutations, abnormal cell differentiation and the inducible gene expression of a tumor suppressor gene product, p53, in various kinds of organisms . In addition, we investigated the influence of microgravity on radiation-induced biological effects in in vitro biochemical reaction systems and in vivo cell culture systems of bacteria and lower eukaryotes . We review here the importance of radiation biology studies on space radiation from the viewpoints of human health and biological evolution, from the beginning of life until today, in the context of environmental genotoxic radiation.

Microbiology, 2002 Jul, 148(Pt 7), 2019 - 27
Mutational analysis of the role of charged residues in target-cell binding, potency and specificity of the pediocin-like bacteriocin sakacin P; Kazazic M et al.; The significance of charged residues for the target-cell binding, potency and specificity of pediocin-like bacteriocins has been studied by site-directed mutagenesis of sakacin P . Most of the charged residues are located in the N-terminal half, which is thought to mediate the initial binding of these bacteriocins to target cells through electrostatic interaction . All the mutated peptides in which the net positive charge was reduced by one (by replacing a charged residue with threonine) exhibited reduced binding to target cells and a 2-15-fold reduction in potency . The least deleterious of these mutations was the removal of the positive charge in position 8 (H8T) . This mutation was, in fact, less deleterious than the conservative His to Lys mutation, indicating that the positive charge in position 8 per se is not of major importance . Somewhat more deleterious was the removal of positive charges at the N- and C-terminal ends (K1T, K43T) . Most deleterious was the elimination of the positive charge at positions 11 and (but to a lesser extent) 12, demonstrating the importance of the cationic patch in the middle of the N-terminal half of pediocin-like bacteriocins . Mutated peptides in which the net positive charge was increased by one were also constructed . Some of these exhibited increased cell binding and a potency that was the same as (44K, i.e . an extra positive charge at the C-terminus), or somewhat greater (T20K) than, that of sakacin P, whereas others (0K, i.e . an extra positive charge at the N-terminus) had reduced potency . Sakacin P contains only one negatively charged residue (Asp17) . This negative charge and its orientation in space were crucial for activity, since the Asp to Asn mutation and (especially) the conservative Asp to Glu mutation were deleterious . Mutations that made the peptide less cationic had, overall, less effect on the potency toward the Carnobacterium piscicola strain than on the potency toward the three other strains tested, whereas the opposite was the case for mutations that made the peptide more cationic . Thus, charged residues in the N-terminal half may - apparently via the initial electrostatic binding of the bacteriocin to target cells - influence the target-cell specificity.

J Leukoc Biol, 2002 Jul, 72(1), 140 - 6
Human monocyte scavenger receptors are pattern recognition receptors for (1-->3)-beta-D-glucans; Rice PJ et al.; Glucans are cell wall constituents of fungi and bacteria that bind to pattern recognition receptors and modulate innate immunity, in part, by macrophage activation . We used surface plasmon resonance to examine the binding of glucans, differing in fine structure and charge density, to scavenger receptors on membranes isolated from human monocyte U937 cells . Experiments were performed at 25 degrees C using a biosensor surface with immobilized acetylated low density lipoprotein (AcLDL) . Inhibition of the binding by polyinosinic acid, but not polycytidylic acid, confirmed the interaction of scavenger receptors . Competition studies showed that there are at least two AcLDL binding sites on human U937 cells . Glucan phosphate interacts with all sites, and the CM-glucans and laminarin interact with a subset of sites . Polymer charge has a dramatic effect on the affinity of glucans with macrophage scavenger receptors . However, it is also clear that human monocyte scavenger receptors recognize the basic glucan structure independent of charge.

Mol Cell Biol, 2002 Aug, 22(15), 5492 - 505
Nrf2 transcription factor, a novel target of keratinocyte growth factor action which regulates gene expression and inflammation in the healing skin wound; Braun S et al.; Keratinocyte growth factor (KGF) is a potent mitogen for epithelial cells, and it promotes survival of these cells under stress conditions . In a search for KGF-regulated genes in keratinocytes, we identified the gene encoding the transcription factor NF-E2-related factor 2 (Nrf2) . Nrf2 is a key player in the cellular stress response . This might be of particular importance during wound healing, where large amounts of reactive oxygen species are produced as a defense against invading bacteria . Therefore, we studied the wound repair process in Nrf2 knockout mice . Interestingly, the expression of various key players involved in wound healing was significantly reduced in early wounds of the Nrf2 knockout animals, and the late phase of repair was characterized by prolonged inflammation . However, these differences in gene expression were not reflected by obvious histological abnormalities . The normal healing rate appears to be at least partially due to an up-regulation of the related transcription factor Nrf3, which was also identified as a target of KGF and which was coexpressed with Nrf2 in the healing skin wound . Taken together, our results reveal novel roles of the KGF-regulated transcription factors Nrf2 and possibly Nrf3 in the control of gene expression and inflammation during cutaneous wound repair.

J Clin Pathol, 2002 Jul, 55(7), 548 - 50
The natural history of a gastric low grade B cell MALT lymphoma followed during 11 years without treatment; Sandmeier D et al.; Low grade B cell mucosa associated lymphoid tissue (MALT) lymphoma of the stomach is usually an indolent tumour that remains localised for a long time before dissemination occurs . MALT appears in the stomach in response to infection by Helicobacter pylori, which is present in 80-90% of cases . The pathogenesis of the evolution from chronic gastritis to malignant lymphoma has not yet been fully explained and the exact role of H pylori in the pathogenesis and progression of gastric lymphoma remains unclear . This report describes the case of a 72 year old woman with a low grade B cell MALT lymphoma localised in the gastric fundus, who refused to be treated for eradication of H pylori . The histological diagnosis of B cell MALT lymphoma was supported by both immunohistochemical and molecular genetic analysis . After 11 years of follow up, this MALT lymphoma remained indolent, without local progression or blastic transformation, and the H pylori infection was still persistent, even though the density of bacteria had decreased drastically . Interestingly, two different clonal immunoglobulin (Ig) gene rearrangements were found in two series of biopsies performed with an interval of 11 years . This case report supports the following notions: (1) H pylori associated gastritis is a risk factor for gastric MALT lymphoma, but might not be sufficient by itself for the progression of the disease, and (2) in the evolution of MALT lymphomas, different cell clones characterised by different Ig rearrangements may emerge.

J Gastroenterol Hepatol, 2002 Jun, 17(6), 651 - 8
Roles of tumor necrosis factor-alpha-receptor type 1 and Fas in the Helicobacter pylori-induced apoptosis of gastric epithelial cells; Hasumi K et al.; OBJECTIVES: Helicobacter pylori (HP) infection has been reported to accelerate the apoptosis of gastric epithelial cells . This bacteria has also been known to enhance the expression levels of molecules such as Fas antigen and a receptor for tumor necrosis factor-alpha-receptor type 1 (TNF-R1) . However, whether Fas and/or TNF-R1 is actually involved in HP-mediated apoptosis has yet to be evaluated . The purpose of this study was therefore to examine the roles of Fas and TNF-R1 in HP-mediated apoptosis . METHODS: Biopsy samples were collected from 10 HP-negative healthy volunteers and from 39 HP-positive ulcer patients . Gastric epithelial cells were obtained from the samples . The cells were then stained with anti-Fas, anti-TNF-R1 and Annexin V, which detected apoptotic cells . The findings were analyzed by three-color flow cytometry . RESULTS: The percentages of apoptotic cells were significantly higher in HP-positive patients than in the controls . In HP-negative controls, almost all of the apoptotic cells lacked both Fas and TNF-R1 . On the other hand, in HP-positive patients, HP upregulated the expression levels of Fas and TNF-R1 and, consequently, enhanced the apoptosis mediated by receptors such as Fas and TNF-R1 . However, even in HP-positive patients, apoptosis was also observed in the cells that lacked both Fas and TNF-R1 . CONCLUSIONS: Fas and TNF-R1 expressed on gastric epithelial cells from HP-infected patients were responsible for the accelerated apoptosis of the cells.

Mol Microbiol, 2002 Jul, 45(1), 9 - 15
Regulation of inducible peroxide stress responses; Mongkolsuk S et al.; Bacteria adapt to the presence of reactive oxygen species (ROS) by increasing the expression of detoxification enzymes and protein and DNA repair functions . These responses are co-ordinated by transcription factors that regulate target genes in response to ROS . We compare three classes of peroxide-sensing regulators: OxyR, PerR and OhrR . In all three cases, peroxides effect changes in the redox status of cysteine residues, but the molecular details are distinct . OxyR is converted into a transcriptional activator by the formation of a disulphide bond between two reactive cysteine residues . PerR is a metalloprotein that functions as a peroxide- sensitive repressor . Oxidation is modulated by metal ion composition and may also involve disulphide bond formation . OhrR represses an organic peroxide resistance protein and mediates derepression in response to organic peroxides . Peroxide sensing in this system requires a single conserved cysteine, which is oxidized to form a cysteine-sulphenic acid derivative.

Biomacromolecules, 2002 Jul-Aug, 3(4), 823 - 7
The "PHB depolymerase inhibitor" of Paucimonas lemoignei is a PHB depolymerase; Reinhardt S et al.; A approximately 35 kDa protein that has been described to be secreted by Paucimonas lemoignei during growth on succinate and to inhibit hydrolysis of denatured (crystalline) poly(3-hydroxybutyrate) (dPHB) by extracellular PHB depolymerases of P . lemoignei (PHB depolymerase inhibitor (PDI)) was purified and characterized . Purified PDI (M(r), 36 199 +/- 45 Da) inhibited hydrolysis of dPHB by two selected purified PHB depolymerases (PhaZ2 and PhaZ5) but did not inhibit the hydrolysis of water-soluble substrates such as p-nitrophenylbutyrate by PhaZ5 and PhaZ2 . PDI revealed a high binding affinity to dPHB although it was not able to hydrolyze the crystalline polymer . However, purified PDI had a high hydrolytic activity if native (amorphous) PHB (nPHB) was used as a substrate . N-terminal sequencing of PDI revealed that it was identical to recently described extracellular PHB depolymerase PhaZ7 which is specific for nPHB and which cannot hydrolyze dPHB . To confirm that the inhibition of hydrolysis of dPHB by PhaZ7 is an indirect surface competition effect at high depolymerase concentration, the activity of PHB depolymerases PhaZ2 and PhaZ5 in the presence of different amounts of protein mixtures was determined . The components of NB or LB medium inhibited hydrolysis of the polymer in a concentration-dependent manner but had no effect on the hydrolysis of p-nitrophenylbutyrate by PHB depolymerases . In combination with PHB depolymerases PhaZ2 and PhaZ5 the protein PhaZ7 ("PDI") enables the bacteria to hydrolyze dPHB and nPHB simultaneously.

J Infect, 2002 May, 44(4), 229 - 35
Apoptosis modulation by mycolic acid, tuberculostearic acid and trehalose 6,6'-dimycolate; Nuzzo I et al.; The object of our study is to demonstrate that some components of M . tuberculosis, such as cord factor or mycolic acid or whole bacteria can prolong cell survival compared to controls . The cells treated with cord factor or mycolic acid at a concentration of 5 microg/ml were 65+/-8% viable reaching 70+/-8% at a concentration of 10 microg/ml . The cells treated with heat killed mycobacteria were 70+/-8% viable; while control cells exhibited a viability 50+/-7% . Conversely, tuberculostearic acid induced early cell death . The results also demonstrated a dose-dependent effect on the viability or induction of macrophage apoptosis . We also showed that prolonged viability of the treated cells with mycolic acid or cord factor (+20+/-4% and +25+/-5%, respectively) was correlated with a significant increase in Bcl-2 expression . The treated cells with whole bacteria presented a Bcl-2 expression of 40+/-6%, while Fas expression was not changed compared to controls . This study confirm that at the site of mycobacterial infection, necrosis, apoptosis or prolonged survival of the cells depend on the quantity and quality of the molecules expressed by the mycobacteria; whether necrosis or apoptosis or prolonged survival is more or less favorable to the host likely depends on several factors regarding the inflammatory and immune response, both markedly stimulated by mycobacteria .

Ann Bot (Lond), 2002 May, 89(5), 503 - 12
Induced systemic resistance (ISR) against pathogens in the context of induced plant defences; Heil M et al.; Induced systemic resistance (ISR) of plants against pathogens is a widespread phenomenon that has been intensively investigated with respect to the underlying signalling pathways as well as to its potential use in plant protection . Elicited by a local infection, plants respond with a salicylic-dependent signalling cascade that leads to the systemic expression of a broad spectrum and long-lasting disease resistance that is efficient against fungi, bacteria and viruses . Changes in cell wall composition, de novo production of pathogenesis-related-proteins such as chitinases and glucanases, and synthesis of phytoalexins are associated with resistance, although further defensive compounds are likely to exist but remain to be identified . In this Botanical Briefing we focus on interactions between ISR and induced resistance against herbivores that is mediated by jasmonic acid as a central signalling molecule . While many studies report cross-resistance, others have found trade-offs, i.e . inhibition of one resistance pathway by the other . Here we propose a framework that explains many of the thus far contradictory results . We regard elicitation separately from signalling and from production, i.e . the synthesis of defensive compounds . Interactions on all three levels can act independently from each other.

Environ Sci Technol, 2002 Jun 15, 36(12), 2736 - 41
Rapid, simple, and accurate method for measurement of VFA and carbonate alkalinity in anaerobic reactors; Lahav O et al.; This paper presents a new simple, rapid, and accurate method suitable for on-site measurement of volatile fatty acids (VFA) and carbonate alkalinity in anaerobic reactors . This titrimetric method involves eight pH observations, and typically, the full procedure takes approximately 15 min . An important feature of the method is a built-in quality control mechanism allowing the user a rapid means of assessing the reliability of the experimental procedure . To evaluate the accuracy of the method, both laboratory-made waters and industrial UASB effluent were tested . High accuracy for both VFA and carbonate alkalinity measurements (error within 2% and 1%, respectively) plus good repetition (average standard deviation of 6.7% and 1.45%, respectively) was obtained . The method takes into account the effects of the phosphate, ammonium, and sulfide weak acid subsystems . Appraisal of the effect of an input error in these subsystems revealed that VFA measurement is fairly insensitive to phosphate and ammonium concentrations . It is, however, sensitive to H2S loss during titration where the sulfide concentration is higher than approximately 10 mg/Las S . With regard to the carbonate alkalinity measurement, error in concentration of either phosphate or sulfide or H2S loss might result in a significant error . Short guidelines for correct execution of the method are given in an appendix.

Crit Rev Oral Biol Med, 2002, 13(2), 132 - 42
Porphyromonas gingivalis lipopolysaccharide signaling in gingival fibroblasts-CD14 and Toll-like receptors; Wang PL et al.; Periodontal disease is the major cause of adult tooth loss and is commonly characterized by a chronic inflammation caused by infection of oral bacteria . Porphyromonas gingivalis (P . gingivalis) is one of the suspected periodontopathic bacteria and is frequently isolated from the periodontal pockets of patients with chronic periodontal disease . The lipopolysaccharide (LPS) of P . gingivalis is a key factor in the development of periodontitis . Gingival fibroblasts, which are the major constituents of gingival connective tissue, may directly interact with bacteria and bacterial products, including LPS, in periodontitis lesions . It is suggested that gingival fibroblasts play an important role in the host responses to LPS in periodontal disease . P . gingivalis LPS enhances the production of inflammatory cytokines such as interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) in gingival fibroblasts . However, the receptor that binds with P . gingivalis LPS on gingival fibroblasts remained unknown for many years . Recently, it was demonstrated that P . gingivalis LPS binds to gingival fibroblasts . It was also found that gingival fibroblasts express CD14, Toll-like receptor 4 (TLR4), and myeloid differentiation primary response gene 88 (MyD88) . P . gingivalis LPS treatment of gingival fibroblasts activates several intracellular proteins, including protein tyrosine kinases, and up-regulates the expression of monocyte chemoattractant protein-1 (MCP-1), extracellular signal-regulated kinase 1 (ERK1), and signal-regulated kinase 2 (ERK2), IL-1 receptor-associated kinase (IRAK), nuclear factor-kappaB (NF-kappaB), and activating protein-1 (AP-1) . These results suggest that the binding of P . gingivalis LPS to CD14 and TLR4 on gingival fibroblasts activates various second-messenger systems . In this article, we review recent findings on the signaling pathways induced by the binding of P . gingivalis LPS to CD14 and Toll-like receptors (TLRs) in gingival fibroblasts.

J Biol Chem, 2002 Oct 4, 277(40), 37711 - 7 Epub 2002 Jul 03.
Simultaneous expression of hCNT1-CFP and hENT1-YFP in Madin-Darby canine kidney cells . Localization and vectorial transport studies; Lai Y et al.; To test the hypothesis that human concentrative and equilibrative nucleoside transporters (hCNT1 and hENT1) are present on the apical and basolateral membrane, respectively, we constructed a Madin-Darby canine kidney (MDCK) cell line that simultaneously and stably expresses recombinant hCNT1 and hENT1 gene products tagged with CFP and YFP fluorescent proteins, respectively . Using a confocal microscope, both hCNT1-CFP and hENT1-YFP were found to be distributed uniformly on the plasma membrane of undifferentiated MDCK cells . Upon differentiation of the MDCK cells on Transwell filter inserts, hCNT1-CFP was visualized exclusively on the apical membrane, whereas hENT1-YFP appeared predominantly on the basolateral membrane . As differentiation proceeded, there was an increase in alkaline phosphatase activity, and activity of hENT1 in the apical compartment decreased while hCNT1 activity remained constant . These results suggest that, on differentiation, hENT1 is sorted to the basolateral membrane . This was confirmed when the hCNT1-mediated uptake of {(3)H}uridine from the apical compartment of the differentiated cells was found to be approximately 20-fold higher and that for hENT1 was approximately 4-fold lower than the corresponding uptake from the basal compartment . As observed in vivo, the net transport of {(3)H}adenosine was from the apical to the basal compartment, whereas that for (14)C-deoxyadenosine was from the basal to the apical compartment . In summary, we have shown for the first time that hCNT1 and hENT1 are expressed in polarized MDCK cells on the apical and basolateral membrane, respectively, allowing vectorial transport in both directions depending on the relative activity (ratio of maximal transporter activity to affinity) of each transporter for their substrates.

J Dent Res, 2002 Apr, 81(4), 241 - 6
Matrilysin (matrix metalloproteinase-7) expression in human junctional epithelium; Uitto VJ et al.; Matrilysin is a matrix metalloproteinase expressed in exocrine and mucosal epithelium in many human tissues . Immunohistochemical staining showed that matrilysin is expressed in suprabasal cells of junctional epithelium facing the teeth and in epithelial cell rests of Malassez . No matrilysin expression was seen in the periodontal pocket tissue . In a tissue culture model mimicking junctional epithelium, matrilysin expression was also observed in suprabasal epithelial cells . Of 13 anaerobic oral bacterial species tested, F . nucleatum, F . necrophorum, P . endodontalis, and P . denticola stimulated matrilysin expression in porcine periodontal ligament epithelial cells from 2.5- to 5.7-fold, compared with untreated cells . The enzyme was localized in intracytoplasmic vesicles that also reacted with antibodies against lysosomal membrane protein h-lamp-1 . The results indicate that matrilysin may play an important role in the normal physiology of junctional epithelium.

Clin Microbiol Rev, 2002 Jul, 15(3), 342 - 54
Cultivation of pathogenic and opportunistic free-living amebas; Schuster FL; Free-living amebas are widely distributed in soil and water, particularly members of the genera Acanthamoeba and NAEGLERIA: Since the early 1960s, they have been recognized as opportunistic human pathogens, capable of causing infections of the central nervous system (CNS) in both immunocompetent and immunocompromised hosts . Naegleria is the causal agent of a fulminant CNS condition, primary amebic meningoencephalitis; Acanthamoeba is responsible for a more chronic and insidious infection of the CNS termed granulomatous amebic encephalitis, as well as amebic keratitis . Balamuthia sp . has been recognized in the past decade as another ameba implicated in CNS infections . Cultivation of these organisms in vitro provides the basis for a better understanding of the biology of these amebas, as well as an important means of isolating and identifying them from clinical samples . Naegleria and Acanthamoeba can be cultured axenically in cell-free media or on tissue culture cells as feeder layers and in cultures with bacteria as a food source . Balamuthia, which has yet to be isolated from the environment, will not grow on bacteria . Instead, it requires tissue culture cells as feeder layers or an enriched cell-free medium . The recent identification of another ameba, Sappinia diploidea, suggests that other free-living forms may also be involved as causal agents of human infections.

ALTEX, 2002, 19 Suppl 1, 76 - 86
{Replacement of mouse and rat antibody production test; comparison of sensitivity between the in vitro and in vivo methods}; Bootz F et al.; Bacteria and viruses may be transmitted through contaminated biological materials such as transplantable tumors, cell lines, sera or other biological material . Currently, the screening of biological material is being done using the mouse or rat antibody production (MAP/RAP) test (serological testing) . We decided to test and validate an alternative assay using polymerase chain reaction (PCR / RT-PCR) technology to detect viral contamination directly in biological material . The aim of this study therefore is the validation of our new PCR assays and the comparison of PCR and MAP test . For 6/14 viruses the conventional PCR, seems to be more sensitive and more specific in detecting murine viruses . In 12/14 viruses the RT-PCR is more sensitive than MAP-test . In 2/14 cases all detection methods have the same sensitivity . Furthermore, PCR screening clearly contributes to the principles of 3R as a replacement technique as it eliminates the need for using animals to screen for murine viruses in biological material.

Am J Gastroenterol, 2002 Jun, 97(6), 1398 - 407
A randomized, double blind, dose-response comparison of balsalazide (6.75 g), balsalazide (2.25 g), and mesalamine (2.4 g) in the treatment of active, mild-to-moderate ulcerative colitis; Levine DS et al.; OBJECTIVE: Balsalazide is a new innovative, mesalamine-containing prodrug that is activated by bacteria in the colon . Balsalazide has been shown previously to be well tolerated and effective in the treatment of acute ulcerative colitis . The aim of this study was to determine the dose-response of balsalazide for efficacy and safety in active, mild-to-moderate ulcerative colitis and to compare this profile with that of mesalamine, pH-dependent, delayed-release tablets . METHODS: A multicenter, randomized, active control, double-blind, double-dummy, dose-response, parallel-group study was performed comparing balsalazide (6.75 g daily), balsalazide (2.25 g daily), and mesalamine (2.4 g daily), administered for 8 wk to 154 patients with active, mild-to-moderate ulcerative colitis as verified by sigmoidoscopy . RESULTS: Eight weeks of treatment with 6.75 g of balsalazide daily provided significantly greater improvement than did balsalazide (2.25 g daily) in rectal bleeding (64.7% {6.75-g balsalazide} vs 32.4% {2.25-g balsalazide}, p < 0.006), stool frequency (58.8% vs 29.4%, p < 0.006), sigmoidoscopic score (78.9% vs 52.5%, p < 0.015), and Physician's Global Assessment (73.7% vs 51.3%, p < 0.03) . The efficacy of balsalazide showed a significantly more rapid onset of action than that of mesalamine (2.4 g daily) (2-wk sigmoidocopic score improvement, 54.7% {6.75-g balsalazide} vs 29.4% {2.4-g mesalamine}, p = 0.006) with numerically greater improvement at 8 wk in five of seven measured signs and symptoms . Balsalazide (6.75 g daily) was well tolerated, and the safety profile did not differ significantly from that of balsalazide (2.25 g daily) or mesalamine . CONCLUSIONS: Eight weeks of treatment with balsalazide (6.75 g daily) is significantly more effective than balsalazide (2.25 g daily) and more rapid in onset than mesalamine (2.4 g daily) in improving signs and symptoms of acute ulcerative colitis . Balsalazide (6.75 g daily) is well tolerated, and the safety profile does not differ from that of balsalazide (2.25 g daily) and mesalamine (2.4 g daily).

Bioresour Technol, 2002 Jul, 83(3), 259 - 61
Effect of thermal and chemical pretreatments on anaerobic ammonium removal in treating septage using the UASB system; Lin CY et al.; This work investigated how thermal and chemical pretreatments influenced the anaerobic ammonium oxidation process in anaerobic digestion using biogranules that had been acclimated to septage . The digestion experiments were performed in serum vials using thermally and chemically pretreated septage as the substrate . The experimental results indicated that heat pretreatment reduced both dinitrogen production and ammonium removal . HCl and NaOH pretreatments increased both values by near 45% and 55% over the control . Alkali addition was more efficient than acid addition in enhancing dinitrogen production and ammonium removal.

Adv Mar Biol, 2002, 42, 1 - 65
Protobranch bivalves; Zardus JD; The subclass Protobranchia comprises more than 600 species of bivalves that occur throughout the world ocean . Mostly deposit feeders in soft sediments, they are abundant in the deep sea . Apomorphies that unite them as a group include gill structure, hinge conformation, shell microstructure, larval development, foot morphology, respiratory pigments, trophic mode and digestion . They are relatively small and highly conserved in form, originating in the Cambrian era . They may represent an ancestral, derived or paraphylectic group of the Bivalvia . The protobranchs include two orders, the Nuculoida and Solemyoida, which previously were classified separately in the subclasses Paleotaxodonta and Cryptodonta, respectively . They are of ecological interest and have a unique functional morphology . They feed mostly under the surface of the sediment with highly modified labial palps, but the degree to which they are selective in diet remains difficult to determine . They are important bioturbators in many soft-sediment assemblages; their feeding and locomotion affects sediment structure and community development . Solemyoids are unusual in inhabiting reducing environments and hydrocarbon seeps and in deriving their nutrition from endosymbiotic chemosynthetic bacteria . A variety of species of protobranchs are found in oceanic trenches, near hydrothermal vents, and in submarine caves . Protobranchs produce a lecithotrophic larval stage, the pericalymma, making their development unique among bivalves . The pericalymma remains in the plankton for a short time and presumably has low dispersal ability . Recruitment may be intermittent . Growth is rapid in post-larvae but decreases with age, though rates may not necessarily be slow, especially in continental shelf species . Life spans are commonly 1 to 2 decades, but deep-sea representatives may grow more slowly and live longer . Bottom fish, seastars and gastropods are their major predators and a few parasites and commensals have been documented . The predominance of protobranchs in deep-sea sediments may be a result of deep-sea origin or displacement from shallow waters by lamellibranchs . Their ability to deposit-feed, digest food extracellularly, and develop by means of lecithotrophic larvae make them particularly well adapted to cold and oligotrophic habitats.

Nutr Cancer, 2001, 41(1-2), 135 - 44
Carnosic acid inhibits proliferation and augments differentiation of human leukemic cells induced by 1,25-dihydroxyvitamin D3 and retinoic acid; Steiner M et al.; Carnosic acid, the polyphenolic diterpene derived from rosemary, is a strong dietary antioxidant that exhibits antimutagenic properties in bacteria and anticarcinogenic activity in various cell and animal models . In the present study, we show that carnosic acid (2.5-10 microM) inhibits proliferation of HL-60 and U937 human myeloid leukemia cells (half-maximal inhibitory concentration = 6-7 microM) without induction of apoptotic or necrotic cell death . Growth arrest occurred concomitantly with a transient cell cycle block in the G1 phase, which was accompanied by an increase in the immunodetectable levels of the universal cyclin-dependent kinase inhibitors p21WAFI and p27Kipl . Carnosic acid caused only a marginal induction of differentiation, as monitored by the capacity to generate superoxide radicals and the expression of cell surface antigens (CD11b and CD14) and receptors for the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine . However, at low concentrations, this polyphenol substantially augmented (100- to 1,000-fold) the differentiating effects of 1,25-dihydroxyvitamin D3 and all-trans retinoic acid . Furthermore, such combinations of carnosic acid and any of these differentiation inducers synergistically inhibited proliferation and cell cycle progression . These results indicate that carnosic acid is capable of antiproliferative action in leukemic cells and can cooperate with other natural anticancer compounds in growth-inhibitory and differentiating effects.

Pediatrics . 2002 Jul;110(1 Pt 1):e4.
No evidence of autoimmunity in 6-year-old children immunized at birth with recombinant hepatitis B vaccine; Belloni C et al.; OBJECTIVES: Taking into account that genetic predisposition, marked by human leukocyte antigen (HLA) class I and II genes, augments the probability of developing an autoimmune disorder after a triggering vaccination, as largely debated, we investigated the frequency of autoantibody production after recombinant hepatitis B vaccine (rHBv) in 6-year-old children immunized at birth to evaluate an association between autoimmune disorders and hepatitis B virus vaccination . METHODS: We investigated the presence of autoantibodies in 210 6-year-old children who were immunized at birth with rHBv: 200 showed anti-hepatitis B surface antigen concentrations > or =10 mUI/mL at seroconversion (responders), and 10 were nonresponders . Data were compared with those obtained in 109 unvaccinated children . All participants were screened for the presence of antinuclear antibodies (ANAs), anti-DNA, antimitochondrial, anti-liver/kidney microsomal, antireticulin, anti-smooth muscle (SMA), and antiribosomal antibodies . All participants were also screened for the presence of antithyroid antibodies, such as antithyroglobulin and antiperoxidase, and for antibodies found in type 1 diabetes, such as tyrosine phosphatase (IA-2A) and glutamic acid decarboxylase (GADA) . HLA typing was extended to all 10 nonresponders . RESULTS: Autoantibodies were found in 16 of the 200 responders: ANAs were found in 12 (6%), smooth muscle antibodies were found in 4 (2.0%), and antireticulin antibodies and endomysial antibodies were found in 1 girl with ANAs . Antithyroid antibodies, IA-2A, and GADA were not present in any of the participants . No significant difference was found in the frequency of autoantibodies between vaccinated and control children . Three of the 10 nonresponder children were SMA-positive (30% vs 2% of responders); they also carried the supratype HLA-C4AQ0,DRB1*0301,DQB1*02 . A family history for autoimmune disorders was present in 3 (18%; 95% confidence interval {CI}: 4.0%-45.6%) of the 16 responder infants with autoantibodies, in 15 (8.4%; 95% CI: 4.6%-13.1%) of responder children without autoantibodies, and in 1 (10%) of the 10 nonsreponder children . CONCLUSIONS: From our data, vaccination with rHBv given during the neonatal period does not seem to increase autoantibody production in a 6-year-old children . Autoantibodies, referred to as natural autoantibodies, can be found in healthy participants, but their significance is unclear . These autoantibodies often cross-react with bacteria or tumor antigens, suggesting their importance in innate immunity . It has been demonstrated in an animal model that self-antigen can promote B-cell accumulation, and that a significant proportion of natural autoantibodies is the product of this self-antigen- dependent process . Consequently, it has been speculated that self-antigens play a positive role in recruiting B cells as a part of innate immunity, but this process carries a potential risk for unregulated growth . Spreading of the immune response is a common theme in organ-specific and systemis autoimmune diseases, and this could be initiated by exogenous agents, in genetically susceptible hosts, owing to molecular mimicry of natural antigen . Moreover, 3 (18%) of the 16 children who had autoantibodies had a family history of autoimmume diseases . Thus, it is apparent that susceptibility to autoimmunity is determined by genetic factors rather than by vaccine challenge . Among all the children considered, only 1 girl (0.5%) developed celiac disease, reflecting the prevalence described in the literature . GADA and IA-2A were not found in our children; this observation is in agreement with data showing that type 1 diabetes risk may not be altered by vaccinations administered during childhood . On the contrary, a high frequency (30%) of autoantibodies, in particular SMA, was observed in the nonresponder children . The 3 SMA-positive children carried the HLA-C4Q0,DRB1*0301,DQB1*02 haplotype, a well-known predisposing factor for autoimmune disorders . On the other hand, the presence of autoantibodies to smooth muscle is known to be common in hepatitis B infection, and, it has been shown that cross-reactive immunity targeting homologous self-protein may partly account for autoantibody production . Although hepatitis B vaccination given during the neonatal period does not increase autoantibody production in 6-year-old immunized children, we deem useful a more prolonged follow-up for these nonresponder children carrying certain HLA haplotypes (such as C4AQ0,DRB1*0301,DQB1*02), particularly because most autoimmune diseases do not develop until later in life.

EMBO J, 2002 Jul 1, 21(13), 3414 - 23
FANCE: the link between Fanconi anaemia complex assembly and activity; Pace P et al.; The Fanconi anaemia (FA) nuclear complex (composed of the FA proteins A, C, G and F) is essential for protection against chromosome breakage . It activates the downstream protein FANCD2 by monoubiquitylation; this then forges an association with the BRCA1 protein at sites of DNA damage . Here we show that the recently identified FANCE protein is part of this nuclear complex, binding both FANCC and FANCD2 . Indeed, FANCE is required for the nuclear accumulation of FANCC and provides a critical bridge between the FA complex and FANCD2 . Disease-associated FANCC mutants do not bind to FANCE, cannot accumulate in the nucleus and are unable to prevent chromosome breakage.

EMBO J, 2002 Jul 1, 21(13), 3225 - 34
Crystal structure of the middle and C-terminal domains of the flagellar rotor protein FliG; Brown PN et al.; The FliG protein is essential for assembly, rotation and clockwise/counter-clockwise (CW/CCW) switching of the bacterial flagellum . About 25 copies of FliG are present in a large rotor-mounted assembly termed the 'switch complex', which also contains the proteins FliM and FliN . Mutational studies have identified the segments of FliG most crucial for flagellar assembly, rotation and switching . The structure of the C-terminal domain, which functions specifically in rotation, was reported previously . Here, we describe the crystal structure of a larger fragment of the FliG protein from Thermotoga maritima, which encompasses the middle and C-terminal parts of the protein (termed FliG-MC) . The FliG-MC molecule consists of two compact globular domains, linked by an alpha-helix and an extended segment that contains a well-conserved Gly-Gly motif . Mutational studies indicate that FliM binds to both of the globular domains, and given the flexibility of the linking segment, FliM is likely to determine the relative orientation of the domains in the flagellum . We propose a model for the organization of FliG-MC molecules in the flagellum, and suggest that CW/CCW switching might occur by movement of the C-terminal domain relative to other parts of FliG, under the control of FliM.

Curr Drug Metab, 2002 Aug, 3(4), 425 - 38
The metabolism and toxicity of quinones, quinonimines, quinone methides, and quinone-thioethers; Monks TJ et al.; Quinones are ubiquitous in nature and constitute an important class of naturally occurring compounds found in plants, fungi and bacteria . Human exposure to quinones therefore occurs via the diet, but also clinically or via airborne pollutants . For example, the quinones of polycyclic aromatic hydrocarbons are prevalent as environmental contaminants and provide a major source of current human exposure to quinones . The inevitable human exposure to quinones, and the inherent reactivity of quinones, has stimulated substantial research on the chemistry and toxicology of these compounds . From a toxicological perspective, quinones possess two principal chemical properties that confer their reactivity in biological systems . Quinones are oxidants and electrophiles, and the relative contribution of these properties to quinone toxicity is influenced by chemical structure, in particular substituent effects . Modification to the quinone nucleus also influences quinone metabolism . This review will therefore focus on the differences in structure and metabolism of quinones, and how such differences influence quinone toxicology . Specific examples will be discussed to illustrate the diverse manner by which quinones interact with biological systems to initiate and propagate a toxic response.

Biochemistry, 2002 Jul 9, 41(27), 8759 - 66
Mutagenesis of Trp(54) and Trp(203) residues on Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase significantly affects catalytic activities of the enzyme; Cheng HL et al.; The possible structural and catalytic functions of the nine tryptophan amino acid residues, including Trp(54), Trp(105), Trp(112), Trp(141), Trp(148), Trp(165), Trp(186), Trp(198), and Trp(203) in Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (Fs beta-glucanase), were characterized using site-directed mutagenesis, initial rate kinetics, fluorescence spectrometry, and structural modeling analysis . Kinetic studies showed that a 5-7-fold increase in K(m) value for lichenan was observed for W141F, W141H, and W203R mutant Fs beta-glucanases, and approximately 72-, 56-, 30-, 29.5-, 4.9-, and 4.3-fold decreases in k(cat) relative to that for the wild-type enzyme were observed for the W54F, W54Y, W141H, W203R, W141F, and W148F mutants, respectively . In contrast, W186F and W203F, unlike the other 12 mutants, exhibited a 1.4- and 4.2-fold increase in k(cat), respectively . W165F and W203R were the only two mutants that exhibited a 4-7-fold higher activity relative to the wild-type enzyme after they were incubated at pH 3.0 for 1 h . Fluorescence spectrometry indicated that all of the mutations on the nine tryptophan amino acid residues retained a folding similar to that of the wild-type enzyme . Structural modeling and kinetic studies suggest that Trp(54), Trp(141), Trp(148), and Trp(203) play important roles in maintaining structural integrity in the substrate-binding cleft and the catalytic efficiency of the enzyme.

Water Res, 2002 Apr, 36(8), 2098 - 108
The effect of UV light on the inactivation of Giardia lamblia and Giardia muris cysts as determined by animal infectivity assay (P-2951-01); Mofidi AA et al.; This study measured the effect of germicidal ultraviolet (UV) light on Giardia lamblia and Giardia muris cysts, as determined by their infectivity in Mongolian gerbils and CD-1 mice, respectively . Reduction of cyst infectivity due to UV exposure was quantified by applying most probable number techniques . Controlled bench-scale, collimated-beam tests exposed cysts suspended in filtered natural water to light from a low-pressure UV lamp . Both G . lamblia and G . muris cysts showed similar sensitivity to UV light . At 3 mJ/cm2, a dose 10-fold lower than what large-scale UV reactors may be designed to provide, > 2-log10 (99 percent) inactivation was observed . These results, combined with previously published data showing other protozoa and bacteria have similar, high sensitivity to UV light, establish that UV disinfection of drinking water is controlled by viruses which may require over 10-fold more UV dose for the same level of control.

Infect Dis Clin North Am, 2002 Jun, 16(2), 377 - 92, x
Diagnostic methods current best practices and guidelines for identification of difficult-to-culture pathogens in infective endocarditis; Houpikian P et al.; Culture-negative endocarditis currently represents a diagnostic challenge for physicians . Traditional methods such as histology, serology, and culture have been improved and new molecular techniques have been developed to improve the detection of difficult-to-culture agents . Serologic tests for the two most frequent etiologic agents, Coxiella burnetii and Bartonella spp, should be performed first because they can usually be identified easily in this way . The sensitivity of culture for intracellular bacteria has been improved by inoculation of samples in shell vials and by the use of novel tissue cell lines . Recently, universal and species-specific primers have been designated to amplify bacterial DNA directly from resected valves, allowing positive identification.

Nat Struct Biol, 2002 Aug, 9(8), 628 - 34
A novel fucose recognition fold involved in innate immunity; Bianchet MA et al.; Anguilla anguilla agglutinin (AAA), a fucolectin found in the serum of European eel, participates in the recognition of bacterial liposaccharides by the animal innate immunity system . Because AAA specifically recognizes fucosylated terminals of H and Lewis (a) blood groups, it has been used extensively as a reagent in blood typing and histochemistry . AAA contains a newly discovered carbohydrate recognition domain present in proteins of organisms ranging from bacteria to vertebrates . The crystal structure of the complex of AAA with alpha-L-fucose characterizes the novel fold of this entire lectin family, identifying the residues that provide the structural determinants of oligosaccharide specificity . Modification of these residues explains how the different isoforms in serum can provide a diverse pathogen-specific recognition.

Invest Ophthalmol Vis Sci, 2002 Jul, 43(7), 2258 - 63
RPE65 is highly uveitogenic in rats; Ham DI et al.; PURPOSE: To examine the hypothesis that RPE65, a protein specific to the retinal pigment epithelium, is uveitogenic in rats . METHODS: Rats of four inbred strains (Lewis, Brown Norway, Fischer, and SHR) were immunized with native or recombinant bovine RPE65, or with S-antigen (S-Ag), emulsified with complete Freund adjuvant, and treated simultaneously with killed Bordetella pertussis bacteria, as indicated . Development of ocular changes was examined and scored both clinically and histologically . RESULTS: Lewis rats immunized with RPE65 showed development of acute and severe inflammatory eye disease that affected most ocular tissues . The minimum uveitogenic dose of RPE65 was similar to that of S-Ag (1 microg per rat), but the changes induced by RPE65 at higher dose ranges were less severe than those induced by S-Ag . Concurrent treatment of the RPE65-immunized rats with B . pertussis bacteria was not critical for disease induction, but enhanced dramatically the pathogenic reaction . Unlike the results with several other retinal proteins, no pinealitis was detected in rats immunized with RPE65 . Fischer (F344) rats resembled Lewis rats in being similarly affected by RPE65 or S-Ag . In contrast, Brown Norway (BN) rats developed severe disease when immunized with RPE65, but showed minimal changes in response to S-Ag . SHR rats responded poorly to disease induced by RPE65, and S-Ag-induced disease failed to develop . CONCLUSIONS: RPE65 is highly uveitogenic in rats, thus suggesting that this molecule could be involved in pathogenic autoimmunity in the human eye.

Comp Biochem Physiol B Biochem Mol Biol, 2002 Jul, 132(3), 611 - 24
Chemical characterization of the oligosaccharides in beluga (Delphinapterus leucas) and Minke whale (Balaenoptera acutorostrata) milk; Urashima T et al.; Carbohydrates were extracted from the milk of a beluga, Delphinopterus leucas (family Odontoceti), and two Minke whales, Balaenoptera acutorostrata (Family Mysticeti), sampled late in their respective lactation periods . Free oligosaccharides were separated by gel filtration and then neutral oligosaccharides were purified by preparative thin layer chromatography and gel filtration, while acidic oligosaccharides were purified by ion-exchange chromatography, gel filtration and high performance liquid chromatography (HPLC) . Their structures were determined by 1H-NMR . In one of the Minke whale milk samples, lactose was a dominant saccharide, with Fuc(alpha1-2)Gal(beta1-4)Glc(2'-fucosyllactose), Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc(lacto-N-neotetraose), GalNAc(alpha1-3){Fuc(alpha1-2)}Gal(beta1-4)Glc(A-tetrasaccharide), Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (para lacto-N-neohexaose), Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (sialyl lacto-N-neotetraose), Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (LST c) and Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (sialyl para lacto-N-neohexaose) also being found in the milk . The second Minke whale sample contained similar amounts of lactose, 2'-fucosyllactose and A-tetrasaccharide, but no free sialyl oligosaccharides . Sialyl lacto-N-neotetraose and sialyl para lacto-N-neohexaose are novel oligosaccharides which have not been previously reported from any mammalian milk or colostrum . These and other oligosaccharides of Minke whale milk may have biological significance as anti-infection factors, protecting the suckling young against bacteria and viruses . The lactose of Minke whale milk could be a source of energy for them . The beluga whale milk contained trace amounts of Neu5Ac(alpha2-3)Gal(beta1-4)Glc(3'-N-acetylneuraminyllactose), but the question of whether it contained free lactose could not be clarified . Therefore, lactose may not be a source of energy for suckling beluga whales.

Tidsskr Nor Laegeforen, 2002 May 10, 122(12), 1218 - 22
{Functional dyspepsia--a psychosomatic disease}; Haug TT; About 4% of consultations in general practice involve patients with upper gastrointestinal complaints . Evidence of peptic ulcer disease is identified in only 20-30% of the patients . No organic explanation of the symptoms is found using endoscopy in 20-50%; these patients are given the diagnosis non-ulcer dyspepsia or functional dyspepsia . Hypersecretion of gastric acid and the bacteria Helicobacter pylori, which seem to be major aetiologic factors in duodenal ulcer, are not important in functional dyspepsia . In these patients gastric motor abnormalities and visceral hypersensitivity are the most important pathophysiological mechanisms . The gastric motility is influenced by stress; there is a strong relationship between anxiety, depression and functional dyspepsia . Antacids, H2-blockers and prokinetics are hardly more effective than placebo in patients with functional dyspepsia, while antidepressants have been proven effective in reducing dyspeptic symptoms . Likewise, psychological treatment like bio-feedback, stress management, interpersonal psychotherapy and cognitive therapy has also been proven effective in reducing dyspeptic symptoms in patients with functional dyspepsia.

J Oral Maxillofac Surg, 2002 Jul, 60(7), 778 - 83
Analysis of human leukocyte antigens in patients with internal derangement of the temporomandibular joint; Henry CH et al.; PURPOSE: Spondyloarthropathy includes the subcategory of reactive arthritis (ReA) . Spondyloarthropathies are commonly associated with certain human leukocyte antigen (HLA) alleles . Because we identified bacteria associated with ReA within the temporomandibular joint (TMJ), we now evaluate the frequency of HLA alleles in patients with TMJ pathology . PATIENTS AND METHODS: HLA typing of 129 patients (121 females and 8 males) performed by standard microcytotoxicity technique . Thirty patients had only class I (HLA-A and -B loci) evaluated . Ninety-nine patients had both class I and class II (HLA-DR loci) evaluated . Identification of alleles at the C locus was not performed . The antigenic frequency in the study group was compared to US white control subjects using a 2-tailed Fisher's exact test with a Bonferroni multiple comparison adjustment . RESULTS: The following class I HLA alleles, -A1 (32%), -A2 (50%), -A3 (33%), -B7 (23%), -B14 (14%), -B35 (20%), and -B44 (36%), including the B7 cross-reactive group (CREG) (49%) and class II alleles -DR1 (25%) and -DR4 (34%), were found to have an increased frequency in our patient group . CONCLUSIONS: Our study shows an increased frequency of several alleles that have been previously associated with arthropathy, and the alleles of the B7 CREG, in patients with TMJ pathology . Patients with these alleles may have an increased risk for the development of internal derangement of the TMJ as a consequence of the bacterial/infectious agents and host interactions with the subsequent cytokine/inflammatory response being influenced by their HLA phenotype .

Science, 2002 Jun 28, 296(5577), 2373 - 6
Nitrate controls on iron and arsenic in an urban lake; Senn DB et al.; Aquatic ecosystems are often contaminated by multiple substances . Nitrate, a common aquatic pollutant, strongly influenced the cycling of arsenic (As) under anoxic conditions in urban Upper Mystic Lake (Massachusetts, USA) by oxidizing ferrous iron {Fe(II)} to produce As-sorbing particulate hydrous ferric oxides and causing the more oxidized As(V), which is more particle-reactive than As(III) under these conditions, to dominate . This process is likely to be important in many natural waters.

J Clin Microbiol, 2002 Jul, 40(7), 2425 - 30
Histologic and genotypic characterization of a novel Mycobacterium species found in three cats; Appleyard GD et al.; Three cases of feline atypical mycobacteriosis from different geographical regions in North America were characterized by large clusters of filamentous bacteria visible on hematoxylin-and-eosin-stained tissue sections . PCR amplification demonstrated the presence of Mycobacterium-specific nucleic acid in samples of skin lesions from these cases . PCR-assisted cloning and DNA sequence analysis of a 541-bp length of the Mycobacterium 16S rRNA gene generated DNA sequences which were >95% identical, suggesting that the three isolates were closely related . Two of the sequences were 99% identical and may represent the same species . Alignment with comparable 16S rRNA gene sequences from 66 Mycobacterium species and partially characterized isolates highlighted similarities (>94%) with Mycobacterium bohemicum, Mycobacterium haemophilum, Mycobacterium ulcerans, Mycobacterium avium subsp . avium, and isolate IWGMT 90242 . Parsimony analysis of sequence data suggested relatedness to M . leprae . Significant molecular genetic and pathobiological differences between these three similar isolates and other known species of mycobacteria suggested that the organisms may not have been described previously and that these cases may represent a new form of mycobacterial disease in cats . We suggest the term "Mycobacterium visibilis" to describe the organism from which the two nearly identical sequences were obtained.

Appl Environ Microbiol, 2002 Jul, 68(7), 3634 - 8
Enrichment double-antibody sandwich indirect enzyme-linked immunosorbent assay that uses a specific monoclonal antibody for sensitive detection of Ralstonia solanacearum in asymptomatic potato tubers; Caruso P et al.; Sensitive and specific routine detection of Ralstonia solanacearum in symptomless potato tubers was achieved by efficient enrichment followed by a reliable double-antibody sandwich indirect enzyme-linked immunosorbent assay based on the specific monoclonal antibody 8B-IVIA . This monoclonal antibody reacted with 168 typical R . solanacearum strains and did not recognize 174 other pathogenic or unidentified bacteria isolated from potato . The optimized protocol included an initial enrichment step consisting of shaking the samples in modified Wilbrink broth for 72 h at 29 degrees C . This step enabled specific detection by the enzyme-linked immunosorbent assay of 1 to 10 CFU of R . solanacearum per ml of initial potato extract . Analysis of 233 commercial potato lots by this method provided results that coincided with the results of conventional methods.

Appl Environ Microbiol, 2002 Jul, 68(7), 3606 - 13
Changes in bacterioplankton community structure and activity with depth in a eutrophic lake as revealed by 5S rRNA analysis; Dominik K et al.; The community structure of bacterioplankton was studied at different depths (0 to 25 m) of a temperate eutrophic lake (Lake Plusssee in northern Germany) by using comparative 5S rRNA analysis . The relative amounts of taxonomic groups were estimated from 5S rRNA bands separated by high-resolution electrophoresis . Comparison of partial 5S rRNA sequences enabled detection of changes in single taxa over space and during seasons . Overall, the bacterioplankton community was dominated by 3 to 14 abundant (>4% of the total 5S rRNA) taxa . In general, the number of 5S rRNA bands (i.e., the number of bacterial taxa) decreased with depth . In the fall, when thermal stratification and chemical stratification were much more pronounced than they were in the spring, the correlation between the depth layers and the community structure was more pronounced . Therefore, in the fall each layer had its own community structure; i.e., there were different community structures in the epilimnion, the metalimnion, and the hypolimnion . Only three 5S rRNA bands were detected in the hypolimnion during the fall, and one band accounted for about 70% of the total 5S rRNA . The sequences of individual 5S rRNA bands from the spring and fall were different for all size classes analyzed except two bands, one of which was identified as Comamonas acidivorans . In the overall analysis of the depth profiles, the diversity in the epilimnion contrasted with the reduced diversity of the bacterioplankton communities in the hypolimnion, and large differences occurred in the composition of the epilimnion at different seasons except for generalists like C . acidivorans.

Curr Opin Immunol, 2002 Aug, 14(4), 466 - 70
Dendritic cells and immunity to leishmaniasis and toxoplasmosis; Scott P et al.; There is increased recognition that dendritic cells (DCs) are an important source of the IL-12 required to initiate protective immunity to protozoa, such as Leishmania and Toxoplasma . This article reviews the advances made in the last two years in understanding the pathways that lead to DC activation after infection with these organisms . Interestingly, there appear to be differences in the DC activation pathways utilized by these two intracellular protozoa which also may differ from the pathways utilized by bacteria.

Curr Opin Immunol, 2002 Aug, 14(4), 444 - 51
The role of IFN-gamma in the outcome of chlamydial infection; Rottenberg ME et al.; Chlamydia are intracellular bacteria which infect many vertebrates, including humans . They cause a myriad of severe diseases, ranging from asymptomatic infection to pneumonia, blindness or infertility . IFN-gamma plays an important role in defense against acute infection and in the establishment of persistence . Chlamydia have evolved mechanisms to escape IFN-gamma functions . IFN-gamma-mediated effector mechanisms may involve effects on the metabolism of tryptophan or iron, on the inducible NO synthase (iNOS), on the secretion of chemokines and adhesion molecules or on the regulation of T-cell activities . IFN-gamma is secreted by the innate and the adaptive arms of the immune system . Within the former, Chlamydia-infected macrophages express IFN-gamma that in turn mediates resistance to infection . IFN-alpha/beta are pivotal for both IFN-gamma- and iNOS-mediated resistance to chlamydial infection in macrophages.

Respir Care, 2002 Jul, 47(7), 818 - 22
The pharmacologic approach to airway clearance: mucoactive agents; Rubin BK; The airway mucosa responds to infection and inflammation in a variety of ways . This response often includes surface mucous (goblet) cell and submucosal gland hyperplasia and hypertrophy, with mucus hypersecretion . Products of inflammation, including neutrophil-derived deoxyribonucleic acid (DNA) and filamentous actin (F-actin), effete cells, bacteria, and cell debris, all contribute to mucus purulence and, when this mucus is expectorated it is called sputum . Mucoactive medications are intended to serve one of 2 purposes; either to increase the ability to expectorate sputum or to decrease mucus hypersecretion . Mucoactive medications have been classified according to their proposed mechanisms of action . Increased knowledge of the properties of mucus has given us tools to better understand the mechanisms of airway disease and mucoactive therapy . Expectorants are thought to increase the volume or hydration of airway secretions . Systemic hydration and classic expectorants have not been demonstrated to be clinically effective . Modifiers of airway water transport are being clinically investigated as expectorants . Mucolytics degrade polymers in secretions . The classic mucolytics have free thiol groups to degrade mucin . Peptide mucolytics break pathologic filaments of neutrophil-derived DNA and actin in sputum . Nondestructive mucolysis includes mucin dispersion by means of charge shielding . Mucokinetics are medications that increase mucociliary efficiency or cough efficiency . Cough flow can be increased by bronchodilators in patients with airway hyperreactivity . Abhesives such as surfactants decrease mucus attachment to the cilia and epithelium, augmenting both cough and mucociliary clearance . Mucoregulatory agents reduce the volume of airway mucus secretion and appear to be especially effective in hypersecretory states such as bronchorrhea, diffuse panbronchiolitis, and some forms of asthma . Mucoregulatory agents include anti-inflammatory agents (indomethacin, glucocorticosteroids), anticholinergic agents, and some macrolide antibiotics . Classifying mucoactive agents should help us to develop and evaluate new types of therapy and to better direct therapy toward the patients who are most likely to benefit.

Br J Nutr, 2002 May, 87 Suppl 2, S231 - 9
Non-digestible oligosaccharides and defense functions: lessons learned from animal models; Buddington RK et al.; Animals are constantly exposed to a diversity of health challenges and the gastrointestinal tract (GIT) is a major, if not the principal, site of exposure . Animal models and a limited number of human clinical studies have shown that the assemblages and metabolic activities of the resident bacteria are important determinants of the effectiveness of the various host defense mechanisms and thereby influence the ability of animals to respond to health challenges . The assemblages of bacteria resident in the GIT provide a first line of defense that can exclude invading pathogens, reduce the proliferation of opportunistic pathogens already resident in the GIT, and reduce the availability, carcinogenicity, or toxicity of noxious chemicals . The mucosa of the GIT is a second, multilayered line of defense that includes the mucous and other secretions, the epithelial cells, and immune-associated cells scattered within and under the epithelium . The final line of defense contends with pathogens or noxious chemicals that transcend the mucosal barrier and enter the host and consists of the innate and acquired components of the systemic immune system and the xenobiotic metabolizing enzymes . The lactic acid producing bacteria (LAB) are considered to be immunomodulatory and directly or indirectly influence the GIT and systemic defense functions . Corresponding with this, supplementing the diet with inulin, oligofructose, or other nondigestible oligosaccharides that increase the densities and metabolic capacities of the LAB enhances defense mechanisms of the host, increases resistance to various health challenges, and accelerates recovery of the GIT after disturbances.

Biol Bull, 2002 Jun, 202(3), 296 - 305
Self-organized fish schools: an examination of emergent properties; Parrish JK et al.; Heterogeneous, "aggregated" patterns in the spatial distributions of individuals are almost universal across living organisms, from bacteria to higher vertebrates . Whereas specific features of aggregations are often visually striking to human eyes, a heuristic analysis based on human vision is usually not sufficient to answer fundamental questions about how and why organisms aggregate . What are the individual-level behavioral traits that give rise to these features? When qualitatively similar spatial patterns arise from purely physical mechanisms, are these patterns in organisms biologically significant, or are they simply epiphenomena that are likely characteristics of any set of interacting autonomous individuals? If specific features of spatial aggregations do confer advantages or disadvantages in the fitness of group members, how has evolution operated to shape individual behavior in balancing costs and benefits at the individual and group levels? Mathematical models of social behaviors such as schooling in fishes provide a promising avenue to address some of these questions . However, the literature on schooling models has lacked a common framework to objectively and quantitatively characterize relationships between individual-level behaviors and group-level patterns . In this paper, we briefly survey similarities and differences in behavioral algorithms and aggregation statistics among existing schooling models . We present preliminary results of our efforts to develop a modeling framework that synthesizes much of this previous work, and to identify relationships between behavioral parameters and group-level statistics.

Syst Appl Microbiol, 2002 Apr, 25(1), 68 - 73
Contrasting nifD and ribosomal gene relationships among Mesorhizobium from Lotus oroboides in northern Mexico; Qian J et al.; PCR screens for length variation in a 5' portion of 23S ribosomal RNA and in the 3' end of the 16S rRNA-23S rRNA internal transcribed spacer (ITS) region indicated that nodule bacteria from a Mexican population of Lotus oroboides were diverse on a local scale . Three 23S rRNA length variants and five ITS length variants were detected among the 22 isolates . Sequencing of nearly full-length 16S rRNA genes in three isolates indicated that they fell into the genus Mesorhizobium, but comprised two distinct groups . Two isolates were closely related to M . loti LMG 6125T, while the other isolate clustered with an assemblage of Mesorhizobium taxa that included M . amorphae, M . plurifarium and M . huakuii . However, a phylogenetic tree based on 715 bp of the nitrogenase alpha-subunit (nifD) gene was significantly discordant with the relationships inferred from rRNA sequences . Two isolates that were nearly identical for 16S rRNA had nifD genes that varied at 2% of sites, and one of these nifD sequences was identical to that of another isolate with a strongly divergent 16S rRNA gene . A plasmid screen followed by Southern hybridization indicated that only one of these strains harbored a plasmid-borne nifD gene . These results imply that gene transfer events have altered the distribution of nifD sequences among lineages within this natural population of Mesorhizobium strains.

Syst Appl Microbiol, 2002 Apr, 25(1), 100 - 8
Flow cytometric detection and quantification of heterotrophic nanoflagellates in enriched seawater and cultures; Guindulain Rifa T et al.; A flow cytometric protocol to detect and enumerate heterotrophic nanoflagellates (HNF) in enriched waters is reported . At present, the cytometric protocols that allow accurate quantification of bacterioplankton cannot be used to quantify protozoa for the following reasons: i) the background produced by the bacterial acquisitions does not allow the discrimination of protozoa at low abundance, ii) since the final protozoan fluorescence is much higher than the bacterioplankton fluorescence (more than 35 fold) the protozoa acquisitions lie outside the range . With an increase in the fluorescence threshold and a reduction of the fluorescence detector voltage, low fluorescence particles (bacteria) are beneath the detection limits and only higher fluorescence particles (most of them heterotrophic nanoflagellates) are detected . The main limitation for the application of the cytometric protocol developed is that a ratio of bacteria/HNF below 1000 is needed . At higher ratios, the background of larger cells of bacterioplankton makes it difficult to discriminate protozoa . The proposed protocol has been validated by epifluorescence microscopy analyzing both a mixed community and two single species of HFN: Rhynchomonas nasuta and Jakoba libera . Taking into account the required bacteria/HNF ratio cited above, the results provide evidence that the flow cytometric protocol reported here is valid for counting mixed communities of HNF in enriched seawater and in experimental micro or mesocosms . In the case of single species of HNF previous knowledge of the biological characteristics of the protist and how they can affect the effectiveness of the flow cytometric count is necessary.

J Am Dent Assoc, 2002 Jun, 133 Suppl, 14S - 22S
Periodontal disease and cardiovascular disease: epidemiology and possible mechanisms; Genco R et al.; BACKGROUND: Many early epidemiologic studies reported an association between periodontal disease and cardiovascular disease . However, other studies found no association or nonsignificant trends . This report summarizes the evidence from epidemiologic studies and studies that focused on potential contributing mechanisms to provide a more complete picture of the association between periodontal and heart disease . TYPES OF STUDIES REVIEWED: The authors summarize the longitudinal studies reported to date, because they represent the highest level of evidence available regarding the connection between periodontal disease and heart disease . The authors also review many of the case-control and cross-sectional studies published, as well as findings from clinical, animal and basic laboratory studies . RESULTS: The evidence suggests a moderate association--but not a causal relationship--between periodontal disease and heart disease . Results of some case-control studies indicate that subgingival periodontal pathogenic infection may be associated with myocardial infarction . Basic laboratory studies point to the biological plausibility of this association, since oral bacteria have been found in carotid atheromas and some oral bacteria may be associated with platelet aggregation, an event important for thrombosis . Animal studies have shown that atheroma formation can be enhanced by exposure to periodontal pathogens . CONCLUSIONS: The accumulation of epidemiologic, in vitro, clinical and animal evidence suggests that periodontal infection may be a contributing risk factor for heart disease . However, legitimate concerns have arisen about the nature of this relationship . These are early investigations . Since even a moderate risk contributed by periodontal disease to heart disease could contribute to significant morbidity and mortality, it is imperative that further studies be conducted to evaluate this relationship . One particularly important study to be carried out is the investigation of a possible clinically meaningful reduction in heart disease resulting from the prevention or treatment of periodontal disease.

Nefrologia, 2002, 22(2), 196 - 8
{Recurrent episodes of acidosis with encephalopathy in a hemodialysis program patient with short bowel syndrome}; Angelet P et al.; We present a case of a patient with short bowel syndrome in a hemodialysis program, with recurrent episodes of serious acidosis . The presence of a D-lactic acidosis peak secondary to bacterial overgrowth in the intestine was discovered during an acute episode of acidosis, with neurological affection . The detection of acidosis in predialysis measurements and the acute episodes of acidosis, made it necessary to administer bicarbonate to the patient and give him additional hemodialysis sessions.

Eur J Biochem, 2002 Jul, 269(13), 3296 - 303
Herbaspirillum seropedicae signal transduction protein PII is structurally similar to the enteric GlnK; Machado Benelli E et al.; PII-like proteins are signal transduction proteins found in bacteria, archaea and eukaryotes . They mediate a variety of cellular responses . A second PII-like protein, called GlnK, has been found in several organisms . In the diazotroph Herbaspirillum seropedicae, PII protein is involved in sensing nitrogen levels and controlling nitrogen fixation genes . In this work, the crystal structure of the unliganded H . seropedicae PII was solved by X-ray diffraction . H . seropedicae PII has a Gly residue, Gly108 preceding Pro109 and the main-chain forms a beta turn . The glycine at position 108 allows a bend in the C-terminal main-chain, thereby modifying the surface of the cleft between monomers and potentially changing function . The structure suggests that the C-terminal region of PII proteins may be involved in specificity of function, and nonenteric diazotrophs are found to have the C-terminal consensus XGXDAX(107-112) . We are also proposing binding sites for ATP and 2-oxoglutarate based on the structural alignment of PII with PII-ATP/GlnK-ATP, 5-carboxymethyl-2-hydroxymuconate isomerase and 4-oxalocrotonate tautomerase bound to the inhibitor 2-oxo-3-pentynoate.

Am J Pharmacogenomics, 2001, 1(3), 223 - 38
Cystic fibrosis and the use of pharmacogenomics to determine surrogate endpoints for drug discovery; Eidelman O et al.; Cystic fibrosis (CF) is caused by a mutation in the CFTR gene, encoding a chloride channel . For the most common mutation, Delta F508, the basis of the deficit is the failure of the mutant CFTR channel protein to traffic properly to the apical plasma membrane of the affected epithelial cell . The trafficking failure results in loss of the cyclic adenosine monophosphate (cAMP)-activated chloride channel function of the CFTR protein in the plasma membrane . The lung is the principal site affecting patient morbidity and mortality in CF . The main reason is that the CF airway epithelial cells also secrete high levels of the proinflammatory cytokine interleukin (IL)-8, resulting in massive cellular inflammation, infection, tissue damage and lung destruction . The relationship between the trafficking defect, the loss of chloride channel activity, and inflammation is not known . However, gene therapy of CF lung epithelial cells with the wild-type CFTR gene can repair the chloride channel defect, as well as suppress the intrinsic hypersecretion of IL-8 . Repair of both defective channels and high IL-8 secretion can also be effected by treatment with the candidate CF drug CPX, which is in clinical trials in CF patients . CPX acts by binding to the mutant CFTR protein, and helps the protein to mature and gain access to the plasma membrane . CPX also suppresses the synthesis and secretion of IL-8 from CF epithelial cells, presumably by virtue of its repair of the trafficking defect of mutant CFTR . To guide pharmacogenomic experiments we have therefore hypothesized that the genomic signature of CF epithelial cells treated with CPX should resemble the signature of the same cells repaired by gene therapy . We have developed two algorithms for identifying genes modified by repair of CFTR defects . The GRASP algorithm uses a statistical test to identify the most profoundly changing genes . The GENESAVER algorithm allows us to identify those genes whose pattern of expression changes in-phase or out-of-phase with IL-8 secretion by CF cells . For the latter algorithm we modified IL-8 secretion from CF cells by treatment with wild-type CFTR, with CPX, or by exposure to bacteria . The results have supported the hypothesis, and have provided a basis for considering the common pharmacogenomic expression signature as a surrogate endpoint for CF drug discovery . Significantly, the nature of the hypothesis, as well as the algorithm developed for this study, can be easily applied to pharmacogenomic studies with other goals.

Spectrochim Acta A Mol Biomol Spectrosc, 2002 May, 58(7), 1523 - 33
Attenuated total reflection Fourier transform infrared spectroscopy of redox transitions in photosynthetic reaction centers: comparison of perfusion- and light-induced difference spectra; Iwaki M et al.; Chemically induced Fourier transform infrared difference spectra associated with redox transitions of several primary electron donors and acceptors in photosynthetic reaction centers (RCs) have been compared with the light-induced FTIR difference spectra involving the same cofactors . The RCs are deposited on an attenuated total reflection (ATR) prism and form a film that is enclosed in a flow cell . Redox transitions in the film of RCs can be repetitively induced either by perfusion of buffers poised at different redox potentials or by illumination . The perfusion-induced ATR-FTIR difference spectra for the oxidation of the primary electron donor P in the RCs of the purple bacteria Rb . sphaeroides and Rp . viridis and P700 in the photosystem 1 of Synechocystis 6803, as well as the Q(A)/Q(A) transition of the quinone acceptor (Q(A)) in Rb . sphaeroides RCs are reported for the first time . They are compared with the light-induced ATR-FTIR difference spectra P+Q(A)/PQ(A) for the RCs of Rb . sphaeroides and P700+/P700 for photosystem 1 . It is shown that the perfusion-induced and light-induced ATR-FTIR difference spectra recorded on the same RC film display identical signal to noise ratios when they are measured under comparable conditions . The ATR-FTIR difference spectra are very similar to the equivalent FTIR difference spectra previously recorded upon photochemical or electrochemical excitation of these RCs in the more conventional transmission mode . The ATR-FTIR technique requires a smaller amount of sample compared with transmission FTIR and allows precise control of the aqueous environment of the RC films.

Curr Top Microbiol Immunol, 2002, 268, 1 - 22
Molecular evolution of proteasomes; Volker C et al.; Proteasomes are large, multisubunit proteases that are found, in one form or another, in all domains of life and play a critical role in intracellular protein degradation . Although they have substantial structural similarity, the proteasomes of bacteria, archaea, and eukaryotes show many differences in architecture and subunit composition . This article discusses possible paths by which proteasomes may have evolved from simple precursors to the highly complicated and diverse complexes observed today.

Microb Ecol, 2002 Aug, 44(2), 154 - 63 Epub 2002 Jun 26.
Distribution of capsulated bacterioplankton in the North Atlantic and North Sea; Stoderegger KE et al.; In laboratory experiments, bacterioplankton were incubated under different nutrient conditions, and the percentage of bacteria exhibiting a polysaccharidic capsule (capsulated bacteria) and that of CTC (cyanotetrazolium chloride)-positive and therefore metabolically highly active bacteria were determined . In these seawater cultures amended with nutrients more than 95% of the CTC-positive cells exhibited a capsule . During two cruises, one to the North Atlantic and one to the North Sea, we investigated the distribution of capsulated bacteria throughout the water column . Capsulated bacteria were generally more abundant in eutrophic surface waters than in deeper layers or more oligotrophic regions . In the upper 100 m of the North Atlantic, about 6-14% of the total bacterioplankton community was capsulated, while in the layers below 100 m depth, 97% of the bacteria lacked a visible capsule . The percentage of capsulated bacteria correlated with bacterial abundance and production, and chlorophyll a concentration . Also, the bioavailability of DOC (dissolved organic carbon), estimated by the ratio between bacterial production and DOC concentration, significantly correlated with the percentage of capsulated bacteria . In the North Sea, the contribution of capsulated bacteria to the total number of bacteria decreased from the surface (3 m depth) to the near-bottom (25-35 m) layers from 20% to 14% capsulated bacteria . In the nearshore area of the North Sea, about 27% of the bacteria exhibited a capsule . Overall, a pronounced decrease in the contribution of capsulated bacteria to the total bacterial abundance was detectable from the eutrophic coastal environment to the open North Atlantic . Using this epifluorescence-based technique to enumerate capsulated bacterioplankton thus allowed us to routinely assess the number of capsulated bacteria even in the oceanic water column . Based on the data obtained in this study we conclude that almost all metabolically highly active bacteria exhibit a capsule, but also some of the metabolically less active cells express a polysaccharide capsule detectable with this method.

Biorheology, 2002, 39(1-2), 183 - 91
The influence of exercise on the composition of developing equine joints; van de Lest CH et al.; An overview is given of the direct and long-term effects of exercise on the biochemical characteristics of cartilage and subchondral bone, and on the metabolic activity of chondrocytes in the juvenile horse . In the experimental setup 43 foals were reared until weaning at 5 months of age under similar conditions, except for the type and amount of exercise . Fifteen foals remained at pasture (Pasture group and also control group), 14 foals were kept in box stalls (Box group), and 14 foals were kept in the same box stalls but were subjected daily to an increasing number of gallop sprints (Training group) . After weaning 8 foals from each group were euthanised . All remaining 19 animals were housed together in a loose box with access to a small paddock to study a possible reversibility of exercise-induced effects . Post mortem subchondral bone and cartilage samples were collected and analysed for bone morphogenic enzymes, matrix composition, chondrocyte metabolic activity, and bone mineral density.It resulted that lack of exercise leads to a retardation of the normal development of the joint . This is largely compensated for when afterwards a more normal exercise regimen is followed . Most parameters in the Training group approximated those of the pastured foals at age 5 months . However, at age 11 months there were indications for a reduced performance of the investigated tissues in this group.It is concluded that regular, sub-maximal loading, as occurred in the Pasture group, seems best for an optimal development of the musculoskeletal tissues . The combination of short bouts of heavy exercise superimposed on a basic box rest regimen appears to have adverse effects on long-term viability of the tissues and may hence lead to an impaired resistance to injury.

J Cell Sci, 2002 Jul 15, 115(Pt 14), 3015 - 25
Ihh enhances differentiation of CFK-2 chondrocytic cells and antagonizes PTHrP-mediated activation of PKA; Deckelbaum RA et al.; Indian Hedgehog (Ihh), a member of the hedgehog (HH) family of secreted morphogens, and parathyroid hormone-related peptide (PTHrP) are key regulators of cartilage cell (chondrocyte) differentiation . We have investigated, in vitro, the actions of HH signalling and its possible interplay with PTHrP using rat CFK-2 chondrocytic cells . Markers of chondrocyte differentiation {alkaline phosphatase (ALP) activity, and type II (Col2a1) and type X collagen (Col10a1) expression} were enhanced by overexpression of Ihh or its N-terminal domain (N-Ihh), effects mimicked by exogenous administration of recombinant N-terminal HH peptide . Moreover, a missense mutation mapping to the N-terminal domain of Ihh (W160G) reduces the capacity of N-Ihh to induce differentiation . Prolonged exposure of CFK-2 cells to exogenous N-Shh (5x10(-9) M) in the presence of PTHrP (10(-8) M) or forskolin (10(-7) M) resulted in perturbation of HH-mediated differentiation . In addition, overexpression of a constitutively active form of the PTHrP receptor (PTHR1 H223R) inhibited Ihh-mediated differentiation, implicating activation of protein kinase A (PKA) by PTHR1 as a probable mediator of the antagonistic effects of PTHrP . Conversely, overexpression of Ihh/N-Ihh or exogenous treatment with N-Shh led to dampening of PTHrP-mediated activation of PKA . Taken together, our data suggest that Ihh harbors the capacity to induce rather than inhibit chondrogenic differentiation, that PTHrP antagonizes HH-mediated differentiation through a PKA-dependent mechanism and that HH signalling, in turn, modulates PTHrP action through functional inhibition of signalling by PTHR1 to PKA.

J Bacteriol, 2002 Jul, 184(14), 3941 - 6
An AraC/XylS family member at a high level in a hierarchy of regulators for phenol-metabolizing enzymes in Comamonas testosteroni R5; Teramoto M et al.; Comamonas testosteroni strain R5 expresses a higher level of phenol-oxygenating activity than any other bacterial strain so far characterized . The expression of the operon encoding multicomponent phenol hydroxylase (mPH), which is responsible for the phenol-oxygenating activity, is controlled by two transcriptional regulators, PhcS and PhcR, in strain R5 . In this study, we identified a third transcriptional regulator for the mPH operon (PhcT) that belongs to the AraC/XylS family . While the disruption of phcT in strain R5 significantly reduced the expression of the mPH operon, it did not eliminate the expression . However, the disruption of phcT in strain R5 increased the expression of phcR . The phenol-oxygenating activity was abolished by the disruption of phcR, indicating that PhcT alone was not sufficient to activate the expression of the mPH operon . The disruption of phcS has been shown in our previous study to confer the ability of strain R5 to express the mPH operon in the absence of the genuine substrate for mPH . PhcT was not involved in the gratuitous expression . Strain R5 thus possesses a more elaborate mechanism for regulating the mPH operon expression than has been found in other bacteria.

J Bacteriol, 2002 Jul, 184(14), 3848 - 55
Mutations in the CCGTTCACA DnaA box of Mycobacterium tuberculosis oriC that abolish replication of oriC plasmids are tolerated on the chromosome; Dziadek J et al.; The origin of replication (oriC) region in some clinical strains of Mycobacterium tuberculosis is a hot spot for IS6110 elements . To understand how clinical strains with insertions in oriC can replicate their DNA, we characterized the oriC regions of some clinical strains . Using a plasmid-based oriC-dependent replication assay, we showed that IS6110 insertions that disrupted the DnaA box sequence CCGTTCACA abolished oriC activity in M . tuberculosis . Furthermore, by using a surface plasmon resonance technique we showed that purified M . tuberculosis DnaA protein binds native but not mutant DnaA box sequence, suggesting that stable interactions of the DnaA protein with the CCGTTCACA DnaA box are crucial for replication of oriC plasmids in vivo . Replacement by homologous recombination of the CCGTTCACA DnaA box sequence of the laboratory strain M . tuberculosis H37Ra with a mutant sequence did not result in nonviability . Together, these results suggest that M . tuberculosis strains have evolved mechanisms to tolerate mutations in the oriC region and that functional requirements for M . tuberculosis oriC replication are different for chromosomes and plasmids.

Kidney Int, 2002 Jul, 62(1), 106 - 15
Cloning and expression of rat caspase-6 and its localization in renal ischemia/reperfusion injury; Singh AB et al.; BACKGROUND: Caspase-6 is an important member of the executioner caspases in the caspase family of cell death proteases . The executioner caspases are the major active caspases detected in apoptotic cells and are generally considered to mediate the execution of apoptosis by cleaving and inactivating intracellular proteins . However, the complete characterization of mRNA and protein of caspase-6 in rat and its expression in normal kidney and in disease state has not been previously elucidated . METHODS: A rat kidney cortex lambdagt10 cDNA library was screened to isolate the full-length caspase-6 cDNA . The recombinant caspase-6 protein was characterized by expression in bacteria and by transient transfection in mammalian cells . The expression in various tissues was analyzed by Northern blot, and localization in normal and ischemic kidney was performed by immunohistochemistry . RESULTS: The predicted amino acid sequence of rat caspase-6 contains 277 amino acids, with two potential glycosylation sites, an integrin binding site (KGD), the caspase active site pentapeptide QACRG and the caspase family signature, HX2-4(S,C) X4(L,I,V,M,F)2(S,T)HG (HVDADCFVCVFLSHG) . Rat caspase-6 is unique among known caspases by possessing a relatively long 5' untranslational region . Among various tissues tested, cas-pase-6 was expressed in varying levels in kidney, liver, spleen, heart, muscle, testis, and lung . Bacterial expression of recombinant rat caspase-6 resulted in production of both of the pro-form and active form of the enzyme suggesting autoactivation . Transient overexpression of rat caspase-6 in COS-1 cells induced DNA fragmentation, a hallmark of apoptosis . We also examined the localization and expression of caspase-6 by immunohistochemistry in kidneys subjected to 40 minutes of ischemia followed by 24 hours of reperfusion injury . Normal kidney showed mostly cytoplasmic and some nuclear staining of the tubules . Kidneys 24 hours after 40 minutes of ischemia showed more intense and diffused cytoplasmic staining with prominent nuclear staining, indicating increased expression and translocation from the cytoplasm to the nuclei . The staining in glomeruli was negative in both normal and ischemic kidney . CONCLUSIONS: These studies demonstrate cloning, expression and characterization of the full-length rat caspase-6 and its localization in normal kidneys and kidneys subjected to ischemia/reperfusion injury . Since caspase-6 is involved in the degradation of nuclear matrix proteins and in activation of caspase-3, it may play an important role during renal ischemic injury.

Biochemistry, 2002 Jul 2, 41(26), 8342 - 50
Mutational analysis of endonuclease V from Thermotoga maritima; Huang J et al.; Endonuclease V nicks damaged DNA at the second phosphodiester bond 3' to inosine, uracil, mismatched bases, or abasic (AP) sites . Alanine scanning mutagenesis was performed in nine conserved positions of Thermotoga maritima endonuclease V to identify amino acid residues involved in recognition or endonucleolytic cleavage of these diverse substrates . Alanine substitution at D43, E89, and D110 either abolishes or substantially reduces inosine cleavage activity . These three mutants gain binding affinity for binding to double-stranded or single-stranded inosine substrates in the absence of a metal ion, suggesting that these residues may be involved in coordinating catalytic metal ion(s) . Y80A, H116A, and, to a lesser extent, R88A demonstrate reduced affinities for double-stranded or single-stranded inosine substrates or nicked products . The lack of tight binding to a nicked inosine product accounts for the increased rate of turnover of inosine substrate since the product release is less rate-limiting . Y80A, R88A, and H116A fail to cleave AP site substrates . Their activities toward uracil substrates are in the following order: H116A > R88A > Y80A . These residues may play a role in substrate recognition . K139A maintains wild-type binding affinity for binding to double-stranded and single-stranded inosine substrate, but fails to cleave AP site and uracil substrate efficiently, suggesting that K139 may play a role in facilitating non-inosine substrate cleavage.

Biochemistry, 2002 Jul 2, 41(26), 8238 - 44
Conversion of a plant oxidosqualene-cycloartenol synthase to an oxidosqualene-lanosterol cyclase by random mutagenesis; Wu TK et al.; A random mutagenesis/in vivo selection approach was applied to generate and identify mutations that alter the product specificity of oxidosqualene-cycloartenol synthase (CAS) from Arabidopsis thaliana . This work complements previous studies of triterpene cyclase enzymes and was undertaken to provide knowledge of the frequency and locations at which point mutations can alter cyclase product specificity . Random mutations were introduced by treatment with hydroxylamine or passage through a mutator strain of bacteria . Libraries of mutated plasmids carrying the cas1 gene were transformed into a cyclase-deficient strain of Saccharomyces cerevisiae (CBY57) bearing a complementing plasmid (pZS11) carrying an Erg7 gene that encodes wild-type yeast oxidosqualene-lanosterol cyclase and a URA3 marker that could be counterselected by growth in media containing 5-fluoroorotic acid (5-FOA) . This allowed use of a plasmid shuffle to select for cas1 mutants that could substitute for ERG7 activity . Five of approximately 73,000 transformants were observed to grow in media containing 5-FOA but lacking ergosterol . pTKP5-derived plasmids isolated from these transformants were sequenced, revealing five distinct and unique point mutations: Tyr410Cys, Ala469Val, His477Tyr, Ile481Thr, and Tyr532His . Analysis of the nonsaponifiable lipids from CBY57 cells expressing these mutants suggests that the Tyr410Cys and His477Tyr mutants produce lanosterol as the dominant product, whereas the Ala469Val, Ile481Thr, and Tyr532His mutants produce a mixture of lanosterol and achilleol A, a product of monocyclization . Sequence and structural homology modeling of CAS indicate that the observed product specificity-altering mutations occur both within (Tyr410Cys, Ile481Thr, and Tyr532His) and outside of (Ala469Val and His477Tyr) the cyclase active site.

Photochem Photobiol, 2002 Jun, 75(6), 619 - 26
Self-assembly of {Et,Et}-bacteriochlorophyll cF on highly oriented pyrolytic graphite revealed by scanning tunneling microscopy; Moltgen H et al.; The chlorosomal light-harvesting antennae of green phototrophic bacteria consist of large supramolecular aggregates of bacteriochlorophyll c (BChl c) . The supramolecular structure of (3(1)-R/S)-BChl c on highly oriented pyrolytic graphite (HOPG) and molybdenum disulfide (MoS2) has been investigated by scanning tunneling microscopy (STM) . On MoS2, we observed single BChl c molecules, dimers or tetramers, depending on the polarity of the solvent . On HOPG, we observed extensive self-assembly of the dimers and tetramers . We propose C=O...H-O...Mg bonding networks for the observed dimer chains, in agreement with former ultraviolet-visible and infrared spectroscopic work . The BChl c moieties in the tetramers are probably linked by four C=O...H-O hydrogen bonds to form a circle and further stabilized by Mg...O-H bondings to underlying BChl c layers . The tetramers form highly ordered, distinct chains and extended two-dimensional networks . We investigated semisynthetic chlorins for comparison by STM but observed that only BChl c self-assembles to well-structured large aggregates on HOPG . The results on the synthetic chlorins support our structure proposition.

Viral Immunol, 2002, 15(2), 365 - 72
Innate immunity in viral encephalitis: role of C5; Chen N et al.; The complement system is a critical component of both the innate and acquired immune systems . It is important in host defense against viruses, bacteria, and fungi for opsonization and for lysis of pathogens . However, activated complement can also cause tissue damage . There is compelling evidence that complement factors are presented in the central nervous system (CNS) . Complement activation (by any of the three pathways: classical, alternate, and lectin) can lead to inflammation and tissue damage, while at the same time may also restrict certain pathogens in the CNS . C5a is formed by proteolytic cleavage C5 . C5a is considered the most potent proinflammatory mediator, often called an anaphylotoxin . In this communication, we examine the roles of C5 (C5a) in vesicular stomatitis virus (VSV)-induced encephalitis . We found that C5a is produced during VSV infection, but C5-deficient mice had similar pathology as their controls . We concluded that C5 is not a critical factor in mediating the host response during VSV encephalitis.

J Mol Biol, 2002 Jun 21, 319(5), 1223 - 34
Crystal structures of two homologous pathogenesis-related proteins from yellow lupine; Biesiadka J et al.; Pathogenesis-related class 10 (PR10) proteins are restricted to the plant kingdom where they are coded by multigene families and occur at high levels . In spite of their abundance, their physiological role is obscure although members of a distantly related subclass (cytokinin-specific binding proteins) are known to bind plant hormones . PR10 proteins are of special significance in legume plants where their expression patterns are related to infection by the symbiotic, nitrogen-fixing bacteria . Here we present the first crystal structures of classic PR10 proteins representing two homologues from one subclass in yellow lupine . The general fold is similar and, as in a birch pollen allergen, consists of a seven-stranded beta-sheet wrapped around a long C-terminal helix . The mouth of a large pocket formed between the beta-sheet and the helix seems a likely site for ligand binding . The shape of the pocket varies because, in variance with the rigid beta-sheet, the helix shows unusual conformational variability consisting in bending, disorder, and axial shifting . A surface loop, proximal to the entrance to the internal cavity, shows an unusual structural conservation and rigidity in contrast to the high glycine content in its sequence . The loop is different from the so-called glycine-rich P-loops that bind phosphate groups of nucleotides, but it is very likely that it does play a role in ligand binding in PR10 proteins . (c) 2002 Elsevier Science Ltd.

Chemosphere, 2002 May, 47(7), 717 - 23
Bioassay-directed chemical analysis of River Elbe surface water including large volume extractions and high performance fractionation; Reineke N et al.; A bioassay-directed fractionation and identification (toxicity identification evaluation procedure) was performed on extracts of 10 1 River Elbe water samples . The experimental method included a SDB-1 solid phase extraction followed by RP-HPLC fractionation and subfractionation . Chemical analysis by GC-MS as well as acute toxicity testing using a luminescent bacteria assay were conducted in the respective fractions . Many substances were identified, among which were pesticides and pharmaceuticals, but many compounds remained unknown.

Chemosphere, 2002 May, 47(7), 695 - 9
A microcosm test for potential mineralization of chlorinated compounds under coupled aerobic/anaerobic conditions; Lyew D et al.; In this study, the feasibility of using a mineralization test under coupled aerobic/anaerobic conditions was demonstrated . The coupling of anaerobic methanogenic and aerobic methanotrophic conditions in a microcosm required the presence of both a carbon source for anaerobic metabolism and oxygen for aerobic metabolism . These requirements were fulfilled by using a slow hydrolyzing organic matter along with intermittent addition of oxygen to the bottle headspace . Perchloroethylene (PCE) mineralization tests confirmed the effectiveness of the proposed methodology as well as PCE mineralization under coupled conditions.

J Anim Sci, 2002 Jun, 80(6), 1405 - 12
Efficacy of intramuscular treatment of beef cows with oxytetracycline to reduce mastitis and to increase calf growth; Lents CA et al.; Spring-calving multiparous Angus x Hereford cows were used to determine the efficacy of intramuscular treatment with oxytetracycline to reduce the incidence of mastitis-causing bacteria, decrease milk somatic cell counts (SCC), and increase calf growth . During 2 yr, milk samples were collected from each quarter from a total of 319 cows at 8 to 14 d after calving and at weaning, to determine the presence of bacteria and SCC . A California mastitis test (CMT) was performed on milk from each quarter of each cow at the initial sample collection . Cows with a CMT score of 1, 2, or 3 in at least one quarter, were randomly assigned to receive either an intramuscular injection of oxytetracycline (n = 63) or the control vehicle (n = 60), and cows with a CMT score of 0 or trace in all four quarters were not treated (n = 196) . Calf weights were determined at birth, early lactation, and weaning . The number of somatic cells in milk and the percentage of quarters that were infected increased as CMT score increased (P < 0.01) . The presence of mastitis-causing bacteria at calving increased (P < 0.05) the incidence of infection at weaning . The presence of mastitis-causing bacteria at weaning was associated with increased SCC for quarters and average SCC for cows (P < 0.01) . Average SCC per cow at weaning increased (P < 0.05) as the number of infected quarters per cow increased . Treatment did not alter (P > 0.10) the percentage of cows or quarters infected with mastitis-causing bacteria or SCC of cows or quarters at weaning . Average SCC per cow was negatively correlated (P < 0.05) with calf weights at early lactation, but not with weaning weights of calves . Treatment did not influence (P > 0.10) calf weights at early lactation or at weaning . Cows with one or more dry quarters after calving had calves that weighed less at early lactation and weaning than cows with four functional quarters (P < 0.01) . Intramuscular oxytetracycline treatment of beef cows that had CMT scores of 1 or greater after calving did not reduce intramammary infection rates or increase calf weights at weaning.

Implant Dent, 2002, 11(2), 170 - 5
Growth of bioactive surfaces on dental implants; Manero JM et al.; Some metallic materials, such as pure titanium, Ti-6Al-4V, are used for dental and orthopedic implants under load-bearing conditions . However, they do not form a chemical bond with bone, which would achieve a good implant-bone fixation in service . In recent works, it has been demonstrated that an in vitro, chemically deposited, bone-like apatite layer with bone-bonding ability could be induced on a titanium surface . By reproducing that chemical procedure, in this work, a dense bone-like apatite layer was formed on the surface of the titanium in simulated body fluid . In addition, the different steps and kinetics of the layer-formation have been studied, because the observation of the samples in the wet state by means of the environmental scanning electron microscope has allowed the observation in situ of the apatite deposition process over a number of days . One of the most important features of the present study is that it can be carried out on a single titanium sample and the process is not interrupted at any stage . One of the main drawbacks of this chemical method is that the samples covered with apatite are susceptible to contamination by bacteria . The behavior of different types of antibiotics used to avoid this contamination has also been studied using the environmental scanning electron microscope . Finally, osteoblast cells have been cultured on the apatite-coated titanium samples to assess their biocompatibility.

Nippon Rinsho, 2002 Jun, 60(6), 1202 - 7
{Characteristic of Norwalk gastroenteritis and its prevention}; Honma S; The application of recently developed molecular technique revealed the important role of the Norwalk virus(NV) . The major role of the NV as agents of food and water-related outbreaks of gastroenteritis has now been recognized in the world . Moreover, a part of NV is also documented as important agents of gastroenteritis in childhood and infancy . A high rate of food-related viral gastroenteritis outbreaks was observed following ingestion of shellfish in Japan . Specific methods are not available for the prevention or control of NV gastroenteritis . Hand washing will be the most effective to decrease transmission within a family and a hospital . To control food-borne diseases a different strategy is necessary with virus and bacteria.

Rinsho Byori, 2002 May, 50(5), 449 - 54
{Reviews for Gram stain}; Yamanaka K; The Gram stain is a rapid examination method providing useful information for diagnosis and treatment of infection . However, a molecular biology inspection and rapid immunological method have been developed, and gram stains have been disregarded recently . Therefore, I explain the usefulness of gram staining for rapid diagnosis, rapid treatment, and the economy . Moreover, I will explain gram stain method "Bartholomew & Mittwer(B&M)" which replaces the Hucker method.

Prog Cardiovasc Dis, 2002 May-Jun, 44(6), 469 - 78
Systemic and local inflammation in patients with unstable atherosclerotic plaques; Willerson JT; The response to injury in the vasculature and the heart is inflammation . Atherosclerosis is often the result of injury followed by inflammation and atherosclerosis . Vascular and myocardial infections from various pathogens, including viruses, bacteria, chlamydia, and other infections result in vascular inflammation and almost certainly play a role in the development of atherosclerosis and acute coronary heart disease syndromes in at least some patients . Current evidence favors prior exposure to multiple pathogens as most likely playing a role in initiating inflammation and contributing to atherosclerosis . Genetic predisposition is almost certainly an important factor in the development of inflammation, impaired endothelial vascular repair, vascular infection, thrombosis, and atherosclerosis . The aging process itself is most likely associated with altered vascular and myocardial defense mechanisms predisposing to inflammation . The oxidation of cholesterol and low-density lipoprotein (LDL) leads to the production of oxidized radicals that promote vascular inflammation . Interventional injury, including angioplasty and stenting, causes endothelial inflammation, thrombosis, and fibroproliferation . Systemic evidence of inflammation identifies patients at high risk of future coronary events, including those who appear to be healthy initially as well as those with stable and unstable coronary heart disease syndromes . Increases in serum C-reactive protein (CRP) identify individuals at risk for future vascular events, including unstable angina, acute myocardial infarction, acute cerebrovascular accident, and sudden death . Similarly, systemic elevations in serum troponin 1, serum amyloid-like protein, fibrinogen, and interleukins-1, 2, 6, 8, and 18 identify patients with unstable angina and non-Q-wave myocardial infarction at increased risk for future coronary events . The presence of vascular inflammation may be detected by identifying temperature heterogeneity within plaques that demonstrate inflammation . In the future, the local evaluation of atherosclerotic plaques to detect the presence of inflammation coupled to measurements of systemic markers of inflammation, such as C-reactive protein, may help identify patients at increased risk and allow both local and systemic therapies that reduce their risk and prevent the development of acute coronary syndromes in at least some patients .

Acta Crystallogr D Biol Crystallogr, 2002 Jul, 58(Pt 7), 1198 - 200 Epub 2002 Jun 20.
Crystallization of a stringent response factor from Aquifex aeolicus; Kristensen O et al.; The crystallization of a key enzyme from Aquifex aeolicus with suggested bifunctional activity, acting as an exopolyphosphatase and a guanosine pentaphosphate phosphohydrolase, is reported . Native data were collected to below 2 A resolution from an orthorhombic crystal with unit-cell parameters a = 50.8, b = 70.3, c = 90.9 A . Methionine residues were introduced by mutation and deliberate oxidation of the protein allowed us to produce additional crystal forms with reproducible diffraction ability and increased phasing potential . This is the first report on the crystallization of a member of the Ppx/GppA phosphatase family.

FEMS Microbiol Lett, 2002 Jun 18, 212(1), 127 - 32
Regulation of the Legionella mip-promotor during infection of human monocytes; Wieland H et al.; The opportunistic pathogen Legionella pneumophila, the etiologic agent of Legionnaires disease, is able to invade and multiply intracellularly in human macrophages . This process is controlled by several bacterial virulence factors . As recently demonstrated, one of these virulence factors, the macrophage infectivity potentiator (Mip) protein, is important for invasion and proper intracellular establishment of L . pneumophila in macrophages and protozoa . Knockout mutants devoid of a functional mip-gene enter host cells much less effectively but intracellular replication is not affected . Using a P(mip)-green fluorescent protein reporter construct in L . pneumophila substrain Corby, P(mip) was recently shown to be constitutively active in replicating bacteria . A stringent regulation during the infection process could not be observed, neither in intracellular nor in BYE broth-grown bacteria . For enhanced temporal and quantitative resolution, we examined the activity of mip on RNA level in order to detect short transient regulatory events . Our results show that P(mip) of L . pneumophila is temporarily repressed directly after invasion of the monocytic human cell line MonoMac 6 and regains activity after 24 h of intracellular replication.

FEMS Microbiol Lett, 2002 Jun 18, 212(1), 29 - 34
Identification of Weissella species by the genus-specific amplified ribosomal DNA restriction analysis; Jang J et al.; The Weissella species have been increasingly isolated from many food and meat samples . For rapid and systematic identification of Weissella species, an amplified ribosomal DNA restriction analysis (ARDRA) has been developed . This method employs both polymerase chain reaction (PCR) and restriction pattern analysis: first, PCR using the genus-specific primers that were designed from 16S rDNA sequence produces a 725-bp band only from Weissella species and next, restriction analysis using MnlI, MseI, and BceAI restriction enzymes identifies these Weissella-specific PCR fragments to the species level . This ARDRA method clearly identified six Weissella strains isolated from kimchi as well as 10 validly described Weissella type strains . Therefore, these results suggest that this ARDRA is a rapid and reliable method for identification of Weissella species.

Cochrane Database Syst Rev . 2002;(2):CD001480.
Pneumococcal vaccines for preventing otitis media; Straetemans M et al.; BACKGROUND: Acute otitis media (AOM) is one of the most common diseases in early infancy and childhood . Long term effects of recurrent episodes of otitis media, rapid emergence of drug resistant bacteria associated with AOM worldwide and huge estimated direct and indirect annual costs associated with otitis media have emphasized the need for an effective vaccination program to prevent episodes of AOM . OBJECTIVES: The object of this review was to assess the effect of pneumococcal vaccination in preventing AOM in children up to 12 years of age . SEARCH STRATEGY: We searched the Cochrane Acute Respiratory Infection Group specialised register (last update, 26th April 2001), the Cochrane Library (Issue 4, 2000), MEDLINE (January 1966-August 2000) and reference list of all studies and review articles retrieved . We also contacted two vaccine manufacturers and first or corresponding authors of some included studies . SELECTION CRITERIA: Randomised controlled clinical trials of pneumococcal vaccination with prevention of AOM as outcome in children aged 12 years or younger and a follow-up of at least six months . DATA COLLECTION AND ANALYSIS: Five reviewers independently assessed trial quality and two reviewers extracted data . Two study authors were contacted . MAIN RESULTS: Eight trials on pneumococcal polysaccharide vaccine (PPV) and two trials on pneumococcal conjugate vaccine (PCV) were included . The highest efficacy of PPV was found in children aged 24 months and older: the rate ratio after adjustment for study was 0.833 {95%CI: 0.625-0.970} . The PPV has little effect on the prevention of AOM in children without documented prior episodes of AOM and only a moderate effect in the group of children with documented AOM episodes prior to vaccination . The results of the two PCV trials in healthy infants, which followed children from the age of two months until two years of age, could not be pooled because of lack of data . Both studies showed that the risk of recurrent disease decreased with 9% in the group of children receiving the PCV together with other childhood vaccinations at 2,4,6 and 14 months of age: Study Black et al 2000 : risk ratio=0.91{95%CI:0.86-0.96}; Study Eskola et al 2001: risk ratio=0.90 {95%CI:0.73-1.12} . REVIEWER'S CONCLUSIONS: Based on the currently available results of the effectiveness of pneumococcal vaccination for the prevention of AOM, a large scale use of pneumococcal vaccination for this indication is not recommended . The results of currently ongoing trials could provide more information whether pneumococcal vaccines are effective in specific high-risk (otitis-prone) populations.

Environ Sci Technol, 2002 Jun 1, 36(11), 2436 - 42
Anaerobic methane oxidation in a landfill-leachate plume; Grossman EL et al.; The alluvial aquifer adjacent to Norman Landfill, OK, provides an excellent natural laboratory for the study of anaerobic processes impacting landfill-leachate contaminated aquifers . We collected groundwaters from a transect of seven multilevel wells ranging in depth from 1.3 to 11 m that were oriented parallel to the flow path . The center of the leachate plume was characterized by (1) high alkalinity and elevated concentrations of total dissolved organic carbon, reduced iron, and methane, and (2) negligible oxygen, nitrate, and sulfate concentrations . Methane concentrations and stable carbon isotope (delta13C) values suggest anaerobic methane oxidation was occurring within the plume and at its margins . Methane delta13C values increased from about -54 per thousand near the source to > -10 per thousand downgradient and at the plume margins . The isotopic fractionation associated with this methane oxidation was -13.6+/-1.0 per thousand . Methane 13C enrichment indicated that 80-90% of the original landfill methane was oxidized over the 210-m transect . First-order rate constants ranged from 0.06 to 0.23 per year, and oxidation rates ranged from 18 to 230 microM/y . Overall, hydrochemical data suggest that a sulfate reducer-methanogen consortium may mediate this methane oxidation . These results demonstrate that natural attenuation through anaerobic methane oxidation can be an important sink for landfill methane in aquifer systems.

Anaesth Intensive Care, 2002 Jun, 30(3), 338 - 40
Washing of gloved hands in antiseptic solution prior to central venous line insertion reduces contamination; Kocent H et al.; Glove contamination at the time a central venous catheter is handled is highly undesirable and likely to increase the risk of subsequent line infection . This study was designed to determine how frequently gloves become contaminated during central venous line insertion and to demonstrate the value of glove decontamination immediately prior to handling of the central venous catheter During twenty routine internal jugular catheter insertions the sterility of the operator's gloved fingertips (just prior to handling the intravenous catheter) was assessed by touching the fingertips onto blood agar plates . The gloved hands were then rinsed in chlorhexidine/alcohol and after drying were placed onto a further plate . Contamination was detected in 55% of the prewash plates but in none of the postwash plates . Procedures performed by less experienced resident staff had a higher contamination rate despite there being no evident breach of sterile technique . It is likely that glove contamination results from the persistance of bacteria within the deeper layers of the skin, despite surface disinfection . These bacteria may be released by manipulation of the skin when identifying landmarks . This hypothesis was supported by a subsequent observation that gloves were more highly contaminated after firm touching of the skin rather than light touching . Glove contamination during central line insertion is frequent . Catheter contamination rates could be reduced (without risk or additional cost) by rinsing gloved hands in a solution of chlorhexidine (0.5%) in alcohol (70%) prior to handling the catheter.

Antisense Nucleic Acid Drug Dev, 2002 Apr, 12(2), 71 - 8
Antisense-mediated inhibition of ICAM-1 expression: a therapeutic strategy against inflammation of human periodontal tissue; Nedbal W et al.; Periodontal diseases, such as gingivitis and periodontitis, are caused by a mixed infection by several types of bacteria in the dental plaque, causing a chronic inflammation of the gingival mucosa . Inflammatory processes in conjunction with immune responses to bacterial attacks are generally protective . In profound periodontitis, however, hyperresponsiveness and hypersensitivity of the immune system are counterproductive because of the destruction of the affected periodontal connective tissues . The intercellular adhesion molecule type 1 (ICAM-1) plays a key role in the onset and manifestation of inflammatory responses . Thus, inhibition of ICAM-1 expression could be of therapeutic relevance for the treatment of destructive periodontitis . Here, antisense oligonucleotides (AS-ON) directed against ICAM-1 suppress protein expression and mRNA levels specifically and effectively in primary human endothelial cells of different tissue origin . Moreover, downregulation of ICAM-1 expression is also observed in AS-ON-transfected inflamed gingival mucosal tissue of patients with periodontal diseases . This work strongly suggests exploiting the local topical application of ICAM-1-directed AS-ON as a therapeutic tool against inflammatory processes of the human gingiva.

Lik Sprava, 2002, (2), 81 - 3
{Clinical efficacy and roentgen results of laser therapy in combined treatment of patients with newly diagnosed pulmonary tuberculosis}; Savyts'ka AV; Efficiency was studied of use of laseropuncture in a complex treatment of 80 patients with pulmonary tuberculosis first diagnosed . The results of studies made showed that use of laseropuncture in the treatment complex promote significantly the dissipation of the intoxicatory and bronchopulmonary syndromes, the bacteria no longer recovered, the time of closure of the cavities of decomposition getting shortened, the indices for the function of external breathing improved.

Appl Microbiol Biotechnol, 2002 Jun, 59(1), 79 - 85 Epub 2002 Apr 12.
Degradation of phenanthrene, methylphenanthrenes and dibenzothiophene by a Sphingomonas strain 2mpII; Nadalig T et al.; Strain Sphingomonassp . (2MPII), isolated from marine sediment, was able to utilize phenanthrene (P) or 2-methylphenanthrene (2MP) as the sole carbon source . However, 9-methylphenanthrene (9MP) and dibenzothiophene (DBT) were weakly degraded . The degradation rates of 9MP and DBT increased in the presence of 2MP, whilst the degradation rate of 2MP increased in the presence of 9MP . However, the presence of DBT inhibited the degradation of 2MP . DBT sulfone, a DBT metabolite, was not assimilated by the bacteria and its presence also decreased the degradation rate of 2MP.

Mycorrhiza, 2002 Jun, 12(3), 131 - 7 Epub 2002 Mar 27.
Use of different nitrogen sources by the edible ectomycorrhizal mushroom Cantharellus cibarius; Rangel-Castro JI et al.; The growth of three strains of Cantharellus cibarius on liquid media containing ammonium, nitrate and bovine serum albumin (BSA) in different combinations was determined . The most readily utilisable source of N was ammonium . BSA utilisation was limited compared with media containing ammonium . Growth on nitrate was also poor, suggesting a limited capacity of C . cibarius to metabolise this nitrogen source . There was some indication of considerable intraspecific variation within C . cibarius in the utilisation of nitrogen sources . Possible links between atmospheric nitrogen deposition and the observed decrease of sporocarp formation by C . cibarius in Europe are discussed . We highlight the potential ecological significance of bacteria associated with C . cibarius which may circumvent the need for fungal extracellular enzymes to access complex nitrogen sources.

Extremophiles, 2002 Jun, 6(3), 253 - 61 Epub 2002 Mar 13.
Planococcus antarcticus and Planococcus psychrophilus spp . nov . isolated from cyanobacterial mat samples collected from ponds in Antarctica; Reddy GS et al.; Thirteen orange-pigmented bacteria associated with cyanobacterial mat samples collected from four different lakes in McMurdo, Antarctica, were isolated . Twelve of the isolates, which were coccoid in shape, were very similar and possessed all the characteristics of the genus Planococcus and represented a new species, which was assigned the name Planococcus antarcticus sp . nov . (CMS 26or(T)) . Apart from the phenotypic differences, P . antarcticus differed from all reported species of Planococcus by more than 2.5% at the 16S rRNA gene sequence level . In addition, at the DNA-DNA hybridization level, it exhibited very little similarity either with P . mcmeekinii (30%-35%), P . okeanokoites (26%-29%), or CMS 53or(T) (15%-25%), the three species with which it is closely related at the rRNA gene sequence level (2.5%-2.9%) . P . antarcticus also showed only 2.5% difference in its 16S rRNA gene sequence compared with the P . alkanoclasticus sequence . But it was distinctly different from P . alkanoclasticus, which exists only as rods, is mesophilic and phosphatase positive, can hydrolyze starch, cannot utilize succinate, glutamate, or glucose, and cannot acidify glucose . Most important, P . antarcticus and P . alkanoclasticus varied distinctly in their fatty acid composition in that C(15:0), C(15:1), C(16:0), iso-C(16:1), and C(17:0) were present only in P . antarcticus but absent in P . alkanoclasticus . CMS 53or(T), the thirteenth isolate, was also identified as a new species of Planococcus and was assigned the name Planococcus psychrophilus sp . nov . This species was distinctly different from all the reported species, including the new species P . antarcticus, with respect to a number of phenotypic characteristics . At the 16S rRNA gene sequence level, it was closely related to P . okeanokoites (98.1%) and P . mcmeekinii (98%), but with respect to the DNA-DNA hybridization, the similarity was only 35%-36% . The type strain of P . antarcticus is CMS 26or(T) (MTCC 3854; DSM 14505), and that of P . psychrophilus is CMS 530r(T) (MTCC 3812; DSM 14507).

Eur J Gastroenterol Hepatol, 2002 Jun, 14(6), 641 - 8
A survey of infectious agents as risk factors for primary sclerosing cholangitis: are Chlamydia species involved?
Ponsioen CY, Defoer J, Ten Kate FJ, Weverling GJ, Tytgat GN, Pannekoek Y, Wertheim-Dillen PM.
OBJECTIVES: The aetiology of primary sclerosing cholangitis (PSC) is unknown, and the role of micro-organisms has been studied only to a limited extent . We tested the hypothesis that past or persisting infection with common viruses or atypical bacteria might play a role in genetically susceptible hosts . DESIGN: Case-control study . METHODS: Serological screening for antibodies against 22 viruses as well as Chlamydia spp . and Mycoplasma pneumoniae was carried out in 41 well-established PSC patients . All 5110 sera tested in 1997 for these micro-organisms at our laboratory served as a background reference group . Subsequently, Chlamydia anti-lipopolysaccharide (LPS) antibodies were determined by enzyme-linked immunosorbent assay (ELISA) in the PSC group and in three race-matched control groups (inflammatory bowel disease (IBD) group, n = 35; non-IBD patients group, n = 39; healthy blood donor group, n = 40) . Subtyping in Chlamydia trachomatis and C . pneumoniae serotypes by specific anti-major outer membrane protein (MOMP) assays was carried out in the four groups . Immunohistochemical staining using specific markers for chlamydiae was carried out on liver biopsies of 14 PSC patients . RESULTS: There was a markedly elevated seroprevalence of Chlamydia-LPS antibodies compared with the 1997 reference group . The odds ratios (ORs) for the presence of immunoglobulin G, immunoglobulin M and immunoglobulin A antibodies for the PSC patients versus the control group were 2.4 (95% confidence interval (CI) 1.1 to 5.4), 1.9 (95% CI 0.9 to 4.0) and 6.7 (95% CI 3.0 to 17.0), respectively . All other micro-organisms tested showed normal antibody profiles that did not differ from the 1997 reference group . The seroprevalence of Chlamydia-anti-LPS antibodies was elevated markedly in the PSC patients compared with the IBD, non-IBD and blood donor groups . The outcomes in the C . trachomatis and C . pneumoniae anti-MOMP assays did not correlate with the anti-LPS-positive PSC sera . The actual presence of Chlamydia bodies in liver tissue could not be demonstrated . CONCLUSION: Our findings suggest an association between PSC and (previous) infection with Chlamydia.

Proc Natl Acad Sci U S A, 2002 Jun 25, 99(13), 8506 - 11 Epub 2002 Jun 18.
All intermediates of the arsenate reductase mechanism, including an intramolecular dynamic disulfide cascade; Messens J et al.; The mechanism of pI258 arsenate reductase (ArsC) catalyzed arsenate reduction, involving its P-loop structural motif and three redox active cysteines, has been unraveled . All essential intermediates are visualized with x-ray crystallography, and NMR is used to map dynamic regions in a key disulfide intermediate . Steady-state kinetics of ArsC mutants gives a view of the crucial residues for catalysis . ArsC combines a phosphatase-like nucleophilic displacement reaction with a unique intramolecular disulfide bond cascade . Within this cascade, the formation of a disulfide bond triggers a reversible "conformational switch" that transfers the oxidative equivalents to the surface of the protein, while releasing the reduced substrate.

J Virol, 2002 Jul, 76(14), 7082 - 93
Analysis of a temperature-sensitive mutant rotavirus indicates that NSP2 octamers are the functional form of the protein; Taraporewala ZF et al.; Evidence that NSP2 plays a role in packaging and replication comes from studies on tsE(1400), a rotavirus mutant with a temperature-sensitive (ts) lesion in the NSP2 gene . Cells infected with tsE and maintained at nonpermissive temperature contain few replication-assembly factories (viroplasms) or replication intermediates and produce virus particles that are mostly empty . Sequence analysis has indicated that an A152V mutation in NSP2 is responsible for the ts phenotype of tsE . To gain insight into the effect of the mutation on the octameric structure and biochemical activities of tsE NSP2, the protein was expressed in bacteria and purified to homogeneity . Analytical ultracentrifugation showed that tsE NSP2 formed octamers which, like those formed by wild-type (wt) NSP2, undergo conformational change into more compact structures upon binding of nucleotides . However, exposure to Mg(2+) and the nonpermissive temperature caused disruption of the tsE octamers and yielded the formation of polydisperse NSP2 aggregates, events not observed with wt octamers . Biochemical analysis showed that the RNA-binding, helix-destabilizing and NTPase activities of tsE NSP2 were significantly less at the nonpermissive temperature than at the permissive temperature . In contrast, these activities for wt NSP2 were higher at the nonpermissive temperature . Our results indicate that the octamer is the fully functional form of NSP2 and the form required for productive virus replication . The propensity of tsE NSP2 to form large aggregates provides a possible explanation for the inability of the protein to support packaging and/or replication in the infected cell at the nonpermissive temperature.

Genetics, 2002 Jun, 161(2), 651 - 60
Frequent germline mutations and somatic repeat instability in DNA mismatch-repair-deficient Caenorhabditis elegans; Tijsterman M et al.; Mismatch-repair-deficient mutants were initially recognized as mutation-prone derivatives of bacteria, and later mismatch repair deficiency was found to predispose humans to colon cancers (HNPCC) . We generated mismatch-repair-deficient Caenorhabditis elegans by deleting the msh-6 gene and analyzed the fidelity of transmission of genetic information to subsequent generations . msh-6-defective animals show an elevated level of spontaneous mutants in both the male and female germline; also repeated DNA tracts are unstable . To monitor DNA repeat instability in somatic tissue, we developed a sensitive system, making use of heat-shock promoter-driven lacZ transgenes, but with a repeat that puts this reporter gene out of frame . In genetic msh-6-deficient animals lacZ+ patches are observed as a result of somatic repeat instability . RNA interference by feeding wild-type animals dsRNA homologous to msh-2 or msh-6 also resulted in somatic DNA instability, as well as in germline mutagenesis, indicating that one can use C . elegans as a model system to discover genes involved in maintaining DNA stability by large-scale RNAi screens.

Vet Immunol Immunopathol, 2002 Sep 10, 87(3-4), 109 - 21
Inflammatory cytokines and antigen presenting cell activation; Murtaugh MP et al.; Immune responses are stimulated in response to threats against health . In animals, defense against infectious agents, particularly rapidly growing viruses and bacteria, requires an immediate response to limit growth and dissemination, and then stimulation of a more prolonged, specific immunity to prevent re-infection . The process by which animals meet the dual needs of an immediate response to danger and initiation of long-term protection is substantially influenced by inflammatory cytokines produced primarily by macrophages and professional antigen presenting cells (APCs) . Inflammatory cytokines mobilize the immune system in response to danger and increase the efficiency of an immune response as effectors of APC function . Here we review the evidence for the involvement of inflammatory cytokines in immune induction and as mediators of APC activity, with a particular emphasis on swine and on the induction of immunity at mucosal surfaces . The vast majority of infections occur at mucosal surfaces of the enteric, respiratory and reproductive tracts, and induction of protective immunity at these sites is particularly challenging . Induction of immunity at mucosal surfaces of the small intestine is greatly facilitated by the oral adjuvant, cholera toxin (CT) . CT potentiates inflammatory cytokine and costimulatory molecule expression in macrophages, and stimulates humoral and cell-mediated immune responses both locally and systemically . These observations are consistent with the hypothesis that activation of APCs is a key step in the induction of antigen-specific immunity, and that inflammatory cytokine expression is a hallmark of activated APC function . The efficacy of vaccine adjuvants, particularly in the context of mucosal immunity, may be determined by their ability to induce a controlled inflammatory response in gut-associated lymphoid tissue, characterized by the expression of various costimulatory molecules and inflammatory cytokines . Thus, elucidation of the patterns of inflammatory cytokine expression and features of APC activation will help to facilitate the rational development of more efficacious vaccines.

Eur J Biochem, 2002 Jun, 269(12), 2941 - 50
The trans-sialidase from the african trypanosome Trypanosoma brucei; Montagna G et al.; Trypanosoma brucei is the cause of the diseases known as sleeping sickness in humans (T . brucei ssp . gambiense and ssp . rhodesiense) and ngana in domestic animals (T . brucei brucei) in Africa . Procyclic trypomastigotes, the tsetse vector stage, express a surface-bound trans-sialidase that transfers sialic acid to the glycosylphosphatidylinositol anchor of procyclin, a surface glycoprotein covering the parasite surface . Trans-sialidase is a unique enzyme expressed by a few trypanosomatids that allows them to scavenge sialic acid from sialylated compounds present in the infected host . The only enzyme extensively characterized is that of the American trypanosome T . cruzi (TcTS) . In this work we identified and characterized the gene encoding the trans-sialidase from T . brucei brucei (TbTS) . TbTS genes are present at a small copy number, at variance with American trypanosomes where a large gene family is present . The recombinant TbTS protein has both sialidase and trans-sialidase activity, but it is about 10 times more efficient in transferring than in hydrolysing sialic acid . Its N-terminus contains a region of 372 amino acids that is 45% identical to the catalytic domain of TcTS and contains the relevant residues required for catalysis . The enzymatic activity of mutants at key positions involved in the transfer reaction revealed that the catalytic sites of TcTS and TbTS are likely to be similar, but are not identical . As in the case of TcTS and TrSA, the substitution of a conserved tryptophanyl residue changed the substrate specificity rendering a mutant protein capable of hydrolysing both alpha-(2,3) and alpha-(2,6)-linked sialoconjugates.

Commun Dis Public Health, 2002 Mar, 5(1), 78 - 86
One hundred years of virology: a chief's perspective; Foster JR; The ubiquitous nature of viruses has had its impact throughout the living world . Virus disease can be found in higher animals, birds, plants, arthropods, protozoa and bacteria . Viruses are no modern phenomenon, although it is only in the last 50 or so years that a fuller knowledge of their biological, chemical and physical properties has emerged . This short account recalls the development of human virology in particular, from the first discovery of a 'filterable virus' in 1892 to the spectacular technological breakthroughs during the 1950's and 1960's, leading to the molecular virology of today . This account was written to accompany an exhibition of artefacts displayed during the IBMS Congress in Birmingham in September 2001.

Arch Microbiol, 2002 Jul, 178(1), 36 - 44 Epub 2002 Apr 23.
Involvement of the Sinorhizobium meliloti leuA gene in activation of nodulation genes by NodD1 and luteolin; Sanjuan-Pinilla JM et al.; The role of leucine biosynthesis by Sinorhizobium meliloti in the establishment of nitrogen-fixing symbiosis with alfalfa ( Medicago sativa) was investigated . The leuA gene from S . meliloti, encoding alpha-isopropylmalate synthase, which catalyses the first specific step in the leucine biosynthetic pathway, was characterized . S . melilotiLeuA(-) mutants were Leu auxotrophs and lacked alpha-isopropylmalate synthase activity . In addition, leuA auxotrophs were unable to nodulate alfalfa . Alfalfa roots did not seem to secrete enough leucine to support growth of leucine auxotrophs in the rhizosphere . Thus, this growth limitation probably imposes the inability to initiate symbiosis . However, in addition to the leucine auxotrophy, leuA strains were impaired in activation of nodulation genes by the transcriptional activator NodD1 in response to the plant flavone luteolin . By contrast, nod gene activation by NodD3, which does not involve plant-derived inducers, was unaffected . Our results suggest that a leucine-related metabolic intermediate may be involved in activation of nodulation genes by NodD1 and luteolin . This kind of control could be of relevance as a way to link bacterial physiological status to the response to plant signals and initiation of symbiosis.

Arch Orthop Trauma Surg, 2002 Jun, 122(5), 259 - 61 Epub 2002 Apr 06.
Efficacy of primary wound cultures in long bone open extremity fractures: are they of any value?
Valenziano CP, Chattar-Cora D, O'Neill A, Hubli EH, Cudjoe EA.
The management of long bone open extremity fractures has included initial wound cultures, antibiotics, operative debridement, and fracture repair, if indicated . The value of initial wound cultures is unclear . We examined whether primary wound cultures predict which wounds will become infected, and whether bacterial growth on primary wound cultures correlates with bacteria cultured from infected wounds . This prospective study involved patients presenting to a regional trauma center . Before any interventions were performed, initial aerobic and anaerobic cultures of the wounds of 117 consecutive open extremity fractures grades I-III were obtained . The results of these cultures were correlated with the development of a wound infection, and if an infection occurred, the organism grown from the infected wound was compared with any organism grown from the primary wound cultures . Of the initial cultures, 76% (89/117) did not demonstrate any growth, while the other 24% (28/117) only grew skin flora . There were only 7 (6%) wound infections, and 71% (5/7) initially did not grow any organisms . Of the isolates that grew from the initial cultures, none were the organisms that eventually led to wound infections . The use of primary wound cultures in open extremity injuries has no value in the management of patients suffering long bone open extremity fractures.

J Biol Chem, 2002 Aug 30, 277(35), 32195 - 201 Epub 2002 Jun 17.
Functional knockout of the adenosine 5'-phosphosulfate reductase gene in Physcomitrella patens revives an old route of sulfate assimilation; Koprivova A et al.; The reduction of adenosine 5'-phosphosulfate (APS) to sulfite catalyzed by adenosine 5'-phosphosulfate reductase is considered to be the key step of sulfate assimilation in higher plants . However, analogous to enteric bacteria, an alternative pathway of sulfate reduction via phosphoadenosine 5'-phosphosulfate (PAPS) was proposed . To date, the presence of the corresponding enzyme, PAPS reductase, could be neither confirmed nor excluded in plants . To find possible alternative routes of sulfate assimilation we disrupted the adenosine 5'-phosphosulfate reductase single copy gene in Physcomitrella patens by homologous recombination . This resulted in complete loss of the correct transcript and enzymatic activity . Surprisingly, the knockout plants grew on sulfate as the sole sulfur source, and the concentration of thiols in the knockouts did not differ from the wild type plants . However, when exposed to a sublethal concentration of cadmium, the knockouts were more sensitive than wild type plants . When fed {(35)S}sulfate, the knockouts incorporated (35)S in thiols; the flux through sulfate reduction was approximately 50% lower than in the wild type plants . PAPS reductase activity could not be measured with thioredoxin as reductant, but a cDNA and a gene coding for this enzyme were detected in P . patens . The moss Physcomitrella patens is thus the first plant species wherein PAPS reductase was confirmed on the molecular level and also the first organism wherein both APS- and PAPS-dependent sulfate assimilation co-exist.

Antimicrob Agents Chemother, 2002 Jul, 46(7), 2069 - 76
Phagocytosis affects biguanide sensitivity of Acanthamoeba spp; Noble JA et al.; The incidence of Acanthamoeba keratitis, a disease associated with contact lens wear, has been in apparent decline with the advent of multipurpose contact lens solutions . The concentrations of the biguanides chlorhexidine digluconate (CHX) and particularly polyhexamethylene biguanide (PHMB) included in multipurpose solutions (MPSs) are sublethal for amoebae . We evaluated by flow cytometry the effects of these two biguanides on phagocytosis of particles and the survival of trophozoites of Acanthamoeba castellanii and A . polyphaga . Trophozoites of A . castellanii and A . polyphaga (10(6)/ml) were exposed to solutions of 5 and 50 microg of PHMB and CHX per ml in the presence and absence of particles (i.e., heat-killed yeasts and bacteria and latex beads) . In addition, trophozoites were exposed to particles treated with these concentrations of the two biguanides . In the absence of particles, trophozoites of A . polyphaga appeared to be more resistant to the biguanides than those of A . castellanii . In the presence of particles, the rates of survival of both species were decreased . In most instances, particles treated with sublethal concentrations of both biguanides that were adsorbed onto the particles reduced the incidence of phagocytosis . Particles present in MPSs in contact lens cases may be involved in the decreased incidence of Acanthamoeba keratitis.

Vet Microbiol, 2002 Jul 22, 87(4), 279 - 89
Piscirickettsiosis and piscirickettsiosis-like infections in fish: a review; Mauel MJ et al.; Piscirickettsia salmonis was the first "rickettsia-like" bacteria to be recognized as a pathogenic agent of fish . Since the first reports of piscirickettsiosis emerged from Chile in the late 1980s, Piscirickettsia-like bacteria have been recognized with increasing frequency in a variety of fish species, from both fresh and saltwaters around the world . Although the first reported incidents of Piscirickettsia were in salmonids, Piscirickettsia-like bacteria are now being frequently associated with disease syndromes in non-salmonid fish . Mortalities have occurred in white seabass (Atactoscion noblis), black seabass (Dicentrarchus sp.), tilapia (Oreochromis, Tilapia and Sarotherodon spp.) and blue-eyed plecostomus (Panaque suttoni) . Piscirickettsiosis and piscirickettsiosis-like diseases have affected aquaculture productivity, profitability, the species of fish compatible with commercial rearing, and transportation of fish from site to site . Piscirickettsiosis and syndromes caused by similar bacteria are an emerging disease complex that will increasingly inhibit fish production.

Biochemistry, 2002 Jun 25, 41(25), 7859 - 65
Stoichiometries of protein-protein/DNA binding and conformational changes for the transition-state regulator AbrB measured by pseudo cell-size exclusion chromatography-mass spectrometry; Cavanagh J et al.; We have developed on-line pseudo cell-size exclusion chromatography-mass spectrometry (PsC-SEC-MS) for the rapid, real time analyses of noncovalently bound protein complexes . The methodology can be used to determine constituent components of such complexes, as well as exact stoichiometries . Furthermore, it enables the efficient determination of gross conformational changes upon complexation . The power of the new approach is demonstrated in the analysis of the global transition-state regulator AbrB and its complex with a target DNA sequence from the promoter sinIR . Using PsC-SEC-MS, we confirm that AbrB is assembled as a homotetramer and not as a homohexamer as previously suggested . Additionally, we show that AbrB binds to the sinIR DNA target element as a homotetramer, affording a 4:1 protein:DNA stoichiometry . Finally, we demonstrate that when the complex binds to sinIR, the hydrodynamic volume (size) of the complex is notably reduced compared to that of the apoprotein, indicating a protein conformational change.

J Theor Biol, 2002 Apr 21, 215(4), 385 - 97
A lifetime perspective on foraging and mortality; Yearsley J et al.; Food intake carries many potential risks which may impair an animal's reproductive success not only in the current breeding cycle, but also for the rest of its lifetime . We examine the lifetime trade-off between the costs and benefits of food intake by presenting a simple animal foraging model, where each unit of food eaten carries with it a risk of mortality . We show that the optimal food intake rate over an animal's lifetime, for both semelparous and iteroparous animals, is not maximal . Instead, animals are required to strike a balance between the immediate reproductive benefits of gathering food and the future reproductive costs incurred by the food's mortality risk . This balance depends upon the lifespan of the animal as well as the nature of the risk . Different mortality risks are compared and it is shown that a mortality risk per unit time spent foraging is not, in general, equivalent to a mortality risk per unit of food consumed . The results suggest that a mortality risk per unit of food consumed, such as that presented by the presence of a toxin or of a parasite in the diet, has important consequences for feeding behaviour and is a possible factor involved in food intake regulation .

Proc Nutr Soc, 2001 Nov, 60(4), 427 - 35
Regulation of mucosal immune responses in effector sites; Bailey M et al.; In human disease and rodent models, immune responses in the intestinal mucosa can be damaging . Damage is characterised by villus atrophy, crypt hyperplasia and reduced ability to digest and absorb nutrients . In normal individuals active responses to harmless environmental antigens associated with food and commensal bacteria are controlled by the development of immunological tolerance . Similar pathological changes occur in piglets weaned early from their mothers . Active immune responses to food antigens are observed in these piglets, and we and others have hypothesised that the changes occur as a result of transient allergic immune responses to novel food or bacteria antigens . The normal mechanism for producing tolerance to food antigens may operate at induction (Peyer's patches and mesenteric lymph nodes) or at the effector stage (intestinal lamina propria) . In our piglet studies immunological tolerance occurs despite the initial active response . Together with evidence from rodents, this observation suggests that active responses are likely to be controlled at the effector stage, within the intestinal lamina propria . Support for this mechanism comes from the observation that human and pig intestinal T-cells are susceptible to apoptosis, and that this process is accelerated by antigen . We suggest that the role of the normal mature intestinal lamina propria is a balance between immunological effector and regulatory function . In neonatal animals this balance develops slowly and is dependant on contact with antigen . Immunological insults such as weaning may tip the balance of the developing mucosal immune system into excessive effector or regulatory function resulting in transient or chronic allergy or disease susceptibility.

Biomaterials, 2002 Jul, 23(14), 2979 - 88
Physical and chemical processing for a human dura mater substitute; Dufrane D et al.; OBJECT: Allogenic human fascia lata used in neurosurgery, as dura mater substitute, can be associated with a risk of viral and bacterial transmission . Chemical and physical procedures, developed to inactivate virus and bacteria, have been applied to fascia lata . The aim of this study consists in the evaluation of the biological properties of this treated graft . METHODS: Grafts were treated with solvent detergents, freeze-dried for conservation and gamma irradiated (25,000 Gy) for sterilization . The indirect toxicity evaluation was performed by extraction method, according to the International Standard Organization (ISO) . First, the cytotoxic effect of each extracts incubated in the presence of human fibroblasts (WI38) was quantitatively assessed by measuring the cell growth, the viability (succinate dehydrogenase activity, MTT), the membrane integrity (uptake of the neutral red by viable cells, NR) as well as the release of lactate dehydrogenase in the culture medium . Second, confocal laser scanning microscopy (CLSM) was used to assess the direct contact between human primary fibroblasts and graft . CLSM was performed at days 3 and 7 after cells loading . RESULTS: No acute cytotoxicity was observed for chemically processed allografts . Cells loaded on the graft have demonstrated a good growth and spreading . CONCLUSIONS: Human fascia lata secured against conventional and non-conventional agents is a fully biocompatible alternative to the available dural graft materials.

Mol Microbiol, 2002 Jun, 44(5), 1377 - 85
Mutual dependence of the expression of the cell differentiation regulatory protein HetR and the global nitrogen regulator NtcA during heterocyst development; Muro-Pastor AM et al.; Heterocyst differentiation in the cyanobacterium Anabaena sp . strain PCC 7120 depends on both the global nitrogen regulator NtcA and the cell differentiation regulatory protein HetR, and induction of hetR upon nitrogen step-down depends on NtcA . The use of two out of the four transcription start points (tsps) described for the hetR gene (those located at positions -728 and -271) was found to be dependent on NtcA, and the use of the tsp located at position -271 was also dependent on HetR . Thus, autoregulation of hetR could take place via the activation of transcription from this tsp . Expression of ntcA in nitrogen-fixing cultures was higher than in cells growing in the presence of ammonium or nitrate, and high expression of ntcA under nitrogen deficiency resulted from an increased use of tsps located at positions -180 and -49 . The induction of the use of these tsps did not take place in ntcA or hetR mutant strains . These results indicate a mutual dependency in the induction of the regulatory genes hetR and ntcA that takes place in response to nitrogen step-down in Anabaena cells . Expression of the hetC gene, which is also involved in the early steps of heterocyst differentiation, from its NtcA-dependent tsp was, however, not dependent on HetR.

Biofizika, 2002 May-Jun, 47(3), 531 - 8
{Effect of combination of high-voltage pulses and other physical factors on the intensity of reproduction of Anabaena flos aquae}; Boiko NI et al.; It was shown that the influence of high-voltage pulses (intensity of electric field 40-120 kV/cm) on cyanobacteria Anabaena flos aquae can be different and depends on the mode of treatment . It was found that in the range of pulse durations less than 1 microsecond with a pulse repetition rate of up to 500 Hz, and final temperature in the working chamber (60 +/- 1) degree C, a gradual full and irreversible inactivation of cyanobacteria independent of the pulse shape takes place . At the same time, a rapid (but slower than by the action of high-voltage pulses) heating without treatment with high-voltage impulses at the same final temperature leads to an inactivation of 80% of initial cyanobacteria followed by growth of bacteria up to 700% and more.

Prikl Biokhim Mikrobiol, 2002 May-Jun, 38(3), 292 - 4
{Changes in the antigenic properties of Azospirillum brasilense lipopolysaccharide on addition of tris(hydroxymethyl)-aminomethane into the culture medium}; Burygin GL et al.; Addition of tris(hydroxymethyl)-aminomethane (Tris) into the culture medium of Azospirillum brasilense sp245 changes the antigenic properties of the lipopolysaccharide (LPS) isolated from the external membrane of the bacteria . LPS preparations from the bacteria grown in the presence of Tris have been analyzed by immunodiffusion, using monospecific antibodies . The disappearance of the precipitation band corresponding to one of the two O-specific polysaccharides of the LPS (O-PS1) and changes in the electrophoretic profile have been revealed . However, only minor differences in absorption spectra of products of O-PS1 reaction with phenol/sulfuric acid have been demonstrated between the bacteria grown under standard conditions and in the presence of Tris.

J Korean Med Sci, 2002 Jun, 17(3), 302 - 6
The effect of the repeated subcultures of Helicobacter pylori on adhesion, motility, cytotoxicity, and gastric inflammation; Kim SS et al.; In vitro subcultures of bacteria can lead to genetic and phenotypic changes . This study aimed at investigating the effect of repeated subcultures on the adhesion, motility, cytotoxicity, and gastric inflammation caused by Helicobacter pylori . H.pylori SS1 strain was subcultured 64 times on agar plates containing Brucella broth and 5% bovine calf serum . The adhesion, motility, cytotoxicity, and gastric inflammation produced in Mongolian gerbils were compared between the first and 64th subcultured strain . The adhesion rates, following 3 hr exposure of AGS cells to either the first strain or the 64th-transferred strain, were 21% and 12%, respectively . The motility of the 64th-transferred strain decreased significantly when compared to the 1st strain (9.1 mm vs . 15.1 mm) . The cytotoxicity index tended to be higher in the first strain than in the 64th-transferred strain (73.7% vs . 69.2%) . The initial infection rate on the gerbils showed no difference between the two strains . However, chronic gastric inflammation of the first strain-infected gerbils was somewhat more severe than that of the 64th-transferred strain-infected gerbils . Therefore, the use of repeatedly subcultured strains of H . pylori in virulence experiments can lead to different results from those of the original strain.

J Biol Chem, 2002 Sep 6, 277(36), 33284 - 90 Epub 2002 Jun 14.
Oxidant-induced vascular endothelial growth factor expression in human keratinocytes and cutaneous wound healing; Sen CK et al.; Neutrophils and macrophages, recruited to the wound site, release reactive oxygen species by respiratory burst . It is commonly understood that oxidants serve mainly to kill bacteria and prevent wound infection . We tested the hypothesis that oxidants generated at the wound site promote dermal wound repair . We observed that H(2)O(2) potently induces vascular endothelial growth factor (VEGF) expression in human keratinocytes . Deletion mutant studies with a VEGF promoter construct revealed that a GC-rich sequence from bp -194 to -50 of the VEGF promoter is responsible for the H(2)O(2) response . It was established that at microm concentrations oxidant induces VEGF expression and that oxidant-induced VEGF expression is independent of hypoxia-inducible factor (HIF)-1 and dependent on Sp1 activation . To test the effect of NADPH oxidase-generated reactive oxygen species on wound healing in vivo, Rac1 gene transfer was performed to dermal excisional wounds left to heal by secondary intention . Rac1 gene transfer accelerated wound contraction and closure . Rac1 overexpression was associated with higher VEGF expression both in vivo as well in human keratinocytes . Interestingly, Rac1 gene therapy was associated with a more well defined hyperproliferative epithelial region, higher cell density, enhanced deposition of connective tissue, and improved histological architecture . Overall, the histological data indicated that Rac1 might be an important stimulator of various aspects of the repair process, eventually enhancing the wound-healing process as a whole . Taken together, the results of this study indicate that wound healing is subject to redox control.

FEBS Lett, 2002 Jun 19, 521(1-3), 31 - 5
Circular dichroism spectroscopy of fluorescent proteins; Visser NV et al.; Circular dichroism (CD) spectra have been obtained from several variants of green fluorescent protein: blue fluorescent protein (BFP), enhanced cyan fluorescent protein (CFP), enhanced green fluorescent protein (GFP), enhanced yellow fluorescent protein (YFP), all from Aequorea victoria, and the red fluorescent protein from the coral species Discosoma (DsRed) . We demonstrate that CD spectra in the spectral fingerprint region of the chromophore yield spectra that after normalization are not coincident with the normalized absorbance spectra of GFP, YFP and DsRed . On the other hand, the CD spectra of BFP and CFP coincide with the absorbance spectra . The resolution of absorption and CD spectra into Gaussian bands confirmed the location of the different electronic band positions of GFP and YFP as reported in the literature using other techniques . In the case of BFP and CFP the location of Gaussian bands provided information of the vibrational progression of the electronic absorption bands . The CD spectrum of DsRed is anomalous in the sense that the major CD band has a clear excitonic character . Far-UV CD spectra of GFP confirmed the presence of the high beta-sheet content of the polypeptide chain in the three-dimensional structure.

Mol Microbiol, 2002 Jun, 44(6), 1493 - 506
Polymerase chain reaction-based mutageneses identify key transporters belonging to multigene families involved in Na+ and pH homeostasis of Synechocystis sp . PCC 6803; Wang HL et al.; Primary ion pumps and antiporters exist as multigene families in the Synechocystis sp . PCC 6803 genome and show very strong homologies to those found in higher plants . The gene knock-outs of five putative Na+/H+ antiporters (slr1727, sll0273, sll0689, slr1595 and slr0415) and seven cation ATPases (sll1614, sll1920, slr0671-72, slr0822, slr1507-08-09, slr1728- 29 and slr1950) in the model cyanobacterium were performed in this study relying on homologous recombination with mutagenenic fragments constructed using a fusion polymerase chain reaction (PCR) approach . The impacts of these gene knock-outs were evaluated in terms of Na+ and pH, and light-induced acidification and alkalization that are asso-ciated with inorganic carbon uptake . Two of the five putative antiporter mutants exhibit a characteristic interplay between the pH and Na+ dependence of growth, but only one of the antiporters appears to be necessary for high NaCl tolerance . On the other hand, the mutation of one of the two copper-trafficking ATPases produces a cell line that shows acute NaCl sensitivity . Additionally, disruptions of a putative Ca2+-ATPase and a gene cluster encoding a putative Na+-ATPase subunit also cause high NaCl sensitivity . The findings and possible mechanisms are discussed in relation to the potential roles of these transporters in Synechocystis sp . PCC 6803.

Cell Microbiol, 2002 Jun, 4(6), 341 - 55
Modulation of Brucella-induced macropinocytosis by lipid rafts mediates intracellular replication; Watarai M et al.; Intracellular replication of Brucella requires the VirB complex, which is highly similar to conjugative DNA transfer systems . In this study, we show that Brucella internalizes into macrophages by swimming on the cell surface with generalized membrane ruffling for several minutes, after which the bacteria are enclosed by macropinosomes . Lipid raft-associated molecules such as glycosylphosphatidylinositol (GPI)-anchored proteins, GM1 gangliosides and cholesterol were selectively incorporated into macropinosomes containing Brucella . In contrast, lysosomal glycoprotein LAMP-1 and host cell transmembrane protein CD44 were excluded from the macropinosomes . Removing GPI-anchored proteins from the macrophage surface and cholesterol sequestration markedly inhibited the VirB-dependent macropinocytosis and intracellular replication . Our results suggest that the entry route of Brucella into the macrophage determines the intracellular fate of the bacteria that is modulated by lipid raft microdomains.

J Endod, 2002 Jun, 28(6), 433 - 7
Scanning electron microscopy study of the adhesion of Prevotella nigrescens to the dentin of prepared root canals; Yang SE et al.; The purpose of this study was to evaluate the effect of the presence or absence of amorphous, irregular smear layers on the adhesion of Prevotella nigrescens, to the dentin of the root canal by using scanning electron microscopy (SEM) . Human incisors extracted within 7 days, with no cavities, no fractures, and no evidence of calcification of the canal, were selected . After cutting the crown portion at the CEJ, root canal preparation was undertaken by using a modified crown-down technique with Profile and Gates Glidden drills . Ten milliliters of physiologic saline solution (groups 1 and 4), 10 ml of 3.5% NaOCl (groups 2 and 5), or 10 ml of NaOCl and 10 ml of 0.5 M EDTA (groups 3 and 6) were used as irrigation solution while preparing the canal . After vertical sectioning and ethylene oxide gas sterilization, samples (groups 1, 2, and 3) were immersed in brain-heart infusion broth with yeast extract, hemin, and menadione, inoculated with P . nigrescens (ATCC 33563), and incubated for 3 h at 37 degrees C . All samples were prepared for and observed with SEM . The data were analyzed by using t test and one-way ANOVA . Smear layer was observed to cover the entire root canal surface after root canal preparation . Smear layer was removed and the entrances of dentinal tubules opened widely after applying 3.5% NaOCl and 0.5 M EDTA . A significantly greater number of bacteria were found to adhere to those teeth in which a smear layer was present (p < 0.05) . Given that the smear layer produced during root canal preparation promoted adhesion and colonization of P . nigrescens to the dentin matrix, it might also increase the likelihood of canal reinfection.

Infect Immun, 2002 Jul, 70(7), 3529 - 38
Clearance of Helicobacter pylori Infection and Resolution of Postimmunization Gastritis in a Kinetic Study of Prophylactically Immunized Mice; Garhart CA et al.; Patients infected with Helicobacter pylori mount an immune response which fails to clear the infection and may contribute to disease . Mice can be protected by immunization . To further characterize the H . pylori-mouse model, stomachs of unimmunized or intranasally immunized C57BL/6 mice were quantitatively cultured 3 days and 1, 2, 4, 8, 16, 32, and 52 weeks after challenge with H . pylori . At 3 days and 1 week after challenge, colonization was the same in the immunized and unimmunized mice . By 2 weeks after challenge, the immunized mice had a >2-log decrease in bacterial load, and at all later time points, they either were culture negative or had at least a 2-log decrease in bacterial load . Gastritis in the immunized mice peaked at 1 to 2 weeks after challenge and was characterized by a mixed inflammatory infiltrate and epithelial proliferation centered at the transition between corpus and antrum . By 52 weeks postchallenge, the gastric histology in the immunized mice was not different from that in control unchallenged mice . The unimmunized group began to show a reduction in bacterial load as early as 16 weeks after challenge, and by 52 weeks seven of eight unimmunized mice had developed gastritis and reduced bacterial loads . These results indicate that prophylactic immunization does not prevent colonization by H . pylori but enables mice to clear the infection or significantly reduce the number of colonizing bacteria . The reduction in bacterial load is associated with gastric inflammation that subsides over time.

J Struct Biol, 2002 Jan-Feb, 137(1-2), 41 - 53
Generalized Crick equations for modeling noncanonical coiled coils; Offer G et al.; Crick envisaged the alpha-helical coiled coil to result from systematic bending of an alpha-helix such that every seventh residue was structurally equivalent, and he derived equations for the coordinates of the backbone atoms . Crick's predictions were vindicated experimentally and coiled-coil sequences were shown to have hydrophobic residues alternately spaced 3 and 4 residues apart . Nonetheless, in some coiled coils such canonical heptad repeats are interrupted by inserts of 3 or 4 residues generating decad and hendecad motifs . The supercoiling of the coiled coils varies with the sequence pattern, being left- or right-handed in purely heptad-based or hendecad-based motifs, respectively . To model coiled coils with a mixture of motifs, we describe how Crick's equations can be modified for cases where the pitch is not constant . Using the analogy of the bending of a beam, we took the tilt angle to change linearly with distance along the major helix and the pitch of a motif to be affected by neighboring motifs depending on the rigidity of the alpha-helical strands . We tested our approach by fitting the two-, three-, and four-stranded noncanonical coiled coils of GrpE, hemagglutinin, and tetrabrachion . The backbone atoms of the model and crystal structures agreed with root mean square deviations of <1.1 A . (c) 2002 Elsevier Science (USA).

Z Naturforsch {C}, 2002 Mar-Apr, 57(3-4), 263 - 71
Studies on the biosynthesis of striatal-type diterpenoids and the biological activity of herical; Anke T et al.; Feeding experiments with specifically 13C-labeled glucose disclosed that the diterpenoid part of the striatals/striatins is formed via the mevalonate pathway, whereas the pentose moiety originates either via glucuronic acid (70%) or the pentose phosphate cycle (30%) . Application of radioactively labeled herical to cultures of Hericium ramosum demonstrates the pivotal role of this cyathane-xyloside in striatal biosynthesis . Herical inhibits a large spectrum of fungi and bacteria and shows cytotoxic and hemolytic properties.

Z Naturforsch {C}, 2002 Mar-Apr, 57(3-4), 257 - 62
The himanimides, new bioactive compounds from Serpula himantoides (Fr.) Karst; Aqueveque P et al.; In a screening of basidiomycete cultures from Chile for the production of antibiotics we identified a Serpula himantoides strain as a producer of metabolites inhibiting the growth of bacteria and fungi . Bioactivity guided purification resulted in the isolation of four new antibiotics . Their structures were elucidated by spectroscopic methods . All four compounds are succinimide and maleimide derivatives, of which two are N-hydroxylated.

Cell Commun Adhes, 2001, 8(4-6), 231 - 6
Role of cytoskeletal elements in the recruitment of Cx43-GFP and Cx26-YFP into gap junctions; Thomas T et al.; Cytoskeletal elements may be important in connexin transport to the cell surface, cell surface gap junction plaque formation and/or gap junction internalization . In this study, fluorescence recovery after photobleaching was used to examine the role of microfilaments and microtubules in the recruitment and coalescence of green fluorescent protein-tagged Cx43 (Cx43-GFP) or yellow fluorescent tagged-Cx26 (Cx26-YFP) into gap junctions in NRK cells . In untreated cells, both Cx26-YFP and Cx43-GFP were recruited into gap junctions within photobleached areas of cell-cell contact within 2 hrs . However, disruption of microfilaments with cytochalasin B inhibited the recruitment and assembly of both Cx26-YFP and Cx43-GFP into gap junctions within photobleached areas . Surprisingly, disruption of microtubules with nocodazole inhibited the recruitment of Cx43-GFP into gap junctions but had limited effect on the transport and clustering of Cx26-YFP into gap junctions within the photobleached regions of cell-cell contact . These results suggest that the recruitment of Cx43-GFP and Cx26-YFP to the cell surface or their lateral clustering into gap junctions plaques is dependent in part on the presence of intact actin microfilaments while Cx43-GFP was more dependent on intact microtubules than Cx26-YFP.

Cell Mol Biol (Noisy-le-grand), 2002 Jun, 48(4), 359 - 71
Lake Baikal: a unique place to study evolution of sponges and their stress response in an environment nearly unimpaired by anthropogenic perturbation; Efremova SM et al.; Lake Baikal is considered as a unique place to study evolution . In this review, we report on recent data on the evolution of endemic freshwater sponges of this ancient lake . Nucleotide sequence data support the idea that these sponges are of monophyletic origin and evolved from Spongillidae . Baikalian sponges form the dominating biomass in the benthos of the lake . Data on the expression of the biomarker heat shock protein 70, revealed that the endemic sponge species of Lake Baikal are useful as bioindicators to assess the anthropogenic impact on the lake.

Cell Microbiol, 2002 May, 4(5), 285 - 96
Reduced activation of inflammatory responses in host cells by mouse-adapted Helicobacter pylory isolates; Philpott DJ et al.; Helicobacter pylori strains that harbour the Cag pathogenicity island (Cag PAI) induce interleukin (IL)-8 secretion in gastric epithelial cells, via the activation of NF- kappa B, and are associated with severe inflammation in humans . To investigate the influence of Cag PAI-mediated inflammatory responses on H . pylori adaptation to mice, a selection of H . pylori clinical isolates (n = 12) was cag PAI genotyped and tested in co-culture assays with AGS gastric epithelial cells, and in mouse colonization studies . Six isolates were shown to harbour a complete cag PAI and to induce NF- kappa B activation and IL-8 secretion in AGS cells . Of the eight isolates that spontaneously colonized mice, six had a cag PAI(-) genotype and did not induce pro-inflammatory responses in these cells . Mouse-to-mouse passage of the two cag PAI(+) -colonizing strains yielded host-adapted variants that infected mice with bacterial loads 100-fold higher than those of the respective parental strains (P= 0.001) . These mouse-adapted variants were affected in their capacity to induce pro-inflammatory responses in host cells, yet no changes in cag PAI gene content were detected between the strains by DNA microarray analysis . This work provides evidence for in vivo selection of H . pylori bacteria with a reduced capacity to induce inflammatory responses and suggests that such bacteria are better adapted to colonize mice.

J Allergy Clin Immunol, 2002 Jun, 109(6 Suppl), S525 - 32
Environmental factors influencing the development and progression of pediatric asthma; von Mutius E; Recent data underscore the importance of environmental factors in the sensitization of children to certain allergens and the development of asthma . Maternal smoking and family (especially maternal) history of atopy appear to be risk factors for persistent sensitization and development of asthma . Indeed, exposure to tobacco smoke in utero significantly increases asthma risk and influences the timing of sensitization . It must be stated that any smoking at home has consequences for the development of asthma and other respiratory conditions . In addition, reports of possible protective effects of specific environmental conditions suggest that exposure to certain stimuli may reduce or block the development and progression of asthma . Attendance at a day care center early in life appears to offer protective effects against wheezing, as do early episodes of rhinitis, herpes, and measles . Children raised on a farm also have a decreased prevalence of atopic diseases . The protective effect of contact with livestock and poultry is consistent among several studies . Although the pathophysiologic mechanisms involved remain undefined, studies suggest that exposure to endotoxin and other components of bacteria may play an important role in protecting against childhood atopic diseases . Whether in utero exposure is beneficial remains to be determined.

Lancet Infect Dis, 2002 May, 2(5), 273 - 80
Role of nitric oxide in HIV-1 infection: friend or foe?
Torre D, Pugliese A, Speranza F.
Nitric oxide (NO) is an important biologically active molecule that plays a key part in host defence against bacteria, protozoa, and tumour cells . NO has antiviral effects against several DNA viruses, such as murine poxvirus, herpes simplex virus, and Epstein-Barr virus, and some RNA viruses, such as coxsackievirus . In several studies, in vitro and in vivo, overproduction of NO has been noted in the presence of HIV-1 infection . Furthermore, increased NO production may contribute directly to the pathogenesis of HIV-1-associated dementia . The mechanisms of virus infection mediated by NO may be related to: direct antiviral effects of NO; impairment of antiviral defence mediated by T-helper-1 immune response by suppressing T-helper-1 functions; NO-induced cytotoxic effects by oxidative injury with cellular and organ dysfunctions; and NO-induced oxidative stress leading to rapid viral evolution with productions of drug-resistant and immunologically tolerant mutants . By contrast, there is some evidence of NO activity--directly, indirectly, or both--decreasing or blocking HIV-1 replication, through inhibition of viral enzymes, such as reverse transcriptase, protease, or cellular nuclear transcription factor (NF-kappa B) and long-terminal repeat-driven transcription . Therefore, although NO surely plays an important part in HIV-1 infection, that role is sometimes helpful and other times damaging to the host . Future challenges are to learn more about the beneficial and harmful effects of NO in HIV-1 infection, and how to selectively inhibit excessive NO production or to use NO-releasing drugs to decrease viral replication . This review discusses the role of NO in the pathogenesis of HIV-1 infection, inasmuch as its role against HIV-1 is unequivocal in inhibiting or increasing viral replication.

Gene, 2002 May 15, 290(1-2), 141 - 51
Cysteine and tyrosine-rich 1 (CYYR1), a novel unpredicted gene on human chromosome 21 (21q21.2), encodes a cysteine and tyrosine-rich protein and defines a new family of highly conserved vertebrate-specific genes; Vitale L et al.; A novel human gene has been identified by in-depth bioinformatics analysis of chromosome 21 segment 40/105 (21q21.1), with no coding region predicted in any previous analysis . Brain-derived DNA complementary to RNA (cDNA) sequencing predicts a 154-amino acid product with no similarity to any known protein . The gene has been named cysteine and tyrosine-rich protein 1 gene (symbol cysteine and tyrosine-rich 1, CYYR1) . The CYYR1 messenger RNA was found by Northern blot analysis in a broad range of tissues (two transcripts of 3.4 and 2.2 kb) . The gene consists of four exons and spans about 107 kb, including a very large intron of 85.8 kb . Analysis of expressed sequence tags shows high CYYR1 expression in cells belonging to the amine precursor uptake and decarboxylation system . We also cloned the cDNA of the murine ortholog Cyyr1, which was mapped by a radiation hybrid panel on chromosome 16 within the region corresponding to that containing the respective human homolog on chromosome 21 . Sequence and phylogenetic analysis led to identification of several genes encoding CYYR1 homologous proteins . The most prominent feature identified in the protein family is a central, unique cysteine and tyrosine-rich domain, which is strongly conserved from lower vertebrates (fishes) to humans but is absent in bacteria and invertebrates.

FEBS Lett, 2002 Apr 24, 517(1-3), 251 - 6
Involvement of TIRAP/MAL in signaling for the activation of interferon regulatory factor 3 by lipopolysaccharide; Shinobu N et al.; Infections of bacteria and viruses induce host defense reactions known as innate responses that include the production of cytokines and chemokines . The production of type I interferon (IFN) is known to be induced by viral double-stranded (ds) RNA or bacterial lipopolysaccharide (LPS) . Although important functions for the transcription factors NF-kappaB and interferon regulatory factor-3 (IRF-3) are indicated, the molecular signals leading to the activation of IFN genes have yet to be elucidated . We provide several lines of evidence that LPS and dsRNA trigger distinct intracellular signals upstream . Notably, our investigation revealed a critical function for TIRAP/MAL, a signaling adapter for Toll-like receptor (TLR) 4, in LPS-induced but not dsRNA-induced activation of IRF-3 . These results highlight cross-talk between TLR-mediated and virus/dsRNA-induced signals resulting in activation of the IFN system.

FEBS Lett, 2002 Apr 24, 517(1-3), 185 - 9
Two distinct classes of FixJ binding sites defined by in vitro selection; Ferrieres L et al.; The phosphorylated FixJ transcriptional activator is key to nitrogen fixation in Sinorhizobium meliloti, switching both the nifA and fixK genes on . Previously no consensus picture emerged concerning the nature of FixJ binding sites . Here we used in vitro DNA selection in order to systematically characterise FixJ binding sequences . This led to the definition of two classes of sites . Class I sites share the CTAAGTAGTTTCCC sequence found in the fixK promoter, whereas class II sites are defined by a GTAMGTAG consensus octamer . Our results indicate that FixJ approximately P binds DNA following two distinct binding modes.

FEBS Lett, 2002 Apr 24, 517(1-3), 87 - 91
Expression of hetN during heterocyst differentiation and its inhibition of hetR up-regulation in the cyanobacterium Anabaena sp . PCC 7120; Li B et al.; The hetN gene plays an important role in heterocyst differentiation and pattern formation . An immunoblotting study showed that the hetN gene in Anabaena sp . PCC 7120 was expressed in vegetative cells grown with combined nitrogen . After a switch to a medium without combined nitrogen, hetN expression first declined and was then followed by a rapid increase in its product, HetN, which was only present in mature heterocysts . HetN is located on both thylakoid membranes and plasma membranes as determined by immunoblotting using purified membranes . Overexpression of hetN completely prevented hetR up-regulation under nitrogen-deprivation conditions, suggesting that its role in pattern control may depend on its inhibition of hetR expression.

FEBS Lett, 2002 Apr 24, 517(1-3), 32 - 6
Carbohydrate carriers affect adhesion of H . pylori to immobilized Leb-oligosaccharide; Kojima N et al.; The present study involved comparison of adhesion of Helicobacter pylori KH202 to immobilized Le(b)-oligosaccharide carried on different carriers, i.e . Leb-oligosaccharide conjugated with polyacrylamide, bovine serum albumin, and dipalmitoylphosphatidylethanolamine (Le(b)-PAA, Le(b)-BSA, and Le(b)-DPPE) . All of the Le(b)-oligosaccharide-carrying neoglycoconjugates served as ligands for H . pylori . However, H . pylori required 10-fold and 100-fold quantities of Le(b)-antigen to adhere to Le(b)-PAA and to Le(b)-DPPE in comparison to the quantity of Le(b)-antigen needed to adhere to Le(b)-BSA, respectively . H . pylori adhesion to Le(b)-PAA and Le(b)-DPPE was clearly inhibited by Le(b)-oligosaccharide, but adhesion to Le(b)-BSA was hardly inhibited by the oligosaccharide . Therefore, the carbohydrate carrier affects the affinity of H . pylori KH202 toward Le(b)-antigen, although the bacteria recognize Le(b)-antigen regardless of the carbohydrate carrier.

Curr Biol, 2002 Jun 4, 12(11), 934 - 7
The TBP-inhibitory domain of TAF145 limits the effects of nonclassical transcriptional activators; Cheng JX et al.; Many genes in bacteria and eukaryotes are activated by "regulated recruitment" . According to that picture, a transcriptional activator binds cooperatively to DNA with the transcriptional machinery, and the constitutively active polymerase then spontaneously transcribes the gene . An important class of experiments that helped develop this model is called the "activator by-pass" experiment . In one version of such an experiment, the ordinary activator-transcriptional machinery interaction is replaced by a heterologous interaction . For example, fusing any of several DNA binding domains to Gal11, a component of the yeast mediator complex, creates a powerful activator of genes bearing the corresponding DNA binding sites . Here, we describe a simple modification of the yeast transcriptional machinery that extends the success of similar experiments involving other mediator components . The results reinforce parallels between regulation of enzymes involved in transcription and in other cellular processes.

Avian Dis, 2002 Apr-Jun, 46(2), 442 - 6
Investigation of the presence of Ornithobacterium rhinotracheale in chickens in Turkey and determination of the seroprevalance of the infection using the enzyme-linked immunosorbent assay; Turan N et al.; In this study, the presence of Ornithobacterium rhinotracheale infection in the avian population in the Marmara and the Western Black Sea region was investigated . Trachea samples were randomly obtained from 96 chickens sent to slaughterhouses . The seroprevalance of the infection was determined in 384 blood sera . Ninety-six of these 384 samples belonged to animals from which trachea samples were obtained . Eleven (11.46%) O . rhinotracheale were isolated in 96 trachea samples taken from 10 different flocks brought to the slaughterhouse . Serotype A was the predominant serotype among the 11 isolates of O . rhinotracheale . One isolate could not be serotyped . O . rhinotracheale antibodies were detected in 251 (64.4%) of the 384 sera, while 55 (14.3%) and 78 (20.3%) were suspected and negative, respectively.

J Med Entomol, 2002 May, 39(3), 417 - 26
Influence of nonsystemic transmission on the epidemiology of insect borne arboviruses: a case study of vesicular stomatitis epidemiology in the western United States; Lord CC et al.; Nonsystemic transmission, where a pathogen is transmitted between infected and uninfected vectors without the vertebrate host becoming viremic, may provide an explanation for transmission in systems where the vertebrate hosts have been difficult to identify . This transmission pathway had been previously demonstrated for tick-borne viruses and bacteria, but the recent demonstration for Simulium and vesicular stomatitis virus is the first for a blood-feeding insect . The epidemiology of vesicular stomatitis viruses has been difficult to understand, and nonsystemic transmission may be important . We use mathematical formulations of the basic reproduction number, R(0), to compare systemic and nonsystemic transmission . The absence of a latent period before host infectiousness in nonsystemic transmission may allow a more rapid increase in prevalence in the biting flies early in the development of a new outbreak . Aggregation of flies between hosts and at favored feeding sites on hosts will be important, but further data on nonsystemic transmission as a function of space and time are required to fully assess this pathway . The data needed to compare the two pathways and their relative roles in virus epidemiology are discussed.

Compend Contin Educ Dent, 2002 May, 23(5), 465 - 8, 470, 472 passim; quiz 476
The oral/dental/craniofacial complex as a model for inflammatory disease; Van Dyke TE; Many inflammatory diseases, including periodontitis, are the result of uncontrolled destruction of host tissues by the host's own inflammatory response . Periodontitis as well as other inflammatory diseases are initiated by bacteria that results from host cell-mediated tissue destruction . The study of inflammation of the oral cavity and craniofacial complex provides an excellent window to the understanding of more complex and difficult to study conditions . In this article, the inflammatory process in periodontitis is reviewed with an emphasis on control pathways that naturally limit inflammation.

Microb Ecol, 2002 Aug, 44(2), 164 - 74 Epub 2002 Jun 13.
Diel patterns of UVBR-induced DNA damage in picoplankton size fractions from the Gulf of Aqaba, Red Sea; Boelen P et al.; This study focuses on the impact of natural levels of UVBR (ultraviolet-B radiation: 280 to 315 nm) on bacterio- and phytoplankton (<10 microm) from the Gulf of Aqaba, Red Sea . Incident biologically effective doses (BEDs) and attenuation of biologically effective radiation in the water column were measured using a DNA biodosimeter . UVBR-induced DNA damage was measured as cyclobutane pyrimidine dimers (CPDs), using an antibody directed to CPDs followed by chemiluminescent detection . Depth profiles of DNA damage were determined in two plankton size fractions (0.2 to 0.8 microm and 0.8 to 10 microm) collected down to 50 m depth . Furthermore, accumulation and removal of CPDs were monitored in surface plankton samples during several daily cycles . Small plankton (plankton <10 microm) composition was determined by flow cytometry . The plankton community in the Gulf of Aqaba was dominated by nonphototrophic bacteria and the free-living prochlorophyte Prochlorococcus spp . (<0.8 microm) . In general, no DNA damage could be detected in dosimeter DNA below 15 m . In contrast, DNA damage (up to 124 CPD Mnucl-1) could be detected in all bacterio- and phytoplankton samples . DNA damage accumulated throughout the day, indicating that plankton in the Gulf of Aqaba undergo UVBR stress via CPD induction . Although the numbers of CPDs decreased during darkness, both size fractions showed some residual DNA damage at the end of the night . This suggests that dark repair processes did not remove all CPDs, or that part of the plankton community was incapable of repair at all . CPD levels in the two size fractions showed no significant differences in situ . During full solar radiation exposures (samples incubated in bags), more CPDs were detected in the smaller (0.2 to 0.8 microm) size fraction as compared to the larger (0.8 to 10 microm) size fraction . In these experiments, initial plankton composition was significantly different from the field samples . This implies that a shift in the population structure or irradiance conditions can lead to a significant change in UVBR sensitivity . In conclusion, the results show that the picoplankton-dominated phyto- and bacterioplankton communities in the clear surface waters from the Gulf of Aqaba undergo UVBR stress . Repair pathways are not sufficient to eliminate damage during or after UVBR exposure hours, suggesting photomortality as a potential loss parameter of the plankton community.

Proc Natl Acad Sci U S A, 2002 Jun 11, 99(12), 7980 - 5
Crystal structure of conserved hypothetical protein Aq1575 from Aquifex aeolicus; Shin DH et al.; The crystal structure of a conserved hypothetical protein, Aq1575, from Aquifex aeolicus has been determined by using x-ray crystallography . The protein belongs to the domain of unknown function DUF28 in the Pfam and PALI databases for which there was no structural information available until now . A structural homology search with the DALI algorithm indicates that this protein has a new fold with no obvious similarity to those of other proteins of known three-dimensional structure . The protein reveals a monomer consisting of three domains arranged along a pseudo threefold symmetry axis . There is a large cleft with approximate dimensions of 10 A x 10 A x 20 A in the center of the three domains along the symmetry axis . Two possible active sites are suggested based on the structure and multiple sequence alignment . There are several highly conserved residues in these putative active sites . The structure based molecular properties and thermostability of the protein are discussed.

Proc Natl Acad Sci U S A, 2002 Jun 11, 99(12), 7872 - 6
In vitro formation of a c-type cytochrome; Daltrop O et al.; C-type cytochromes are essential for almost all organisms; they are characterized by the covalent attachment of heme to protein through two thioether bonds to a Cys-Xaa-Xaa-Cys-His peptide motif . Here we show, contrary to opinion of 30 years standing, that a c-type cytochrome can form from heme and apoprotein in vitro under mild conditions and in the absence of any biosynthesis apparatus . This reaction occurs provided formation of a disulfide bond within the Cys-Xaa-Xaa-Cys-His motif is avoided . There are important implications for understanding in vivo cytochrome c assembly.

Proc Natl Acad Sci U S A, 2002 Jun 11, 99(12), 7866 - 71
In vitro and in vivo studies of a VEGF121/rGelonin chimeric fusion toxin targeting the neovasculature of solid tumors; Veenendaal LM et al.; Vascular endothelial growth factor (VEGF) plays a key role in the growth and metastasis of solid tumors . We generated a fusion protein containing VEGF(121) linked by a flexible G(4)S tether to the toxin gelonin (rGel) and expressed this as a soluble protein in bacteria . Purified VEGF(121)/rGel migrated as an 84-kDa homodimer under nonreducing conditions . VEGF(121)/rGel bound to purified, immobilized Flk-1, and the binding was competed by VEGF(121) . Both VEGF(121)/rGel and VEGF(121) stimulated cellular kinase insert domain receptor (KDR) phosphorylation . The VEGF(121)/rGel fusion construct was highly cytotoxic to endothelial cells overexpressing the KDR/Flk-1 receptor . The IC(50) of the construct on dividing endothelial cells expressing 10(5) or more KDR/Flk-1 receptors per cell was 0.5-1 nM, as compared with 300 nM for rGel itself . Dividing endothelial cells overexpressing KDR were approximately 60-fold more sensitive to VEGF(121)/rGel than were nondividing cells . Endothelial cells overexpressing FLT-1 were not sensitive to the fusion protein . Human melanoma (A-375) or human prostate (PC-3) xenografts treated with the fusion construct demonstrated a reduction in tumor volume to 16% of untreated controls . The fusion construct localized selectively to PC-3 tumor vessels and caused thrombotic damage to tumor vessels with extravasation of red blood cells into the tumor bed . These studies demonstrate the successful use of VEGF(121)/rGel fusion construct for the targeted destruction of tumor vasculature in vivo.

Plant J, 2002 Jan, 29(1), 61 - 2
Involvement of the Arabidopsis alpha-DOX1 fatty acid dioxygenase in protection against oxidative stress and cell death; De Leon IP et al.; alpha-dioxygenases (alpha-DOXs) catalyze the primary oxygenation of fatty acids into a newly identified group of oxylipins . Here we show that expression of the Arabidopsis alpha-DOX1 gene is induced in response to both incompatible and compatible bacterial infections . However, the level of alpha-DOX1 mRNA and dioxygenase activity appears earlier and reaches higher values when infection promotes a hypersensitive reaction . Furthermore, whereas gene expression is confined to necrotic lesions during the hypersensitive response, it occurs throughout the chlorotic area during a compatible interaction . Accumulation of alpha-DOX1 transcripts is impaired in SA-compromised plants and induced by SA and by chemicals generating nitric oxide (NO), intracellular superoxide or singlet oxygen, three signals mediating host cell death . Transgenic plants with altered levels of alpha-dioxygenase react like wild-type plants to a compatible pathogen . In contrast, plants with reduced activity develop a more rapid and severe necrotic response than wild-type plants to incompatible bacteria and paraquat treatment, respectively, and a milder response when alpha-DOX1 is overproduced . Our results suggest that plant alpha-dioxygenases are used to generate lipid-derived molecules for a process that protects plant tissues from oxidative stress and cell death.

Crit Rev Food Sci Nutr, 2002, 42(3 Suppl), 279 - 84
Inhibition of Helicobacter pylori adhesion to human gastric mucus by a high-molecular-weight constituent of cranberry juice; Burger O et al.; A high-molecular-weight constituent of cranberry juice has been found to inhibit the sialyllactose specific adhesion of Helicobacter pylori strains to immobilized human mucus, erythrocytes, and cultured gastric epithelial cells . Different isolates of H . pylori differ in their affinity to the cranberry juice constituent . Cranberry juice may also inhibit adhesion of bacteria to the stomach in vivo, and may prove useful for the prevention of stomach ulcer that is caused by H . pylori.

Waste Manag Res, 2002 Apr, 20(2), 143 - 9
Plant available nitrogen from anaerobically digested sludge and septic tank sludge applied to crops grown in the tropics; Sripanomtanakorn S et al.; Agricultural land is an attractive alternative for the disposal of biosolids since it utilises the recyclable nutrients in the production of crops . In Thailand and other tropical regions, limited field-study information exists on the effect of biosolids management strategies on crop N utilisation and plant available N (PAN) of biosolids . A field study was conducted to quantify the PAN of the applied biosolids, and to evaluate the N uptake rates of some tropical crops . Sunflower (Helianthus annuus) and tomato (Lycopersicon esculentum) were chosen in this study . Two types of biosolids used were: anaerobically digested sludge and septic tank sludge . The soil is acid sulfate and is classified as Sulfic Tropaquepts with heavy clay in texture . The anaerobically digested sludge applied rates were: 0, 156 and 312 kg N ha(-1) for the sunflower plots, and 0, 586, and 1172 kg N ha(-1) for the tomato plots . The septic tank sludge applied rates were: 0, 95 and 190 kg N ha(-1) for the sunflower plots, and 0, 354 and 708 kg N ha(-1) for the tomato plots, respectively . The results indicated the feasibility of applying biosolids to grow tropical crops . The applications of the anaerobically digested sludge and the septic tank sludge resulted in the yields of sunflower seeds and tomato fruits and the plant N uptakes comparable or better than that applied with only the chemical fertiliser . The estimated PAN of the anaerobically digested sludge was about 27-42% of the sludge organic N during the growing season . For the septic tank sludge, the PAN was about 15-58% of the sludge organic N . It is interesting to observe that an increase of the rate of septic tank sludge incorporated into this heavy clay soil under the cropping system resulted in the decrease of N mineralisation rate . This situation could cause the reduction of yield and N uptake of crops.

Folia Microbiol (Praha), 2002, 47(2), 131 - 6
Alpha,beta-unsaturated 4-oxo acids in heterocylic synthesis . II . Behavior of 3-(1,1-dioxadibenzothein-4-oyl)acrylic acid towards carbon nucleophiles under Michael reaction condition; Wasfy AA et al.; 3-(1,1-Dioxadibenzothien-4-oyl)acrylic acid (1) was condensed with compounds containing active methylene groups under Michael reaction conditions to furnish the Michael adducts (lactones 2a-c, lactams 3a-c, ketones 4a,b) . The behavior of these adducts toward the action of hydrazine hydrate were investigated . The compounds were tested for their biological properties.

Mol Biol Cell, 2002 Jun, 13(6), 2106 - 19
Novel ATPase of SNF2-like protein family interacts with androgen receptor and modulates androgen-dependent transcription; Rouleau N et al.; Nuclear receptors, including the androgen receptor (AR), regulate target cell transcription through interaction with auxiliary proteins to modify chromatin structure . We describe herein a novel AR-interacting protein, termed ARIP4, that has structural features typical of the SNF2-like protein family . With regard to the Snf2 domain, the closest homolog of ARIP4 is the ATRX protein . ARIP4 is a nuclear protein and comprises 1466 amino acids . It interacts with AR in vitro and in cultured yeast and mammalian cells . ARIP4 can be labeled with 8-azido-{gamma-32P}ATP and exhibits DNA-dependent ATPase activity . Like several ATP-dependent chromatin remodeling proteins, ARIP4 generates superhelical torsion within linear DNA fragments in an ATP-dependent manner . With a stably integrated target promoter, ARIP4 elicits a modest enhancement of AR-dependent transactivation . In transient cotransfection assays, ARIP4 modulates AR function in a promoter-dependent manner; it enhances receptor activity on minimal promoters, but does not activate more complex promoters . ARIP4 mutants devoid of ATPase activity fail to alter DNA topology and behave as trans-dominant negative regulators of AR function in transient assays.

J Biol Chem, 2002 Sep 13, 277(37), 33978 - 86 Epub 2002 Jun 10.
Microaerophilic conditions permit to mimic in vitro events occurring during in vivo Helicobacter pylori infection and to identify Rho/Ras-associated proteins in cellular signaling; Cottet S et al.; Molecular dissection of the mechanisms underlying Helicobacter pylori infection suffers from the lack of in vitro systems mimicking in vivo observations . A system was developed whereby human epithelial cells (Caco-2) grown as polarized monolayers and bacteria can communicate with each other under culture conditions optimal for each partner . Caco-2 cells grown on filter supports were inserted in a vertical position into diffusion chambers equilibrated with air and 5% CO(2) at their basolateral surface (aerophilic conditions) and 5% CO(2), 5% O(2), 90% N(2) (microaerophilic conditions) in the apical compartment . Remarkably, the epithelial polarized layer was stable under these asymmetric culture conditions for at least 24 h, and the presence of Caco-2 cells was necessary to maintain H . pylori growth . In contrast to previous studies conducted with non-polarized Caco-2 cells and other cell lines kept under aerophilic conditions, we found H . pylori-dependent stimulation of cytokine secretion (MCP-1 (monocyte chemoattractant protein-1), GRO-alpha (growth-regulated oncogene-alpha), RANTES (regulated on activation normal T cell expressed and secreted)) . This correlated with nuclear translocation of NF-kappaB p50 and p65 subunits . Tyrosine phosphorylation of nine cellular proteins was induced or enhanced; we identified p120(RasGAP), p190(RhoGAP), p62dok (downstream of tyrosine kinases), and cortactin as H . pylori-inducible targets . Moreover, reduction of H . pylori urease expression was observed in adherent bacteria as compared with bacteria in suspension . In addition to mimicking several observations seen in the inflamed gastric mucosa, the novel in vitro system was allowed to underscore complex cellular events not seen in classical in vitro analyses of microaerophilic bacteria-epithelial cell cross-talk.

J Bacteriol, 2002 Jul, 184(13), 3530 - 8
Roles for sigma factors in global circadian regulation of the cyanobacterial genome; Nair U et al.; The circadian clock of the unicellular cyanobacterium Synechococcus elongatus PCC 7942 imposes a global rhythm of transcription on promoters throughout the genome . Inactivation of any of the four known group 2 sigma factor genes (rpoD2, rpoD3, rpoD4, and sigC), singly or pairwise, altered circadian expression from the psbAI promoter, changing amplitude, phase angle, waveform, or period . However, only the rpoD2 mutation and the rpoD3 rpoD4 and rpoD2 rpoD3 double mutations affected expression from the kaiB promoter . A striking differential effect was a 2-h lengthening of the circadian period of expression from the promoter of psbAI, but not of those of kaiB or purF, when sigC was inactivated . The data show that separate timing circuits with different periods can coexist in a cell . Overexpression of rpoD2, rpoD3, rpoD4, or sigC also changed the period or abolished the rhythmicity of PpsbAI expression, consistent with a model in which sigma factors work as a consortium to convey circadian information to downstream genes.

Curr Opin Microbiol, 2002 Jun, 5(3), 318 - 22
Genomics to fluxomics and physiomics - pathway engineering; Sanford K et al.; Developments in microanalytical methods are enabling quantitative measurement of multiple metabolic fluxes and, in conjunction with transcript and proteomic profiling, are revolutionizing the ability of researchers to manipulate metabolism through pathway engineering in a variety of species . We review recent literature on the advances in genomics, proteomics, fluxomics and computational modeling focused on metabolic pathway engineering applications.

Biochemistry, 2002 Jun 18, 41(24), 7725 - 31
Vinylphosphonate internucleotide linkages inhibit the activity of PcrA DNA helicase; Bertram RD et al.; During the past 5 years a great deal of structural and biochemical information has given us a detailed insight into the molecular mechanism of action of the PcrA DNA helicase and challenged previous notions about the molecular mechanism of action of helicases in general . Despite this wealth of information the mechanisms of the interaction of helicases with their DNA substrates and their unidirectional translocation along ssDNA are poorly understood . In this study, we synthesized a chemically modified DNA substrate with reduced backbone rotational flexibility and minimal steric hindrance and studied its effect on the activity of the monomeric 3'-5' DNA helicase, PcrA . Our results show that a single modification on the backbone of the translocating strand is sufficient to inhibit the activity of PcrA helicase, suggesting that rotational flexibility of the backbone is important for efficient unwinding.

Med Hypotheses, 2002 May, 58(5), 411 - 5
Alcohol consumption in the absence of adequate nutrition may lead to activation of the glyoxylate cycle in man; Kokavec A et al.; The consumption of alcohol prior to food intake results in alcohol metabolism occurring in the liver, and the liver is often damaged in chronic alcoholics . This paper highlights the possibility that alcohol consumption in the absence of adequate nutrition after an extended period of time may lead to activation of the glyoxylate cycle, an energy pathway associated with the conversion of fat into carbohydrate which until recently was thought to only exist in plants and bacteria .

Med Hypotheses, 2002 May, 58(5), 382 - 5
Probiotics as an adjuvant to detoxification protocols; Brudnak MA; Autism is a developmental disease characterized by a spectrum of symptoms ranging from decreased verbal skills and social withdrawal, to repetitive behavior and violent outbursts . Genetic analysis has yielded a few potentially interesting genes, however no clear linkage has been established . For this reason, it has been suggested that the etiology of autism may involve multiple loci . This, in large part, explains why so many different theories abound . One such theory is that of mercury poisoning . Environmentally acquired mercury, either through some causal contact or through vaccination, has been postulated as the culprit . Mercury is thought to be exerting its neurological effect on the brain . The standard treatment has been to apply chelating agents in an attempt to extricate the mercury . One missing component in the treatment is the utilization of the body's own detoxification mechanisms . Arguably the largest detoxification component of the body, the endogenous enteric bacteria are an enormous reservoir, which can be constantly and safely replenished . This paper discusses the use of high-dose probiotics as an adjuvant for detoxification protocols with an emphasis on use in autistics .

J Comp Pathol, 2002 May, 126(4), 243 - 53
Post-mortem findings and causes of death of harbour porpoises (Phocoena phocoena) stranded from 1990 to 2000 along the coastlines of Belgium and Northern France; Jauniaux T et al.; Between the years 1990 and 2000, an attempt was made to determine the causes of death of 55 harbour porpoises stranded along the Belgian and northern French coasts . From 1990 to 1996, only five carcasses were collected as against seven in 1997, eight in 1998, 27 in 1999 and eight in 2000 . The sex ratio was normal and most of the animals were juvenile . The most common findings were emaciation, severe parasitosis and pneumonia . A few cases of fishing net entanglement were observed . The main microscopical lesions were acute pneumonia, massive lung oedema, enteritis, hepatitis and gastritis . Encephalitis was observed in six cases . No evidence of morbillivirus infection was detected . Pneumonia was associated with bacteria or parasites, or both . The causes of death and the lesions were similar to those previously reported in other countries bordering the North Sea . The cause of the increased numbers of carcasses in 1999 was unclear but did not include viral epizootics or net entanglement . A temporary increase in the porpoise population in the southern North Sea may have been responsible .

Gastroenterol Nurs, 2002 May-Jun, 25(3), 114 - 9
Establishing the shelf life of flexible colonoscopes; Riley R et al.; This study was designed to establish a shelf life for processed (peracetic acid) flexible colonoscopes and to demonstrate the adequacy of manual cleaning procedures . The study challenges current practice in Australia where endoscopes are routinely reprocessed prior to use if the endoscope has been sitting at least overnight . The design was a simulated study in the clinical environment and involved artificial contamination of a colonoscope, cleaning validation, and a time series analysis after processing with peracetic acid in a Steris System 1 processor and drying with 100% isopropyl alcohol . The main outcome measure was the presence of bacteria in the internal channels of the colonoscope after cleaning and at 24 and 168 hours after processing with peracetic acid . The findings suggest that colonoscopes may be left for up to 1 week before needing to be reprocessed before use, provided all channels are thoroughly reprocessed and dried, resulting in cost savings and reduced wear and tear on the instruments . The findings also demonstrate the impact of providing feedback to staff on a regular basis about the efficacy of their cleaning techniques.

Microbiology, 2002 Jun, 148(Pt 6), 1947 - 57
Molecular characterization and endosymbiotic localization of the gene encoding D-ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) form II in the deep-sea vestimentiferan trophosome; Elsaied H et al.; To better understand the contribution of micro-organisms to the primary production in the deep-sea gutless tubeworm Lamellibrachia sp., the 16S-rDNA-based phylogenetic data would be complemented by knowledge of the genes that encode the enzymes relevant to chemoautotrophic carbon fixation, such as D-ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO; EC 4.1.1.39) . To phylogenetically characterize the autotrophic endosymbiosis within the trophosome of the tubeworm Lamellibrachia sp., bulk trophosomal DNA was extracted and analysed based on the 16S-rRNA- and RuBisCO-encoding genes . The 16S-rRNA- and RuBisCO-encoding genes were amplified by PCR, cloned and sequenced . For the 16S rDNA, a total of 50 clones were randomly selected and analysed directly by sequencing . Only one operational taxonomic unit resulted from the 16S rDNA sequence analysis . This may indicate the occurrence of one endosymbiotic bacterial species within the trophosome of the Lamellibrachia sp . used in this study . Phylogenetic analysis of the 16S rDNA showed that the Lamellibrachia sp . endosymbiont was closely related to the genus Rhodobacter, a member of the alpha-Protebacteria . For the RuBisCO genes, only the form II gene (cbbM) was amplified by PCR . A total of 50 cbbM clones were sequenced, and these were grouped into two operational RuBisCO units (ORUs) based on their deduced amino acid sequences . The cbbM ORUs showed high amino acid identities with those recorded from the ambient sediment bacteria . To confirm the results of sequence analysis, the localization of the symbiont-specific 16S rRNA and cbbM sequences in the Lamellibrachia sp . trophosome was visualized by in situ hybridization (ISH), using specific probes . Two types of cells, coccoid and filamentous, were observed at the peripheries of the trophosome lobules . Both the symbiont-specific 16S rDNA and cbbM probes hybridized at the same sites coincident with the location of the coccoid cells, whereas the filamentous cells showed no cbbM-specific signals . The RuBisCO form I gene (cbbL) was neither amplified by PCR nor detected by ISH . This is the first demonstration of chemoautotrophic symbiosis in the deep-sea gutless tubeworm, based on sequence data and in situ localization of both the 16S-rRNA- and RuBisCO-encoding genes.

Microbiology, 2002 Jun, 148(Pt 6), 1767 - 76
A novel extracytoplasmic phenol oxidase of Streptomyces: its possible involvement in the onset of morphogenesis; Endo K et al.; Exogenous addition of copper stimulates cellular differentiation in Streptomyces spp . Several lines of evidence suggested a parallel correlation between the stimulatory effect of copper and phenol-oxidizing enzyme activities in Streptomyces griseus . Here a novel extracytoplasmic phenol oxidase (EpoA) associated with cellular development of this organism was identified and characterized . EpoA activity, examined by an in-gel stain procedure with N,N'-dimethyl-p-phenylenediamine sulfate as a substrate, was repressed by glucose and induced by copper supplied in the medium . The enzyme activity was abolished and markedly reduced in the mutants forA-factor biosynthesis and amfR, respectively, which suggested that the activity of the enzyme depends on those essential regulators for morphogenesis in S . griseus . EpoA protein was purified to homogeneity and the N-terminal amino acid sequence was determined . A homologous sequence identified in the genomic database of Streptomyces coelicolorA3(2) was used as a probe to clone the complete epoA gene of S . griseus . The deduced amino acid sequence of EpoA revealed that the mature protein with a molecular mass of 34 kDa was preceded by a signal peptide consisting of 34 aa, consistent with EpoA being a secreted enzyme . EpoA was predicted to be a laccase-type oxidase by not only the sequence similarity, but its substrate selectivity, oxidizing not tyrosine but dihydroxyphenylalanine (DOPA) to generate melanin pigment . Introduction of epoA on a plasmid partially restored both the EpoA activity and aerial mycelium productivity in an A-factor-deficient mutant . Exogenous supplementation of a substance synthesized by purified EpoA from DOPA stimulated cellular differentiation in S . griseus and several other species . Ultrafiltration indicated that the molecular mass of the putative stimulant synthesized by EpoA is between 500 and 1000 Da.

Microbiology, 2002 Jun, 148(Pt 6), 1667 - 77
Role of the Legionella pneumophila rtxA gene in amoebae; Cirillo SL et al.; Legionella pneumophila infects humans, causing Legionnaires' disease, from aerosols generated by domestic and environmental water sources . In aquatic environments L . pneumophila is thought to replicate primarily in protozoa . A 'repeats in structural toxin' (RTX) gene, rtxA, from L . pneumophila was identified recently that plays a role in entry and replication in human macrophages and also has the ability to infect mice . However, the role of this gene in the interaction of L . pneumophila with environmental protozoa and its distribution in different Legionella species has not been examined . Southern analyses demonstrated that rtxA is present in all L . pneumophila isolates tested and correlates with species that have been shown to cause disease in humans . To evaluate the importance of rtxA in the interaction with protozoa a series of studies was carried out in an environmental host for L . pneumophila, Acanthamoeba castellanii . The L . pneumophila rtxA gene plays a role in both adherence and entry into A . castellanii similar to that observed in human monocytic cells . Furthermore, it was found that rtxA is involved in intracellular survival and trafficking . In addition to demonstrating involvement of rtxA in the interaction of L . pneumophila with host cells, these data support a role for this gene both during disease in humans and in environmental reservoirs.

Am J Physiol Cell Physiol, 2002 Jul, 283(1), C155 - 68
An ancient prevertebrate Na+-nucleoside cotransporter (hfCNT) from the Pacific hagfish (Eptatretus stouti); Yao SY et al.; The human concentrative (Na+-linked) plasma membrane transport proteins hCNT1, hCNT2, and hCNT3 are pyrimidine nucleoside-selective (system cit), purine nucleoside-selective (system cif), or broadly selective for both pyrimidine and purine nucleosides (system cib), respectively . All have orthologs in other mammalian species and belong to a gene family (CNT) that has members in insects, nematodes, pathogenic yeast, and bacteria . Here, we report the cDNA cloning and functional characterization of a CNT family member from an ancient marine prevertebrate, the Pacific hagfish (Eptatretus stouti) . This Na+-nucleoside symporter, designated hfCNT, is the first transport protein to be characterized in detail in hagfish and is a 683-amino acid residue protein with 13 predicted transmembrane helical segments (TMs) . hfCNT was 52, 50, and 57% identical in sequence to hCNT1, hCNT2, and hCNT3, respectively . Similarity to hCNT3 was particularly marked in the TM 4-13 region . When produced in Xenopus oocytes, hfCNT exhibited the transport properties of system cib, with uridine, thymidine, and inosine apparent K(m) values of 10-45 microM . The antiviral nucleoside drugs 3'-azido-3'-deoxythymidine, 2',3'-dideoxycytidine, and 2',3'-dideoxyinosine were also transported . Simultaneous measurement of uridine-evoked currents and radiolabeled uridine uptake under voltage-clamp conditions gave a Na+-to-uridine coupling ratio of 2:1 (cf . 2:1 for hCNT3 and 1:1 for hCNT1/2) . The apparent K50 value for Na+ activation was >100 mM . A 50:50 chimera between hfCNT and hCNT1 (TMs 7-13 of hfCNT replaced by those of hCNT1) exhibited hCNT1-like cation interactions, establishing that the structural determinants of cation stoichiometry and binding affinity were located within the carboxy-terminal half of the protein . The high degree of sequence similarity between hfCNT and hCNT3 may indicate functional constraints on the primary structure of the transporter and suggests that cib-type CNTs fulfill important physiological functions.

J Mol Biol, 2002 May 3, 318(3), 837 - 45
Water contributes actively to the rapid crossing of a protein unfolding barrier; Jacob MH et al.; The cold-shock protein CspB folds rapidly in a N <= => U two-state reaction via a transition state that is about 90% native in its interactions with denaturants and water . This suggested that the energy barrier to unfolding is overcome by processes occurring in the protein itself, rather than in the solvent . Nevertheless, CspB unfolding depends on the solvent viscosity . We determined the activation volumes of unfolding and refolding by pressure-jump and high-pressure stopped-flow techniques in the presence of various denaturants . The results obtained by these methods agree well . The activation volume of unfolding is positive (Delta V(++)(NU)=16(+/-4) ml/mol) and virtually independent of the nature and the concentration of the denaturant . We suggest that in the transition state the protein is expanded and water molecules start to invade the hydrophobic core . They have, however, not yet established favorable interactions to compensate for the loss of intra-protein interactions . The activation volume of refolding is positive as well (Delta V(++)(NU)=53(+/-6) ml/mol) and, above 3 M urea, independent of the concentration of the denaturant . At low concentrations of urea or guanidinium thiocyanate, Delta V(++)(UN) decreases significantly, suggesting that compact unfolded forms become populated under these conditions . (c) 2002 Elsevier Science Ltd.

Arch Biochem Biophys, 2002 May 15, 401(2), 145 - 54
Diversity of properties among catalases; Switala J et al.; Catalases from 16 different organisms including representatives from all three phylogenetic clades were purified and characterized to provide a comparative picture of their respective properties . Collectively the enzymes presented a diverse range of activities and properties . Specific activities ranged from 20,700 to 273,800 units per milligram of protein and maximal turnover rates ranged from 54,000 to 833,000 per second . The effective concentrations of common catalase inhibitors, cyanide, azide, hydroxylamine, aminotriazole, and mercaptoethanol, varied over a 100- to 1000-fold concentration range, and a broad range of sensitivities to heat inactivation was observed . Michaelis-Menten kinetics were approximately followed only at the low substrate concentrations . At high H(2)O(2) concentrations, inactivation of small-subunit enzymes resulted in lower velocities than what were predicted, whereas large-subunit enzymes had velocities higher than predicted . Kinetic constants such as K(m) and V(max) for catalases must be labeled as "apparent." (c) 2002 Elsevier Science (USA).

Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 987 - 93
Okibacterium fritillariae gen . nov., sp . nov., a novel genus of the family Microbacteriaceae; Evtushenko LI et al.; Okibacterium fritillariae gen . nov., sp . nov . (type strain VKM Ac-2059T = IFO 16404T) is proposed for aerobic, oxidase- and catalase-positive, coryneform bacteria isolated from seeds of Fritillaria ruthenica Wikstr . and Clematis recta L . Strains of the new genus form a distinct branch within the phylogenetic cluster composed of members of the family Microbacteriaceae and are characterized by B-type peptidoglycan containing amino acids glycine, glutamate, homoserine, alanine and lysine, the glycolyl type of muramic acid, the major menaquinones MK-10 and MK-11, the principal phospholipids phosphatidylglycerol and diphosphatidylglycerol, and a DNA G+C content of approximately 67 mol %.

Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 693 - 8
'Candidatus mycoplasma haemodidelphidis' sp . nov., 'Candidatus mycoplasma haemolamae' sp . nov . and Mycoplasma haemocanis comb . nov., haemotrophic parasites from a naturally infected opossum (Didelphis virginiana), alpaca (Lama pacos) and dog (Canis familiaris): phylogenetic and secondary structural relatedness of their 16S rRNA genes to other mycoplasmas; Messick JB et al.; The 16S rRNA sequence of newly characterized haemotrophic bacteria in an opossum (Didelphis virginiana) and alpaca (Lama pacos) was determined . In addition, the 16S rRNA sequence of a haemotrophic parasite in the dog (Canis familiaris) was determined . Sequence alignment and evolutionary analysis as well as secondary structural similarity and signature nucleotide sequence motifs of their 16S rRNA genes, positioned these organisms in the genus Mycoplasma . The highest scoring sequence similarities were 16S rRNA genes from haemotrophic mycoplasma species (Haemobartonella and Eperythrozoon spp.) . However, the lack of several higher-order structural idiosyncrasies used to define the pneumoniae group, suggests that these organisms and related haemotrophic mycoplasmas represent a new group of mycoplasmas . It is recommended that the organisms be named 'Candidatus Mycoplasma haemodidelphidis', 'Candidatus Mycoplasma haemolamae' and Mycoplasma haemocanis comb . nov., to provide some indication of the target cell and host species of these parasites, and to reflect their phylogenetic affiliation.

Tsitologiia, 2002, 44(2), 218 - 23
{Aging as a metagenetic process}; Vakhmin IuB; Genomes of eukaryotic cells are so complicated that spontaneous processes lead inevitably to a continuous formation of egoistic genetic elements from the normal ones . These elements convert the intracellular Cosmos into Chaos and therefore they can be named chaonogenes . They behave as endogenous genetic parasites and are able to evaluate . The rate of their evolution is very rapid, which unevitably results in senescence and death of not only cells and multicellular organisms but also of populations and species, because chaonogens are transmitted from somatic cells to gametes . Populations of chaonogenes are very sensitive to environmental changes, and different sets of intracellular or extracellular changes are commonly used in nature to put obstracles in deleterious evolution of chaonogenes or to stop their evolution . These changes can be moderate (as at mitosis) or crude (as at meiosis), or they can be predicted (as programmed biochemical changes in the course of mitosis, meiosis and gametogenesis) or unpredicred (mutations, somatic crossingover, random association of gamets), but in all the cases they lead eventually to some degree of rejuvenation . In somatic cell populations, the process of senescence in slowed down by means of epigenetically determined changes and mitotic divisions, at which both kinds of changes (programmed and accidental) are moderate, and for this reason only a small part of dividing cells dies . At meiosis both kinds of changes are so acute that the majority of cells die, but the formation of gametes and zygotes becomes almost completely rejuvenated . Only mutations leading to very acute changes in intracellular conditions (whose products act on chaonogenes similarly as new antibiotics on bacteria) can save aging populations of multicellular organisms from death (as do L . N . Gumilev's "mutations of passionarity"), and only accidentally appearing "catastrophic" macromutations can give rise to new (and, of the same time, young) species . It is concluded that the induction of acute temporal biochemical changes in the inner environment is to slow down processes of human senescence and to lead to rejuvenation.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(1), 123 - 127
The Expression of Adenoviral Mediated gfp Reporter Gene in Tumor Cells Induced and Regulated by Irradiation; Wei DY et al.; In order to establish a radiation inducible gene expression system for cancer gene therapy, the promoter sequence of radiation-inducible Egr-1 gene was amplified from genomic DNA of BALB/c mouse with PCR method, and linked to gfp reporter gene . Then the pEgr-gfp expression cassette was subcloned into an adenoviral shuttle plasmid to generate recombinant adenovirus of AdEgr-GFP by using a novel, high efficient method of homologous recombination in bacteria . After infection with AdEgr-GFP, MM45T.Li tumor cells were exposed to different doses of gamma-irradiation from 0 Gy to 15 Gy in vitro . The percentage of GFP expression positive cells increased greatly in a dose-dependent manner as detected by FACS and Western blot analysis . For in vivo study, AdEgr-GFP were injected intratumorally, and tumor site received different doses of local gamma-irradiation 48 h after injection, and after 8 h the tumor samples were biopsed for investigating the GFP expression . Tumor tissue image analysis revealed that gamma-irradiation could markedly increase GFP expression in a dose-dependent manner as compared with that of non-irradiated control group . Our results indicate that the irradiation can effectively control adenoviral-mediated GFP expression in tumor cells via Egr-1 promoter, and these data laid basis for further gene radiotherapy study.

Phytochemistry, 2002 Jul, 60(5), 437 - 40
The function of trehalose biosynthesis in plants; Wingler A; Trehalose (alpha-D-glucopyranosyl-1,1-alpha-D-glucopyranoside) occurs in a large variety of organisms, ranging from bacteria to invertebrate animals, where it serves as an energy source or stress protectant . Until recently, only few plant species, mainly desiccation-tolerant 'resurrection' plants, were considered to synthesise trehalose . Instead of trehalose, most other plants species accumulate sucrose as major transport sugar and during stress . The ability to synthesize sucrose has probably evolved from the cyanobacterial ancestors of plastids and may be linked to photosynthetic function . Although most plant species do not appear to accumulate easily detectable amounts of trehalose, the discovery of genes for trehalose biosynthesis in Arabidopsis and in a range of crop plants suggests that the ability to synthesise trehalose is widely distributed in the plant kingdom . The apparent lack of trehalose accumulation in these plants is probably due to the presence of trehalase activity . After inhibition of trehalase, trehalose synthesis can be detected in Arabidopsis . Since trehalose induces metabolic changes, such as an accumulation of storage carbohydrates, rapid degradation of trehalose may be required to prevent detrimental effects of trehalose on the regulation of plant metabolism . In addition, the precursor of trehalose, trehalose-6-phosphate, is probably involved in the regulation of developmental and metabolic processes in plants.

Curr Top Med Chem, 2002 Jul, 2(7), 701 - 15
Therapy and prevention of parasitic insects in veterinary medicine using imidacloprid; Mencke N et al.; Ectoparasitic insects play a major role in veterinary medicine . The flea, especially the cat flea (Ctenocephalides felis felis Bouch 1835) is the most important ectoparasite worldwide . The cat flea parasitizes not only on dogs and cats but also on other warm-blooded animals including humans . The veterinary importance of flea infestation are dermatological conditions due to allergic reactions to antigens in the flea saliva and the transmission of infectious agents like bacteria, viruses and helminths . Insecticides used in veterinary medicine today have to fulfil criteria of elimination of a existing flea infestation (therapy) and prevention (prophylaxis) of new infestation for weeks . Imidacloprid is a compound of the chemical class of CNI (chloronicotinyl insecticides syn . neonicotinoids) that fits these criteria . The high selectivity towards the site of action within insects together with the high safety margin on mammals allowed to develop imidacloprid as an insecticide for agricultural use and finally for the application as a veterinary medicine . The major features of imidacloprid chemistry, toxicology and the development and use as a veterinary medicinal remedy are described.

J Mol Biol, 2002 May 31, 319(2), 463 - 77
Crystal structure of the autocatalytic initiator of glycogen biosynthesis, glycogenin; Gibbons BJ et al.; Glycogen is an important storage reserve of glucose present in many organisms, from bacteria to humans . Its biosynthesis is initiated by a specialized protein, glycogenin, which has the unusual property of transferring glucose from UDP-glucose to form an oligosaccharide covalently attached to itself at Tyr194 . Glycogen synthase and the branching enzyme complete the synthesis of the polysaccharide . The structure of glycogenin was solved in two different crystal forms . Tetragonal crystals contained a pentamer of dimers in the asymmetric unit arranged in an improper non-crystallographic 10-fold relationship, and orthorhombic crystals contained a monomer in the asymmetric unit that is arranged about a 2-fold crystallographic axis to form a dimer . The structure was first solved to 3.4 A using the tetragonal crystal form and a three-wavelength Se-Met multi-wavelength anomalous diffraction (MAD) experiment . Subsequently, an apo-enzyme structure and a complex between glycogenin and UDP-glucose/Mn2+ were solved by molecular replacement to 1.9 A using the orthorhombic crystal form . Glycogenin contains a conserved DxD motif and an N-terminal beta-alpha-beta Rossmann-like fold that are common to the nucleotide-binding domains of most glycosyltransferases . Although sequence identity amongst glycosyltransferases is minimal, the overall folds are similar . In all of these enzymes, the DxD motif is essential for coordination of the catalytic divalent cation, most commonly Mn2+ . We propose a mechanism in which the Mn2+ that associates with the UDP-glucose molecule functions as a Lewis acid to stabilize the leaving group UDP and to facilitate the transfer of the glucose moiety to an intermediate nucleophilic acceptor in the enzyme active site, most likely Asp162 . Following transient transfer to Asp162, the glucose moiety is then delivered to the final acceptor, either directly to Tyr194 or to glucose residues already attached to Tyr194 . The positioning of the bound UDP-glucose far from Tyr194 in the glycogenin structure raises questions as to the mechanism for the attachment of the first glucose residues . Possibly the initial glucosylation is via inter-dimeric catalysis with an intra-molecular mechanism employed later in oligosaccharide synthesis .

Fungal Genet Biol, 2002 Jun, 36(1), 71 - 83
Endopolygalacturonase is encoded by a multigene family in the basidiomycete Chondrostereum purpureum; Williams HL et al.; The basidiomycete Chondrostereum purpureum produces several plant cell wall-degrading enzymes, including endopolygalacturonase (endoPG) . Degenerate oligonucleotide primers were designed according to conserved regions of endoPG genes from various fungi, plants, and bacteria and used to amplify members of this gene family from C . purpureum . Four different amplification products showed significant similarity to known endoPGs and were used as hybridization probes to screen a library of genomic DNA sequences and to retrieve five full-length endoPG genes (epgA, epgB1, epgB2, epgC, and epgD) . The identities between the deduced polypeptides for epgA, epgB1, epgC, and epgD ranged from 61.8 to 80.0%, while the deduced polypeptides for epgB1 and epgB2 shared 97.1% identity . Phylogenetic analysis suggested that the duplication of existing endoPG genes occurred after the divergence of the ascomycetes and basidiomycetes . C . purpureum is the first basidiomycete fungus for which the endoPG gene family has been described.

Environ Res, 2002 May, 89(1), 58 - 65
Airborne fungi and endotoxin concentrations in different areas within textile plants in Taiwan: a 3-year study; Su HJ et al.; Bioaerosols have been found in many occupational environments, including animal feeding houses, poultry slaughter houses, and cotton textile plants . This study was undertaken to examine a group of bioaerosols, the endotoxins, fungi, and bacteria, inside two textile factories over 3 years in Taiwan, where temperature and humidity are usually high year-round . Airborne dust was collected with filter cassettes attached to personal pumps and analyzed by the Kinetic Limulus Assay with Resistant-parallel-line Estimation . For fungi and bacteria determination, samples were collected using duplicated single-stage impactors, and organisms were counted after incubation . Endotoxin was the major contamination inside textile plants . Indoor levels were substantially higher than outdoor concentrations by 63- to 278-fold . The average values of fungi inside and outside the plants were not significantly different . Airborne bacteria levels were higher inside the plants as compared to outside . The carding sites, using only cotton, had extremely high endotoxin levels, greater than those at sites using synthetic fibers . Cotton, may be a major source of endotoxin contamination . In conclusion, the early stage of textile processing seems to generate high endotoxin and bacteria contamination . Priorities should be given to occupational hygiene programs for workers at various sites in textile plants . 2002 Elsevier Science (USA)

Biochem Biophys Res Commun, 2002 May 10, 293(3), 892 - 8
AMP-activated protein kinase kinase: detection with recombinant AMPK alpha1 subunit; Hamilton SR et al.; The AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase important for the responses to metabolic stress . It consists of a catalytic alpha subunit and two non-catalytic subunits, beta and gamma, and is regulated both by the allosteric action of AMP and by phosphorylation of the alpha and beta subunits catalyzed by AMPKK(s) and autophosphorylation . The Thr172 site on the alpha subunit has been previously characterized as an activating phosphorylation site . Using bacterially expressed AMPK alpha1 subunit proteins, we have explored the role of Thr172-directed AMPKKs in alpha subunit regulation . Recombinant alpha1 subunit proteins, representing the N-terminus, have been expressed as maltose binding protein (MBP) 6x His fusion proteins and purified to homogeneity by Ni(2+) chromatography . Both wild-type alpha1(1-312) and alpha1(1-312)T172D are inactive when expressed in bacteria, but the former can be fully phosphorylated (1 mol/mol) on Thr172 and activated by a surrogate AMPKK, CaMKKbeta . The corresponding AMPKalpha1(1-392), an alpha construct containing its autoinhibitory sequence, can be similarly phosphorylated, but it remains inactive . In an insulinoma cell line, either low glucose or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) treatment leads to activation and T172 phosphorylation of endogenous AMPK . Under the same conditions of cell incubation, we have identified an AMPKK activity that both phosphorylates and activates the recombinant alpha1(1-312), but this Thr172-directed AMPKK activity is unaltered by low glucose or AICAR, indicating that it is constitutively active . (c) 2002 Elsevier Science (USA).

Clin Exp Rheumatol, 2002 Mar-Apr, 20(2), 127 - 32
The incidence and clinical characteristics of Mycobacterium tuberculosis infection among systemic lupus erythematosus and rheumatoid arthritis patients in Korea; Yun JE et al.; OBJECTIVES: The aim of this study was to describe the incidence and clinical characteristics of Mycobacterium tuberculosis infection in SLE and RA patients in Korea where the prevalence rate of active pulmonary tuberculosis in a general population is relatively higher than in Western countries . PATIENTS: We reviewed the medical records of 283 SLE and 284 RA patients retrospectively and then assessed the incidence, risk factors, and clinical characteristics of active tuberculous infection . We then compared the results for the two different groups . RESULTS: Tuberculosis was documented in 15 SLE and 7 RA patients with an incidence rate of 7.9/1,000 patient-years and 2.3/1,000 patient-years, respectively (p = 0.003) . SLE-associated tuberculosis cases included 3 of miliary tuberculosis, 7 of pulmonary tuberculosis (including 1 case of diffuse pulmonary involvement with meningitis) predominantly involving two or more lobes at the mid-/lower lungfield, and 5 extra-pulmonary forms (joint, bone, kidney, larynx, pleura) . All of the RA-associated tuberculosis cases were pulmonary forms with the majority being localized to single lobe, and only one case had a past history of tuberculosis, whereas a past history of tuberculosis and a longer duration of the underlying disease were significantly correlated with the development of tuberculosis in the SLE patients . Major organ involvement, the mean daily dosage of prednisolone, and a history of over 30 mg of daily prednisolone were not related to the development of tuberculosis . However, when we took only those patients taking corticosteroid until the diagnosis of tuberculosis for analysis, SLE patients with tuberculosis showed a higher daily dosage of prednisolone than those without tuberculosis . CONCLUSION: Taken together, the characteristics of tuberculosis in SLE patients were: (1) a higher incidence rate, (2) more frequent extra-pulmonary involvement, (3) more extensive pulmonary involvement, and (4) a higher relapse rate than in rheumatoid arthritis . Thus, the contributory role of M . tuberculosis infection in the morbidity and mortality of patients with SLE must be emphasized, especially in areas in which this bacteria is endemic.

Magy Onkol, 2001, 45(1), 39 - 44
{Clinical features and guidelines of treatment of mucosa-associated lymphoid tissue lymphoma of stomach}; Schneider T et al.; The mucosa-associated lymphoid tissue (MALT) lymphoma is a very indolent disease . Its most common site is the stomach . The lymphoma begins as a reactive lymphocyte accumulation mostly due to an infection of Helicobacter pylori (HP) . Through repeated mutations this tissue is transformed into the characteristic MALT lymphoma . At the time of the diagnosis the lymphoma is usually localised, but in one third of the patients the disease has already been disseminated . There are not any commonly accepted guidelines of therapy concerning this primary gastric MALT lymphoma, but certain general tendencies have already been defined . In the early disease the aim of the treatment is curative with the preservation of the stomach as much as possible . In a considerable number of cases, when the surface of the stomach is affected by HP, one can achieve histological and molecular biologic remission after eliminating the bacteria . However, there is no such therapeutic consequence to be expected in case of a deeply invasive tumour . The optimal treatment of patients of this group as well as those whose disease is resistant to HP eradication treatment together with those who are HP negative is radiotherapy or surgery with chemotherapy . In this latter case quality of life becomes worse . In an advanced case cure is impossible and chemotherapy is the most effective to ease the patient's state.

Trends Plant Sci, 2002 Jun, 7(6), 251 - 6
Flagellin perception: a paradigm for innate immunity; Gomez-Gomez L et al.; There are surprising similarities between how animals and plants perceive pathogens . In animals, innate immunity is based on the recognition of pathogen-associated molecular patterns . This is mediated by the Toll-like receptor (TLR) family, which rapidly induce the innate immunity response, a first line of defence against infectious disease . Plants have highly sensitive perception systems for general elicitors and they respond to these stimuli with a defence response . One of these general elicitors is flagellin, the main component of the bacterial flagellum . Genetic analysis in Arabidopsis has shown that FLS2, which encodes a receptor-like kinase, is essential for flagellin perception . FLS2 shares homology with the TLR family, and TLR5 is responsible for flagellin perception in mammals.

J Immunother, 2002 Mar-Apr, 25 Suppl 1, S35 - 41
Construction and analysis of new vector systems with improved interleukin-18 secretion in a xenogeneic human tumor model; Goto H et al.; Interleukin (IL)-18 plays an important role in enhancing cellular immunity against cancer and bacteria . We constructed retroviral and adenoviral vectors that show improved secretion of bioactive murine IL-18 that could further enhance antitumor immunity in a murine model . Secretion of bioactive IL-18 was facilitated by fusing the leader sequences of prepro-parathyroid hormone (PTH) or IL-1 receptor antagonist (IL-1ra) to the 5; end of the mature murine IL-18 cDNA . Transfectants established by the retroviral vector carrying IL-1ra/IL-18 hybrid showed about 100-fold more IL-18 production and interferon (IFN)-gamma induction from splenocytes when compared with those carrying PTH/IL-18 hybrid . Repeated intraperitoneal injection of an adenoviral vector with IL-1ra/IL-18 hybrid ligated to IL-18 (Ad.IL-1ra.IL-18) successfully prevented establishment of human colon cancer cells in the abdominal cavity of mice . Treatment with Ad.IL-1ra.IL-18 was associated with significantly elevated levels of serum IL-18 and IFN- gamma . IL-18 administration also enhanced the cytostatic activity of peritoneal exudate cells against cancer cells . These improved viral vectors, which efficiently produce bioactive IL-18, could be used as a useful tool for cancer gene therapy.

Proc Natl Acad Sci U S A, 2002 Jun 11, 99(12), 7974 - 9 Epub 2002 Jun 04.
Preferential cleavage of plasmid-based R-loops and D-loops by Drosophila topoisomerase IIIbeta; Wilson-Sali T et al.; The topoisomerase (topo) III enzymes are found in organisms ranging from bacteria to humans, yet the precise cellular function of these enzymes remains to be determined . We previously found that Drosophila topo IIIbeta can relax plasmid DNA only if the DNA is first hypernegatively supercoiled . To investigate the possibility that topo IIIbeta requires a single-stranded region for its relaxation activity, we formed R-loops and D-loops in plasmids . In addition to containing a single-stranded region, these R-loops and D-loops have the advantage of being covalently closed and supercoiled, thus allowing us to assay for supercoil relaxation . We found that topo IIIbeta preferentially cleaves, rather than relaxes, these substrates . The cleavage of the R-loops and D-loops, which is primarily in the form of nicking, occurs to a greater extent at a temperature that is lower than the optimal temperature for relaxation of hypernegatively supercoiled plasmid . In addition, the cleavage can be readily reversed by high salt or high temperature, and the products fail to enter the gel in the absence of proteinase K treatment and are not observed with an active-site Y332F mutant of topo IIIbeta, indicating that the cleavage is mediated by a topoisomerase . We mapped the cleavage to the unpaired strand within the loop region and found that the cleavage occurs along the length of the unpaired strand . These studies suggest that the topo III enzyme behaves as a structure-specific endonuclease in vivo, providing a reversible DNA cleavage activity that is specific for unpaired regions in the DNA.

Trends Genet, 2002 May, 18(5), 245 - 8
Recent emergence of the modern genetic code: a proposal; Syvanen M; This article proposes that the genetic code was not fully formed before the divergence of life into three kingdoms . Rather, at least arginine and tryptophan evolved after the diversification of archaea, bacteria and eukaryotes, and were spread by horizontal gene transfer . Evidence for this hypothesis is based on data suggesting that enzymes for biosynthesis of arginine and tryptophan, and for arginine tRNA ligase, have shorter divergence times than the underlying lineages . Also, many of these genes display "star" phylogenies . This proposal is an extension of the idea that the genetic code was unified because of the evolutionary pressure from horizontal gene transfer . These considerations further undermine the need to postulate the existence of a "last common ancestor"; a simpler model would be that multiple lineages gave rise to life today.

Immunology, 2002 Jun, 106(2), 273 - 83
Host responses to Renibacterium salmoninarum and specific components of the pathogen reveal the mechanisms of immune suppression and activation; Grayson TH et al.; During infection, Renibacterium salmoninarum survives within the pronephric macrophages of salmonid fish . Therefore, to study the initial phases of the interaction we infected macrophages with live bacteria and analysed the responses of host and pathogen . It was found that the expression of msa encoding the p57 antigen of R . salmoninarum, was constitutive, while the expression of hly and rsh, encoding haemolysins, and lysB and grp was reduced after infection . Macrophages showed a rapid inflammatory response in which the expression of interleukin-1beta (IL-1beta), major histocompatibility complex class II (MHC II), inducible cyclo-oxygenase (Cox-2), and inducible nitric oxide synthase (iNOS) was enhanced, but tumour necrosis factor-alpha (TNF-alpha) expression was greatly reduced initially and then increased . After 5 days, except for TNF-alpha and MHC II, expression returned to levels approaching those of uninfected macrophages . We propose that R . salmoninarum survives initial contact with macrophages by avoiding and/or interfering with TNF-alpha-dependent killing pathways . The effects of specific R . salmoninarum components were studied in vivo by injecting fish with DNA vaccine constructs expressing msa, hly, rsh, lysB, or grp . We found that msa reduced the expression of IL-1beta, Cox-2, and MHC II but stimulated TNF-alpha while hly, rsh and grp stimulated MHC II but down-regulated TNF-alpha . Constructs expressing hly or lysB stimulated iNOS expression and additionally, lysB stimulated TNF-alpha . The results show how p57 suppresses the host immune system and suggest that the immune mechanisms for the containment of R . salmoninarum infections rely on MHC II- and TNF-alpha-dependent pathways . Moreover, prolonged stimulation of TNF-alpha may contribute to the chronic inflammatory pathology of bacterial kidney disease.

Immunology, 2002 Jun, 106(2), 144 - 58
T-cell activation occurs simultaneously in local and peripheral lymphoid tissue following oral administration of a range of doses of immunogenic or tolerogenic antigen although tolerized T cells display a defect in cell division; Smith KM et al.; How the mucosal immune system promotes active immunity against harmful organisms but tolerance to commensal bacteria or dietary antigens is poorly understood . Thus, the antigen-presenting cell (APC), site of antigen presentation, and effector mechanisms responsible for oral priming and tolerance remain unclear . Characterizing differences between oral priming and tolerance may improve the exploitation of oral tolerance for therapeutic applications and aid the design of oral vaccines . To address these questions we compared the mucosal and systemic activation and localization of antigen-specific T cells during the induction of oral priming and tolerance . Activation marker expression and cell division by tg T cells was determined in conjunction with their anatomical location . These studies show that after feeding, T cells are activated in both peripheral and local lymphoid tissues within 6 hr, irrespective of the presence of adjuvant . Subsequently, T-cell accumulation can be detected simultaneously in peripheral and mesenteric lymph nodes and Peyer's patches within 24 hr of feeding, but only after 3 days post feeding in the lamina propria . Primed and tolerized T cells adopted similar phenotypes as assessed by activation marker expression . However, within the mesenteric lymph nodes (MLN) tolerized T cells underwent significantly fewer divisions than primed T cells . Thus, T-cell activation and expansion occurs throughout the animal after feeding a range of doses of antigen, irrespective of whether priming or tolerance is the eventual outcome . However, the presence of an adjuvant enhances clonal expansion in the MLN while tolerized T cells display defective cell division.

Helicobacter, 2002 Jun, 7(3), 170 - 4
Production of chemokines and reactive oxygen species by human neutrophils stimulated by Helicobacter pylori; Shimoyama T et al.; BACKGROUND: Bacteria have different characteristics in stimulation of human neutrophils to produce reactive oxygen species (ROS) and chemokines . This study examined the ability of Helicobacter pylori to induce production of ROS and chemokines by human neutrophils . METHODS: H . pylori strains (1.5 x 108 CFU/ml) were cocultured with 5 x 104 neutrophils isolated from healthy subjects . Samples were incubated with human serum with or without IgG antibodies to H . pylori . ROS production was measured using luminol-dependent chemiluminescence (LmCL), and the concentrations of chemokines (IL-8, RANTES, MIP-1alpha and MCP-1) were measured by ELISA . RESULTS: The mean of the highest LmCL (peak height; PH) value stimulated by H . pylori was 3318 in the absence of serum . PH increased to 4687 when incubated with anti-H . pylori antibody-positive sera (p <.001) but antibody-negative sera did not affect LmCL response . The mean final concentration of IL-8 produced in the absence of serum was 142.6 pg/ml . Increased IL-8 production was seen by addition of antibody positive serum (p <.01) . IL-8 production was not significantly correlated with production of ROS . On the other hand, H . pylori stimulation did not induce neutrophil production of RANTES, MIP-1alpha or MCP-1 . CONCLUSIONS: H . pylori was capable of inducing IL-8 production by human neutrophils, but not C-C chemokines . Production of C-X-C dominant chemokine by neutrophils is consistent with the pathological characteristics of H . pylori-induced gastritis, where persistent neutrophil infiltration is present.

Aliment Pharmacol Ther, 2002 Jul, 16 Suppl 4, 53 - 8
Review article: a critical approach to new forms of treatment of Crohn's disease and ulcerative colitis; Seegers D et al.; Most patients with inflammatory bowel disease can be managed with conventional immunosuppressive therapy . The choice of agents to prevent relapses of inflammatory bowel disease must be based on efficacy, toxicity and cost . Studies in animal models of inflammatory bowel disease indicate that chronic intestinal inflammation results from enhanced immune responses to bacteria that are present normally in the lumen . Loss of tolerance, an abnormal function or defective healing of the mucosal barrier may all give raise to chronic intestinal inflammation . This hypothesis is the basis of new therapies aimed at either decreasing the levels of luminal bacterial antigens and/or selectively blocking detrimental mucosal immune responses . Anti-TNF is an example of this novel approach that is very effective in Crohn's disease . The use of biological therapy is costly, however, and the long-term complications are not yet known . The recent increase of tuberculosis in patients treated with anti-TNF indicates that careful monitoring is necessary . It is clear that the new forms of treatment may play an important role in tailoring the appropriate drug to a specific group of patients . However, for the time being, fine-tuning in the use of conventional immunosuppression is necessary . New knowledge in the pharmacogenetics of these compounds allows improvements to be made in their use . It is to be hoped that a critical approach in the use of current and future drugs, taking into account the advances in the aetiopathogenesis of inflammatory bowel disease, will contribute to the quality of life of patients with inflammatory bowel disease.

Aliment Pharmacol Ther, 2002 Jul, 16 Suppl 4, 35 - 9
Review article: medical treatment of active Crohn's disease; Scribano ML et al.; Crohn's disease, a heterogeneous inflammatory process that can affect various sites in the gut, presents an ongoing management challenge for the clinician . The treatment of active disease and complications is one of the main goals in the therapy of this disease . New therapies are aimed at delivering the active compounds to the diseased site, reduction or suppression of enteral flora and modulation of more focal targets within the immune response . The use of antibiotics in the therapy of Crohn's disease is gaining popularity, on the grounds that intestinal bacteria may play a role in the pathogenesis of Crohn's disease lesions . Metronidazole is one of the most widely used antibiotics, especially in the treatment of perianal disease . Corticosteroids are the mainstays of medical treatment in active Crohn's disease and induce the remission of symptoms in about 60-80% of patients . The use of immunosuppressive agents, such as cyclosporine and methotrexate, in patients with active disease resistant to standard therapy has gained acceptance in recent years . With new therapies the outlook for patients with Crohn's disease is more optimistic than it has been for a long time.

J Dairy Res, 2002 Feb, 69(1), 1 - 12
Complement factor B and the alternative pathway of complement activation in bovine milk; Rainard P; The contribution of the alternative pathway of complement activation to the capacity of normal milk to deposit C3 fragments on bacteria was tested by attempting to block C3 deposition with antibodies to the alternative pathway component factor B (fB) . Factor B was purified and antibodies of the IgY class, which does not activate mammalian complement, were obtained from the egg yolk of immunized laying hens . These antibodies specifically inhibited the deposition of C3 . This inhibition and the absence of deposition of C4 demonstrated that C3 deposition in normal milk resulted from the activation of the alternative pathway . Antibodies raised in rabbit were used to develop an ELISA for measuring fB concentrations in milk . The mean concentration of fB was 2.06 microg/ml (+/- 0.18, SEM), 0.57% of the mean value found in serum (360 microg/ml) . This proportion was comparable to that of serum albumin (0.63% of serum value) but less than the proportion of C3 in milk (2.71%) . Nevertheless, fB was apparently not a limiting factor for the functioning of the alternative pathway, since addition of purified fB to normal milk did not improve C3 deposition . In serum, mild heat-treatment (56 degrees C for 3 min or 50 degrees C for 45 min) blocked the alternative pathway and destroyed fB, as shown by loss of antigenicity in ELISA . In milk, mild heat-treatment did not abrogate C3 deposition, and fB was protected, retaining its functionality and antigenicity . Heating at 56 degrees C for at least 45 min was necessary to completely inhibit C3 deposition in normal milk.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2002, 37(4), 725 - 35
Application of anaerobic digested residues on safe food production; Shi YJ et al.; Experiments were conducted in pot culture and field plots to study the effects of Anaerobic Digested Residues (ADR) on nitrate accumulation in leaf vegetables, which is critical for the safety of food . The results showed that compared to chemical fertilizer, ADR could decrease the nitrate accumulation in rape and spinach . Furthermore, nitrate content in plant tissue was increased with the increase of percentage of chemical nitrogen in the mixture of chemical fertilizer and ADR . A comparison of spraying digested slurry with irrigation showed that spraying method could reduce the nitrate content of rape, however, a reverse result was found in spinach . The nitrate accumulation in rape affected by ADR was more apparent in high fertility soil than that in low fertility one . To regulate the nitrate accumulation in plant, it was more apparent in rape under greenhouse cultivation, while more apparent in spinach under open-air cultivation . The results demonstrated that the ADR was effective in the safe food production and it may convert the technology to be more profitable.

Naturwissenschaften, 2002 Feb, 89(2), 84 - 8
Molecular structures and associations of humic substances in the terrestrial environment; Simpson AJ et al.; Here we show, for the first time, evidence of the primary molecular structures in humic substances (HS), the most abundant naturally occurring organic molecules on Earth, and their associations as mixtures in terrestrial systems . Multi-dimensional nuclear magnetic resonance (NMR) experiments show us that the major molecular structural components in the mixtures operationally defined as HS are aliphatic acids, ethers, esters and alcohols; aromatic lignin derived fragments; polysaccharides and polypeptides . By means of diffusion ordered spectroscopy, distinct diffusion coefficients consistent with relatively low molecular weight molecules were observed for all the components in the mixtures, and saccharides were the largest single class of component present . Liquid chromatography NMR confirmed that HS components can be easily separated and nuclear Overhauser effect (NOE) enhancements support the finding that the components are of relatively low molecular weight <approximately 2,000 Da . The widely recognized properties of HS, i.e., characteristics indicative of crosslinked, macromolecular networks, can now be explained as aggregation of mixtures, most likely instigated by complexation with metal cations.

Braz J Med Biol Res, 2002 Jun, 35(6), 645 - 50
Genomics and X-ray microanalysis indicate that Ca2+ and thiols mediate the aggregation and adhesion of Xylella fastidiosa; Leite B et al.; The availability of the genome sequence of the bacterial plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, is accelerating important investigations concerning its pathogenicity . Plant vessel occlusion is critical for symptom development . The objective of the present study was to search for information that would help to explain the adhesion of X . fastidiosa cells to the xylem . Scanning electron microscopy revealed that adhesion may occur without the fastidium gum, an exopolysaccharide produced by X . fastidiosa, and X-ray microanalysis demonstrated the presence of elemental sulfur both in cells grown in vitro and in cells found inside plant vessels, indicating that the sulfur signal is generated by the pathogen surface . Calcium and magnesium peaks were detected in association with sulfur in occluded vessels . We propose an explanation for the adhesion and aggregation process . Thiol groups, maintained by the enzyme peptide methionine sulfoxide reductase, could be active on the surface of the bacteria and appear to promote cell-cell aggregation by forming disulfide bonds with thiol groups on the surface of adjacent cells . The enzyme methionine sulfoxide reductase has been shown to be an auxiliary component in the adhesiveness of some human pathogens . The negative charge conferred by the ionized thiol group could of itself constitute a mechanism of adhesion by allowing the formation of divalent cation bridges between the negatively charged bacteria and predominantly negatively charged xylem walls.

Annu Rev Biochem, 2002, 71, 165 - 89 Epub 2001 Nov 09.
Eukaryotic ribonuclease P: a plurality of ribonucleoprotein enzymes; Xiao S et al.; Ribonuclease P (RNase P) is an essential endonuclease that acts early in the tRNA biogenesis pathway . This enzyme catalyzes cleavage of the leader sequence of precursor tRNAs (pre-tRNAs), generating the mature 5' end of tRNAs . RNase P activities have been identified in Bacteria, Archaea, and Eucarya, as well as organelles . Most forms of RNase P are ribonucleoproteins, i.e., they consist of an essential RNA subunit and protein subunits, although the composition of the enzyme in mitochondria and chloroplasts is still under debate . The recent purification of the eukaryotic nuclear RNase P has demonstrated a significantly larger protein content compared to the bacterial enzyme . Moreover, emerging evidence suggests that the eukaryotic RNase P has evolved into at least two related nuclear enzymes with distinct functions, RNase P and RNase MRP . Here we review current information on RNase P, with emphasis on the composition, structure, and functions of the eukaryotic nuclear holoenzyme, and its relationship with RNase MRP.

Front Biosci, 2002 Jun 01, 7, d1516 - 24
Cancer gene therapy: 'delivery, delivery, delivery' ; Greco O et al.; Gene therapy for cancer treatment represents a promising approach that has shown selectivity and efficacy in experimental systems as well as clinical trials . Some major problems remain to be solved before this strategy becomes routinely adopted in the clinic, one of the main challenges being the improvement of gene delivery . Namely, the development of DNA vectors characterized by maximum efficiency and minimal toxicity will define the success of gene therapy and its chances of being accepted by public and clinicians . A number of issues need to be considered . The "magic" vector should be targeted, protected from degradation and immune attack, and safe for the recipient and the environment . Moreover, it should express the therapeutic gene for as long as required, in an appropriately regulated fashion . Vehicles such as retroviruses, adenoviruses and liposomes have been adopted in clinical studies, with varying results . New therapeutic modalities are also being explored in order to overcome the limitation of poor gene transfer and patient toxicity, including bacteria, adeno-associated and herpes simplex viruses, lentiviruses, cationic polymer-DNA complexes and electroporation . Some of the delivery systems tested in preclinical and clinical models are reviewed in this article, with particular attention to the targeting of the tumor environment.

Biochim Biophys Acta, 2002 Jun 3, 1597(2), 173 - 92
Glucosamine-6-phosphate synthase--the multi-facets enzyme; Milewski S; L-Glutamine: D-fructose-6-phosphate amidotransferase, known under trivial name of glucosamine-6-phosphate synthase, as the only member of the amidotransferase subfamily of enzymes, does not display any ammonia-dependent activity . This enzyme, catalysing the first committed step in a pathway leading to the eventual formation of uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), is an important point of metabolic control in biosynthesis of amino sugar-containing macromolecules . The molecular mechanism of reaction catalysed by GlcN-6-P synthase is complex and involves both amino transfer and sugar isomerisation . Substantial alterations to the enzyme structure and properties have been detected in different neoplastic tissues . GlcN-6-P synthase is inflicted in phenomenon of hexosamine-induced insulin resistance in diabetes . Finally, this enzyme has been proposed as a promising target in antifungal chemotherapy . Most of these issues, especially their molecular aspects, have been extensively studied in recent years . This article provides a comprehensive overview of the present knowledge on this multi-facets enzyme.

FEBS Lett, 2002 Jun 5, 520(1-3), 47 - 52
Insulators prevent transcriptional interference between two promoters in a double gene construct for transgenesis; Hasegawa K et al.; In transgenesis, the expression of two transgenes is often subject to mutual interference by each of the two expression cassettes when they are driven by different transcriptional regulatory elements in a single construct . To study this problem, we constructed vectors consisting of two expression units, one contains a strong ubiquitous promoter and the other contains a tissue-specific transcriptional element . The expression pattern of each transgene was examined in transfected cell lines and also in transgenic mice . In both cases, two expression units in a single construct were expressed in an independent manner and were controlled by their respective regulatory element only if we placed insulators at both ends of one expression unit . These results indicate that usage of insulators is a valuable tool for transfection of double gene constructs in transgenesis.

FEMS Microbiol Lett, 2002 May 7, 210(2), 165 - 72
The neuA/flmD gene cluster of Helicobacter pylori is involved in flagellar biosynthesis and flagellin glycosylation; Josenhans C et al.; Helicobacter pylori possesses a gene (HP0326/JHP309) homologous to neuA of other bacteria, encoding a cytidyl monophosphate-N-acetylneuraminic acid synthetase-homologous enzyme in its N-terminal portion . We analysed the function of this gene, which is controlled by a flagellar class 2 sigma(54) promoter, in flagellar biosynthesis . HP0326/JHP309 actually represents a bicistronic operon consisting of a neuA and a flmD-like putative glycosyl transferase gene . An isogenic flmD mutant synthesized basal bodies but no filaments, was non-motile, and expressed severely reduced amounts of a FlaA flagellin of reduced molecular mass . FlaA flagellin was found to be glycosylated in its exported form within the flagellar filament, but not inside the cytoplasm . Glycosylated FlaA was not detectable in the flmD mutant . Together with other genes in the H . pylori genome, a proposed function of the neuA/flmD gene products could be to provide a pathway for glycosylation of flagellin and other extracytoplasmic molecules during type III secretion.

Toxicol Lett, 2002 Jun 14, 132(2), 109 - 15
DNA single strand breaks in peripheral blood lymphocytes induced by three nitroimidazole derivatives; Rodriguez Ferreiro G et al.; Tinidazole (TNZ), ornidazole (ONZ) and metronidazole (MTZ) are antiparasitic drugs (nitroimidazole derivatives) that have proven to be effective against Trichomonas vaginalis, Entoamoeba histolytica, Giardia lamblia and Helicobacter pylori . The reduction of the nitro group and the generation of short-lived reactive intermediates are the basis of its parasiticidal activity . This reduction is associated with its mutagenic activity in bacteria, although in mammalian cells DNA damage seems to be related to the production of reactive oxygen species (ROS) . Using alkaline single cell electrophoresis, a significant increase in single strand breaks and alkali labile sites in human peripheral blood lymphocytes (PBL) exposed to MTZ, ONZ and TNZ at 10, 100 and 500 microg/ml is observed . MTZ causes less damage, especially at higher concentrations, when compared with TNZ, the most harmful of the drugs tested . These findings suggest that primary damage is induced under aerobic conditions and confirms that these nitroimidazoles are DNA damaging agents.

Trends Genet, 2002 Jun, 18(6), 291 - 4
Base composition bias might result from competition for metabolic resources; Rocha EP et al.; The GC content of bacterial genomes varies from 25 to 75%, but the reason for this variation is unclear . Here, we show that genomes of bacteria that rely on their host for survival (obligatory pathogens or symbionts) tend to be AT rich . Furthermore, we have analysed bacterial phages, plasmids and insertion sequences, which might also be regarded as 'intracellular pathogens', and show that they too are significantly richer in AT than their hosts . We suggest that the higher energy cost and limited availability of G and C over A and T/U could be a basis for the understanding of these differences.

Curr Allergy Asthma Rep, 2002 Jul, 2(4), 309 - 15
Immunologic aspects of otitis media; Bernstein JM; The middle ear cleft is a modified gas pocket which functions normally when the gas contents are regulated by a normal eustachian tube, resulting in equalization of middle ear pressure to that of the environment . The most important regulator of this middle ear pressure is the eustachian tube, a critical passageway from the nasopharynx into the middle ear . Any alteration of eustachian tube mucociliary function caused by virus, allergy, pollutants, or alteration of the normal homeostasis of the nasopharynx will result in eustachian tube obstruction . This, in turn, leads to underventilation of the middle ear, and transudation of fluid . If bacteria or virus or viral-bacterial interaction leads to infectious disease of the middle ear, an immune response is produced as a result of the inflammatory response, allowing lymphocytes and antigen-presenting cells to enter into the middle-ear mucosa . This article summarizes the immunologic reactivity in the middle ear following a viral-bacterial inflammatory reaction in the middle-ear mucosa . Although secretory IgA is critical for protection of the nasopharynx, its function in the middle ear has still not been resolved . The evidence strongly suggests that IgG1 and IgG3 subclasses are responsible for eradication of middle ear pathogens . Finally, a review of alternative approaches to the prevention of otitis media is briefly discussed in this critical period of emergence of resistant bacteria to available antibioticsPublication Types:

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