Microorganism

J Biol Chem, 2003 Jun 13, 278(24), 21517 - 25 Epub 2003 Apr 03.
Inhibition of proteasome activity induces concerted expression of proteasome genes and de novo formation of Mammalian proteasomes; Meiners S et al.; The 26 S proteasome is a high molecular mass proteinase complex that is built by at least 32 different protein subunits . Such protease complexes in bacteria and yeast are systems that undergo a highly sophisticated network of gene expression regulation . However, regulation of mammalian proteasome gene expression has been neglected so far as a possible control mechanism for the amount of proteasomes in the cell . Here, we show that treatment of cells with proteasome inhibitors and the concomitant impairment of proteasomal enzyme activity induce a transient and concerted up-regulation of all mammalian 26 S proteasome subunit mRNAs . Proteasome inhibition in combination with inhibition of transcription revealed that the observed up-regulation is mediated by coordinated transcriptional activation of the proteasome genes and not by post-transcriptional events . Our experiments also demonstrate that inhibitor-induced proteasome gene activation results in enhanced de novo protein synthesis of all subunits and in increased de novo formation of proteasomes . This phenomenon is accompanied by enhanced expression of the proteasome maturation factor POMP . Thus, our experiments present the first evidence that the amount of proteasomes in mammalia is regulated at the transcriptional level and that there exists an autoregulatory feedback mechanism that allows the compensation of reduced proteasome activity.

Appl Environ Microbiol, 2003 Apr, 69(4), 2201 - 8
Interstrain inhibition in the sweet potato pathogen Streptomyces ipomoeae: purification and characterization of a highly specific bacteriocin and cloning of its structural gene; Zhang X et al.; Strains of the sweet potato soil rot pathogen Streptomyces ipomoeae had previously been divided into three groups based on their ability to inhibit one another during pairwise cocultivation . While group I strains are not antagonistic to members of the other groups, group II and group III strains produce separate substances that are inhibitory to strains outside their respective cognate groups . Here, we purified the group III inhibitory substance from the culture supernatant of a representative strain and found that it consists of a single 10-kDa cationic protein which is bacteriolytic for S . ipomoeae group I and II strains but which showed no inhibitory function against other streptomycetes or other bacterial genera tested . The structural gene for the inhibitor was cloned from a chromosomal library of the producing strain, and while the gene sequence revealed that the inhibitor is initially made in a larger precursor form, the deduced mature protein showed no significant homology to other known proteins . Our results demonstrate that S . ipomoeae group III inhibitory activity is manifested in the form of a highly specific, potentially novel bacteriocin, which we have designated ipomicin.

Appl Environ Microbiol, 2003 Apr, 69(4), 1980 - 9
Effect of signal compounds and incubation conditions on the culturability of freshwater bacterioplankton; Bruns A et al.; The effect of signal compounds and of different incubation conditions on the culturability (i.e., the fraction of all cells capable of growth) of natural bacterioplankton from the eutrophic lake Zwischenahner Meer was investigated over a period of 20 months . Numbers of growing cells were determined by the most-probable-number technique in liquid media containing low concentrations (10 micro M) of the signal compounds N-(oxohexanoyl)-DL-homoserine lactone, N-(butyryl)-DL-homoserine lactone, cyclic AMP (cAMP), or ATP . cAMP was the most effective signal compound, leading to significantly increased cultivation efficiencies of up to 10% of the total bacterial counts . Microautoradiography with {2,8-(3)H}cAMP, combined with fluorescence in situ hybridization, demonstrated that cAMP was taken up by 18% of all cells . The bacterial cAMP uptake systems had a very low K(m) value of </=1 nM . Analysis of the cultured bacteria by 16S rRNA gene fingerprinting showed that different bacterial phylotypes were recovered in the presence and in the absence of cAMP . Consequently, the addition of cAMP caused a stimulation of otherwise nonculturable bacteria . Phylogenetically different bacteria were also recovered at different temperatures and oxygen partial pressures . Throughout the study period, mainly members of the beta-subclass of the Proteobacteria were cultivated . In addition, some members of the Actinomycetales were enriched . Quantification by culture-independent fluorescence in situ hybridization demonstrated that beta-Proteobacteria and Actinomycetales also dominated the natural bacterioplankton assemblage . Sequence comparison revealed that two members of the Actinomycetales which reached high numbers in the natural bacterioplankton assemblage could actually be enriched by our cultivation approach.

Appl Environ Microbiol, 2003 Apr, 69(4), 1959 - 66
5-keto-D-gluconate production is catalyzed by a quinoprotein glycerol dehydrogenase, major polyol dehydrogenase, in gluconobacter species; Matsushita K et al.; Acetic acid bacteria, especially Gluconobacter species, have been known to catalyze the extensive oxidation of sugar alcohols (polyols) such as D-mannitol, glycerol, D-sorbitol, and so on . Gluconobacter species also oxidize sugars and sugar acids and uniquely accumulate two different keto-D-gluconates, 2-keto-D-gluconate and 5-keto-D-gluconate, in the culture medium by the oxidation of D-gluconate . However, there are still many controversies regarding their enzyme systems, especially on D-sorbitol and also D-gluconate oxidations . Recently, pyrroloquinoline quinone-dependent quinoprotein D-arabitol dehydrogenase and D-sorbitol dehydrogenase have been purified from G . suboxydans, both of which have similar and broad substrate specificity towards several different polyols . In this study, both quinoproteins were shown to be identical based on their immuno-cross-reactivity and also on gene disruption and were suggested to be the same as the previously isolated glycerol dehydrogenase (EC 1.1.99.22) . Thus, glycerol dehydrogenase is the major polyol dehydrogenase involved in the oxidation of almost all sugar alcohols in Gluconobacter sp . In addition, the so-called quinoprotein glycerol dehydrogenase was also uniquely shown to oxidize D-gluconate, which was completely different from flavoprotein D-gluconate dehydrogenase (EC 1.1.99.3), which is involved in the production of 2-keto-D-gluconate . The gene disruption experiment and the reconstitution system of the purified enzyme in this study clearly showed that the production of 5-keto-D-gluconate in G . suboxydans is solely dependent on the quinoprotein glycerol dehydrogenase.

Appl Environ Microbiol, 2003 Apr, 69(4), 1884 - 9
Identification of Streptomyces coelicolor proteins that are differentially expressed in the presence of plant material; Langlois P et al.; Streptomyces coelicolor and Lemna minor were used as a model to study the modulation of bacterial gene expression during plant-streptomycete interactions . S . coelicolor was grown in minimal medium with and without L . minor fronds . Bacterial proteomes were analyzed by two-dimensional gel electrophoresis, and a comparison of the two culture conditions resulted in identification of 31 proteins that were induced or repressed by the presence of plant material . One-half of these proteins were identified by peptide mass fingerprinting by using matrix-assisted laser desorption ionization-time of flight mass spectrometry . The induced proteins were involved in energetic metabolism (glycolysis, pentose phosphate pathway, oxidative phosphorylation), protein synthesis, degradation of amino acids, alkenes, or cellulose, tellurite resistance, and growth under general physiological or oxidative stress conditions . The repressed proteins were proteins synthesized under starvation stress conditions . These results suggest that root exudates provide additional carbon sources to the bacteria and that physiological adaptations are required for efficient bacterial growth in the presence of plants.

Genomics, 2003 Apr, 81(4), 356 - 60
Reversible gene inactivation in the mouse; Mallo M et al.; Gene-inactivation techniques in the mouse have become an essential tool for modern biomedical research . Both ubiquitous and tissue-specific inactivation are possible with current approaches, and recent developments facilitate a temporal control of the inactivation process . However, one of the limitations of current procedures is that inactivation is irreversible . We have produced complete and reversible inactivation of the Hoxa2 gene in the mouse using the control elements of the tetracycline-resistance operon . We show that a Hoxa2 allele containing tetracycline operator (tetO) sequences is susceptible to controlled regulation by tTS, a chimeric molecule containing the tetracycline repressor and a transcriptional repressing domain . This inhibition was specific to the tetO-modified allele, did not affect neighboring genes, and was reversible by administration of doxycycline to the pregnant female . This procedure allows the production of gene inactivation that is complete, is reversible, and can be controlled at the spatial and temporal levels.

Bioresour Technol, 2003 Aug, 89(1), 57 - 62
The effect of temperature fluctuations on psychrophilic anaerobic sequencing batch reactors treating swine manure; Masse DI et al.; Under northern climatic conditions, a temporary decrease in the temperature of anaerobic reactors treating swine manure is likely to happen at the farm . The objective of this study was to evaluate the impact of temperature fluctuations, between 10 and 20 degrees C, on the stability and performance of psychrophilic anaerobic sequencing batch reactors (ASBRs) treating swine manure . Methane yield decreased from 0.266+/-0.014 l/g of total chemical oxygen demand (TCOD) fed to the ASBRs at 20 degrees C to 0.218+/-0.022 and 0.080+/-0.002 l/g TCOD (fed) at 15 and 10 degrees C, respectively . Soluble chemical oxygen demand (SCOD) reduction decreased from 94.2+/-1.1% at 20 degrees C to 78.8+/-3.0% at 15 degrees C and 60.4+/-6.4% at 10 degrees C . Total COD removal also tended to decrease as temperature was lowered, but difference between operating temperatures was not as pronounced . A lower methanogenic activity in the ASBRs operated at 10 degrees C probably favoured quiescent conditions during the settling period, thereby increasing physical removal of the TCOD through sedimentation of the solids with the biomass . When the operating temperature was increased back to 15 and 20 degrees C, methane yield and SCOD reduction improved, but reactor performance remained significantly (P<0.05) lower than that achieved before the cycles at 10 degrees C . Results from this experiment nevertheless suggested that fluctuation in the operating temperature of psychrophilic ASBRs should only have temporary effects on the performance and stability of the process.

Toxicol Lett, 2003 Apr 11, 140-141, 391 - 401
Long-term effects of heavy metals and microelements on nematode assemblage; Bakonyi G et al.; Effects of Cd, Cr, Se and Zn at a maximum rate of 270 mg kg(-1) were studied on a nematode assemblage after 6-10 years of application . Winter wheat, sunflower, sorrel, barley and rape were grown on the experimental field . Cd had a moderate effect on nematodes in spite of the fact that this element significantly decreased plant biomass . Cr was harmful to plants only in the first year of the study . However, Cr decreased Aporcelaimellus density and maturity index, increased Pratylenchus density and bacterial-fungal ratio, changed the c-p structure and feeding type composition . Se proved to be very toxic at a concentration of 11 mg kg(-1) (NH(4)-acetate+EDTA soluble form) . Some advantageous effects of Zn were found in the first year . These disappeared later on . Remarkable between-year fluctuations of the nematode assemblage composition were observed.

Environ Int, 2003 Jun, 29(2-3), 213 - 39
Ecological effects of particulate matter; Grantz DA et al.; Atmospheric particulate matter (PM) is a heterogeneous material . Though regulated as un-speciated mass, it exerts most effects on vegetation and ecosystems by virtue of the mass loading of its chemical constituents . As this varies temporally and spatially, prediction of regional impacts remains difficult . Deposition of PM to vegetated surfaces depends on the size distribution of the particles and, to a lesser extent, on the chemistry . However, chemical loading of an ecosystem may be determined by the size distribution as different constituents dominate different size fractions . Coating with dust may cause abrasion and radiative heating, and may reduce the photosynthetically active photon flux reaching the photosynthetic tissues . Acidic and alkaline materials may cause leaf surface injury while other materials may be taken up across the cuticle . A more likely route for metabolic uptake and impact on vegetation and ecosystems is through the rhizosphere . PM deposited directly to the soil can influence nutrient cycling, especially that of nitrogen, through its effects on the rhizosphere bacteria and fungi . Alkaline cation and aluminum availability are dependent upon the pH of the soil that may be altered dramatically by deposition of various classes of PM . A regional effect of PM on ecosystems is linked to climate change . Increased PM may reduce radiation interception by plant canopies and may reduce precipitation through a variety of physical effects . At the present time, evidence does not support large regional threats due to un-speciated PM, though site-specific and constituent-specific effects can be readily identified . Interactions of PM with other pollutants and with components of climate change remain important areas of research in assessment of challenges to ecosystem stability.

J Comput Biol, 2003, 10(1), 83 - 93
Temporal changes in phosphoglycerate kinase coding sequences: a quantitative measure; Chattopadhyay S et al.; The ratio of the average of the square of the number of the nucleotides to that of the random sequence of the same strand bias is proposed as a quantitative measure of evolution in some coding DNA sequences . Applying this measure to the phosphoglycerate kinase gene we observe a monotonic rise of the ratio with evolution . We present an interpretation of this data on some bacteria.

J Vet Med B Infect Dis Vet Public Health, 2003 Mar, 50(2), 90 - 4
Differentiation of twelve Actinobacillus pleuropneumoniae serotypes by outer membrane lipoprotein gene-based restriction fragment length polymorphism; Cho WS et al.; Twelve Actinobacillus pleuropneumoniae serotypes were differentiated by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)-amplified fragments from the outer membrane lipoprotein (omlA) gene . All 12 reference serotypes and 80 field isolates produced the expected 950-base pair (bp) fragment of the omlA gene by PCR . Combining the RFLP patterns obtained with SfaNI, Bst71I, AluI, NciI, nine distinct patterns were observed in the 12 serotype reference strains . The PCR-based RFLP analysis of omlA genes allows differentiation among the 12 serotypes, with the exception of group 1 (serotypes 1, 9 and 11), and group 2 (serotypes 2 and 8) . When the PCR products from the 70 field isolates were subjected to RFLP analysis, 68 showed the same RFLP patterns as their respective serotype reference strain . Two isolates that could not be typed had the same RFLP patterns as those of serotype 5 . These results suggest that PCR-based RFLP analysis of the omlA genes may be of value in differentiating among 12 A . pleuropneumoniae serotypes.

Russ J Immunol, 2002 Oct, 7(3), 219 - 28
New approaches to immunoprevention and immunotherapy of neoplasms; Glushkov AN; The problems in the creation of vaccines for the prevention and treatment of neoplasms and the perspectives of their application are discussed . The positive and negative properties of the conjugates of the chemical carcinogens with proteins, DNA and enterotoxins, as well as the properties of the monoclonal anti-idiotype antibodies to the carcinogens are being analyzed . We are describing here a new technology of the anti-carcinogen vaccine production that we have developed . We suggest using transgenic plants and bacteria carrying the Fv-genes of antibodies for the passive immunoprevention of cancer diseases . We assume that lymphoproliferative neoplasms mainly arise from the clones carrying membrane receptors for the carcinogens . Therefor, here it is proposed to use the xenogenic anticarcinogens antibodies for the active immunotherapy of leukemia and other lymphoproliferative neoplasms.

Indian J Med Res, 2002 Oct, 116, 140 - 4
Effect of rifampicin & isoniazid on cytochrome P-450 in mycobacteria; Ramachandran G et al.; BACKGROUND & OBJECTIVES: Rifampicin and isoniazid are the most important first line drugs used in the treatment of tuberculosis . These drugs are also used in combination with other medications to treat co-infections . It, therefore, becomes important to study the effect of these drugs on the drug metabolizing system, namely, cytochrome P-450, not only in the host but also in the bacteria . We report the effect of rifampicin and isoniazid on the cytochrome P-450 activity in Mycobacterium smegmatis and M . tuberculosis H37Rv . METHODS: Subinhibitory concentrations of rifampicin and isoniazid were added to the organisms after they had attained the growth phase and cytochrome P-450 activity was estimated in the membranous fractions of the bacteria at different time points . RESULTS: Rifampicin was able to significantly enhance cytochrome P-450 in both M . smegmatis and M . tuberculosis H37Rv . Isoniazid was found to inhibit cytochrome P-450 in M . tuberculosis H37Rv, while there seemed to be no effect in M . smegmatis . INTERPRETATION & CONCLUSION: We report here the effect of rifampicin and isoniazid on mycobacterial cytochrome P-450 . These findings are similar to those found in eukaryotic organisms . The role of mycobacterial cytochrome P-450 in the metabolism of drugs within the bacteria needs to be elucidated.

Sheng Wu Gong Cheng Xue Bao, 2002 Nov, 18(6), 703 - 8
{Catalysis of lyase-isomerase PecE/PecF for several apophycobiliproteins}; Zhu JP et al.; Phycoerythrocyanin(PEC) lyase-isomerase PecE/PecF from Mastigocladus laminosus is the specific enzyme for biosynthesis of PEC alpha-subunit(alpha-PEC) . In this work, the specificity of PecE/PecF on substrate apoproteins was reported . PecE/PecF could catalyse the reconstitution of phycocyanobilin(PCB) with apoproteins of alpha-PEC from two different subspecies of Mastigocladus laminosus, as well the site-directed mutated apoprotein of alpha-PEC with Trp at 128 to Phe in vitro, but could not catalyse the reconstitution of PCB with apoprotein of phycocyanin alpha-subunit(alpha-CPC) from Mastigocladus laminosus . The surfactant Triton X-100 had no effect for the reconstitution of alpha-PEC, while it could improve the reconstitution of PCB with apoprotein of alpha-CPC.

J Eukaryot Microbiol, 2003 Jan-Feb, 50(1), 43 - 8
Trichomonas vaginalis: in vitro attachment and internalization of HIV-1 and HIV-1-infected lymphocytes; Rendon-Maldonado J et al.; Sexually transmitted diseases (STDs) caused by bacteria and protozoa play an important role in the epidemiology of human immunodeficiency virus (HIV-1) infection . Human trichomoniasis, produced by the protozoan parasite Trichomonas vaginalis, is one of the most common STDs, and is a cause of mucosal lesions in the urogenital tract, which may increase the risk for HIV infection . However, there are no reports concerning the outcome of in vitro interactions between HIV particles and trichomonads . Therefore, we incubated T . vaginalis with three subtypes of HIV-1 (A, B, and D), as well as with HIV-1-infected lymphocytes, and analyzed the interactions with immunofluorescence microscopy and transmission electron microscopy . Our results demonstrated that HIV-1 particles attach and are incorporated into T . vaginalis through endocytic vesicles and are degraded within cytoplasmic vacuoles in approximately 48 h . There was no ultrastructural evidence of HIV-1 replication in trichomonads . These results demonstrated that trichomonads may internalize and harbor HIV-1 particles for short periods of time . In addition, under in vitro conditions, T . vaginalis ingests and digests HIV-1-infected lymphocytes.

Adv Biochem Eng Biotechnol, 2003, 78, 75 - 96
The effect of plants on the degradation and toxicity of petroleum contaminants in soil: a field assessment; Banks MK et al.; A field project located at the US Naval Base at Port Hueneme, California was designed to evaluate changes in contaminant concentrations and toxicity during phytoremediation . Vegetated plots were established in petroleum (diesel and heavy oil) contaminated soil and were evaluated over a two-year period . Plant species were chosen based on initial germination studies and included native California grasses . The toxicity of the impacted soil in vegetated and unvegetated plots was evaluated using Microtox, earthworm, and seed germination assays . The reduction of toxicity was affected more by contaminant aging than the establishment of plants . However, total petroleum hydrocarbon concentrations were lower by the end of the study in the vegetated plots when compared to the unvegetated soil . Although phytoremediation is an effective approach for cleaning-up of petroleum contaminated soil, a long-term management plan is required for significant reductions in contaminant concentrations.

Quintessence Int, 2003 Jan, 34(1), 61 - 70
Identification of hierarchical factors to guide clinical decision making for successful long-term pulp capping; Murray PE et al.; OBJECTIVE: Clinicians have few quantitative studies that rank the in vivo pulp capping effects of commonly used restorative materials . These factors were investigated to provide guidance to clinicians . METHOD AND MATERIALS: One hundred sixty-one standardized pulp-exposed cavities were prepared in nonhuman primate teeth . Exposed pulps were capped with calcium hydroxide, resin-modified glass-ionomer cements, and resin composites . Teeth were collected from 7 to 720 days to observe a full range of responses . Pulpal reactions were categorized according to the standards set by the International Standards Organization . Bacteria were detected with McKay's stain . RESULTS: The incidence of bacterial microleakage was 19.7% with resin composite, 21.1% with resin-modified glass-ionomer cement, and 47.0% with calcium hydroxide . The severity of pulpal inflammation increased with the presence of bacteria or tunnel defects . The severity of pulpal inflammation prevented dentinal bridge formation at varying levels: slight for resin composite and resin-modified glass-ionomer cement, and severe with calcium hydroxide . The incidence of severe inflammation or pulpal necrosis was 7.9% with resin composite, 10.6% with calcium hydroxide, and 10.5% with resin-modified glass-ionomer cement . Other variables, such as pulpal exposure width and tertiary dentin formation, were not highly correlated to pulpal inflammation . CONCLUSION: Pulp capping with resin composite provided the lowest incidence of bacterial microleakage, the lowest levels of pulpal inflammation, and the lowest incidence of necrosis.

J Physiol Pharmacol, 2003 Mar, 54(1), 33 - 51
Triple eradication therapy counteracts functional impairment associated with Helicobacter pylori infection in Mongolian gerbils; Brzozowski T et al.; Gastric Helicobacter pylori (Hp) infection in Mongolian gerbils is an established experimental model of gastric carcinogenesis resulting from the long-term Hp infection but functional aspects accompanying this Hp-induced progression from gastritis to the cancer, especially changes in gastric acid secretion, gastric blood flow (GBF) and gastrin-somatostatin link have been little studied . It is unclear whether Hp eradication therapy alters the functional and the histopathological changes in this animal model of Hp-infection . We examined the effects of intragastric (i.g.) inoculation of Mongolian gerbils with Hp strain (cagA+ vacA+, 5 x 10(6) CFU/ml) that had been isolated from a patient with gastric ulcer as compared to those induced by vehicle (saline) in gerbils with or without gastric fistula (GF) at 1.2, 4, 6, 9, 12 and 30 wks upon gastric inoculation with this bacteria . An attempt was made to evaluate the influence of anti-Hp triple therapy with omeprazole, amoxicillin and tinidazol on gastric Hp-infection and Hp-induced functional impairment of the gastric mucosa . Gastric mucosal biopsy specimens were taken for the assessment of the morphological changes and the presence of Hp infection using rapid urease test (CLO-test) and the density of Hp-colonization were assessed by counting of the number of bacterial colonies per plate . Gastric blood flow (GBF) was measured by H2-gas clearance technique and the venous blood and the gastric content were collected for the measurement of plasma gastrin levels and the gastric luminal somatostatin level by radioimmunoassay (RIA) . The Hp in gastric mucosa was detected in all animals by culture and rapid urease test at various periods upon Hp inoculation . Basal gastric acid in non-infected conscious gerbils with GF reached the level of about 28 +/- 4 micromol/h and this was reduced by over 50% immediately upon the Hp-inoculation and persisted for time intervals tested up to 30 wk . Early lesions were seen 4 wks after the Hp-inoculation and consisted of chronic gastritis with thickened gastric mucosal foldings and elongated interfoveolar ridges . Edema and congestion as well as significant mucosal inflammatory infiltration with lymphoid infiltrate in lamina propria of the mucosa occurred in all infected gerbils . Adenomatous hyperplasia with cellular atypia was observed at 12 wk upon Hp-inoculation together with increased mitotic activity and numerous apoptotic bodies formation, while lamina propria was reduced leaving dilated atypical gastric gland situated "back-to-back" . This glandular atypia failed to show lamina propria or submucosa infiltration corresponding to gastric intraepithelial neoplasia . The GBF in Hp-infected gerbils was significantly lower, and a 6-7 fold increase in plasma gastrin levels combined with a significant fall in gastric luminal somatostatin contents observed at all tested periods as compared to vehicle-controls and these effects were counteracted by anti-Hp triple therapy . We conclude that: 1) . Hp-infection in Mongolian gerbils in early stages before adenocarcinoma formation results in the development of typical functional and pathological changes such as suppression of gastric secretion and impairment of both, gastric mucosal microcirculation and gastrin-somatostatin link, and 2) . this deleterious influence of Hp on gastric morphology and gastric functions is greatly attenuated in gerbils treated with Hp-eradication therapy.

Gastric Cancer, 2003, 6(1), 1 - 7
Helicobacter pylori infection and gastric carcinogenesis in animal models; Tatematsu M et al.; The effects of helicobacter pylori infection on gastric disorders have been proven by many epidemiological and experimental studies . To explore the relationships between h . Pylori infection and gastric carcinogenesis, many factors, including host responses, environmental status, and the virulence factors of the bacteria should be taken into account . Mongolian gerbils ( meriones unguiculatus) can be easily infected with h . Pylori, and provide an excellent in-vivo experimental model to clarify the role of h . Pylori in active gastritis, peptic ulcers, intestinal metaplasia, and gastric carcinoma . Studies have revealed that h . Pylori infection markedly enhances all histological types of gastric cancers in gerbils treated with a chemical carcinogen . Eradication reduced the enhancing effect of h . Pylori on gastric carcinogenesis, whereas a high-salt diet synergistically enhanced the effect of h . Pylori . Various factors involving inflammation, cell proliferation, and cell differentiation could be examined with this experimental model to help elucidate this mechanisms of gastric carcinogenesis.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Apr, 35(4), 311 - 6
Targeting strategies in cancer gene therapy; Wang JH et al.; Targeting to the tumor tissues can improve the therapeutic effect of gene transfer by preventing damage of healthy tissues and decreasing the risk of germ line transduction . Although targeting seems not important for intratumoral gene delivery, it becomes crucial when systemic gene transfer is performed . Targeted gene therapy of malignancies can be achieved through targeted gene delivery or targeted gene transcription . Recent advances in targeted delivery include the successful use of bifunctional crosslinkers to target adenoviral and retroviral vectors, inserting short targeting peptides and larger polypeptide-binding domains into the coat proteins of a number of different viral vectors, and replication-competent vectors which have been shown to be promise as anti-cancer agents . Some other non-viral therapeutic agents, including receptor-mediated DNA or liposome-DNA complex, and bacteria vehicles have also been developed . Some of these delivery systems are currently in clinical trials . For targeted and regulable gene transcription, tissue or tumor specific promoters and some manual regulatory systems are used to regulate therapeutic gene expression . Antisense oligonucleotides, some ribozyme and DNAzyme molecules are developed to inactivate genes that are essential to the development of many tumors.

Plant Cell, 2003 Apr, 15(4), 845 - 53
Increased lysine synthesis coupled with a knockout of its catabolism synergistically boosts lysine content and also transregulates the metabolism of other amino acids in Arabidopsis seeds; Zhu X et al.; To elucidate the relative significance of Lys synthesis and catabolism in determining Lys level in plant seeds, we expressed a bacterial feedback-insensitive dihydrodipicolinate synthase (DHPS) in a seed-specific manner in wild-type Arabidopsis as well as in an Arabidopsis knockout mutant in the Lys catabolism pathway . Transgenic plants expressing the bacterial DHPS, or the knockout mutant, contained approximately 12-fold or approximately 5-fold higher levels, respectively, of seed free Lys than wild-type plants . However, the combination of these two traits caused a synergistic approximately 80-fold increase in seed free Lys level . The dramatic increase in free Lys in the knockout mutant expressing the bacterial DHPS was associated with a significant reduction in the levels of Glu and Asp but also with an unexpected increase in the levels of Gln and Asn . This finding suggested a special regulatory interaction between Lys metabolism and amide amino acid metabolism in seeds . Notably, the level of free Met, which competes with Lys for Asp and Glu as precursors, was increased unexpectedly by up to approximately 38-fold in the various transgenic and knockout plants . Together, our results show that Lys catabolism plays a major regulatory role in Lys accumulation in Arabidopsis seeds and reveal novel regulatory networks of seed amino acid metabolism.

J Bacteriol, 2003 Apr, 185(8), 2582 - 91
Signal transduction protein P(II) is required for NtcA-regulated gene expression during nitrogen deprivation in the cyanobacterium Synechococcus elongatus strain PCC 7942; Fadi Aldehni M et al.; The transcription factor of the cyclic AMP receptor protein/FNR family, NtcA, and the P(II) signaling protein play central roles in global nitrogen control in cyanobacteria . A dependence on P(II) for NtcA-regulated transcription, however, has not been observed . In the present investigation, we examined alterations in gene expression following nitrogen deprivation in Synechococcus elongatus strain PCC 7942 and specifically the roles of NtcA and P(II) . Global changes in de novo protein synthesis following combined-nitrogen deprivation were visualized by in vivo {(35)S}methionine labeling and two-dimensional polyacrylamide gel electrophoresis analysis . Nearly all proteins whose synthesis responded specifically to combined-nitrogen deprivation in wild-type cells of S . elongatus failed to respond in P(II)- and NtcA-deficient mutants . One of the proteins whose synthesis was down-regulated in a P(II)- and NtcA-dependent manner was RbcS, the small subunit of RubisCO . Quantification of its mRNA revealed that the abundance of the rbcLS transcript following combined-nitrogen deprivation rapidly declined in wild-type cells but not in P(II) and NtcA mutant cells . To investigate further the relationship between P(II) and NtcA, fusions of the promotorless luxAB reporter genes to the NtcA-regulated glnB gene were constructed and these constructs were used to transform wild-type cells and P(II)(-) and NtcA(-) mutants . Determination of bioluminescence under different growth conditions showed that NtcA represses gene expression in the presence of ammonium in a P(II)-independent manner . By contrast, NtcA-dependent activation of glnB expression following combined-nitrogen deprivation was impaired in the absence of P(II) . Together, these results suggest that under conditions of combined-nitrogen deprivation, the regulation of NtcA-dependent gene expression requires the P(II) signal transduction protein.

Sci Total Environ, 2003 Apr 15, 305(1-3), 169 - 76
Bioaerosol characteristics in hospital clean rooms; Li CS et al.; Bioaerosol characteristics were evaluated in hospital clean rooms with different class levels . For total particles, an airborne particle counter was used to determine the particle size ranges (0.1, 0.2, 0.3, 0.5, 1 and 5 microm) for air inlets and patient beds . An Andersen 1-STG sampler was used for bacterial and fungal collection . For aerosol characteristics, it was found that some air inlet particle levels were higher than 100000/foot(3) in class 100000 clean rooms . In addition, it was clearly demonstrated that particle concentrations in patient beds were much higher than those for air inlets . Human activity might play a role in these particle concentration differences . Moreover, it was demonstrated that bacterial and fungal concentrations ranged from 1 to 423 and from 0 to 319 CFU/m(3), respectively . For class 100 clean rooms, no particles were ever found . In addition, bacterial concentrations were found to be in the range of 0-32 CFU/m(3) and there were no fungal aerosols . For operating rooms of class 10000, some of the particle levels observed were higher than 10000/foot(3) . Furthermore, the average level of bacterial aerosols was 88 with a range of 13-336 CFU/m(3) . In addition, fungal levels ranged from 0 to 51 with a mean value of 4 CFU/m(3) . It was indicated that bacterial levels were higher than fungal ones, which might be related to human sources . Moreover, there were weak relationships among class level, particle concentration and bioaerosol levels.

FEMS Microbiol Lett, 2003 Mar 28, 220(2), 271 - 5
Activation of the complement cascade by Bordetella pertussis; Barnes MG et al.; Bordetella pertussis must survive the defenses of the human respiratory tract including the complement system . The BrkA (Bordetella resistance to killing) protein prevents killing by the antibody-dependent classical pathway . In this study, the ability of B . pertussis to activate the human complement cascade by other pathways was examined . B . pertussis was not killed in serum depleted of C2, however serum depleted for factor B killed B . pertussis as efficiently as intact serum, suggesting complement activation occurred exclusively by the classical pathway . B . pertussis was not killed by serum depleted of antibody, suggesting the bacteria fail to activate the antibody-independent branches of the classical pathway, including the mannose binding lectin pathway . Mutants lacking the terminal trisaccharide of lipopolysaccharide retained the complement-resistant phenotype, suggesting this structure does not influence activation of complement.

Int J Parasitol, 2003 Mar, 33(3), 257 - 67
Programmed cell death in trypanosomatids and other unicellular organisms; Debrabant A et al.; In multicellular organisms, cellular growth and development can be controlled by programmed cell death (PCD), which is defined by a sequence of regulated events . However, PCD is thought to have evolved not only to regulate growth and development in multicellular organisms but also to have a functional role in the biology of unicellular organisms . In protozoan parasites and in other unicellular organisms, features of PCD similar to those in multicellular organisms have been reported, suggesting some commonality in the PCD pathway between unicellular and multicellular organisms . However, more extensive studies are needed to fully characterise the PCD pathway and to define the factors that control PCD in the unicellular organisms . The understanding of the PCD pathway in unicellular organisms could delineate the evolutionary origin of this pathway . Further characterisation of the PCD pathway in the unicellular parasites could provide information regarding their pathogenesis, which could be exploited to target new drugs to limit their growth and treat the disease they cause.

J Am Chem Soc, 2003 Apr 9, 125(14), 4012 - 3
Hybridization-based unquenching of DNA hairpins on au surfaces: prototypical "molecular beacon" biosensors; Du H et al.; There is a keen interest in developing techniques for rapid genetic analysis that do not require labeling of an analyte . Here we demonstrate that fluorophore-tagged DNA hairpins attached to gold films can function as immobilized "molecular beacons" . Two DNA hairpins incorporating portions of the Staphlococcus aureus FemA and mecR methicillin-resistance genes were attached to a gold substrate . Upon exposure to the complement, a approximately 26-fold increase in fluorescence intensity was measured corresponding to a 96 +/- 5% quenching efficiency . Studies with nonspecific DNA indicate that DNA hairpins immobilized on a gold surface retain their ability to bind complementary DNA sequences selectively.

J Int Acad Periodontol, 2002 Jul, 4(3), 110 - 4
The future of periodontology: new treatments for a new era; Tonetti MS; Scientific evidence that has been emerging over the last decade has provided the basis for a radical change in the understanding, diagnosis and treatment of periodontal diseases . A new era in periodontal research and practice is being defined along the following lines: Better understanding of disease . Emerging needs . Novel treatment strategies and therapeutics . Choice between multiple therapeutic modalities . The purpose of this manuscript is to critically discuss changes in understanding and to provide a rationale to change daily practice.

J Int Acad Periodontol, 2002 Jul, 4(3), 101 - 9
The periodontal infection-systemic disease link: a review of the truth or myth; Paquette DW; Observational studies indicate periodontal infections as a risk factor for systemic conditions like cardiovascular disease and preterm low birth weight . This paper reviews and argues the biological plausibility for a periodontal infection-systemic disease link and reviews the available experimental data from animal models and human intervention trials . Five principal lines of evidence can be used to explain the biological plausibility of a link . First, infection in general has been implicated in the pathogenesis of both atherosclerosis and preterm delivery . Periodontal infection secondly causes transient and low-grade bacteraemias and endotoxaemias in patients . Thirdly, periodontal infection promotes systemic inflammatory and immune responses that may play roles in disease . Periodontal pathogens express specific virulence factors that can affect atherogenic or parturition events . Lastly, periodontal pathogens have also been isolated from non-oral tissues like atheromatous plaques . Experimental data derived from rodent and pig models indicate that infection or bacteraemias with the periodontal pathogen, Porphyromonas gingivalis, can increase atheroma size or reduce litter weights as compared to controls . While human intervention data are lacking for patients at risk for cardiovascular disease, early data indicate that periodontal therapy administered to pregnant mothers with periodontitis can reduce the incidence of preterm low birth weight deliveries . Nevertheless, more and larger intervention trials are needed before we can fully accept periodontal infection as a true risk factor in the causal pathways of cardiovascular disease and preterm low birth weight.

J Antibiot (Tokyo), 2003 Jan, 56(1), 24 - 9
Core structure in roselipins essential for eliciting inhibitory activity against diacylglycerol acyltransferase; Tomoda H et al.; Fungal roselipins, discovered as inhibitors of diacylglycerol acyltransferase (DGAT), consist of three parts; highly methylated C20 fatty acid, mannose and arabinitol . Demannosyl and/or dearabinitoyl roselipins were prepared chemically or enzymatically . Demannnosyl roselipins conserved the DGAT inhibitory activity, but the others lost the activity, indicating that the arabinitoyl fatty acid core is essential for eliciting the activity.

Genetika, 2003 Feb, 39(2), 244 - 9
{Expression analysis of proteins encoded by genes of the tag7/tagL (PGRP-S,L) family in human peripheral blood cells}; Kibardin AV et al.; Various types of human blood cells were tested for expression of the Tag7/PGRP-SA and TagL/PGRP-L proteins, which belong to the family of proteins possessing the lysozyme-like peptidoglycan recognition protein (PGRP) domain . Expression regulation by several factors was demonstrated.

Nat Rev Immunol, 2003 Apr, 3(4), 331 - 41
Anatomical basis of tolerance and immunity to intestinal antigens; Mowat AM; The intestinal immune system has to discriminate between harmful and beneficial antigens . Although strong protective immunity is essential to prevent invasion by pathogens, equivalent responses against dietary proteins or commensal bacteria can lead to chronic disease . These responses are normally prevented by a complex interplay of regulatory mechanisms . This article reviews the unique aspects of the local microenvironment of the intestinal immune system and discuss how these promote the development of regulatory responses that ensure the maintenance of homeostasis in the gut.

J Exp Med, 2003 Apr 7, 197(7), 845 - 60 Epub 2003 Mar 31.
B cell receptor-independent stimuli trigger immunoglobulin (Ig) class switch recombination and production of IgG autoantibodies by anergic self-reactive B cells; Phan TG et al.; In both humans and animals, immunoglobulin (Ig)G autoantibodies are less frequent but more pathogenic than IgM autoantibodies, suggesting that controls over Ig isotype switching are required to reinforce B cell self-tolerance . We have used gene targeting to produce mice in which hen egg lysozyme (HEL)-specific B cells can switch to all Ig isotypes (SWHEL mice) . When crossed with soluble HEL transgenic (Tg) mice, self-reactive SWHEL B cells became anergic . However, in contrast to anergic B cells from the original nonswitching anti-HEL x soluble HEL double Tg model, self-reactive SWHEL B cells also displayed an immature phenotype, reduced lifespan, and exclusion from the splenic follicle . These differences were not related to their ability to Ig class switch, but instead to competition with non-HEL-binding B cells generated by VH gene replacement in SWHEL mice . When activated in vitro with B cell receptor (BCR)-independent stimuli such as anti-CD40 monoclonal antibody plus interleukin 4 or lipopolysaccharide (LPS), anergic SWHEL double Tg B cells proliferated and produced IgG anti-HEL antibodies as efficiently as naive HEL-binding B cells from SWHEL Ig Tg mice . These results demonstrate that no intrinsic constraints to isotype switching exist in anergic self-reactive B cells . Instead, production of IgG autoantibodies is prevented by separate controls that reduce the likelihood of anergic B cells encountering BCR-independent stimuli . That bacteria-derived LPS could circumvent these controls may explain the well-known association between autoantibody-mediated diseases and episodes of systemic infection.

Development, 2003 May, 130(10), 2139 - 48
Isolation of COV1, a gene involved in the regulation of vascular patterning in the stem of Arabidopsis; Parker G et al.; The molecular mechanisms that control the ordered patterning of vascular tissue development in plants are not well understood . Several models propose a two-component system for vascular differentiation . These components include an inducer of vascular tissue development and an inhibitor that prevents the formation of vascular bundles near pre-existing bundles . We have identified two recessive allelic mutants in Arabidopsis, designated continuous vascular ring (cov1), that display a dramatic increase in vascular tissue development in the stem in place of the interfascicular region that normally separates the vascular bundles . The mutant plants exhibited relatively normal vascular patterning in leaves and cotyledons . Analysis of the interaction of cov1 with a known auxin signalling mutant and direct analysis of auxin concentrations suggests that cov1 affects vascular pattering by some mechanism that is independent of auxin . The COV1 protein is predicted to be an integral membrane protein of unknown function, highly conserved between plants and bacteria . In plants, COV1 is likely to be involved in a mechanism that negatively regulates the differentiation of vascular tissue in the stem.

Microb Pathog, 2003 Apr, 34(4), 187 - 94
The Legionella pneumophila GacA homolog (LetA) is involved in the regulation of icm virulence genes and is required for intracellular multiplication in Acanthamoeba castellanii; Gal-Mor O et al.; Legionella pneumophila, the causative agent of legionnaires' disease, is a broad-host-range facultative intracellular pathogen . Thus far, 24 genes (icm/dot genes) required for L . pneumophila intracellular growth, have been discovered . In this study, a deletion substitution was constructed in the L . pneumophila homolog of the gacA response regulator (letA) and its involvement in L . pneumophila pathogenicity and icm/dot gene expression was characterized . The letA mutant constructed had no intracellular growth defect when analyzed in HL-60 derived human macrophages, but it was found to be severely attenuated for intracellular growth in the protozoan host Acanthamoeba castellanii . The growth defect in amoebae was fully complemented by introducing the L . pneumophila letA gene on a plasmid . In addition, the LetA regulator was found to be involved in the expression of three icm genes (icmT, icmP and icmR) . The level of expression of the icmT::lacZ and icmR::lacZ fusions was found to be higher, while the level of expression of the icmP::lacZ fusion was found to be lower when analyzed in the letA mutant strain, in comparison to the wild-type strain . We concluded that LetA has a moderate effect on icm/dot gene expression, but it probably plays a major role in the expression of L . pneumophila genes required for intracellular growth in protozoan hosts . A similar host specific phenotype was previously described for the RpoS sigma factor and the type II general secretion system of L . pneumophila .

Microb Pathog, 2003 Apr, 34(4), 179 - 86
Differential expression of the invasion-associated locus B (ialB) gene of Bartonella bacilliformis in response to environmental cues; Coleman SA et al.; Bartonella bacilliformis is the causative agent of the biphasic human disease, Oroya fever . During the primary disease phase, up to 100% of the circulating erythrocytes can be parasitized and 80% lysed . During the secondary phase of this disease, bacterial invasion shifts to endothelial cells lining the vasculature . B . bacilliformis is transferred between human hosts by the sandfly, Lutzomyia verrucarum . To investigate the regulation of ialB by environmental cues signaling vector-to-host transmission; nuclease protection assays were performed to compare the amount of ialB mRNA in bacteria subjected to temperature shift, pH change, oxidative stress, or hemin limitation . The amount of ialB mRNA increased by 223-310% in acid-treated samples and decreased by 28-39% in base-treated samples as compared to bacteria kept at pH 7.2 . B . bacilliformis samples showed a 56-63% and 74-80% decrease in ialB mRNA when shifted to 37 degrees C from growth temperatures of 20 and 30 degrees C, respectively . Oxidative stress (1 mM H(2)O(2)) and hemin limitation had no significant effect on mRNA levels . Determination of IalB protein amounts using SDS-PAGE and immunoblotting showed the greatest amounts of IalB under acidic conditions or at 20 degrees C . The least amount of IalB was synthesized under basic conditions or at 37 degrees C . The viability of wild-type B . bacilliformis under the various experimental culture conditions was determined and found not to affect ialB mRNA amounts in these experiments . Finally, we compared the survival of wild-type and ialB mutant B . bacilliformis and found no difference in the viability of these two strains, demonstrating that IalB does not aid bacterial survival under these conditions .

Virology, 2003 Mar 15, 307(2), 341 - 57
Expression and characterization of a recombinant novel reverse transcriptase of a porcine endogenous retrovirus; Avidan O et al.; The study of porcine endogenous retroviruses (PERVs) becomes increasingly important due to the potential use of pig cells, tissues, and organs as a source for xenogenic cell therapy and xenotransplantation into humans . Consequently, we have constructed a plasmid that induces in bacteria the synthesis of a soluble and highly active reverse transcriptase (RT) of PERV-B . The purified PERV RT was studied biochemically in comparison with the RT of murine leukemia virus (MLV), because of the high-sequence homology between these two RTs . The data show that in several properties the two enzymes are similar, particularly regarding the monomeric subunit composition of the proteins in solution, the high resistance to deoxynucleoside analogues, and the pattern of RNA cleavage by the ribonuclease H activity (RNase H) of the RTs . However, in several cases there are apparent differences between the two RTs, most notable the divalent cation preference (Mn(+2) versus Mg(+2)) in the DNA polymerase reactions . As already shown for viral PERV RT, the novel recombinant PERV RT exhibits a relatively high resistance to several deoxynucleoside analogue inhibitors, suggesting that they might not be very efficient in inhibiting the replication of PERV virions . Therefore, the availability of large amounts of the recombinant RT can be useful for a wide screening of novel drugs against infectious PERV.

Arch Biochem Biophys, 2003 Apr 15, 412(2), 231 - 41
Evolutionary conservation of physical and functional interactions between phospholipase D and actin; Kusner DJ et al.; Phospholipase D (PLD) enzymes from bacteria to mammals exhibit a highly conserved core structure and catalytic mechanism, but whether protein-protein interactions exhibit similar commonality is unknown . Our objective was to determine whether the physical and functional interactions of mammalian PLDs with actin are evolutionarily conserved among bacterial and plant PLDs . Highly purified bacterial and plant PLDs cosedimented with mammalian skeletal muscle alpha-actin, indicating direct interaction with F-actin . The binding of bacterial PLD to G-actin exhibited two affinity states, with dissociation constants of 1.13 pM and 0.58 microM . The effects of actin on the activities of bacterial and plant PLDs were polymerization dependent; monomeric G-actin inhibited PLD activity, whereas polymerized F-actin augmented PLD activity . Actin modulation of bacterial and plant PLDs demonstrated kinetic characteristics, efficacies, and potencies similar to those of human PLD1 . Thus, physical and functional interactions between PLD and actin in PLD family members from bacteria to mammals are highly conserved throughout evolution.

Ann Intern Med, 2003 Apr 1, 138(7), 550 - 9
Gene vaccines; Srivastava IK et al.; Gene vaccines are a new approach to immunization and immunotherapy in which, rather than a live or inactivated organism (or a subunit thereof), one or more genes that encode proteins of the pathogen are delivered . The goal of this approach is to generate immunity against diseases for which traditional vaccines and treatments have not worked, to improve vaccines, and to treat chronic diseases . Gene vaccines make use of advances in immunology and molecular biology to more specifically tailor immune responses (cellular or humoral, or both) against selected antigens . They are still under development in research and clinical trials . The mechanisms for inducing cellular (as opposed to humoral) responses against a particular antigen have been elucidated . Gene vaccines provide a means to generate specific cellular responses while still generating antibodies, if desired . In addition, by delivering only the genes that encode the particular proteins against which a protective or therapeutic immune response is desired, the potential limitations and risks of certain other approaches can be avoided . This article describes the rationale for, immunologic mechanisms involved in, and design of gene vaccines under development . Preclinical and clinical studies of these vaccines are discussed for various clinical applications, focusing on infectious diseases.

J Int Acad Periodontol, 2001 Apr, 3(2), 31 - 7
A transmission electron microscopy study of supragingival calculus; Galgut PN et al.; In this study the ultrastructure of human dental calculus was investigated using transmission electron microscopy (TEM) . The aim of the study was to observe and clarify the calcification process and the part played by dental plaque in this process . Supragingival calculus from 10 patients was harvested and processed . It was shown that both calculus, and dental plaque within non-mineralised lacunae in calculus, are heterogeneous . Furthermore it was shown that the calculus/dental plaque interface was distinct, although also variable . As calcification occurred apparently independently of dental plaque in some places and when calcification of dental plaque bacteria occurred, it was also varied, it was concluded that supragingival calculus is not simply 'mineralised plaque' but a complex heterogeneous material consisting of numerous phases of mineralisation and non-mineralised areas . The role of dental plaque in the mineralisation process is unclear . However, calculus was not observed arising out of mineralising dental plaque . Mineralising dental calculus and maturing dental plaque are two distinct and different phases occurring simultaneously, albeit with great variability.

J Int Acad Periodontol, 2000 Oct, 2(4), 115 - 9
Removal of supragingival plaque in an intraoral model by use of the Sonicare toothbrush; Stanford CM et al.; This study was performed to evaluate plaque removal efficacy of a mechanical toothbrush in a controlled in vivo trial . The study used enamel sections, obtained from extracted human teeth, to evaluate the efficacy of supragingival plaque removal by a mechanical powered toothbrush in various modes of use . Enamel sections were positioned in milled depressions on metal extensions of a maxillary partial denture that bilaterally extended along the buccal corridors . Four sections were used per side, positioned zero, 2 or 3mm from the surface of the metal extension . The prosthesis was worn for 16 hours, whereupon four enamel sections (two per side) were removed . Intra-oral brushing was then performed for 5 or 15 seconds on the remaining four sections . The total of adherent bacteria was then assayed from all enamel sections . Ten to twenty trials were performed (n=4 pairs/trial) for each set of variables . When the electric toothbrush was used as a manual brush ('off'), it was equivalent in plaque removal to a conventional toothbrush (Oral-B 35; P = 0.49) . However, when the powered brush was in its active mode ('on'), it removed more bacteria (P < 0.0001); this efficacy was observed even when the bristle tips were at a distance from the plaque surface.

J Int Acad Periodontol, 1999 Oct, 1(4), 117 - 20
Effect of amine fluoride-stannous fluoride containing toothpaste (Meridol) on plaque and gingivitis in adults: a six-month clinical study; Shapira L et al.; Treatments that aim to reduce the accumulation of bacteria on dental surfaces are considered today as the most efficient way of treating gingivitis and the resultant destruction of periodontal tissues . Mechanical plaque removal was found to be very effective in the control of gingival inflammation for most patients, but the supplement of toothpastes and mouthwashes with anti-plaque properties may benefit individuals with unperfected plaque control . The aim of the present study was to investigate the long-term effect of amine fluoride/stannous fluoride (AmF/SnF2) toothpaste on plaque levels and gingivitis in adults, as an additional measure to standard oral hygiene . One hundred and three adult subjects with gingivitis were divided into two randomised groups . One group used NaF toothpaste (control) and the second group AmF/SnF2 (Meridol) toothpaste . The subjects were examined every two months under double-blind conditions . The recorded parameters were plaque index (PlI), gingival index (GI) and bleeding on probing . The differences in PlI between baseline and the end of the study were statistically significant for both treatments, and no significant differences were found between the two treatment groups . There was a significant drop in GI between baseline and the concluding examination only for the AmF/SnF2 group, but the differences between the two treatment groups were not significant . A significant reduction in the percentage of bleeding sites during the six-month study period was found in the AmF/SnF2 group, but not in the control group . The results of the present study suggest that the long-term use of AmF/SnF2 toothpaste in adults may be of benefit to gingival health.

J Int Acad Periodontol, 1999 Oct, 1(4), 110 - 6
Epidemiology of periodontal diseases: an update; Papapanou PN; This article provides a brief review of findings from epidemiological studies of periodontal disease that have been generated over the past five years . In line with the conclusions of the 1996 World Workshop in Periodontics, the recent data support the concept that early onset periodontitis appears to be rather infrequent, while advanced adult periodontitis, leading to severe loss of supporting periodontal tissues and tooth loss, does not exceed a prevalence of 10-15% in most populations . However, a number of important issues remain unresolved . It is still not clear whether the prevalence of these diseases has shown an overall decline in recent years . In fact, retention of teeth in older age may contribute to an increase . Analytical epidemiological studies have been increasingly successful in identifying a handful of risk factors for disease onset and progression . These include colonisation at high levels by certain subgingival bacteria, environmental exposures such as cigarette smoking, and systemic conditions such as diabetes mellitus . Importantly, the molecular basis of host susceptibility has recently begun to be unraveled . Research efforts are now focused on creating multi-factorial models to assess the risk for disease, prior to the development of irreversible damage . Importantly, the role of periodontal infections as a modifier of systemic health is being increasingly explored.

J Periodontol, 2003 Feb, 74(2), 219 - 24
Constitutionally hyperreactive neutrophils in periodontitis; Fredriksson MI et al.; BACKGROUND: Neutrophils constitute the first line of host defense, but their extracellular release of oxygen radicals and proteases may cause tissue destruction . Conflicting results have been reported on the neutrophilic production of oxygen radicals in periodontal patients . These may vary with different forms of periodontitis, the pathway of neutrophil activation studied, and the influence of smoking . METHODS: Venous blood was taken from 15 patients with severe chronic periodontitis, treated with scaling, with or without surgery . They were matched by age and gender to periodontally healthy controls; all participants were non-smokers . Peripheral neutrophils from the participants were activated with IgG-opsonized bacteria, complement-opsonized zymosan, or phorbol myristate acetate (PMA) . The total release of oxygen radicals was measured with luminol-enhanced chemiluminescence (CL) and the extracellular release of oxygen radicals with isoluminol-enhanced CL . Antibodies to endotoxin and the concentrations of interleukin (IL)-8 and acute phase proteins in plasma were analyzed to see if these parameters were related to hyperreactivity of the neutrophils . RESULTS: Neutrophils from periodontal patients had significantly higher CL when activated via the FcgammaR pathway, measured as both total generation of oxygen radicals and extracellular generation . The neutrophil generation of oxygen radicals was also higher in patients when complement-opsonized zymosan or PMA was used, but these differences were less pronounced . No significant differences in plasma proteins were found . CONCLUSION: Our findings suggest that the increased CL from peripheral neutrophils in periodontitis may be related to a constitutionally greater responsiveness of the Fcgamma-receptor.

Dtsch Tierarztl Wochenschr, 2003 Feb, 110(2), 73 - 5
The effect of outdoor production on the seroprevalence of Lawsonia intracellularis in growing-finishing pigs in a large pig production unit infected with endemic porcine proliferative enteropathy; Bona B et al.; The study was performed in a large Croatian production unit from May 2000 till June 2002 . Blood samples form late-pregnant gilts were tested by indirect immunofluorescence antibody (IFA) serum assay for Lawsonia intracellularis . The offspring of 301 positively tested gilts were dislocated after the nursery phase either to indoor or outdoor growing-finishing facilities . Ten percent of these animals (142 indoor, 143 outdoor raised pigs) were tested at 2, 6, 10, 14, 18, 22 and 26 weeks of age for seroprevalence of Lawsonia intracellularis . All offspring of IFA positive gilts were seronegative at 2 and 6 weeks of age . At 10 weeks of age 71.1% (101 animals) of indoor and 32.8% (47 animals) of outdoor pigs were tested positive (P < 0.05) . While at 14 weeks of age 71.1% of indoor raised pigs showed seropositivity, the seropositivity declined in outdoor units to 7.6% (P < 0.01) . At weeks 18 (52.1%), 22 (47.8%) and 26 (21.7%) indoor raised pigs still showed marked seropositivity and but their outdoor raised counterparts returned to seronegativity.

J Comput Chem, 2003 Apr 30, 24(6), 741 - 53
Mechanistic insights into oxidosqualene cyclizations through homology modeling; Schulz-Gasch T et al.; 2,3-Oxidosqualene cyclases (OSC) are key enzymes in sterol biosynthesis . They catalyze the stereoselective cyclization and skeletal rearrangement of (3S)-2,3-oxidosqualene to lanosterol in mammals and fungi and to cycloartenol in algae and higher plants . Sequence information and proposed mechanism of 2,3-oxidosqualene cyclases are closely related to those of squalene-hopene cyclases (SHC), which represent functional analogs of OSCs in bacteria . SHCs catalyze the cationic cyclization cascade converting the linear triterpene squalene to fused ring compounds called hopanoids . High stereoselectivity and precision of the skeletal rearrangements has aroused the interest of researchers for nearly half a century, and valuable data on studying mechanistic details in the complex enzyme-catalyzed cyclization cascade has been collected . Today, interest in cyclases is still unbroken, because OSCs became targets for the development of antifungal and hypocholesterolemic drugs . However, due to the large size and membrane-bound nature of OSCs, three-dimensional structural information is still not available, thus preventing a complete understanding of the atomic details of the catalytic mechanism . In this work, we discuss results gained from homology modeling of human OSC based on structural information of SHC from Alicyclobacillus acidocaldarius and propose a structural model of human OSC . The model is in accordance with previously performed experimental studies with mechanism-based suicide inhibitors and mutagenesis experiments with altered activity and product specificity . Structural insight should strongly stimulate structure-based design of antifungal or cholesterol-lowering drugs .

FASEB J, 2003 Apr, 17(6), 644 - 51
A new approach to treat tissue destruction in periodontitis with chemically modified dextran polymers; Escartin Q et al.; Periodontitis are diseases of the supportive tissues of the teeth provoked by bacteria and characterized by gingival inflammation and bone destruction . We have developed a new strategy to repair tissues by administrating agents (RGTA) that mimic heparan sulfates by protecting selectively some of the growth factors naturally present within the injured tissue and interfering with inflammation . After periodontitis induction in hamsters, the animals were left untreated or received weekly i.m . injections of RGTA1507 at a dose of 100 microg/kg, 400 microg/kg, 1.5 mg/kg, or 15 mg/kg for 4 wk . RGTA treatment significantly reduced gingival tissue inflammation, thickened the pocket epithelium by increasing cell proliferation, and enhanced collagen accumulation in the gingiva . A marked reduction in bone loss was observed, resulting from depression of osteoclasia and robust stimulation of bone formation at the dose of 1.5 mg/kg . RGTA treatment for 8 wk at this dose reversed macroscopic bone loss, sharply contrasting with the extensive bone destruction in the untreated animals . RGTA treatment decreased gelatinase A (MMP-2) and B (MMP-9) pro-forms in gingival tissues . Our data indicate that a 4 wk treatment dose-dependently attenuated gingival and bone manifestations of the disease, whereas a longer treatment restored alveolar bone close to controls . By modulating and coordinating host responses, RGTA has unique therapeutic properties and is a promising candidate for the treatment of human periodontitis.

Photochem Photobiol Sci, 2003 Feb, 2(2), 118 - 23
Replicated mesocosm study on the role of natural ultraviolet radiation in high CDOM, shallow lakes; Perez AP et al.; The role of ultraviolet radiation on shallow, high CDOM (colored dissolved organic matter) lakes was investigated during two consecutive summers (1999 and 2000) in replicated mesocosms (rectangular fiberglass tanks) . Each tank (volume: 300 L; depth: 40 cm) was covered with a layer (approximately 3 cm) of sediment from lake El Toro (40 degrees 14' S; 70 degrees 22' W) and filled with filtered water . The experimental design consisted of two treatments: full natural radiation (UV-exposed) and natural radiation without ultraviolet radiation (UV-shielded) . UV-exposed and UV-shielded treatments differed in most studied variables as revealed by repeated measures ANOVA . UV-exposed tanks displayed lower CDOM levels (dissolved absorbance) of lower average molecular size (absorbance ratio between 250 and 365 nm), higher bacterial biomass, and lower chlorophyll a concentration . The effect on consumers (rotifers and crustaceans) was less noticeable . The results are consistent with UV stimulation of bacteria production mediated by higher rates of CDOM photobleaching, and the photoinhibition of planktonic algae . Thus, a major effect of UVR in shallow, high CDOM ecosystems appears to be the stimulation of heterotrophic pathways and a simultaneous inhibition of photoautotrophs.

Eksp Klin Gastroenterol . 2003;(1):57, 183.
{Role of dysbiotic disruption in etiology and pathogenesis of irritable bowel syndrome}; Ruchkina IN et al.; Based on the obtained data, it was established that the development of dysbiotic changes in the large and small intestines after enteric infections is a cause for exacerbation of the irritable bowel syndrome.

Extremophiles, 2003 Apr, 7(2), 159 - 68 Epub 2003 Jan 10.
Purification and characterization of 5'-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus: substrate specificity and primary structure analysis; Cacciapuoti G et al.; 5'-Methylthioadenosine phosphorylase (MTAP) was purified to homogeneity from the hyperthermophilic archaeon Pyrococcus furiosus . The protein is a homoexamer of 180 kDa . The enzyme is highly thermoactive, with an optimum temperature of 125 degrees C, and extremely thermostable, retaining 98% residual activity after 5 h at 100 degrees C and showing a half-life of 43 min at 130 degrees C . In the presence of 100 mM phosphate, the apparent T(m) (137 degrees C) increases to 139 degrees C . The enzyme is extremely stable to proteolytic cleavage and after incubation with protein denaturants, detergents, organic solvents, and salts even at high temperature . Thiol groups are not involved in the catalytic process, whereas disulfide bond(s) are present, since incubation with 0.8 M dithiothreitol significantly reduces the thermostability of the enzyme . N-Terminal sequence analysis of the purified enzyme is 100% identical to the predicted amino acid sequence of the gene PF0016 from the partially sequenced P . furiosus genome . The deduced amino acid sequence of the gene revealed a high degree of identity (52%) with human MTAP . Nevertheless, unlike human MTAP, MTAP from P . furiosus is not specific for 5'-methylthioadenosine, since it phosphorolytically cleaves adenosine, inosine, and guanosine . The calculated k(cat)/ K(m) values for 5'-methylthioadenosine and adenosine, about 20-fold higher than for inosine and guanosine, indicate that 6-amino purine nucleosides are preferred substrates of MTAP from P . furiosus . The structural features and the substrate specificity of MTAP from P . furiosus document that it represents a 5'-methylthioadenosine-metabolizing enzyme different from those previously characterized among Archaea, Bacteria, and Eukarya . The functional and structural relationships among MTAP from P . furiosus, human MTAP, and two putative MTAPs from P . furiosus and Sulfolobus solfataricus are discussed here for the first time.

Extremophiles, 2003 Apr, 7(2), 123 - 30 Epub 2003 Jan 16.
Arsenic resistance in the archaeon "Ferroplasma acidarmanus": new insights into the structure and evolution of the ars genes; Gihring TM et al.; Arsenic resistance in the acidophilic iron-oxidizing archaeon " Ferroplasma acidarmanus" was investigated . F . acidarmanus is native to arsenic-rich environments, and culturing experiments confirm a high level of resistance to both arsenite and arsenate . Analyses of the complete genome revealed protein-encoding regions related to known arsenic-resistance genes . Genes encoding for ArsR (arsenite-sensitive regulator) and ArsB (arsenite-efflux pump) homologues were found located on a single operon . A gene encoding for an ArsA relative (anion-translocating ATPase) located apart from the arsRB operon was also identified . Arsenate-resistance genes encoding for proteins homologous to the arsenate reductase ArsC and the phosphate-specific transporter Pst were not found, indicating that additional unknown arsenic-resistance genes exist for arsenate tolerance . Phylogenetic analyses of ArsA-related proteins suggest separate evolutionary lines for these proteins and offer new insights into the formation of the arsA gene . The ArsB-homologous protein of F . acidarmanus had a high degree of similarity to known ArsB proteins . An evolutionary analysis of ArsB homologues across a number of species indicated a clear relationship in close agreement with 16S rRNA evolutionary lines . These results support a hypothesis of arsenic resistance developing early in the evolution of life.

Int Immunol, 2003 Apr, 15(4), 525 - 34
Antigen-specific, CD4+CD25+ regulatory T cell clones induced in Peyer's patches; Tsuji NM et al.; Since intestine is exposed to numerous exogenous antigens such as food and commensal bacteria, the organ bears efficient mechanisms for establishment of tolerance and induction of regulatory T cells (T(reg)) . Intestinal and inducible T(reg) include T(r)1-like and T(h)3 cells whose major effector molecules are IL-10 and transforming growth factor (TGF)-beta . These antigen-specific T(reg) are expected to become clinical targets to modify the inflammatory immune response associated with allergy, autoimmune diseases and transplantation . In the present study, we characterized the antigen-specific T(reg) induced in the intestine by orally administering high-dose beta-lactoglobulin (BLG) to BALB/c mice . Seven days after feeding, only Peyer's patch (PP) cells among different organs exerted significant suppressive effect on antibody production upon in vitro BLG stimulation . This suppressive effect was also prominent in six BLG-specific CD4(+) T cell clones (OPP1-6) established from PP from mice orally administered with high doses of BLG and was partially reversed by antibodies to TGF-beta . Intravenous transfer of OPP2 efficiently suppressed BLG-specific IgG1 production in serum following immunization, indicating the role of such T(reg) in the systemic tolerance after oral administration of antigen (oral tolerance) . OPP clones secrete TGF-beta, IFN-gamma and low levels of IL-10, a cytokine pattern similar to that secreted by anergic T cells . OPP clones bear a CD4(+)CD25(+) phenotype and show significantly lower proliferative response compared to T(h)0 clones . This lower response is recovered by the addition of IL-2 . Thus, antigen-specific CD4(+)CD25(+) T(reg), which have characteristics of anergic cells and actively suppress antibody production are induced in PP upon oral administration of protein antigen.

J Pineal Res, 2003 May, 34(4), 233 - 41
Non-vertebrate melatonin; Hardeland R et al.; Melatonin has been detected in bacteria, eukaryotic unicells, macroalgae, plants, fungi and various taxa of invertebrates . Although precise determinations are missing in many of these organisms and the roles of melatonin are still unknown, investigations in some species allow more detailed conclusions . Non-vertebrate melatonin is not necessarily circadian, and if so, not always peaking at night, although nocturnal maxima are frequently found . In the cases under study, the major biosynthetic pathway is identical with that of vertebrates . Mimicking of photoperiodic responses and concentration changes upon temperature decreases have been studied in more detail only in dinoflagellates . In plants, an involvement in photoperiodism seems conceivable but requires further support . No stimulation of flowering has been demonstrated to date . A participation in antioxidative protection might be possible in many aerobic non-vertebrates, although evidence for a contribution at physiological levels is mostly missing . Protection from stress by oxidotoxins or/and extensions of lifespan have been shown in very different organisms, such as the dinoflagellate Lingulodinium, the ciliate Paramecium, the rotifer Philodina and Drosophila . Melatonin can be taken up from the food, findings with possible implications in ecophysiology as well as for human nutrition and, with regard to high levels in medicinal plants, also in pharmacology.

Biochem J, 2003 May 15, 372(Pt 1), 33 - 40
Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy; Calleja V et al.; To be able to detect in situ changes in protein conformation without perturbing the physiological environment would be a major step forward in understanding the precise mechanism occurring in protein interaction . We have developed a novel approach to monitoring conformational changes of proteins in intact cells . A double-labelled fluorescent green fluorescent protein-yellow fluorescent protein (GFP-YFP) fusion protein has been constructed, allowing the exploitation of enhanced-acceptor-fluorescence (EAF)-induced fluorescence resonance energy transfer (FRET) . Additionally, a novel fusion partner, YFP(dark), has been designed to act as a sterically hindered control for EAF-FRET . Any conformational changes will cause a variation in FRET, which, in turn, is detected by fluorescence lifetime imaging microscopy ("FLIM") . Protein kinase B (PKB)/Akt, a key component of phosphoinositide 3-kinase-mediated signalling, was selected for this purpose . Although conformational changes in PKB/Akt consequent to lipid binding and phosphorylation have been proposed in models, its behaviour in intact cells has not been tractable . We report here that platelet-derived-growth-factor ("PDGF") stimulation of NIH3T3 cells expressing the GFP-Akt-YFP construct resulted in a loss of FRET at the plasma membrane and hence a change in PKB/Akt conformation . We also show that the GFP-Akt-YFP construct conserves fully its functional integrity . This novel approach of monitoring the in situ conformational changes has broad application for other members of the AGC kinase superfamily and other proteins.

J Org Chem, 2003 Apr 4, 68(7), 2529 - 39
Discovering nature's diverse pathways to vitamin B12: a 35-year odyssey; Scott AI; The chronology of the discoveries along the pathway of vitamin B(12) biosynthesis is reviewed from a personal perspective, including discussion of the most recent finding that two pathways to B(12) exist-one aerobic and one anaerobic-which differ mainly in the ring contraction mechanisms that convert porphyrin to corrin.

Photochem Photobiol Sci, 2002 Apr, 1(4), 225 - 36
UV-induced DNA damage and repair: a review; Sinha RP et al.; Increases in ultraviolet radiation at the Earth's surface due to the depletion of the stratospheric ozone layer have recently fuelled interest in the mechanisms of various effects it might have on organisms . DNA is certainly one of the key targets for UV-induced damage in a variety of organisms ranging from bacteria to humans . UV radiation induces two of the most abundant mutagenic and cytotoxic DNA lesions such as cyclobutane-pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) and their Dewar valence Isomers . However, cells have developed a number of repair or tolerance mechanism to counteract the DNA damage caused by UV or any other stressors . Photoreactivation with the help of the enzyme photolyase is one of the most important and frequently occurring repair mechanisms in a variety of organisms . Excision repair, which can be distinguished into base excision repair (BER) and nucleotide excision repair (NER), also plays an important role in DNA repair in several organisms with the help of a number of glycosylases and polymerases, respectively . In addition, mechanisms such as mutagenic repair or dimer bypass, recombinational repair, cell-cycle checkpoints, apoptosis and certain alternative repair pathways are also operative in various organisms . This review deals with UV-induced DNA damage and the associated repair mechanisms as well as methods of detecting DNA damage and its future perspectives.

Kansenshogaku Zasshi, 2003 Feb, 77(2), 110 - 5
{Evaluation of rapid identification method for Mycobacterium tuberculosis complex using the immunochromatographic slide test kit}; Hasegawa M et al.; Capilia TB, a lateral flow immunochromatographic slide test kit for directly identifying Mycobacterium tuberculosis complex (MTC), was evaluated by using culture-positive specimens from Mycobacteria Growth Indicator Tubes (MGIT) . Sputum specimens from patients suspected of having tuberculosis were treated with NALC-NaOH and cultivated in MGIT960 . Liquid specimens were collected from the positive tubes and directly inoculated with Capilia TB . Liquid specimens were also directly tested with AccuProbe . Of the organisms isolated from the 100 MGIT positive tubes, M . tuberculosis complex was identified in 49 (49%) tubes with Capilia TB and not identified in 51 (51%) with Capilia TB . Mycobacterium avium-intracellulare complex (MAC) was identified in 46 (46%) with AccuProbe MAC and other acid-fast bacteria were identified in 5 (5%) by DNA-DNA hybridization method . There were one tube in which M . tuberculosis complex was detected with Capilia TB and M . tuberculosis complex was not detected with AccuProbe MTC, but no tubes in which M . tuberculosis complex was detected with AccuProbe MTC and M . tuberculosis complex was not detected with Capilia TB . Capilia TB is excellent in sensitivity and specificity and very suitable for rapid diagnosis of tuberculosis and is considered to contribute to public health intervention measures taken for the tuberculosis control in Japan.

J Infect Dis, 2003 Apr 1, 187(7), 1126 - 36 Epub 2003 Mar 19.
Type I interferon induction pathway, but not released interferon, participates in the maturation of dendritic cells induced by negative-strand RNA viruses; Lopez CB et al.; Signaling through toll-like receptors (TLRs) is essential for dendritic cell (DC) maturation induced by bacteria and other pathogens . The mechanism for virus-induced DC maturation is not known . By use of pairs of live viruses with different abilities to induce the interferon (IFN) pathway, a strong correlation between DC maturation and the ability of the virus to induce type I IFN synthesis was demonstrated . The secreted IFN was not necessary, nor was it sufficient to induce full DC maturation . Intracellular viral replication is necessary for this process, and the transcription factor nuclear factor-kappaB was crucial for cytokine induction . The double-stranded RNA-dependent protein kinase was not essential for DC maturation . Similar to TLR-induced DC maturation, after virus infection, separate pathways for the induction of cytokine secretion and the up-regulation of major histocompatibility complex and costimulatory molecules were activated . It was demonstrated that these pathways have different sensitivities to the presence of viral stimulus.

Minerva Stomatol, 2002 Dec, 51(11-12), 495 - 9
{Risk factors in periodontal disease}; Sartori S et al.; Some life styles and systemic pathologies represent risk factors for the periodontal disease . Cigarette smoking in particular increases the speed of progression of periodontitis . Other conditions such as diabetes mellitus, presence of genetic markers and some bacteria are significantly correlated with the periodontal disease.

J Leukoc Biol, 2003 Apr, 73(4), 493 - 501
Fever-like thermal conditions regulate the activation of maturing dendritic cells; Tournier JN et al.; Fever is one of the most frequent clinical signs encountered in pathology, especially with respect to infectious diseases . It is currently thought that the role of fever on immunity is limited to activation of innate immunity; however, its relevance to activation of adaptive immunity remains unclear . Dendritic cells (DCs) that behave as sentinels of the immune system provide an important bridge between innate and adaptive immunity . To highlight the role of fever on adaptive immunity, we exposed murine bone marrow-derived lipopolysaccharide (LPS)- or live bacteria-maturing DCs over a 3-h period to 37 degrees C or to fever-like thermal conditions (39 degrees C or 40 degrees C) . At these three temperatures, we measured the kinetics of cytokine production and the ability of DCs to induce an allogeneic mixed lymphocyte reaction . Our results show that short exposure of DCs to temperatures of 39 degrees C or 40 degrees C differentially increased the secretion of interleukin (IL)-12p70 and decreased the secretion of IL-10 and tumor necrosis factor alpha by maturing DCs . These fever-like conditions induced a regulation of cytokine production at the single-cell level . In addition, short-term exposed LPS-maturing DCs to 39 degrees C induced a stronger reaction with allogeneic CD4(+) T cells than maturing DCs incubated at 37 degrees C . These results provide evidence that temperature regulates cytokine secretion and DC functions, both of which are of particular importance in bacterial diseases.

Biochem Biophys Res Commun, 2003 Apr 4, 303(2), 640 - 4
Binding and internalization of Helicobacter pylori VacA via cellular lipid rafts in epithelial cells; Kuo CH et al.; In this study we investigated the roles of lipid rafts and glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the process of VacA binding and internalization into epithelial cells . Vacuolating activity analysis in AGS, CHO cells, and a CHO-derived line that highly expresses GPI-linked fasI proteins indicated the significance of cholesterol and GPI-APs for VacA activity . Flow cytometric analysis along with VacA-cholesterol co-extraction experiments showed a cholesterol-dependent manner for VacA cell-binding activity, while GPI-APs were not related to it . Differential detergent extraction and fractionation in sucrose density gradient showed co-association of VacA and fasI with rafts on cell membranes . Subcellular distribution of fasI visualized by confocal microscope suggested that fasI trafficked via a newly defined endocytic pathway for GPI-APs in the derived line . Upon VacA intoxication, VacA was visualized to co-migrate along with fasI and finally induced vacuolation coupled with dramatic redistribution of fasI molecules . These results suggest that VacA exploits rafts for docking and entering the cell via the endocytic pathway of GPI-APs.

Biochem Biophys Res Commun, 2003 Apr 4, 303(2), 475 - 82
One-step on-column affinity refolding purification and functional analysis of recombinant human VDAC1; Shi Y et al.; The outer mitochondrial membrane porin, voltage-dependent anion-selective channel (VDAC), is believed to play an important role in mediating mitochondria-dependent apoptosis . However, detailed structure-function studies of VDAC have been hindered by the difficulties to obtain a soluble, correctly folded, and fully active form of the recombinant VDAC and its mutant variants due to its transmembrane nature . Here we report a high-throughput one-step chromatographic procedure in purification of recombinant human VDAC1 (rhVDAC1) protein overexpressed in bacteria . The improved methodology could generate a large quantity of rhVDAC1 with correct folding in terms of the secondary structure, with full biological activities in mediating cytochrome c release and in interaction with Bcl-X(L) . The method will significantly benefit genetic, biochemical, and structural studies of this critical channel protein.

Alcohol Clin Exp Res, 2003 Mar, 27(3), 503 - 8
Phagocytosis and production of reactive oxygen species by peripheral blood phagocytes in patients with different stages of alcohol-induced liver disease: effect of acute exposure to low ethanol concentrations; Parlesak A et al.; BACKGROUND: In rodents, the development of alcoholic liver disease (ALD) after chronic alcohol feeding was shown to depend on the activity of enzymes that are necessary for production of reactive oxygen species (ROS) in phagocytes . The aim of this study was to determine the formation of ROS by resting and challenged phagocytes of patients with different stages of ALD in the presence of ethanol concentrations commonly found in the blood of alcohol abusers . PATIENTS AND METHODS: The release of ROS and the phagocytosis of bacteria by neutrophils and monocytes obtained from 60 patients, who were categorized in three groups due to the severity of ALD, were compared to that of 28 healthy controls . ROS release by these phagocytes was measured after challenging with endotoxin and the addition of ethanol (22 and 44 mM) . RESULTS: Resting neutrophils but not monocytes from patients with severe stages of ALD produced significantly more ROS than those of healthy controls . Basal values of ROS production from neutrophils correlated closely to markers of the severity of ALD . ROS formation was depressed dose-dependently by ethanol in the healthy controls but not in alcohol abusers . CONCLUSIONS: Changes in the ROS metabolism of phagocytes found in this study might contribute to both the development of ALD and the impaired immune response occurring in patients with severe ALD.

Pancreas, 2003 Apr, 26(3), 274 - 8
Adenovirus-mediated gene transfer in the treatment of pancreatic cancer; Pan X et al.; INTRODUCTION: Suicide gene therapy is a new experimental form of cancer chemotherapy that is currently being evaluated in human trials.AIM: To evaluate the killing effects of the cytosine deaminase (CD) gene mediated by an adenovirus vector on human pancreatic carcinoma in vitro and in vivo . METHODOLOGY: The CD gene was cloned into pAdTrack-CMV-CD, pAdTrack-CMV-CD, and pAdEasy-1, which underwent recombination in bacteria BJ5183 . The newly recombinant Ad-CD containing green fluorescent protein was propagated in 293 cells and purified by cesium chloride gradient centrifugation . The human pancreatic carcinoma cell line PaTu8988/SW1990 was infected with this virus, and then 5-fluorocytosine (5-FC) was added; XTT assay was used to estimate relative numbers of viable cells . An in vivo model of pancreatic cancer was established by injecting 1.0 x 10(7) PaTu8988/SW1990 cells subcutaneously in Balb/c nude mice . When tumors were palpable, Ad-CD was injected into each tumor, and 5-FC was administered . The positive clones were selected by endonuclease digestion of the combinations, and the concentration of viral liquids containing the CD gene was 2 x 10(11) pfu/mL . RESULTS: It was found that significant cytotoxic activities were possessed by 5-FC for CD gene-transduced PaTu8988/SW1990 cells, but there was little effect on the nontransduced pancreatic carcinoma cells . The antitumor effect was observed in PaTu8988/SW1990 xenografts from nude mice with in situ CD gene transduction . CONCLUSION: These results indicate that the CD gene mediated by adenovirus has a high level of infectivity and is efficient for gene therapy for pancreatic carcinoma.

J Neurosci, 2003 Mar 15, 23(6), 2170 - 81
Synchronized formation and remodeling of postsynaptic densities: long-term visualization of hippocampal neurons expressing postsynaptic density proteins tagged with green fluorescent protein; Ebihara T et al.; To explore mechanisms governing the formation and remodeling of postsynaptic density (PSD), we used dissociated cultures of hippocampal neurons isolated from transgenic embryos expressing green fluorescent protein (GFP)-tagged PSD proteins PSD-Zip45 (Homer 1c) and PSD-95 . Expression of GFP-tagged PSD molecules was stable, and the remodeling process of PSDs could be followed for >1 week . A higher expression level of GFP-PSD-Zip45 enabled us to quantitatively analyze the amount of PSD-Zip45 clusters during development . Repetitive imaging of the same cell populations between 11 and 17 d in culture revealed an increase of the average PSD-Zip45 cluster density from 0.32 to 0.73/microm . Newly generated dendrites rapidly acquired GFP-PSD-Zip45 clusters, and their density reached the level of parental dendrites within a few days . Temporal profiles of GFP-PSD-Zip45 cluster density showed a variety of patterns . Some dendrites showed a monotonous increase of clusters, whereas others showed complex patterns, including short decremental stages . Analysis of long-term remodeling of PSD-95-GFP clusters confirmed that the decremental stages were not specific to the PSD-Zip45 clusters . Comparison of the temporal profiles of the cluster density among neurons indicated synchronization of both GFP-PSD-Zip45 and PSD-95 clustering within individual cells . Furthermore, activation of cAMP-dependent protein kinase suppressed the decremental stages of cluster remodeling . These observations suggest the presence of signaling mechanisms that can induce synchronized addition or elimination of PSD proteins throughout dendritic arborization of a single neuron.

Gene, 2003 Mar 13, 306, 37 - 44
Molecular cloning and characterization of a novel gene family of four ancient conserved domain proteins (ACDP); Wang CY et al.; We have recently cloned four novel human genes that encode the ancient conserved domain proteins (ACDP) . The full-length cDNA sequence of ACDP1 consists of 5898 bp and encodes a predicted protein of 951 amino acids (AA) . The transcript for ACDP2 has 4058 bp of cDNA sequence, encoding a protein of 875 AA . ACDP3 contains 3113 bp of cDNA sequence and encodes a putative protein of 707 AA . ACDP4 contains 4765 bp of cDNA sequence and encodes a protein of 775 AA . The ACDP genes belong to a highly conserved new gene family . The conserved region showed 62.8% of nucleotide sequence identity, and 65.5% of AA identity with 92% of AA homologies among ACDP members . The conserved domain is also found in genes from evolutionarily divergent species from bacteria, yeast, Caenorhabditis elegans, and Drosophila melanogaster to mammals . All ACDP genes except ACDP1 have a ubiquitous expression pattern while ACDP1 expression is restricted to the brain and testis . Immunofluorescence staining of premeablized HeLa cells showed that ACDP proteins are predominantly localized in the nucleus . Sequence homology analyses revealed AA property and structural homologies between the ACD domain and cyclin molecules.

Int Endod J, 2003 Feb, 36(2), 106 - 16
Histomorphometric analysis of odontoblast-like cell numbers and dentine bridge secretory activity following pulp exposure; Murray PE et al.; AIM: The purpose of this study was to collect quantitative information about the numbers and dentine bridge secretory activity of odontoblast-like cells following dental pulp exposure . METHODOLOGY: The numbers and secretory activity of odontoblast-like cells were measured histomorphometrically between 7 days and 2 years in 161 pulp-exposed nonhuman primate teeth . The area of dentine bridges and the dimensions of cavity preparations were measured . The density of odontoblast-like cells and subjacent reorganizing tissue cells were measured beneath dentine bridge formation . The presence of operative dentine debris and tunnel defects in bridges was noted . Pulp inflammation was categorized according to ISO standards . Bacteria were detected using McKay's stain . RESULTS: The area of dentine bridges was mediated by the density and secretory activity of odontoblast-like cells over time . The cell density of subjacent reorganizing tissue was found to be strongly associated with that of odontoblast-like cells . Bacterial microleakage was found to impede dentine bridge secretion by odontoblast-like cells . CONCLUSIONS: Pulp reparative activity occurs naturally beneath capping materials in the absence of bacterial microleakage . The outcome of pulp-capping treatments could be beneficially influenced by concentrating attention on limiting the width of pulp exposure, minimizing pulp injury by limiting the creation of operative debris and placing materials which prevent bacterial microleakage.

Aliment Pharmacol Ther, 2003 Apr 1, 17(7), 895 - 904
A randomized controlled trial of a probiotic, VSL#3, on gut transit and symptoms in diarrhoea-predominant irritable bowel syndrome; Kim HJ et al.; AIM: To investigate the effects of a probiotic formulation, VSL#3, on gastrointestinal transit and symptoms of patients with Rome II irritable bowel syndrome with predominant diarrhoea . METHODS: Twenty-five patients with diarrhoea-predominant irritable bowel syndrome were randomly assigned to receive VSL#3 powder (450 billion lyophilized bacteria/day) or matching placebo twice daily for 8 weeks after a 2-week run-in period . Pre- and post-treatment gastrointestinal transit measurements were performed in all patients . Patients recorded their bowel function and symptoms daily in a diary during the 10-week study, which was powered to detect a 50% change in the primary colonic transit end-point . RESULTS: There were no significant differences in mean gastrointestinal transit measurements, bowel function scores or satisfactory global symptom relief between the two treatment groups, pre- or post-therapy . Differences in abdominal bloating scores between treatments were borderline significant (P = 0.09, analysis of covariance) . Further analysis revealed that abdominal bloating was reduced (P = 0.046) with VSL#3 {mean post- minus pre-treatment score, - 13.7; 95% confidence interval (CI), - 2.5 to - 24.9}, but not with placebo (P = 0.54) (mean post- minus pre-treatment score, - 1.7; 95% CI, 7.1 to - 10.4) . With the exception of changes in abdominal bloating, VSL#3 had no effect on other individual symptoms: abdominal pain, gas and urgency . All patients tolerated VSL#3 well . CONCLUSION: VSL#3 appears to be promising in the relief of abdominal bloating in patients with diarrhoea-predominant irritable bowel syndrome . This is unrelated to an alteration in gastrointestinal or colonic transit.

Drugs, 2003, 63(6), 535 - 53
Fixed-dose combination drugs for tuberculosis: application in standardised treatment regimens; Blomberg B et al.; Short-course chemotherapy is highly efficacious in treating tuberculosis (TB) . However, the length (>/=6 months) and complexity (three or four different drugs) of the treatment makes adherence difficult . Erratic treatment not only fails to cure patients but also creates chronically contagious cases, who may excrete drug-resistant TB bacteria . The Directly Observed Treatment Short-course (DOTS) strategy recommended by WHO provides a comprehensive organisational and infrastructural framework for the rational use of diagnosis, drug supply, as well as case and programme management services, in TB control . WHO and other organisations recommend fixed-dose combination formulations (FDCs) as a further step to facilitate the optimal drug treatment of TB . Using FDCs in TB control will simplify the doctor's prescription and patient's drug intake, as well as the drug supply management of the programme . By preventing monotherapy and facilitating the ingestion of adequate doses of the constituent anti-TB drugs, FDCs are expected to help prevent the emergence of drug resistance.This article presents the international recommendations for the use of FDCs in TB programmes . The fundamental issue is to obtain drug supplies of good quality . A laboratory network for quality testing, including bioavailability testing of FDCs exists, and the recently established Global TB Drug Facility (GDF) supplies quality TB drugs, including 4-drug FDCs, to countries requesting assistance . This articles deals with the requirements for a successful transition to FDC-based treatment . It emphasises the need for appropriately revised programme documentation (programme manual, training modules, treatment guidelines and forms), training of staff at all levels, carefully calculated drug needs, and a plan for the exhaustion of existing stocks of loose tablets and the phasing-in of FDCs at all levels of the programme at the same time . Loose drugs for individualised treatment of patients with adverse effects should be kept at district or central health institutions.

Chemosphere, 2003 Jan, 50(1), 165 - 9
The inhibitory effects of lindane in batch and upflow anaerobic sludge blanket reactors; Erguder TH et al.; In this study, the inhibitory effects of lindane (LIN) on originally unacclimated mixed anaerobic cultures were investigated by anaerobic toxicity assay (ATA) experiments . ATA experiments revealed that 10 mg/l LIN exerted inhibitory effects on anaerobic cultures, which was recoverable . Continuous reactor experiments conducted to determine the inhibitory effects of LIN and the maximum LIN loading rate achievable in two-stage upflow anaerobic sludge blanket (UASB) reactors indicated that anaerobic granular cultures were successfully acclimated to 30 mg/l LIN . The maximum LIN loading rate and minimum hydraulic retention time (HRT) possible for the UASB system were 10 mg/l day and 18 h, respectively, which resulted in the overall chemical oxygen demand (COD) removal efficiency of 89%.

J Biol Chem, 2003 Jun 6, 278(23), 20882 - 9 Epub 2003 Mar 24.
Lectins homologous to those of monocotyledonous plants in the skin mucus and intestine of pufferfish, Fugu rubripes; Tsutsui S et al.; We have characterized pufflectin, a novel mannose-specific lectin, from the skin mucus of the pufferfish, Fugu rubripes . Molecular mass estimations by gel filtration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry and the SDS-PAGE pattern suggest that pufflectin is a homodimer composed of non-covalently associated subunits of 13 kDa . The full-length pufflectin cDNA consists of 527 bp, with 116 amino acid residues deduced from the open reading frame . The amino acid sequence of pufflectin shows no homology with any known animal lectin . Surprisingly, pufflectin shares sequence homology with mannose-binding lectins of monocotyledonous plants and has conserved two of three carbohydrate recognition domains of these plant lectins . The pufflectin gene is expressed in gills, oral cavity wall, esophagus, and skin . In addition, an isoform occurs exclusively in the intestine . Pufflectin differs from mannose-binding lectins purified from the blood plasma of Fugu . Whereas pufflectin did not agglutinate five bacterial species tested, it was demonstrated to bind to the parasitic trematode, Heterobothrium okamotoi . This finding suggests that pufflectin contributes to the parasite-defense system in Fugu.

Infect Immun, 2003 Apr, 71(4), 1995 - 2001
Anaplasma phagocytophilum reduces neutrophil apoptosis in vivo; Scaife H et al.; Ovine neutrophils spontaneously underwent apoptosis during culture in vitro, as assessed by morphological changes and exposure of annexin V binding sites on their cell surfaces . The addition of conditioned medium from concanavalin A-treated ovine peripheral blood mononuclear cells (PBMC) could partially protect against this progression into apoptosis, but dexamethasone and sodium butyrate could not . Actinomycin D accelerated the rate at which ovine neutrophils underwent apoptosis . Neutrophils isolated from sheep experimentally infected with Anaplasma phagocytophilum showed significantly delayed apoptosis during culture ex vivo, and the addition of conditioned medium from PBMC to these cells could not delay apoptosis above the protective effects observed after in vivo infection . The ability of neutrophils from A . phagocytophilum-infected sheep to activate a respiratory burst was increased compared to the activity measured in neutrophils from uninfected sheep, but chemotaxis was decreased in neutrophils from infected sheep . These data are the first demonstration that in vivo infection with A . phagocytophilum results in changes in rates of apoptosis of infected immune cells . This may help explain how these bacteria replicate in these normally short-lived cells.

Antimicrob Agents Chemother, 2003 Apr, 47(4), 1430 - 2
Novel tetracycline resistance determinant from the oral metagenome; Diaz-Torres ML et al.; A major drawback of most studies on how bacteria become resistant to antibiotics is that they concentrate mainly on bacteria that can be cultivated in the laboratory . In the present study, we cloned part of the oral metagenome and isolated a novel tetracycline resistance gene, tet(37), which inactivates tetracycline.

Drug Discov Today, 2003 Apr 1, 8(7), 297 - 306
Targeting RNA: new opportunities to address drugless targets; Zaman GJ et al.; Historically, pharmaceutical industries have focussed on the discovery of compounds that target the protein products of genes . The intermediary product between gene and protein, consisting of RNA, has remained largely unexplored . Several drugs targeting the rRNA of bacteria have been, however, in clinical use for over half a century . One of these drug classes, the aminoglycoside antibiotics, also targets human rRNA, and have been developed as therapeutics for genetic disorders . Targeting at the RNA level is an economical approach to address non-drugable proteins and targets that have failed to give leads by hits in HTS, as it can build on biological knowledge gathered over years . RNA also offers entirely new opportunities for drug development, such as targeting of non-coding RNA sequences.

J Mol Biol, 2003 Apr 4, 327(4), 867 - 84
Hammond behavior versus ground state effects in protein folding: evidence for narrow free energy barriers and residual structure in unfolded states; Sanchez IE et al.; Apparent transition state movement upon mutation or changes in solvent conditions is frequently observed in protein folding and is often interpreted in terms of Hammond behavior . This led to the conclusion that barrier regions in protein folding are broad maxima on the free energy landscape . Here, we use the concept of self-interaction and cross-interaction parameters to test experimental data of 21 well-characterized proteins for Hammond behavior . This allows us to characterize the origin of transition state movements along different reaction coordinates . Only one of the 21 proteins shows a small but coherent transition state movement in agreement with the Hammond postulate . In most proteins the structure of the transition state is insensitive to changes in protein stability . The apparent change in the position of the transition state upon mutation, which is frequently observed in phi-value analysis, is in most cases due to ground-state effects caused by structural changes in the unfolded state . This argues for significant residual structure in unfolded polypeptide chains of many proteins . Disruption of these residual interactions by mutation often leads to decreased folding rates, which implies that these interactions are still present in the transition state . The failure to detect Hammond behavior shows that the free energy barriers encountered by a folding polypeptide chain are generally rather narrow and robust maxima for all experimentally explorable reaction coordinates .

Pediatr Int, 2003 Feb, 45(1), 31 - 4
Therapeutic effect of spiramycin in brucellosis; Geyik MF et al.; OBJECTIVE: This study was undertaken to investigate the usefulness of spiramycin in treatment for brucellosis in an animal model . METHODS: Eighty-four Sprague-Dawley rats were infected by intraperitoneal injection of Brucella melitensis suspension . Seven days after inoculation, four rats were selected randomly, killed and spleen cultures and Brucella standard tube agglutination test were carried out . All four rats were found to be infected . Eighty adult rats were randomly divided into four groups of 20 rats each . Tap water was given to the first group . Rifampicin 50 mg/kg per day and doxycycline 40 mg/kg per day were given to the second group, spiramycin 50 mg/kg per day orally was given to the third group, and a combination of spiramycin and rifampicin at the same dose and period was given to the fourth group . Duration of therapy regimens in all groups was 21 days . The spleens of all 80 rats were removed aseptically, homogenized, and placed onto Brucella agar plates to determine if viable bacteria were present . RESULTS: Bacterial growth occurred in all of the rats' spleens in the first group and in two rats' spleens in the spiramycin group . Mean colony forming unit (c.f.u.) values were at the highest in the first group . The effectivities of spiramycin and rifampicin-spiramycin were similar to rifampicin-doxycycline . There were no differences in the treatment results between the three groups that received combined rifampicin-doxycycline, rifampicin-spiramycin and only spiramycin (P>0.05) . CONCLUSIONS: The results show that spiramycin cures experimental rat brucellosis and may be an effective alternative in the therapy of human brucellosis.

J Oral Pathol Med, 2003 Apr, 32(4), 219 - 23
Helicobacter pylori DNA in recurrent aphthous stomatitis; Victoria JM et al.; BACKGROUND: Considering not only the fact that recurrent aphthous stomatitis (RAS) and stomach ulcers are immunologically mediated ulcers associated with Helicobacter pylori, but also the recent evidence that anaemia can be associated with both diseases, and the discovery of H . pylori in the oral mucosa led us to hypothesize that this bacteria may be related to RAS pathogenesis . METHODS: Thirty-six consecutive subjects affected by minor and major forms of RAS and 48 healthy volunteers were included in the present study . The nested polymerase chain reaction (PCR) technique was used to detect the presence of H . pylori in the oral lesion, the normal contralateral mucosa of patients affected by RAS and the oral mucosa of control subjects . The chi2- and Fisher's tests were used for statistical analysis . RESULTS: No association between RAS lesions and H . pylori was observed . However, 14 out of 36 (38.9%) of the patients with RAS were found to show the presence of H . pylori DNA in the lesion and/or contralateral mucosa . Sixteen out of 48 (33.3%) of the patients without RAS (control subjects) were positive (P > 0.05) . CONCLUSION: The present study does not give support to the assumption that H . pylori could be involved in RAS development.

Arch Pathol Lab Med, 2003 Mar, 127(3), 289 - 96
Reactivity of resident immunocytes in normal and prepsoriatic skin using an ex vivo skin-explant model system; Curry JL et al.; CONTEXT: While it is well known that both exogenous and endogenous stimuli can trigger appearance of psoriatic lesions, the initial cellular and molecular events mediated by immunocompetent cells normally resident in prepsoriatic (PN) skin are not well understood . Moreover, it is unclear whether there are any fundamentally important differences in the innate immune response of normal healthy skin (NN skin) versus PN skin . Since acute tissue responses to stimuli involve both resident cells and immunocytes recruited rapidly from circulation, it is difficult to discern the contribution of endogenous cells normally present in skin . OBJECTIVE: To solely characterize the reactivity of resident immunocytes using an experimental system . DESIGN: To probe the activation potential of resident immunocytes in NN (n = 18) and PN skin (n = 10), a short-term ex vivo organ culture system containing interleukin (IL)-2 was established and characterized . To mimic exogenous or environmental trigger factors, bacteria-derived superantigens and lipopolysaccharide were added to the skin-explant assays, whereas endogenous trigger factors were investigated using heat shock proteins . RESULTS: Using this skin-explant assay, both NN and PN skin gave rise to an expansion of various T-cell subsets, which could differentially produce various cytokines and a growth factor (keratinocyte growth factor), depending on the stimulus and source of skin . Bacterial superantigens were relatively potent inducers of interferon-gamma, and natural killer-T cells were observed proliferating from PN skin . CONCLUSIONS: Despite relatively few T cells normally residing in either NN or PN skin, initiation of skin explants from both sets of individuals in the presence of IL-2 triggered vigorous T-cell proliferation and cytokine/growth factor release . These results demonstrate the utility of this skin-explant assay system to further investigate quantitative and qualitative immune responses of NN and PN skin.

Biochemistry, 2003 Apr 1, 42(12), 3446 - 56
Structural studies on ADP activation of mammalian glutamate dehydrogenase and the evolution of regulation; Banerjee S et al.; Glutamate dehydrogenase (GDH) is found in all organisms and catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate . Unlike GDH from bacteria, mammalian GDH exhibits negative cooperativity with respect to coenzyme, activation by ADP, and inhibition by GTP . Presented here are the structures of apo bovine GDH, bovine GDH complexed with ADP, and the R463A mutant form of human GDH (huGDH) that is insensitive to ADP activation . In the absence of active site ligands, the catalytic cleft is in the open conformation, and the hexamers form long polymers in the crystal cell with more interactions than found in the abortive complex crystals . This is consistent with the fact that ADP promotes aggregation in solution . ADP is shown to bind to the second, inhibitory, NADH site yet causes activation . The beta-phosphates of the bound ADP interact with R459 (R463 in huGDH) on the pivot helix . The structure of the ADP-resistant, R463A mutant of human GDH is identical to native GDH with the exception of the truncated side chain on the pivot helix . Together, these results strongly suggest that ADP activates by facilitating the opening of the catalytic cleft . From alignment of GDH from various sources, it is likely that the antenna evolved in the protista prior to the formation of purine regulatory sites . This suggests that there was some selective advantage of the antenna itself and that animals evolved new functions for GDH through the addition of allosteric regulation.

Biomed Pharmacother, 2002, 56 Suppl 2, 319s - 326s
Geomagnetics and society interact in weekly and broader multiseptans underlying health and environmental integrity; Cornelissen G et al.; Evidence for the ubiquity and partial endogenicity of about-weekly (circaseptan) components and multiples and/or submultiples thereof (the multiseptans) accumulates as longer and denser records become available . Often attributed to a mere response to the social schedule, circaseptan components now have been documented to characterize environmental variables related to primarily non-photic solar effects . Plausibly, like circadians, circaseptans are anchored in genomes, from bacteria to humans, via both an internal and external evolution . If so, circaseptans, like circadians, may be found in the absence of a 7-day schedule, whereas the social schedule may play a synchronizing role and be responsible for the detection of prominent weekly variations in population statistics . The wobbliness of multiseptans and other components of some environmental time structures (chronomes) may correspond to the wobbliness of multiseptans found in cardiovascular morbidity statistics . Here, the latter stem primarily, but not exclusively, from an extensive database on the incidence of daily calls for an ambulance in Moscow, Russia from 1979-1981 . A modulation of multiseptans and other chronome components of both environmental and biological variables by the about 11-year solar activity cycle (and of other low-frequency signals reviewed elsewhere) may account for prior controversies and scepticism about a variety of non-photic effects on biota . This is notably the case when relatively short series are analyzed without consideration of effects of unassessed long-term variations; this is the task of the new field of chronomics . In the spectral element of the chronomes of geophysical and biospherical variability, there are natural near weeks,apart from any precise 7-day periodicity.

DNA Seq, 2002 Dec, 13(6), 313 - 20
The major component of the cellulosomes of anaerobic fungi from the genus Piromyces is a family 48 glycoside hydrolase; Steenbakkers PJ et al.; Sequencing of two cDNAs from the anaerobic fungi Piromyces equi and Piromyces sp . strain E2 revealed that they both encode a glycoside hydrolase (GH) family 48 cellulase, containing two C-terminal fungal dockerin domains . N-terminal sequencing of the major component of the Piromyces multi-enzyme cellulase/hemicellulase complex, termed the cellulosome, showed that these 80 kDa proteins corresponded to the GH family 48 enzyme . These data show for the first time that GH family 48 cellulases are not confined to bacteria, and that bacterial and fungal cellulosomes share the same pivotal component.

Genes Dev, 2003 Mar 15, 17(6), 774 - 85
Recruitment of terminal protein to the ends of Streptomyces linear plasmids and chromosomes by a novel telomere-binding protein essential for linear DNA replication; Bao K et al.; Bidirectional replication of Streptomyces linear plasmids and chromosomes from a central origin produces unpaired 3'-leading-strand overhangs at the telomeres of replication intermediates . Filling in of these overhangs leaves a terminal protein attached covalently to the 5' DNA ends of mature replicons . We report here the essential role of a novel 80-kD DNA-binding protein (telomere-associated protein, Tap) in this process . Biochemical studies, yeast two-hybrid analysis, and immunoprecipitation/immunodepletion experiments indicate that Tap binds tightly to specific sequences in 3' overhangs and also interacts with Tpg, bringing Tpg to telomere termini . Using DNA microarrays to analyze the chromosomes of tap mutant bacteria, we demonstrate that survivors of Tap ablation undergo telomere deletion, chromosome circularization, and amplification of subtelomeric DNA . Microarray-based chromosome mapping at single-ORF resolution revealed common endpoints for independent deletions, identified amplified chromosomal ORFs adjacent to these endpoints, and quantified the copy number of these ORFs . Sequence analysis confirmed chromosome circularization and revealed the insertion of adventitious DNA between joined chromosome ends . Our results show that Tap is required for linear DNA replication in Streptomyces and suggest that it functions to recruit and position Tpg at the telomeres of replication intermediates . They also identify hotspots for the telomeric deletions and subtelomeric DNA amplifications that accompany chromosome circularization.

J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Mar 25, 786(1-2), 45 - 59
Purification of recombinant BtpA and Ycf3, proteins involved in membrane protein biogenesis in Synechocystis PCC 6803; Schwabe TM et al.; The gene products Ycf3 (hypothetical chloroplast open reading frame) and BtpA (biogenesis of thylakoid protein) are thought to be involved in the biogenesis of the membrane protein complex photosystem I (PSI) from Synechocystis PCC 6803 . PSI consists of 12 different subunits and binds more than 100 cofactors, making it a model protein to study different aspects of membrane protein biogenesis . For a detailed biophysical characterization of Ycf3 and BtpA pure proteins must be available in sufficient quantities . Therefore we cloned the corresponding genes into expression vectors . To facilitate purification we created His-tagged versions of Ycf3 and BtpA in addition to the unmodified forms . Immobilized metal affinity chromatography (IMAC) yielded His-tagged proteins which were used for the production of antibodies . Purification strategies for non-tagged proteins could also be established: Ycf3 could be purified in soluble form using a two-step purification in which ammonium sulfate precipitation was combined with anion-exchange chromatography (IEC) . BtpA had to be purified from inclusion bodies by two-consecutive IEC steps under denaturing conditions . An optimized refolding protocol was established that yielded pure BtpA . In all cases, MALDI-TOF peptide mass fingerprinting (PMF) was used to confirm protein identity . Initially, size exclusion chromatography and CD-spectroscopy were used for biophysical characterization of the proteins . Both Ycf3 and BtpA show homo-oligomerization in vitro . In summary, purification protocols for Ycf3 and BtpA have been designed that yield pure proteins which can be used to probe the molecular function of these proteins for membrane protein biogenesis.

Transgenic Res, 2003 Feb, 12(1), 101 - 9
Chemical-inducible, ecdysone receptor-based gene expression system for plants; Padidam M et al.; We have developed an inducible gene expression system with potential for field application using the ecdysone receptor (EcR) from the spruce budworm and the non-steroidal EcR agonist, methoxyfenozide . Chimeric transcription activators were constructed with EcR ligand binding domain, GAL4 and LexA DNA binding domains, and VP16 activation domain . In the presence of methoxyfenozide, the transcription activators induced expression of the luciferase reporter gene cloned downstream of a promoter containing GAL4A- or LexA-response element and a minimal 35S promoter . Low basal and high induced luciferase expression was optimized by cloning the activator and the reporter genes in different tandem orientations . Many transgenic Arabidopsis and tobacco plants were obtained with little or no basal expression in the absence of methoxyfenozide and inducible expression that was several fold higher than that observed with the constitutive 35S promoter . Moreover, gene expression was controlled over a wide range of methoxyfenozide concentration . Our results demonstrate that the inducible gene expression system based on the spruce budworm EcR ligand binding domain with methoxyfenozide as a ligand is very effective in regulating transgenes in plants . It is suitable for field applications because methoxyfenozide is commercially available and has an exceptional health and environmental safety profile.

Schweiz Arch Tierheilkd, 2003 Feb, 145(2), 70 - 4
{Use and reliability of the Delvo-P-test}; Hassig M; The Delvo-P-test (Gist-brocades nr, Delft, The Netherlands) is based on biological detection of antibiotics in the milk . According to the procedure of the manufacture the test is suited for bulk milk processing factories . Problems arise when the test is used on milk samples of a single cow . Buffers, cells, bacteria, cell contents, and desinfection without using antibiotics may give false positive and false negative results . An error up to 44.8% was detected on single cow milk samples . The test is well suited for bulk milk, where the processing of milk follows a biological procedure as for cheese . The use on single cow milk samples to prove absence of antibiotics is not recommended.

Am J Physiol Heart Circ Physiol, 2003 Jul, 285(1), H277 - 82 Epub 2003 Mar 20.
Do angiotensin-converting enzyme inhibitors directly stimulate the kinin B1 receptor?
Fortin JP, Gobeil F Jr, Adam A, Regoli D, Marceau F.
It has been recently claimed that the human B1 receptors for kinins bind angiotensin-converting enzyme (ACE) inhibitors via a potential zinc-binding domain and are pharmacologically stimulated by these drugs . We verified whether ACE inhibitors stimulate B1 receptors in vitro . The isolated rabbit aorta or mouse stomach responded by negligible contractions to the application of captopril, enalaprilat, or zofenoprilat . The human isolated umbilical vein also failed to respond to enalaprilat . All of these preparations were responsive to the B1 receptor agonists des-Arg9-bradykinin (BK) or Lys-des-Arg9-BK . Furthermore, enalaprilat applied continuously had no significant interaction with the effects of Lys-des-Arg9-BK on the rabbit aorta . Enalaprilat failed to stimulate {3H}arachidonate release, translocate the receptors (confocal microscopy), or stimulate ERK1/2 phosphorylation (immunoblot) in HEK-293 cells stably expressing the rabbit B1 receptor conjugated to yellow fluorescent protein . The phospho-ERK1/2 content of arterial smooth muscle cells of human or rabbit origin was increased by treatment with Lys-des-Arg9-BK but not with enalaprilat . ACE inhibitors do not act as bona fide agonists of the kinin B1 receptors.

Trends Microbiol, 2003 Mar, 11(3), 105 - 8
Hypervirulence and pathogen fitness; Foreman-Wykert AK et al.; Historically, pathogenesis research has focused on the identification and characterization of virulence factors . More recently, 'anti-virulence' genes have been discovered . Mutations in these loci result in a hypervirulent phenotype, as measured by a lower lethal dose, a colonization advantage, reduced clearance or decreased survival time of the host . If these genes function to reduce pathogen virulence, why have they been retained? Multiple hypotheses have been offered to explain this phenomenon.

Drug Dev Ind Pharm, 2003 Feb, 29(2), 121 - 9
Effect of buffers on the properties of microbicidal hydrogels containing monoglyceride as the active ingredient; Kristmundsdottir T et al.; Hydrogel formulations containing the monoglyceride monocaprin have shown potent microbicidal activity against several sexually transmitted viruses and bacteria . It is recommended that formulations for preventing infection in the vagina have a low pH as the HIV virus is inactivated at low pH . The object of the work was to investigate how incorporation of buffers into the hydrogel formulations affects physicochemical properties and microbicidal activity of the active substance . Two series of gels were formulated using carbomer (Carbopol 934) and sodium carboxymethylcellulose (NaCMC) as gel-forming agents . The presence of buffers in the gels caused a lowering in gel viscosity, with carbomer gels being more sensitive to buffer presence than NaCMC gels . To obtain viscosity similar to that of a gel without buffer, the amount of polymer needs to be increased . An increase in the amount of NaCMC by 60-70% is needed to obtain the same viscosity as in gel without buffers; but for carbomer, the amount of polymer needs to be doubled . It appears that the effect of maleate buffer on NaCMC gel formation is greater than that of the citrate/lactate buffer; but for carbopol gels, the effects of the buffer systems tested on gel viscosity were equal . The virucidal activity of NaCMC gel buffered with citrate/lactate buffer against herpes simplex virus type 1 and HIV was not reduced by the presence of buffer . The results show that the presence of buffers in the hydrogel formulations affects gel viscosity, but the virucidal effect of the active compound, monocaprin, is not diminished.

Zh Obshch Biol, 2003 Jan-Feb, 64(1), 65 - 77
{Classification of nucleotide sequences over their frequency dictionaries reveals a relation between the structure of sequences and taxonomy of their bearers}; Gorban' AN et al.; Classification of 16S RNA sequences over their frequency dictionaries, both real ones, and transformed ones was studied . Two entities were considered to be close each other from the point of view of their structure, if their frequency dictionaries were close, in Eucledian metric . A transformation procedure of a frequency dictionary has been implemented that reveals the peculiarities of information structure of a nucleotide sequence . A comparative study of two classification developed over the real frequency dictionary vs . that one developed over the transformed frequency dictionary was carried out . The strong correlation is revealed between the classification and the taxonomy of 16S RNA bearer . For the classes isolated, the information valuable words were identified . These words are the main factors of a difference between the classes . The frequency dictionaries containing the words of the length 3 exhibit the best correlation between a class and a genus . A genus, as a rule, is included into the same class, and the exclusion are sporadic . A development of hierarchy classification over the transformed frequency dictionaries separated one or two taxonomy groups, as each stage of classification . The unexpectedly frequent, or contrary, unexpectedly rare occurred of words (of the length 3) in entities under consideration make the structure difference between the classes of the nucleotide sequences.

Pediatr Res, 2003 Jul, 54(1), 52 - 7 Epub 2003 Mar 19.
Monocyte function in preterm, term, and adult sheep; Kramer BW et al.; The preterm infant has functionally immature monocytes . The effects of common clinical interventions and exposures that might modulate inflammation were evaluated using monocytes isolated from blood of preterm lambs {130 d gestational age (GA)}, near-term lambs (141 d GA), and adult sheep . Endotoxin stimulated hydrogen peroxide production by adult monocytes, but monocytes from 130-d and 141-d GA lambs had a reduced and delayed hydrogen peroxide production . Endotoxin did not decrease apoptosis of monocytes from 130-d and 141-d GA lambs but decreased apoptosis of adult monocytes . Dexamethasone increased the phagocytosis of bacteria and apoptotic cells by adult monocytes by 35% but not by monocytes from 130-d and 141-d GA lambs . Synthetic and natural surfactants and dipalmitoylphosphatidylcholine increased phagocytosis of apoptotic cells by monocytes from preterm, term, and adult sheep . Monocytes from preterm and term lambs differ from adult monocytes in tests of both the initiation and the resolution of inflammation . The reduced phagocytosis of apoptotic cells by monocytes from the preterm may contribute to prolonged inflammation in diseases such as bronchopulmonary dysplasia.

J Immunol, 2003 Apr 1, 170(7), 3782 - 9
Depletion of neutrophils in IL-10(-/-) mice delays clearance of gastric Helicobacter infection and decreases the Th1 immune response to Helicobacter; Ismail HF et al.; Gastric infection with Helicobacter induces a lymphocyte-rich mucosal inflammation that contains a minor population of neutrophilic granulocytes . The function of neutrophils in the local immune response to gastric Helicobacter infection remains unknown . To investigate this issue, we conducted experiments in neutrophil-depleted control wild-type (wt) and IL-10(-/-) mice infected with Helicobacter felis by gastric lavage . Infection of wt mice elicited a mild, focal gastritis and a Helicobacter-specific Th1 immune response . In wt mice Helicobacter colonization of the stomach was persistent and progressively increased during the 29 days of observation . Infection of IL-10(-/-) mice with H . felis elicited a severe chronic gastritis and a greatly enhanced Helicobacter-specific Th1 immune response, as compared with wt mice . After initial colonization, the IL-0(-/-) mice completely cleared Helicobacter from the stomach by day 8 . The gastric inflammation in wt and IL-10(-/-) mice contained modest numbers of neutrophils . The intensity of gastric inflammation and the extent of Helicobacter colonization were similar in control and in neutrophil-depleted wt mice . In contrast, neutrophil depletion of Helicobacter-infected IL-10(-/-) mice decreased the severity of gastritis, modulated the Helicobacter-specific Th1 immune response, and delayed the clearance of bacteria from the stomach . These studies identify a role for neutrophils in the local and systemic immune response to gastric Helicobacter in IL-10(-/-) mice.

Biochem Biophys Res Commun, 2003 Mar 28, 303(1), 223 - 9
Characterization of aldehyde ferredoxin oxidoreductase gene defective mutant in Magnetospirillum magneticum AMB-1; Wahyudi AT et al.; A non-magnetic mutant of Magnetospirillum magneticum AMB-1, designated as NMA21, was generated by mini-Tn5 transposon mutagenesis to identify genes involved in bacterial magnetic particle (BMP) synthesis . Alignment of the DNA sequences flanking the transposon allowed the isolation of an open reading frame (ORF2) within an operon consisting of five genes . The amino acid sequence of ORF2 showed homology with tungsten-containing aldehyde ferredoxin oxidoreductase (AOR) from Pyrococcus furiosus (48% identity and 64% similarity), which functions for aldehyde oxidation . AOR was found to be expressed under microaerobic conditions and localized in the cytoplasm of AMB-1 . Iron uptake and growth of NMA21 were lower than wild type . Transmission electron microscopy (TEM) of NMA21 revealed that no BMPs were completely synthesized, but polyhydroxybutyrate (PHB)-like granules were persistently produced . These results indicate that AOR may contribute to ferric iron reduction during BMP synthesis in M . magneticum AMB-1 under microaerobic respiration.

Ugeskr Laeger, 2003 Mar 3, 165(10), 1039 - 40
{Splenic cysts--pathogenesis, diagnostics and treatment}; Moller AC et al.; A case of a benign noninfectious cyst is reported . Splenic cysts are uncommon . They are diagnosed by ultrasonography, computer tomography, and nuclear magnetic resonance . The cysts are either infectious (e.g . parasitic) or noninfectious (e.g . posttraumatic) . The cystic content should be analysed for bacteria, parasites, and amylase . Noninfectious cysts larger than four cm and/or symptomatic cysts should be treated surgically with laparoscopic decapsulation.

Yi Chuan Xue Bao, 2002, 29(11), 1028 - 33
{Cloning and characterization of a homologous gene of plant class V chitinase from balsampear, Momordica charantia Linn.}; Xiao YH et al.; Balsampear (M . charantia Linn.) is a vegetable crop, highly resistant to pathogens . Chitinases were proposed to play an important role in the defense response of this crop . Based on the N-terminal sequence of a purified balsampear chitinase, a fragment (ChitB), similar to the tobacco class V chitinase gene, was amplified from the leaf RNA using 3'RACE, and the corresponding 5' sequence was further amplified by the Y-RACE method . By joining the two amplified fragments, the full-length cDNA of M . charatica homologous gene of plant class V chitinase (McChi5) was obtained . The 1348 bp cDNA contained an ORF of 1044 bp, which coded for a polypeptide of 347 amino acids . The deduced polypeptide had a predicted molecular weight of 38.3 kD and a pI of 5.77 . Homology analysis demonstrated that, McChi5 protein, which contained a conserved domain of family 18 glycosyl hydrolyse, had the sequence similar to tobacco class V chitinases, several putative chitinases and chitinase-like proteins of Arabidopsis thiliania, and some chitinases from mammals, insects and bacteria . Southern blotting suggested that two copies of McChi5 gene and several homologous genes existed in the M . charatica genome . By RNA dot blotting analysis, expression of the McChi5 gene was detected in cotyledons, roots, stems, and leaves, and it was not induced by wounding treatment . The biological functions and the potential applications of Mochi5 gene were discussed.

Plant Physiol, 2003 Mar, 131(3), 963 - 75
Conformation of a group 2 late embryogenesis abundant protein from soybean . Evidence of poly (L-proline)-type II structure; Soulages JL et al.; Late embryogenesis abundant (LEA) proteins are members of a large group of hydrophilic, glycine-rich proteins found in plants, algae, fungi, and bacteria known collectively as hydrophilins that are preferentially expressed in response to dehydration or hyperosmotic stress . Group 2 LEA (dehydrins or responsive to abscisic acid) proteins are postulated to stabilize macromolecules against damage by freezing, dehydration, ionic, or osmotic stress . However, the structural and physicochemical properties of group 2 LEA proteins that account for such functions remain unknown . We have analyzed the structural properties of a recombinant form of a soybean (Glycine max) group 2 LEA (rGmDHN1) . Differential scanning calorimetry of purified rGmDHN1 demonstrated that the protein does not display a cooperative unfolding transition upon heating . Ultraviolet absorption and circular dichroism spectroscopy revealed that the protein is in a largely hydrated and unstructured conformation in solution . However, ultraviolet absorption and circular dichroism measurements collected at different temperatures showed that the protein exists in equilibrium between two extended conformational states: unordered and left-handed extended helical or poly (L-proline)-type II structures . It is estimated that 27% of the residues of rGmDHN1 adopt or poly (L-proline)-type II-like helical conformation at 12 degrees C . The content of extended helix gradually decreases to 15% as the temperature is increased to 80 degrees C . Studies of the conformation of the protein in solution in the presence of liposomes, trifluoroethanol, and sodium dodecyl sulfate indicated that rGmDHN1 has a very low intrinsic ability to adopt alpha-helical structure and to interact with phospholipid bilayers through amphipathic alpha-helices . The ability of the protein to remain in a highly extended conformation at low temperatures could constitute the basis of the functional role of GmDHN1 in the prevention of freezing, desiccation, ionic, or osmotic stress-related damage to macromolecular structures.

J Bacteriol, 2003 Apr, 185(7), 2383 - 6
VnfY is required for full activity of the vanadium-containing dinitrogenase in Azotobacter vinelandii; Ruttimann-Johnson C et al.; A gene from Azotobacter vinelandii whose product exhibits primary sequence similarity to the NifY, NafY, NifX, and VnfX family of proteins, and which is required for effective V-dependent diazotrophic growth, was identified . Because this gene is located downstream from vnfK in an arrangement similar to the relative organization of the nifK and nifY genes, it was designated vnfY . A mutant strain having an insertion mutation in vnfY has 10-fold less vnf dinitrogenase activity and exhibits a greatly diminished level of (49)V label incorporation into the V-dependent dinitrogenase when compared to the wild type . These results indicate that VnfY has a role in the maturation of the V-dependent dinitrogenase, with a specific role in the formation of the V-containing cofactor and/or its insertion into apodinitrogenase.

J Bacteriol, 2003 Apr, 185(7), 2306 - 14
Heterocyst-specific expression of patB, a gene required for nitrogen fixation in Anabaena sp . strain PCC 7120; Jones KM et al.; The patB gene product is required for growth and survival of the filamentous cyanobacterium Anabaena sp . strain PCC 7120 in the absence of combined nitrogen . A patB::gfp fusion demonstrated that this gene is expressed exclusively in heterocysts . patB mutants have a normal initial pattern of heterocyst spacing along the filament but differentiate excess heterocysts after several days in the absence of combined nitrogen . Expression of hetR and patS, two critical regulators of the heterocyst development cascade, are normal for patB mutants, indicating that patB acts downstream of them in the differentiation pathway . A patB deletion mutant suffers an almost complete cessation of growth and nitrogen fixation within 24 h of combined nitrogen removal . In contrast, a new PatB mutant that is defective in its N-terminal ferredoxin domain, or a previously described mutant that has a frameshift removing its C-terminal helix-turn-helix domain, grows very slowly and differentiates multiple contiguous heterocysts under nitrogen-deficient conditions.

Immunol Lett, 2003 Apr 3, 86(2), 177 - 82
Advanced age negatively influences mesenteric lymph node T cell responses after burn injury; Choudhry MA et al.; While the pathophysiology of burn injury is well established in young adults, the factors that contribute to pathogenesis and increased death in elderly burn patients are not defined . The purpose of this study is to determine the effects of burn injury on mesenteric lymph node (MLN) T cell responses in young and aged mice . MLN is a cluster of lymph nodes that drains various parts of the intestine and is known to play role in clearance bacteria originating from the intestinal lumen . Results presented here suggest a significant suppression in Con A-induced MLN cell proliferation and IL-2 production in uninjured aged mice compared with uninjured young mice . Following 24 h after injury, although, a significant decrease in lymph node cell proliferation and IL-2 production was observed in both young and aged mice compared with their respective sham-injured animals, the suppression was more in aged mice . In addition we found a reduction in IFN-gamma, a Th-1 cytokine by MLN T cells from aged burned mice relative to young burn (P<0.05) or sham-injured mice (P<0.01) . The Th-2 cytokine IL-4, on the other hand, was significantly increased in both young and aged burn-injured mice MLN T cells compared with their respective sham-injured mice . These results show that burn injury causes a greater suppression in MLN T cells ability to proliferate and a more pronounced shift to Th-2 phenotype in aged mice as compared with young mice . Such decreases in T cell functions may impair MLN's ability to clear the bacterial pathogens originating from intestine and thereby contribute to increased pathogenesis in injured host.

Trends Parasitol, 2003 Mar, 19(3), 128 - 33
Reversing Wolbachia-based population replacement; Dobson SL; Genetic manipulation that reduces the competence of a vector population to transmit pathogens would provide a useful tool to complement current control strategies, which are based primarily on the reduction/exclusion of vector populations and the prophylactic/therapeutic treatment of the vertebrate host population . Genetic drive is an important component of vector population replacement strategies, facilitating the replacement of natural populations with a genetically modified population . Genetic drive is reviewed here, emphasizing strategies that would employ infections of intracellular Wolbachia bacteria as a vehicle for population replacement . Also discussed are strategies for the retarding, arresting or reversing of Wolbachia-based population replacement . These strategies are based upon altering the conditions required for transgene invasion and are a prudent safeguard, should unexpected detrimental effects become associated with transgene spread.

J Am Chem Soc, 2003 Mar 26, 125(12), 3493 - 502
Anomalous binding profile of phenylboronic acid with N-acetylneuraminic acid (Neu5Ac) in aqueous solution with varying pH; Otsuka H et al.; Borates are known to interact with carbohydrate moieties expressed on the surface of biological membranes of a variety of cells, viruses, bacteria, and fungi . This study revealed the anomalous binding profile of borate in aqueous solution with N-acetylneuraminic acid (Neu5Ac, sialic acid) as a potential receptor site on the surfaces of biological membranes using (11)B, (1)H, (13)C, and (15)N nuclear magnetic resonance spectroscopies . 3-(Propionamido)phenylboronic acid (PAPBA) was chosen as the model borate compound . The equilibrium constant (K) for Neu5Ac binding to PAPBA was compared with those for glucose, mannose, and galactose, which are the major carbohydrate constituents of glycoproteins and glycolipids expressed on biological membranes . In the Neu5Ac/PAPBA system, the unusual pH dependency of the K values, a decrease in K with increasing pH, was observed, suggesting the formation of a trigonal-formed complex stabilized by the coordination of an amide group of Neu5Ac at the C-5 position to the boron atom, forming intramolecular B-N or B-O bonding . Furthermore, the anomalously high complexing ability at physiological pH 7.4 was confirmed for this system, with the K value 37.6 which is approximately 7 times higher than that for glucose . This exceptionally high value of K at physiological pH, compared to those of other sugars, strongly suggests that the boronic acid selectively recognizes the Neu5Ac residues of the glycosylated components including glycoproteins and gangliosides existing on the surface of the biological membranes.

Cell Mol Biol (Noisy-le-grand), 2002, 48 Online Pub, OL343 - 58
Jenfluor ap--a novel fluorogenic substrate for in situ detection of alkaline phosphatase activity; Halbhuber KJ et al.; Alkaline phosphatase (AP) activity is often targeted in enzyme-related histochemistry as probe enzyme to detect neoplastic cells, as marker for primordial germ cells as well as in preimplantation studies, osteoblast differentiation, phosphate starvation in bacteria, yeast and phytoplankton . Moreover, AP-marker activity is a very useful tool in immunohistochemistry to detect gene sequences, antigens and antibodies . Here we describe a novel high resolution fluorescence method to localize AP-activity in cells and tissue sections based on a naphthol-AS azo coupling procedure (Jenfluor ap) . This method provides amorphous photostable fluorescent final reaction products without any diffusion artifacts which are visible in conventional fluorescence microscopes as well as in confocal laser scanning and near infrared multiphoton laser scanning microscopes . The superiority of the Jenfluor ap method in comparison to the known Fast Red TR salt as well as the ELF stains is discussed.

Curr Diab Rep, 2002 Jun, 2(3), 210 - 5
Hepatic steatosis and type 2 diabetes mellitus; Clark JM et al.; Type 2 diabetes is strongly associated with nonalcoholic fatty liver disease (NAFLD), a spectrum of liver damage that ranges from relatively benign hepatic steatosis to potentially fatal cirrhosis . The severities of insulin resistance and liver damage parallel each other, with the greatest prevalence of cirrhosis occurring in cirrhotics . However, it is unknown whether one of these conditions causes the other, or if both are consequences of another process . Experimental evidence suggests that both insulin resistance and NAFLD result from a chronic inflammatory state . The mechanisms driving this chronic inflammation are unknown but might include the egress of products from intestinal bacteria into the portal blood, liver, and systemic circulation to trigger a sustained inflammatory cytokine response in genetically susceptible individuals . More research is needed to evaluate this hypothesis and to determine the benefits of treatments that interrupt this pathogenic cascade.

Drug Metab Dispos, 2003 Apr, 31(4), 392 - 7
Lipopolysaccharide-mediated modulation of cytochromes P450 in Stat1 null mice; Pan J et al.; Signal transducer and activator of transcription (Stat), a family of transcriptional factors, has been demonstrated to play a critical role in gene regulation in response to inflammatory cytokines, such as interferon and interleukin-6 . Inflammatory cytokines and bacterial endotoxin are known to suppress, in most of cases, the constitutive or induced cytochromes P450 (P450) in animals and humans . However, it is not clear if the suppression of P450 by cytokines is through the Stat-signaling pathway . In the present study, we determined whether Stat1 is involved in lipopolysaccharide (LPS)-mediated modulation of P450 in mouse liver . In both Stat1(+/+) (wild type) and Stat1(-/-) (null) mice, a single dose of LPS treatment (1 mg/kg of body weight, i.p.) significantly reduced the expression of CYP3A11, 2C29, and 1A2 mRNA to 8 to 40% of the control levels as determined by real-time quantitative reverse transcription-polymerase chain reaction . The reduction was supported by Western blot analysis . In contrast, LPS significantly induced the level of CYP4A10 mRNA in both Stat1(+/+) (338% of control) and Stat1(-/-) mice (264% of control) . Although suppression of mRNA levels of CYP2E1, and 2D9 was not observed in either LPS-treated Stat1 null or wild-type animals, LPS treatment resulted in a reduction of CYP2E1 protein content, which was more significant in Stat1(+/+) (23% of control) than in Stat1(-/-) mice (67% of control) . Consistent with this result, the chlorzoxazone 6-hydroxylase and lauric acid 11-hydroxylase activities, as CYP2E1 representative activities, were reduced markedly by LPS in Stat1(+/+) but not in Stat1(-/-) mice . The ethoxyresorufin O-deethylase activity, as a representative CYP1A activity, was also reduced significantly only in LPS-treated Stat1(+/+) mice . These data clearly demonstrate that LPS-mediated modulation of CYP3A11, 2B10, 2C29, 1A2, and 4A10 in mouse liver is Stat1-independent . However, the significant difference between the LPS-treated Stat1(+/+) and Stat1(-/-) mice in the levels of CYP2E1 protein and activity as well as in the activity level of CYP1A suggests that Stat1 may be indirectly involved in the post-transcriptional modulation of these two mouse P450 enzymes.

Drug Metab Dispos, 2003 Apr, 31(4), 356 - 9
Homotropic versus heterotopic cooperativity of cytochrome P450eryF: a substrate oxidation and spectral titration study; Khan KK et al.; P450eryF is the only bacterial P450 to show cooperativity of substrate binding and oxidation . However, the studies reported so far have provided evidence only for homotropic cooperativity of P450eryF but not for heterotropic cooperativity . Therefore, oxidation of 7-benzyloxyquinoline (7-BQ) and 1-pyrenebutanol (1-PB) by P450eryF A245T and spectral binding of 9-aminophenanthrene (9-AP) to wild-type P450eryF were investigated in the presence of various effectors . The addition of steroids and flavones caused no stimulation but rather moderate inhibition of 7-BQ or 1-PB oxidation by P450eryF A245T . However, the binding affinity of 9-AP was significantly increased in the presence of androstenedione or alpha-naphthoflavone (ANF) . A comparative study with CYP3A4 revealed a similar increase in the binding affinity of 9-AP for the enzyme at low ANF concentrations but some competition at higher ANF concentrations . These studies, to our knowledge, provide the first report of heterotropic cooperativity in P450eryF as well as spectroscopic evidence for simultaneous presence of two ligand molecules in the CYP3A4 active site.

Blood Rev, 2003 Jun, 17(2), 111 - 22
Safety of the blood supply: role of pathogen reduction; Wu YY et al.; Even though the blood supply is very safe, the risk of transfusion transmitted disease is not zero . To improve the safety of the blood supply, pathogen reduction (PR) technology has been developed . The principle of most current PR strategies involves modifying DNA or RNA templates and making them inaccessible to DNA or RNA polymerase . Several platforms of pathogen reduction are available including psoralens, alkylating compounds, binary ethyleneimine-like compounds, riboflavin, methylene blue, and solvent-detergent treatment . PR systems have been designed for RBC, plasma, and platelets . PR technology has been found to be effective for a variety of pathogens including lipid-enveloped and non-enveloped viruses, bacteria and parasites . Pre-clinical studies and Phase III clinical trials to evaluate the efficacy and safety of these PR technologies are currently ongoing .

Curr Infect Dis Rep, 2003 Apr, 5(2), 120 - 124
Current Concepts in the Management of Fungal Peritonitis; Salvaggio MR et al.; Fungal pathogens are uncommon isolates in the setting of peritonitis . Secondary peritonitis results from a breach in the gastrointestinal tract with gross contamination of the peritoneum . Peritonitis in patients undergoing peritoneal dialysis represents a unique form of secondary peritonitis often caused by nosocomial bacteria and fungi . Regardless of the clinical circumstances, most cases of fungal peritonitis are caused by Candida species, however, other yeasts and filamentous fungi have been uncommonly reported . Treatment of secondary peritonitis consists of appropriate surgical intervention and systemic antifungal therapy . Systemic antifungals such as amphotericin B or fluconazole are also essential for the treatment of fungal peritonitis in patients who are peritoneal dialysis dependent . Salvage of the peritoneal dialysis catheter may be attempted, however, removal is usually required to achieve cure . Prophylaxis with fluconazole in patients with recurrent gastrointestinal perforations or anastomotic leakages has reduced the incidence of Candida peritonitis . Benefit of nystatin for fungal peritonitis prophylaxis in peritoneal dialysis patients is questionable.

Curr Infect Dis Rep, 2003 Apr, 5(2), 108 - 113
The Occurrence and Prevention of Infections Associated with Gastrointestinal Endoscopy; Lisgaris MV; In the four decades since the introduction of flexible endoscopy into medical practice, nearly 300 cases of human infections or pseudoinfections involving bacteria, fungi, parasites, and viruses have been linked to endoscopic procedures . In the majority of such cases, inadequate cleaning and disinfection techniques during the reprocessing of the instruments or their accessories have been likely contributing factors . Working groups from major gastroenterology societies and infection control organizations have established standards of care for the routine maintenance of endoscopic equipment in order to decrease the rates of infection even further . Since the institution of these standards, rates of transmission of infections to patients have decreased, though have not been completely resolved . This article reviews the available literature on transmission of pathogenic agents through endoscopic procedures, summarizes the current guidelines for the care of endoscopic equipment, and discusses available preventive measures aimed at decreasing the risk of endoscopy-related infections.

Int J Exp Pathol, 2002 Oct, 83(5), 217 - 23
CpG oligodeoxynucleotides accelerate reovirus type 2-triggered insulitis in DBA/1 suckling mice; Hayashi T et al.; We reported previously that reovirus type-2 (Reo-2) triggers T-helper (Th) 1-mediated autoimmune insulitis resulting in temporal impaired glucose tolerance (IGT) approximately 10 days post infection (d.p.i) in suckling DBA/1 mice . We hypothesized that CpG motifs in bacteria may enhance virus-induced insulitis through its content of unmethylated CpG motifs . In the infected mice, the intraperitoneal treatment of synthetic 20-base oligodeoxynucleotides with CpG motifs (CpG ODN) caused increase in cumulative incidence of insulitis with IGT, increased serum interferon (IFN)-gamma concentration, and high frequency of autoantibody against pancreatic islet cells, compared to the infected mice without CpG ODN at 17 d.p.i . Also CD4+ and CD8+ lymphocytes infiltrated in and/or around pancreatic islets in the CpG ODN-treated mice . This evidence suggests that CpG ODN may contribute to accelerate Reo-2-induced autoimmune reaction against pancreatic islet cells via additional effects of Th1 cytokines especially IFN-gamma.

J Hist Dent, 2003 Mar, 51(1), 15 - 8
Levi Spear Parmly: father of dental hygiene and children's dentistry in America; Chernin D et al.; Levi Spear Parmly (1790-1859) was one of the outstanding dental practitioners and teachers in the early part of the eighteenth century, both in America and Europe . He published two highly regarded books, practiced and taught in London for several years, before returning to America, where he became the most eminent dental practitioner in the southern United States . He had great financial success and used his time and money to offer free dental service to children . His major contribution to dental science was the concept that dental caries was caused by external influences related to foreign material on the tooth surface . This was before our knowledge of dental plaque and the action of the oral bacteria . Based upon this concept, he stressed the importance of clean teeth to prevent tooth decay.

Gen Dent, 2002 Jul-Aug, 50(4), 333 - 8
A paradigm shift in the treatment of caries; Steinberg S; This paper presents a shift in the paradigm for treating dental caries from a surgical to a medical approach . Two key points are essential to understanding caries . First, caries is a bacterial infection caused by specific bacteria . Second, before cavitation, caries is a reversible multifactoral process . Two features, caries activity and caries risk, provide a diagnosis and prognosis leading to a treatment plan . Patients receive treatment using a medical model consisting of four steps: bacterial control; reduction of risk levels for at-risk patients; reversal of active sites by remineralization; and follow-up and maintenance.

Adv Dent Res, 2001 Aug, 15, 8 - 13
Epigenetic signals during odontoblast differentiation; Lesot H et al.; Odontoblast terminal differentiation occurs according to a tooth-specific pattern and implies both temporospatially regulated epigenetic signaling and the expression of specific competence . Differentiation of odontoblasts (withdrawal from the cell cycle, cytological polarization, and secretion of predentin/dentin) is controlled by the inner dental epithelium, and the basement membrane (BM) plays a major role both as a substrate and as a reservoir of paracrine molecules . Cytological differentiation implies changes in the organization of the cytoskeleton and is controlled by cytoskeleton-plasma membrane-extracellular matrix interactions . Fibronectin is re-distributed during odontoblast polarization and interacts with cell-surface molecules . A non-integrin 165-kDa fibronectin-binding protein, transiently expressed by odontoblasts, is involved in microfilament reorganization . Growth factors (TGF beta 1, 2, 3/BMP2, 4, and 6), expressed in tooth germs, signal differentiation . Systemically derived molecules (IGF1) may also intervene . IGF1 stimulates cytological but not functional differentiation of odontoblasts: The two events can thus be separated . Immobilized TGF beta 1 (combined with heparin) induced odontoblast differentiation . Only immobilized TGF beta 1 and 3 or a combination of FGF1 and TGF beta 1 stimulated the differentiation of functional odontoblasts over extended areas and allowed for maintenance of gradients of differentiation . Presentation of active molecules in vitro appeared to be of major importance; the BM should fulfill this role in vivo by immobilizing and spatially presenting TGF beta s . Attempts are being made to investigate the mechanisms which spatially control the initiation of odontoblast differentiation and those which regulate its propagation . Analysis of molar development suggested that odontoblast differentiation and crown morphogenesis are interdependent, although the possibility of co-regulation requires further investigation.

J Pediatr, 2003 Mar, 142(3), 263 - 7
Does assessment of pretest probability of disease improve the utility of echocardiography in suspected endocarditis in children?
Michelfelder EC, Ochsner JE, Khoury P, Kimball TR.
OBJECTIVE: To compare the yield rate (YR) of echocardiography when evaluating children with suspected infectious endocarditis (IE) in both the actual clinical setting and in the hypothetic setting where strict clinical criteria are applied.Study design Medical records of 101 children undergoing echocardiography for suspected IE were reviewed . Echocardiograms with positive findings were identified and the actual diagnostic YR was calculated . With the use of clinical criteria proposed by von Reyn (VR), the probability of IE was retrospectively classified as (1) rejected, (2) possible, or (3) probable . Theoretic YR of echocardiography was calculated for each classification . RESULTS: The actual YR of echocardiography was 12% (12/101) . The YR of echocardiography by VR class was 0% in rejected, 20% in possible, and 80% in probable cases (chi(2) = 55.1, P <.0001) . Echocardiography did not change the probability of IE in any patient classified as rejected, but allowed reassignment of disease probability in a significant proportion of patients with possible or probable IE . CONCLUSIONS: The YR of echocardiography was significant when clinical probability of IE was intermediate-to-high, and low, with marginal clinical utility, when clinical probability was low . Strict pretest assessment of disease probability may lead to more effective utilization of echocardiography in this population.

J Biol Chem, 2003 May 16, 278(20), 18434 - 9 Epub 2003 Mar 14.
PAS domains . Common structure and common flexibility; Vreede J et al.; PAS (PER-ARNT-SIM) domains are a family of sensor protein domains involved in signal transduction in a wide range of organisms . Recent structural studies have revealed that these domains contain a structurally conserved alpha/beta-fold, whereas almost no conservation is observed at the amino acid sequence level . The photoactive yellow protein, a bacterial light sensor, has been proposed as the PAS structural prototype yet contains an N-terminal helix-turn-helix motif not found in other PAS domains . Here we describe the atomic resolution structure of a photoactive yellow protein deletion mutant lacking this motif, revealing that the PAS domain is indeed able to fold independently and is not affected by the removal of these residues . Computer simulations of currently known PAS domain structures reveal that these domains are not only structurally conserved but are also similar in their conformational flexibilities . The observed motions point to a possible common mechanism for communicating ligand binding/activation to downstream transducer proteins.

Dermatol Online J . 2003 Mar;9(2):5.
Recent advances in the treatment of leprosy; Ishii N; Leprosy, a chronic infectious disease caused by Mycobacterium leprae, was identified by G . H . A . Hansen in 1873 . The different clinical presentations of the disease are determined by the quality of the host immune response . The bacteria have affinity for the peripheral nerves and are likely the cause of neuropathy, a cardinal manifestation of the disease . WHO recommends a protocol of multidrug therapy (MDT), which effectively controls the disease, hence contributing to the global elimination program . Early detection of leprosy and treatment by MDT are the most important steps in preventing deformity and disability.

Immunobiology, 2003, 207(1), 37 - 42
Viral origin of antiphospholipid antibodies: endothelial cell activation and thrombus enhancement by CMV peptide-induced APL antibodies; Gharavi AE et al.; Our observations and those from others, give further support to our hypothesis that "autoimmune aPL" may be generated by immunization with products from bacteria or viruses after incidental exposure or infection . We also were able to generate APS-like syndrome in a strain of mice susceptible to autoimmunity, indicating that other factors such as genetics are likely to be involved in the development of APS . Furthermore, not all aPL antibodies generated by immunization with bacterial or viral products are pathogenic . Based on the clinical experience and on the numerous reports indicating presence of aPL in a large number of infectious diseases, it may be expected that not all aPL antibodies produced during infection will be pathogenic . We hypothesize that a limited number aPL antibodies induced by certain viral/bacterial products would be pathogenic in certain groups of predisposed individuals . Identification of these bacterial and/ or viral agents may help to find strategies for the prevention of production of aPL "pathogenic" antibodies . Alternatively, free peptides may be used to induce tolerance against aPL production.

Crit Rev Microbiol, 2003, 29(1), 25 - 35
The directed mutation controversy in an evolutionary context; Brisson D; Neo-Darwinists have long held that random mutations produce genetic differences among individuals, and selection increases the frequency of advantageous alleles . In 1988, Cairns et al . claimed that an environmental pressure can cause advantageous mutations to occur in specific genes to alleviate that particular pressure . Directed mutation, as proposed by Cairns, has been all but eradicated from evolutionary thinking . However, more than a decade of research spurred by the Cairns et al . paper has cast doubt on three neo-Darwinian principles: (1) mutations occur independently of the environment, (2) mutations are due to replication errors, and (3) mutation rates are constant . This mini-review explores the history of the controversy and the decade of research that followed so as to place it in an evolutionary context . Several of the cellular mechanisms and models that explain the increased genetic diversity in populations experiencing adverse environmental pressure are described . In most cases it is clear that the increased genetic diversity is due to breakdowns of cellular machinery or alleles evolved for a purpose other than increasing genetic diversity, rather than to cellular systems that have been evolutionarily selected to increase the genetic diversity in times of stress.

Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku, 2002, (120), 6 - 38
{Indoor air and human health--sick house syndrome and multiple chemical sensitivity}; Ando M; The number of complaints about the quality of indoor air has increased during the past two decades . These complaints have been frequent enough that the term "Sick House Syndrome or Sick Building Syndrome" and "Multiple Chemical Sensitivity" has been coined . Complaints are likely related to the increased use of synthetic organic materials in house, furnishing, and consumer products; and the buildings, furnishings, and consumer products; and the decreased ventilation for energy conservation in homes . Approximately thousand volatile chemicals have been identified in indoor air . The main sources of these chemicals are house materials, combustion fumes, cleaning compounds, and paints or stains . Exposure to high levels of these emissions and to others, coupled with the fact that most people spend more time indoors than outdoors, raises the possibility that the risk to human health from indoor air pollution may be potentially greater than the risk posed from outdoor pollutants . The complaints most frequently voiced with respect to Sick House Syndrome are irritations of the eye, nose, and throat; cough and hoarseness of voice; headache and mental fatigue . The syndrome of multiple chemical sensitivities is controversial subject with increasing impact on the field of indoor air quality . The controversy surrounding Multiple Chemical Sensitivity includes its definition, theories of etiology and pathogenesis, diagnostic, and life style . Multiple Chemical Sensitivity is considered the hypothesis that is a disease caused by exposure to many chemically distinct environmental substances at very low.

Immunol Res, 2003, 27(1), 71 - 84
Diverse roles of integrins in human T lymphocyte biology; Miyamoto YJ et al.; T lymphocytes are the primary cells responsible for maintaining the immune system . There are many intricate mechanisms involved in the regulation of T cells and the integrin family of adhesive surface proteins plays a pivotal role in the control of T lymphocyte activation and functions . Integrins are heterodimeric transmembrane proteins that are not merely adhesion molecules but also function in T cell coactivation by providing a scaffold for signaling and cytoskeletal proteins that are adept at transmitting signals from the inside of the cell to the outside ("inside-out signaling") or from the outside of the cell to the inside ("outside-in signaling") . The signaling property of integrins allows for rapid responses to changes in the microenviroment of the lymphocyte . Therefore, whether the T cell needs to adhere or detach, integrins can quickly accommodate either state of the cell . Once cells are guided to sites of infection, inflammation, or antigen presentation, integrins can also participate in the initiation, maintenance, or termination of the response . This review will focus on the aspects of integrin-mediated T cell coactivation, affinity and avidity control of integrins, signaling molecules involved with integrins, association of integrins in lipid microdomains, and negative regulation of integrins.

J Biol Chem, 2003 May 30, 278(22), 20140 - 53 Epub 2003 Mar 10.
Secretory IgA N- and O-glycans provide a link between the innate and adaptive immune systems; Royle L et al.; Secretory IgA (SIgA) is a multi-polypeptide complex consisting of a secretory component (SC) covalently attached to dimeric IgA containing one joining (J) chain . We present the analysis of both the N- and O-glycans on the individual peptides from this complex . Based on these data, we have constructed a molecular model of SIgA1 with all its glycans, in which the Fab arms form a T shape and the SC is wrapped around the heavy chains . The O-glycan regions on the heavy (H) chains and the SC N-glycans have adhesin-binding glycan epitopes including galactose-linked beta1-4 and beta1-3 to GlcNAc, fucose-linked alpha1-3 and alpha1-4 to GlcNAc and alpha1-2 to galactose, and alpha2-3 and alpha2-6-linked sialic acids . These glycan epitopes provide SIgA with further bacteria-binding sites in addition to the four Fab-binding sites, thus enabling SIgA to participate in both innate and adaptive immunity . We also show that the N-glycans on the H chains of both SIgA1 and SIgA2 present terminal GlcNAc and mannose residues that are normally masked by SC, but that can be unmasked and recognized by mannose-binding lectin, by disrupting the SC-H chain noncovalent interactions.

Am J Med, 2003 Feb 15, 114(3), 217 - 23
Eosinophilic meningitis; Lo Re V 3rd et al.; Eosinophilic meningitis is a rare clinical entity that can be useful in narrowing the differential diagnosis of central nervous system disease . It is defined by the presence of 10 or more eosinophils/microL in the cerebrospinal fluid (CSF) or a CSF eosinophilia of at least 10% . The most common cause is invasion of the central nervous system by helminthic parasites, particularly Angiostrongylus cantonensis, but other infections as well as noninfectious conditions may also be associated . This review describes the etiologies of eosinophilic meningitis, focusing primarily on the helminths that cause CSF eosinophilia.

Vet Microbiol, 2003 May 19, 93(2), 133 - 44
Adherence of Actinobacillus pleuropneumoniae to primary cultures of porcine lung epithelial cells; Boekema BK et al.; To study adherence of Actinobacillus pleuropneumoniae to porcine lower respiratory epithelium, a cell culture model was developed using primary cultures of porcine lung epithelial cells (LEC) . Adherence assays were performed and results were compared with data obtained with swine kidney cells (SK6) . A . pleuropneumoniae efficiently adhered to LEC with up to 62 bacteria per cell after 2h of incubation . Reference strain of serotype 3 (R3) adhered better to LEC than reference strains of serotypes 1 (R1), 7 (R7) and 8 (R8) . Overall the adherence to LEC was more rapid and up to 30-fold more efficient than adherence to SK6 cells . In search for the mechanism involved in the adherence event, we tested the effect of LPS which has previously been demonstrated to cause adherence of the pathogen to upper respiratory epithelium . Adherence assays with LPS transposon mutants demonstrated unaltered (mutant with modification in core/lipid A moiety) or even three-fold more adherence (mutants lacking O antigen) compared to the parent micro-organisms . Purified LPS of strains R1, R3, R7 and R8 did not inhibit adherence of R8 to LEC either, suggesting that LPS and particularly the O-antigen are not essential for adherence of A . pleuropneumoniae to LEC . The efficient, LPS-independent adherence of A . pleuropneumoniae to LEC cells indicates that A . pleuropneumoniae may carry different, cell type-specific adhesins and that primary cultures of lower respiratory epithelium are valuable infection models in studying A . pleuropneumoniae pathogenesis.

Environ Sci Technol, 2003 Feb 15, 37(4), 713 - 20
Reductive dehalogenation of halomethanes in iron- and sulfate-reducing sediments . 1 . Reactivity pattern analysis; Kenneke JF et al.; The incorporation of reductive transformations into environmental fate models requires the characterization of natural reductants in sediments and aquifer materials . For this purpose, reactivity patterns (range and relative order of reactivity) for a series of 14 halogenated methanes were measured in iron- and sulfate-reducing sediments and two representative model systems: adsorbed Fe(II)/goethite {Fe(II)ads/alpha-FeOOH} and iron sulfide (FeS) . Both Fe(II)ads and FeS are naturally occurring reductants . The strong similarity in reactivity patterns between the iron- and sulfate-reducing sediments suggests that the two share a common reductant despite their different chemical compositions (i.e., the sulfate-reducing sediment contained FeS) . An orthogonal regression analysis of the halomethane transformation rate data in the sediment and model systems supports the assumption that a common mechanism for halomethane transformation exists between the sediments and the Fe(II)ads/alpha-FeOOH system and further corroborates the conclusion that Fe(II) adsorbed to Fe(III)-containing minerals is the dominant reductant in both sediment systems . Weak (0.5 N) and strong (6.0 N) acid extraction of the sediments indicated that solid-phase Fe(II) was 67% higher in the sulfate-reducing sediment than in the iron-reducing sediment, which is consistent with the observations that the halomethanes were transformed a factor of 3 times faster in the sulfate-reducing sediment and that Fe(II) was the dominant reductant.

Microbiol Immunol, 2003, 47(1), 51 - 5
Serum antibodies of periodontitis patients compared to the lipopolysaccharides of Porphyromonas gingivalis and Fusobacterium nucleatum; Onoue S et al.; Serum antibody titers against the lipopolysaccharides (LPSs) of Porphyromonas gingivalis and Fusobacterium nucleatum were compared between 9 periodontitis patients and 24 healthy persons . The IgG titers against the LPSs of P . gingivalis ATCC 33277(T) and W50 were clearly higher in the patients than in the healthy persons . However, IgM titers against the LPSs of P . gingivalis strains were relatively low, and no significant difference was observed between the patients and healthy persons . On the other hand, IgG and IgM titers against the LPS of Fusobacterium nucleatum JCM 8532(T) in some patients were significantly higher than those in the healthy persons, although the difference in IgG titers was not large compared to that of the LPS of P . gingivalis . These results suggest that the antibody measurement of patients' sera against the LPS of periodontal bacteria can be applied for the diagnosis of periodontitis.

Clin Plast Surg, 2003 Jan, 30(1), 25 - 35, v
Assessing and controlling wound infection; Heggers JP; Quantitatively, wounds harboring bacteria that exceed 105 colony-forming units per gram are considered infected wounds . There are acute wounds and chronic wounds, and the approach to controlling the infection is similar . Although the granulating bed may be avascular, systemic anti-infectives are employed adjunctively to circumvent systemic infection . The main armamentarium of the attack is the use of topical anti-infectives, which invade the bacteria where they reside, and, consequently, reduce their numbers and promote wound healing.

Nat Prod Rep, 2003 Feb, 20(1), 79 - 110
The chalcone synthase superfamily of type III polyketide synthases; Austin MB et al.; This review covers the functionally diverse type III polyketide synthase (PKS) superfamily of plant and bacterial biosynthetic enzymes . from the discovery of chalcone synthase (CHS) in the 1970s through the end of 2001 . A broader perspective is achieved by a comparison of these CHS-like enzymes to mechanistically and evolutionarily related families of enzymes, including the type I and type II PKSs, as well as the thiolases and beta-ketoacyl synthases of fatty acid metabolism . As CHS is both the most frequently occurring and best studied type III PKS, this enzyme's structure and mechanism is examined in detail . The in vivo functions and biological activities of several classes of plant natural products derived from chalcones are also discussed . Evolutionary mechanisms of type III PKS divergence are considered, as are the biological functions and activities of each of the known and functionally divergent type III PKS enzymc families (currently twelve in plants and three in bacteria) . A major focus of this review is the integration of information from genetic and biochemical studies with the unique insights gained from protein X-ray crystallography and homology modeling . This structural approach has generated a number of new predictions regarding both the importance and mechanistic role of various amino acid substitutions observed among functionally diverse type III PKS enzymes.

Int J Med Microbiol, 2003 Feb, 292(7-8), 513 - 25
Subtyping of Mycoplasma pneumoniae isolates based on extended genome sequencing and on expression profiles; Dumke R et al.; Mycoplasma pneumoniae isolates from patients, collected over a period of 12 years in Germany, were characterized by various methods (parameters) including multilocus sequence typing, restriction fragment length polymorphisms, Western blotting with mono-specific antibodies directed against selected proteins or with polyspecific antibodies directed against the Triton X-114-soluble protein fraction, and two-dimensional gel electrophoresis . The results for 91 isolates from Germany, which were complemented with 14 isolates from the USA and 10 isolates from France, clearly showed that M . pneumoniae is a highly uniform species and that most of the isolates could be assigned to one of the two subtypes 1 and 2 . The members of one subtype differ from the other with respect to the sequence of the P1 gene, the ORF6 gene, the P65 gene, and by a typical DNA restriction fragment pattern . We observed four isolates (variants), which seemed identical by the above mentioned criteria, but did not belong to either one of the two subtypes . They showed most of the subtype 2-specific features, but differed in the sequence of the P1 gene and showed a variation in the restriction fragment pattern . The appearance of subtype 1 or 2 over the last 12 years in Germany showed a dominance of subtype 1 between 1989 and 1996 and a dominance of subtype 2 between 1997 and 1998 . The variant (neither subtype 1 nor subtype 2) was only detected in 1991 and 1995 but it had no epidemiological consequences.

Int J Med Microbiol, 2003 Feb, 292(7-8), 453 - 61
Virulence and the heat shock response; Gophna U et al.; The major adaptive response to elevation in temperature is the heat shock response that involves the induction of many proteins--called heat shock proteins . These include chaperones, proteases, alternative sigma factors and other regulatory and structural proteins . The heat shock response is also turned on by other stress conditions, such as oxidative stress or pH changes . Bacterial entry into the host organism involves a significant environmental change, which is expected to induce the heat shock response . Indeed, some of the heat shock proteins are themselves virulence factors while others affect pathogenesis indirectly, by increasing bacterial resistance to host defenses or regulating virulence genes . The cross talk between heat shock and virulence genes is discussed.

Pediatr Infect Dis J, 2003 Mar, 22(3), 244 - 51
Epidemiology of neonatal infections: experience during and after hospitalization; Sinha A et al.; OBJECTIVE: We assessed the epidemiology of neonatal infections coming to medical attention among inpatient and outpatient newborn infants within a defined health care maintenance organization (HMO) population . DESIGN AND METHODS: This was a retrospective cohort study, using automated data from a large health maintenance organization and the hospital where the majority of HMO patients delivered . All infants delivered between October 1, 1990 and March 31, 1998 at the study hospital and receiving postdischarge care at the study HMO for the first 30 days of life were included . The outcomes assessed were 10 neonatal infection syndromes defined according to modified National Nosocomial Infection System criteria . RESULTS: There were 13 224 infants in the study cohort . Of these, 559 infants (4.2%) had 574 infections . Nonpneumonia respiratory infections were most common, accounting for 43% of all infections . Infections diagnosed in the outpatient setting comprised 63% of all infections . More serious infections (bloodstream infection, clinical sepsis and pneumonia) were typically diagnosed in the first few days of life and before nursery discharge . Infants with an infection had a significantly longer length of nursery stay (4.7 excess days among full term infants, P < 0.001), more office visits (397.3 vs . 273.3 per 100, P < 0.001) and more hospital admissions (16.7 vs . 3.6 per 100, P = 0.001) than uninfected infants . CONCLUSION: Infections were common during the first 30 days of life . The majority were noninvasive, were diagnosed in the outpatient setting and were most likely community-acquired . Serious infections were rare, occurred early in the neonatal period and were usually diagnosed before nursery discharge.

Proc Natl Acad Sci U S A, 2003 Apr 1, 100(7), 3820 - 5 Epub 2003 Mar 12.
Purified particulate methane monooxygenase from Methylococcus capsulatus (Bath) is a dimer with both mononuclear copper and a copper-containing cluster; Lieberman RL et al.; Particulate methane monooxygenase (pMMO) is a membrane-bound enzyme that catalyzes the oxidation of methane to methanol in methanotropic bacteria . Understanding how this enzyme hydroxylates methane at ambient temperature and pressure is of fundamental chemical and potential commercial importance . Difficulties in solubilizing and purifying active pMMO have led to conflicting reports regarding its biochemical and biophysical properties, however . We have purified pMMO from Methylococcus capsulatus (Bath) and detected activity . The purified enzyme has a molecular mass of approximately 200 kDa, probably corresponding to an alpha(2)beta(2)gamma(2) polypeptide arrangement . Each 200-kDa pMMO complex contains 4.8 +/- 0.8 copper ions and 1.5 +/- 0.7 iron ions . Electron paramagnetic resonance spectroscopic parameters corresponding to 40-60% of the total copper are consistent with the presence of a mononuclear type 2 copper site . X-ray absorption near edge spectra indicate that purified pMMO is a mixture of Cu(I) and Cu(II) oxidation states . Finally, extended x-ray absorption fine structure data are best fit with oxygennitrogen ligands and a 2.57-A Cu-Cu interaction, providing direct evidence for a copper-containing cluster in pMMO.

Microbiology, 2003 Mar, 149(Pt 3), 773 - 84
Transcription analysis of the dnaA gene and oriC region of the chromosome of Mycobacterium smegmatis and Mycobacterium bovis BCG, and its regulation by the DnaA protein; Salazar L et al.; The regions flanking the Mycobacterium dnaA gene have extensive sequence conservation, and comprise various DnaA boxes . Comparative analysis of the dnaA promoter and oriC region from several mycobacterial species revealed that the localization, spacing and orientation of the DnaA boxes are conserved . Detailed transcriptional analysis in M . smegmatis and M . bovis BCG shows that the dnaN gene of both species and the dnaA gene of M . bovis BCG are transcribed from two promoters, whereas the dnaA gene of M . smegmatis is transcribed from a single promoter . RT-PCR with total RNA showed that dnaA and dnaN were expressed in both species at all growth stages . Analysis of the promoter activity using dnaA-gfp fusion plasmids and DnaA expression plasmids indicates that the dnaA gene is autoregulated, although the degree of transcriptional autorepression was moderate . Transcription was also detected in the vicinity of oriC of M . bovis BCG, but not of M . smegmatis . These results suggest that a more complex transcriptional mechanism may be involved in the slow-growing mycobacteria, which regulates the expression of dnaA and initiation of chromosomal DNA replication.

Int J Mol Med, 2003 Apr, 11(4), 411 - 8
Deciphering the impaired cytokine cascades in chronic leg ulcers (review); Quatresooz P et al.; Chronic leg ulcers are typically wounds that do not heal at a normal rate . Impaired healing appears to be due to primary microvascular changes and it is aggravated by ongoing bacteria-driven vasculitis . The various cytokines identified in experimental wounds are also present in leg ulcers . VEGF is strongly implicated as a promoter of blood vessel growth in patients with venous disease . In addition, there is good evidence of increased expression of bFGF, TGF-beta1, and PDGF in lipodermatosclerosis . All of these growth factors are involved in wound healing . Upregulated TGF-beta1 is probably one of the main causes of the fibrosis observed in lipodermatosclerosis . In leg ulcers, cytokines appear to be trapped in the perivascular fibrinoid deposits . It is not the nature and amount of cytokines that are inadequate in leg ulcers, but rather their spatial distribution . Dermal dendrocytes (DD) are resident factor XIIIa-enriched macrophages . They likely play a role in tissue repair when boosted adequately . New therapies aiming at helping the release of cytokines by DD apparently promote and improve the healing phase.

FASEB J, 2003 Mar, 17(3), 333 - 40
Reappraisal of serum starvation, the restriction point, G0, and G1 phase arrest points; Cooper S; The restriction point in the G1 phase of the mammalian cell cycle is the oldest, best-known, and widely accepted control point regulating division cycle in mammalian cells . Origins of the restriction point and its subsequent history are reanalyzed here . The initial proposal of the restriction point has an alternative explanation, which is that cells arrested with a G1 phase amount of DNA can arise from the inhibition of a process or processes occurring throughout the cell cycle and are not restricted to any particular phase of the cell cycle or specifically related to any event in the G1 phase of the cell cycle . The initial evidence and subsequent analyses require reexamination . It is proposed that the arrest of cells with a particular DNA content equivalent to that in cells in the G1 phase of the division cycle does not mean there is any particular G1 phase control point.

J Appl Microbiol, 2003, 94(4), 738 - 46
Molecular characterization of Oenococcus oeni genes encoding proteins involved in arginine transport; Divol B et al.; AIMS: This work was carried out to complete the sequence of the arc cluster involved in arginine catabolism in Oenococcus oeni, and particularly to characterize the genes encoding proteins involved in arginine transport . METHODS AND RESULTS: Using molecular cloning, two loci encoding proteins involved in the arginine-ornithine antiport were isolated . Their expression patterns were monitored by RT-PCR to study the influence of arginine on their transcription . Polycistronic mRNAs were detected . PCR performed directly on colonies with primer pairs specific of arc genes was used to discriminate strains able/unable to degrade arginine . CONCLUSIONS: Oenococcus oeni contains two arcD loci encoding similar proteins . Their expression is not influenced by arginine and polycistronic messengers were detected . The inability to use arginine is due to a lack of genetic information encoding proteins of the arginine deiminase pathway . SIGNIFICANCE AND IMPACT OF THE STUDY: The constitutive expression of arcD genes points to the positive role of arginine on O . oeni cell growth . The occasional presence of all the arc ABCD genes together in O . oeni strains might provide insights into the growth rate variability within this species.

Anal Quant Cytol Histol, 2003 Feb, 25(1), 8 - 11
P16INK4A as an adjunct test in liquid-based cytology; Bibbo M et al.; OBJECTIVE: To assess the utility of P16INK4A as an adjunct test in liquid-based cytology in cases with equivocal morphologic changes of high grade squamous intraepithelial lesion (HSIL) . STUDY DESIGN: P16INK4A immunoreactivity was investigated in residual ThinPrep material (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) from 30 cases with equivocal diagnoses of HSIL that had corresponding follow-up biopsies . Two control ThinPrep cases were included: 1 HSIL with biopsy-confirmed cervical intraepithelial neoplasia (CIN) 3 and a negative specimen with a corresponding biopsy of squamous metaplasia . The expression of P16INK4A in ThinPrep specimens and corresponding biopsies was scored as previously described . A ThinPrep case was scored positive if it contained > 10 abnormal cells with nuclear and cytoplasmic immunocytochemical staining . Corresponding biopsies were scored as having negative, sporadic, focal or diffuse staining . RESULTS: The P16INK4A antibody assay was positive in 19 of 30 ThinPrep cases (63.3%) . Seventeen of the 19 (89.4%) biopsies corresponding to the positively stained ThinPreps also were positive, with a score of at least focal positivity in the dysplastic regions (2 CIN 1, 4 CIN 2, 11 CIN 3; 2 lesions lost in the tissue recut) . The assay was negative in 11 ThinPreps (36.6%) and 10 biopsies (33.3%) with tissue confirmation of chronic cervicitis (5), squamous metaplasia (2), CIN 1 (3) and 1 lesion lost in the tissue recut . Seventeen of 18 (94.4%) ThinPreps confirmed as high grade lesions upon biopsy showed P16INK4A positivity . The control HSIL case with a CIN 3 biopsy was diffusely positive for P16INK4A, and the control negative case with biopsy diagnosis of squamous metaplasia was negative . Nondysplastic squamous and metaplastic epithelium in 7 biopsies and nondysplastic squamous or metaplastic cells in ThinPrep cases were negative . Sporadic staining of bacteria, inflammatory cells and endocervical cells was noted . CONCLUSION: ThinPrep cases in the equivocal cytologic category with the corresponding tissue biopsy assayed for P16INK4A expression showed that there was utility for this type of testing . A larger series comparing corresponding ThinPrep and tissue biopsies will be undertaken . The role of HPV infection in these cases will also be explored.

Prev Vet Med, 2003 Apr 30, 58(1-2), 1 - 13
A review of M . bovis BCG protection against TB in cattle and other animals species; Suazo FM et al.; Bovine tuberculosis (TB) causes severe economic losses in livestock due to low production, animal deaths and condemnation of carcasses . It is also an important constraint in international trade of animals and animal products . A scientific committee in Great Britain in 1997 concluded that the development of a cattle vaccine would be the best option for long-term control of TB . However, vaccination of cattle currently is not accepted because the vaccine interferes with the skin reaction to the tuberculin test in the field . Efficacy of M . bovis BCG in protecting bovine and other animal species against tuberculous infection has received much study . Vaccination of cattle prevents the spread of the disease in populations by reducing the number and size of the lesions, and the load of bacteria (rather than by preventing infection) . We review the literature about the efficacy of BCG in protecting cattle and other animal species against infection with field strains of M . bovis and discusses its potential use in programs of TB control in high-prevalence populations.

Biochem Pharmacol, 2003 Mar 1, 65(5), 699 - 707
"Multivalent" saccharides: development of new approaches for inhibiting the effects of glycosphingolipid-binding pathogens; Schengrund CL; A number of diseases are initiated by the adherence of viruses, bacteria, or bacterial toxins to cell surface carbohydrates, a number of which are components of glycosphingolipids (GSLs) . Studies of the binding of lectins indicated that many adhered weakly to monomeric carbohydrate ligands . The seminal observation that lectins adhered more strongly to a ligand with multiple carbohydrate binding sites initiated a plethora of studies designed to identify effective "multivalent" carbohydrate ligands for pathogens expressing multiple carbohydrate-binding sites . In addition to more completely defining ligand specificity of the carbohydrate-binding pathogen, identification of "multivalent" carbohydrate ligands has led to studies of their efficacy as pathogen inhibitors . This commentary focuses on pathogens that recognize the carbohydrate portion of GSLs . Because many GSL-binding pathogens have been shown to bind "multivalent" saccharides, approaches for identifying and preparing them as well as methods for characterizing their effectiveness as ligands are reviewed . Also discussed are areas of promise that should be investigated and pitfalls that might be encountered in the development of "multivalent" saccharides as pharmacologic agents.

Life Sci, 2003 Mar 28, 72(18-19), 2055 - 61
The non-neuronal cholinergic system in humans: expression, function and pathophysiology; Wessler I et al.; Acetylcholine, a prime example of a neurotransmitter, has been detected in bacteria, algae, protozoa, and primitive plants, indicating an extremely early appearance in the evolutionary process (about 3 billion years) . In humans, acetylcholine and/or the synthesizing enzyme, choline acetyltransferase (ChAT), have been found in epithelial cells (airways, alimentary tract, urogenital tract, epidermis), mesothelial (pleura, pericardium), endothelial, muscle and immune cells (mononuclear cells, granulocytes, alveolar macrophages, mast cells) . The widespread expression of non-neuronal acetylcholine is accompanied by the ubiquitous presence of cholinesterase and receptors (nicotinic, muscarinic) . Thus, the non-neuronal cholinergic system and non-neuronal acetylcholine, acting as a local cellular signaling molecule, has to be discriminated from the neuronal cholinergic system and neuronal acetylcholine, acting as neurotransmitter . In the human placenta anti-ChAT immunoreactivity is found in multiple subcellular compartments like the cell membrane (microvilli, coated pits), endosomes, cytoskeleton, mitochondria and in the cell nucleus . These locations correspond with the results of experiments where possible functions of non-neuronal acetylcholine have been identified (proliferation, differentiation, organization of the cytoskeleton and the cell-cell contact, locomotion, migration, ciliary activity, immune functions) . In the human placenta acetylcholine release is mediated by organic cation transporters . Thus, structural and functional differences are evident between the non-neuronal and neuronal cholinergic system . Enhanced levels of acetylcholine are detected in inflammatory diseases . In conclusion, it is time to revise the role of acetylcholine in humans . Its biological and pathobiological roles have to be elucidated in more detail and possibly, new therapeutical targets may become available .

Neuron, 2003 Mar 6, 37(5), 731 - 4
Molecular mechanisms of mechanosensation: big lessons from small cells; Blount P; Little is known of molecular mechanisms of human mechanosensation . Only now are candidate eukaryotic sensors being identified . In contrast, bacterial sensors, including mechanosensitive channels, have been cloned, sequenced, reconstituted, and functional mutants characterized . Moreover, crystal structures for bacterial mechanosensitive channels have been resolved and structural gating transitions predicted . These studies give clues to general principles underlying the ability of a membrane protein to sense and respond to perturbations of its lipid environment that may be conserved between bacteria and humans.

Biochemistry, 2003 Mar 18, 42(10), 2916 - 25
A rugged energy landscape mechanism for trapping of transmembrane receptors during endocytosis; Licht SS et al.; Efficient clathrin-mediated endocytosis of transmembrane receptors requires that clathrin-coated pits retain the receptors long enough to allow vesicle formation and internalization . In many cases, however, the receptors can exhibit mean lifetimes in coated pits much shorter than the lifetime of the pit at the plasma membrane . A rugged energy landscape for binding, which produces a broad distribution of residence times, ensures a significant probability of times much greater than the mean and would allow efficient trapping of proteins in these cases . We used fluorescence correlation spectroscopy and total internal reflection microscopy to measure the kinetics of movement of a C5a receptor-yellow fluorescent protein fusion in living cells . These experiments demonstrate that clusters of trapped receptors exhibit fluctuations in fluorescence intensity that vary in time scale over 2 orders of magnitude . Most of the variation in intensity is likely due to the motion of the receptors in the plane of the plasma membrane, although it is not possible to rule out a small contribution from motion orthogonal to the plane of the membrane . The broad time scale distribution of the intensity fluctuations is consistent with a rugged energy landscape mechanism for trapping of the receptors . This mechanism, which allows efficient trapping to coexist with rapid exchange, may also be relevant to other biological processes involving binding in heterogeneous chemical environments.

Lasers Med Sci, 2003, 18(1), 32 - 5
The possibility of detecting subgingival calculus by laser-fluorescence in vitro; Krause F et al.; The aim of the present study was to investigate laser-fluorescence effects on the root surface . The possibility of detecting subgingival calculus should be evaluated to control complete calculus removal after scaling and root planing . Twenty freshly extracted human teeth, partially covered with calculus on the root surface, were fixed on a translation stage . The specimens were irradiated by light from a laser source (diode laser, <1 mW) with a wavelength of 655 nm using the Diagnodent trade mark -system . To simulate the conditions in a periodontal pocket during the measurement procedure, the teeth were covered by a layer of physiological saline solution or blood in order to determine the influence of different inflammatory fluids . The root surface was scanned on a line of about 1 cm in steps of 0.5 mm . The start and end was marked by a notch . The results of the laser-fluorescence detection were compared to clinical and histological findings . Clinically apparent calculus on the root surface was always accompanied with a statistically significant increase of laser-fluorescence values ( p < 0.05) . These values were not influenced by the different fluids ( p < 0.05) . The laser-fluorescence values were reproducible ( p > 0.05) . After scaling, when no debris remained at the root surface, laser-fluorescence values decreased significantly ( p < 0.05) . The histological findings supported the clinical results . In conclusion, the present study indicates that laser-fluorescence values on the root surface are strongly correlated to the presence of calculus . This fluorescence effect can basically be used to develop a new detecting system for subgingival calculus in periodontal pockets.

J Biol Chem, 2003 May 23, 278(21), 18744 - 53 Epub 2003 Mar 07.
The hexokinase of the hyperthermophile Thermoproteus tenax . ATP-dependent hexokinases and ADP-dependent glucokinases, teo alternatives for glucose phosphorylation in Archaea; Dorr C et al.; The phosphorylation of glucose by different sugar kinases plays an essential role in Archaea because of the absence of a phosphoenolpyruvate-dependent transferase system characteristic for Bacteria . In the genome of the hyperthermophilic Archaeon Thermoproteus tenax a gene was identified with sequence similarity to glucokinases of the so-called ROK family (repressor protein, open reading frame, sugar kinase) . The T . tenax enzyme, like the recently described ATP-dependent "glucokinase" from Aeropyrum pernix, shows the typical broad substrate specificity of hexokinases catalyzing not only phosphorylation of glucose but also of other hexoses such as fructose, mannose, or 2-deoxyglucose, and thus both enzymes represent true hexokinases . The T . tenax hexokinase shows strikingly low if at all any regulatory properties and thus fulfills no important control function at the beginning of the variant of the Embden-Meyerhof-Parnas pathway in T . tenax . Transcript analyses reveal that the hxk gene of T . tenax is cotranscribed with an upstream located orfX, which codes for an 11-kDa protein of unknown function . Growth-dependent studies and promoter analyses suggest that post-transcriptional RNA processing might be involved in the generation of the monocistronic hxk message, which is observed only under heterotrophic growth conditions . Data base searches revealed T . tenax hexokinase homologs in some archaeal, few eukaryal, and many bacterial genomes . Phylogenetic analyses confirm that the archaeal hexokinase is a member of the so-called ROK family, which, however, should be referred to as ROK group because it represents a group within the bacterial glucokinase fructokinase subfamily II of the hexokinase family . Thus, archaeal hexokinases represent a second major group of glucose-phosphorylating enzymes in Archaea beside the recently described archaeal ADP-dependent glucokinases, which were recognized as members of the ribokinase family . The distribution of the two types of sugar kinases, differing in their cosubstrate as well as substrate specificity, within Archaea is discussed on the basis of physiological constraints of the respective organisms.

IUBMB Life, 2002 Jun, 53(6), 277 - 81
Protease-activated receptors: a means of converting extracellular proteolysis into intracellular signals; Mackie EJ et al.; Protease-activated receptors (PARs) mediate cellular responses to a variety of extracellular proteases . The four known PARs constitute a subgroup of the family of seven-transmembrane domain G protein-coupled receptors and activate intracellular signalling pathways typical for this family of receptors . Activation of PARs involves proteolytic cleavage of the extracellular domain, resulting in formation of a new N terminus, which acts as a tethered ligand . PAR-1, -3, and -4 are relatively selective for activation by thrombin whereas PAR-2 is activated by a variety of proteases, including trypsin and tryptase . Recent studies in mice genetically incapable of expressing specific PARs have defined roles for PAR-1 in vascular development, and for PAR-3 and -4 in platelet activation, which plays a fundamental role in blood coagulation . PAR-1 has also been implicated in a variety of other biological processes including inflammation, and brain and muscle development . Responses mediated by PAR-2 include contraction of intestinal smooth muscle, epithelium-dependent smooth muscle relaxation in the airways and vasculature, and potentiation of inflammatory responses . The area of PAR research is rapidly expanding our understanding of how cells communicate and control biological functions, in turn increasing our knowledge of disease processes and providing potential targets for therapeutic intervention.

Laryngorhinootologie, 2003 Feb, 82(2), 118 - 22
{Gastroesophageal reflux -- a common illness?}; Issing WJ; For a long time heartburn was not considered a symptom for serious illness . By now, however, it is accepted that the incidence of secondary carcinoma of the esophagus caused by chronic GERD has increased dramatically since the nineteen-seventies . Mechanisms leading to GERD are complex and its incidence is not necessarily pathological . However pathological reflux in the lower esophagus (pH lower than 4 in 6 % of 24 hours), caused by decreased sphinctertonus, impaired peristalsis and clearance of the esophagus, may lead to complications . Helicobacter pylori may play a key role in GERD . There is strong evidence for a protective effect of Hp-infection in the development of GERD . In pangastritis, caused by Hp-infection, gastric acid production is inhibited resulting in a reduction of stomach-acid-concentration . This may be caused by either the chronic infection itself and the resulting atrophy of the stomach-mucosa, by the ammonia-producing HP-bacteria, or an increase in acid re-absorbtion of gastric epithelium . Laryngopharyngeal reflux (LPR) often results in atypical manifestations with oral, pharyngeal, laryngeal, and pulmonary disorders . Laryngopharyngeal reflux is known to contribute to posterior acid laryngitis and laryngeal contact ulceration or granuloma formation, laryngeal cancer, chronic hoarseness, pharyngitis, asthma, pneumonia, nocturnal choking, and dental diseases . Today, PPI are the medication of choice in both acute and long-term (prophylactic) therapy of GERD . The so called "step-up-strategy" of medication is no longer recommended . Here, patients were first treated with antacids, then prokinetics followed by H2-blockers and finally low-dose PPI . Only in the case of persisting symptoms medication was further increased to high-dose PPI therapy . In the past this increase in medication lead to a prolonged healing process and consequently to higher medication costs . Studies have shown that a "step-down"-therapy, beginning with high dose PPI, is highly preferable, since it is much more effective . Depending on the degree of the symptoms, however, medication may also be applied "on-demand" . The BfArM has approved this kind of medication application only for Esomeprazol (Nexium mups 20 mg).

Microbiology, 2003 Feb, 149(Pt 2), 485 - 95
Mutation rates: estimating phase variation rates when fitness differences are present and their impact on population structure; Saunders NJ et al.; Phase variation is a mechanism of ON-OFF switching that is widely utilized by bacterial pathogens . There is currently no standardization to how the rate of phase variation is determined experimentally, and traditional methods of mutation rate estimation may not be appropriate to this process . Here, the history of mutation rate estimation is reviewed, describing the existing methods available . A new mathematical model that can be applied to this problem is also presented . This model specifically includes the confounding factors of back-mutation and the influence of fitness differences between the alternate phenotypes . These are central features of phase variation but are rarely addressed, with the result that some previously estimated phase variation rates may have been significantly overestimated . It is shown that, conversely, the model can also be used to investigate fitness differences if mutation rates are approximately known . In addition, stochastic simulations of the model are used to explore the impact of 'jackpot cultures' on the mutation rate estimation . Using the model, the impact of realistic rates and selection on population structure is investigated . In the absence of fitness differences it is predicted that there will be phenotypic stability over many generations . The rate of phenotypic change within a population is likely, therefore, to be principally determined by selection . A greater insight into the population dynamics of mutation rate processes can be gained if populations are monitored over successive time points.

Microbiology, 2003 Feb, 149(Pt 2), 377 - 88
Identification and in vivo characterization of PpaA, a regulator of photosystem formation in Rhodobacter sphaeroides; Gomelsky L et al.; A regulatory protein, PpaA, involved in photosystem formation in the anoxygenic phototrophic proteobacterium Rhodobacter sphaeroides has been identified and characterized in vivo . Based on the phenotypes of cells expressing the ppaA gene in extra copy and on the phenotype of the ppaA null mutant, it was concluded that PpaA activates photopigment production and puc operon expression under aerobic conditions . This is in contrast to the function of the PpaA homologue from Rhodobacter capsulatus, AerR, which acts as a repressor under aerobic conditions {Dong, C., Elsen, S., Swem, L . R . & Bauer, C . E . (2002) . J Bacteriol 184, 2805-2814} . The expression of the ppaA gene increases several-fold in response to a decrease in oxygen tension, suggesting that the PpaA protein is active under conditions of low or no oxygen . However, no discernible phenotype of a ppaA null mutant was observed under anaerobic conditions tested thus far . The photosystem gene repressor PpsR mediates repression of ppaA gene expression under aerobic conditions . Sequence analysis of PpaA homologues from several anoxygenic phototrophic bacteria revealed a putative corrinoid-binding domain . It is suggested that PpaA binds a corrinoid cofactor and the availability or structure of this cofactor affects PpaA activity.

J Clin Microbiol, 2003 Mar, 41(3), 1266 - 9
Conventional and molecular methods for verification of results obtained with BacT/Alert Nonvent blood culture bottles; Marlowe EM et al.; A strategy comparing molecular and conventional methods for verification of the BacT/Alert nonvent blood culture bottles (Organon Teknika, Durham, N.C.) was performed with seeded isolates . The bottles were evaluated with 12 common organisms from bloodstream infections . Overall, the bottles were equivalent as determined by conventional and molecular methods.

J Clin Microbiol, 2003 Mar, 41(3), 976 - 80
Heterogeneity of cag genotypes in Helicobacter pylori isolates from human biopsy specimens; Tomasini ML et al.; The Helicobacter pylori chromosomal cluster of genes known as the cytotoxin-associated gene (cag) island may have different compositions in infecting strains . In this study, we analyzed 150 single colonies obtained from gastric biopsy specimens from 10 patients infected with cagA-positive H . pylori strains and sweep isolates (isolates harvested with sweep in different points of the plate) from 6 patients infected with cagA-negative strains . Three loci in the cag island (cagA, cagE, and virB11) and the conserved gene glmM (ureC) were investigated by PCR . The levels of anti-H . pylori and anti-CagA antibodies in patient sera were also measured . For subjects infected with cagA-negative strains, all sweep isolates were also negative for cagE and virB11, suggesting the complete absence of the cag island . For subjects infected with cagA-positive strains, most of the isolates were positive for all three genes studied, whereas 24.7% of the isolates had a partial or total deletion of the cag island . cagA, cagE, and virB11 were, respectively, present in 87.3, 77.3, and 90% of the colonies . The deletion of virB11 was always associated with the deletion of cagA and/or cagE . H . pylori colonies with different cag genotypes were isolated within a single gastric biopsy specimen from 3 of the 10 patients and were further characterized by random amplified polymorphic DNA (RAPD) analysis and by sequencing of an arbitrarily selected gene segment . Although the colonies had different cag genotypes, their RAPD profiles were highly similar within each patient, and the nucleotide sequences of the selected gene segment were identical . All of the patients had detectable antibodies against H . pylori, and 9 of 10 had anti-CagA antibodies . In conclusion, we show that a single infecting H . pylori strain may include variable proportions of colony subtypes with different cag genotypes . The extension of our analysis to patients with well-characterized gastric diseases may provide significant information on the relationship between cag genotypes and clinical outcomes of H . pylori infections.

Waste Manag, 2003, 23(1), 37 - 44
The PAF model: an integrated approach for landfill sustainability; Cossu R et al.; Lab-scale tests were set up to investigate different options for achieving sustainability by reducing long-term landfill emissions . The options which have been studied and compared with the traditional anaerobic landfill for unprocessed refuse were: mechanical-biological pretreatment, landfill aeration with forced and with natural advective air flow (semi-aerobic), flushing . Combination of different options have been experimented . The combination of mechanical-biological Pretreatment, Aeration by the semi-aerobic method and Flushing (PAF model) seems to synergize the advantages of the individual options . The research is ongoing and further studies are necessary in order to assess the full-scale applicability of the model.

J Clin Periodontol, 2003 Feb, 30(2), 95 - 101
Ultrasonic dental scaler: associated hazards; Trenter SC et al.; BACKGROUND: The ultrasonic dental scaler is a valuable tool in the prevention of periodontal disease; however, this equipment has a number of hazards with which it is associated . These include heating of the tooth during scaling, vibrational hazards causing cell disruption, possible platelet damage by cavitation, associated electromagnetic fields that can interrupt pacemakers, auditory damage to patient and clinician and the release of aerosols containing dangerous bacteria . OBJECTIVE: To collate the research reported on the various hazards associated with the ultrasonic dental scaler and discuss possible future research areas . DATA SOURCES: The scientific literature was searched using Web of Science, EMBASE and Medline, and the results of these were then hand-searched to eliminate nonrelevant papers . CONCLUSIONS: This review outlines some of the research conducted into these areas of associated hazard in order to assess their significance in the clinical situation, and discusses ideas for future research . Suggestions of recommendations are given, which have been previously investigated for their aid in reducing possible hazards, to ensure the safe working of ultrasonic scalers in the dental practice.

Mol Microbiol, 2003 Mar, 47(6), 1495 - 512
An integrated analysis of the genome of the hyperthermophilic archaeon Pyrococcus abyssi; Cohen GN et al.; The hyperthermophilic euryarchaeon Pyrococcus abyssi and the related species Pyrococcus furiosus and Pyrococcus horikoshii, whose genomes have been completely sequenced, are presently used as model organisms in different laboratories to study archaeal DNA replication and gene expression and to develop genetic tools for hyperthermophiles . We have performed an extensive re-annotation of the genome of P . abyssi to obtain an integrated view of its phylogeny, molecular biology and physiology . Many new functions are predicted for both informational and operational proteins . Moreover, several candidate genes have been identified that might encode missing links in key metabolic pathways, some of which have unique biochemical features . The great majority of Pyrococcus proteins are typical archaeal proteins and their phylogenetic pattern agrees with its position near the root of the archaeal tree . However, proteins probably from bacterial origin, including some from mesophilic bacteria, are also present in the P . abyssi genome.

J Med Microbiol, 2003 Mar, 52(Pt 3), 211 - 6
Medium pH-dependent redistribution of the urease of Helicobacter pylori; Hong W et al.; Helicobacter pylori is an aetiological agent of gastric disease . Although the role of urease in gastric colonization of H . pylori has been shown, it remains unclear as to where urease is located in this bacterial cell . The purpose of this study was to define the urease-associated apparatus in the H . pylori cytoplasm . H . pylori was incubated at both a neutral and an acidic pH in the presence or absence of urea and examined by double indirect immunoelectron microscopy . The density of gold particles for UreA was greatest in the inner portion of the wild-type H . pylori cytoplasm at neutral pH but was greatest in the outer portion at acidic pH . This difference was independent of the presence of urea and was not observed in the ureI-deletion mutant . Also, the eccentric shift of urease in acidic pH was not observed in UreI . After a 2 day incubation period at acidic pH, it was observed that the urease gold particles in H . pylori assembled and were associated with UreI gold particles . Urease immunoreactivity shifted from the inner to the outer portion of H . pylori as a result of an extracellular decrease in pH . This shift was urea-independent and UreI-dependent, suggesting an additional role of UreI in urease-dependent acid resistance . This is the first report of the intracellular transport of molecules in bacteria in response to changes in the extracellular environment.

Mutagenesis, 2003 Mar, 18(2), 145 - 50
Effects of black tea theafulvins on aflatoxin B(1) mutagenesis in the Ames test; Catterall F et al.; Black tea theafulvins, a fraction of thearubigins isolated from black tea aqueous infusions, potentiated the mutagenic activity of the mycotoxin aflatoxin B(1) in the Ames test, in the presence of a hepatic S9 activation system derived from Aroclor 1254-treated rats . In contrast, when the S9 activation system was replaced with isolated microsomes, theafulvins suppressed the mutagenicity of the mycotoxin . When microsomal metabolism was terminated after metabolic activation of the mycotoxin, incorporation of the theafulvins into the activation system reduced the mutagenic activity, whereas if it was added before termination of microsomal activity a potentiation of mutagenic response was observed . In in vitro studies, theafulvins inhibited epoxide hydrolase and glutathione S-transferase activities in a concentration-dependent manner . Finally, the mutagenicity of aflatoxin B(1) was much more pronounced in bacteria that were pre-exposed to theafulvins but from which they were subsequently washed off . It may be inferred from the above studies that the genotoxic synergy between aflatoxin B(1) and black tea theafulvins does not occur during the bioactivation of the carcinogen, but may partly be due to decreased deactivation of the reactive intermediate, aflatoxin B(1) 8,9-oxide, by conjugation with glutathione.

Appl Environ Microbiol, 2003 Mar, 69(3), 1647 - 54
Characterization of the Streptomyces sp . strain C5 snp locus and development of snp-derived expression vectors; DeSanti CL et al.; The Streptomyces sp . strain C5 snp locus is comprised of two divergently oriented genes: snpA, a metalloproteinase gene, and snpR, which encodes a LysR-like activator of snpA transcription . The transcriptional start point of snpR is immediately downstream of a strong T-N(11)-A inverted repeat motif likely to be the SnpR binding site, while the snpA transcriptional start site overlaps the ATG start codon, generating a leaderless snpA transcript . By using the aphII reporter gene of pIJ486 as a reporter, the plasmid-borne snpR-activated snpA promoter was ca . 60-fold more active than either the nonactivated snpA promoter or the melC1 promoter of pIJ702 . The snpR-activated snpA promoter produced reporter protein levels comparable to those of the up-mutated ermE* promoter . The SnpR-activated snpA promoter was built into a set of transcriptional and translational fusion expression vectors which have been used for the intracellular expression of numerous daunomycin biosynthesis pathway genes from Streptomyces sp . strain C5 as well as the expression and secretion of soluble recombinant human endostatin.

Appl Environ Microbiol, 2003 Mar, 69(3), 1442 - 51
Isolation of novel ultramicrobacteria classified as actinobacteria from five freshwater habitats in Europe and Asia; Hahn MW et al.; We describe the first freshwater members of the class Actinobacteria that have been isolated . Nine ultramicro-size (<0.1 microm(3)) strains were isolated from five freshwater habitats in Europe and Asia . These habitats represent a broad spectrum of ecosystems, ranging from deep oligotrophic lakes to shallow hypertrophic lakes . Even when the isolated strains were grown in very rich media, the cell size was <0.1 microm(3) and was indistinguishable from the cell sizes of bacteria belonging to the smaller size classes of natural lake bacterioplankton . Hybridization of the isolates with oligonucleotide probes and phylogenetic analysis of the 16S rRNA gene sequences of the isolated strains revealed that they are affiliated with the class Actinobacteria and the family Microbacteriaceae . The previously described species with the highest levels of sequence similarity are Clavibacter michiganensis and Rathayibacter tritici, two phytopathogens of terrestrial plants . The 16S rRNA gene sequences of the nine isolates examined are more closely related to cloned sequences from uncultured freshwater bacteria than to the sequences of any previously isolated bacteria . The nine ultramicrobacteria isolated form, together with several uncultured bacteria, a diverse phylogenetic cluster (Luna cluster) consisting exclusively of freshwater bacteria . Isolates obtained from lakes that are ecologically different and geographically separated by great distances possess identical 16S rRNA gene sequences but have clearly different ecophysiological and phenotypic traits . Predator-prey experiments demonstrated that at least one of the ultramicro-size isolates is protected against predation by the bacterivorous nanoflagellate Ochromonas sp . strain DS.

Appl Environ Microbiol, 2003 Mar, 69(3), 1428 - 34
Population dynamics of male-killing and non-male-killing spiroplasmas in Drosophila melanogaster; Anbutsu H et al.; The endosymbiotic bacteria Spiroplasma spp . are vertically transmitted through female hosts and are known to cause selective death of male offspring in insects . One strain of spiroplasma, NSRO, causes male killing in Drosophila species, and a non-male-killing variant of NSRO, designated NSRO-A, has been isolated . It is not known why NSRO-A does not kill males . In an attempt to understand the mechanism of male killing, we investigated the population dynamics of NSRO and NSRO-A throughout the developmental course of the laboratory host Drosophila melanogaster by using a quantitative PCR technique . In the early development of the host insect, the titers of NSRO were significantly higher than those of NSRO-A at the first- and second-instar stages, whereas at the egg, third-instar, and pupal stages, the titers of the two spiroplasmas were almost the same . Upon adult emergence, the titers of the two spiroplasmas were similar, around 2 x 10(8) dnaA copy equivalents . However, throughout host aging, the two spiroplasmas showed strikingly different population growth patterns . The titers of NSRO increased exponentially for 3 weeks, attained a peak value of around 4 x 10(9) dnaA copy equivalents per insect, and then decreased . In contrast, the titers of NSRO-A were almost constant throughout the adult portion of the life cycle . In adult females, consequently, the titer of NSRO was significantly higher than the titer of NSRO-A except for a short period just after emergence . Although infection of adult females with NSRO resulted in almost 100% male killing, production of some male offspring was observed within 4 days after emergence when the titers of NSRO were as low as those of NSRO-A . Based on these results, we proposed a threshold density hypothesis for the expression of male killing caused by the spiroplasma . The extents of the bottleneck in the vertical transmission through host generations were estimated to be 5 x 10(-5) for NSRO and 3 x 10(-4) for NSRO-A.

J Neuroimmunol, 2003 Mar, 136(1-2), 172 - 7
Molecular identification and antibody testing of Chlamydophila pneumoniae in a subgroup of patients with HIV-associated dementia complex . Preliminary results; Contini C et al.; Chlamydophila pneumoniae DNA was investigated by polymerase chain reaction (PCR), in cerebrospinal fluid (CSF) specimens from patients suffering from HIV-1-associated dementia complex (HADC) . Four (17.3%) cases of C . pneumoniae infection were identified among 23 HADC individuals with DNA amplification of major outer membrane protein (MOMP) gene and 16S rRNA gene sequences . Sequence analysis revealed significant homologies with C . pneumoniae compared to Chlamydia trachomatis and Chlamydia psittaci . High mean levels of CSF specific anti-C . pneumoniae antibodies and C . pneumoniae antibody specific index values significantly elevated were also found by enzyme-linked immunosorbent assay (ELISA) in these patients . The results suggest a hypothetical role of C . pneumoniae in the pathogenesis or progression of HADC.

FEMS Microbiol Lett, 2003 Feb 28, 219(2), 241 - 8
The response regulator LetA regulates the stationary-phase stress response in Legionella pneumophila and is required for efficient infection of Acanthamoeba castellanii; Lynch D et al.; In order to identify a potential regulator of virulence gene expression in Legionella pneumophila, the L . pneumophila homologue of the response regulator GacA, LetA, was identified and cloned, facilitating the generation of a L . pneumophila letA insertion mutant . The L . pneumophila letA insertion mutant was more sensitive to oxidative and acid stress than the wild-type . The letA mutant exhibited reduced infectivity and was defective for intracellular growth within Acanthamoeba castellanii . Transcription of the rpoS and dotA genes was reduced in the letA mutant . Our data indicate that the response regulator LetA functions as a regulator of the stationary-phase stress response in L . pneumophila and is required for efficient replication within A . castellanii.

FEMS Microbiol Lett, 2003 Feb 28, 219(2), 173 - 9
Investigation of mechanisms involved in phagocytosis of Legionella pneumophila by human cells; Weissgerber P et al.; Legionella pneumophila, the causative agent of Legionnaires' disease, is able to survive and multiply efficiently in a variety of mammalian cells . By using in vitro assays, the uptake of L . pneumophila into monocytes has shown to be mediated, at least in part, through attachment of complement-coated bacteria to complement receptors, but complement-independent phagocytosis could also be demonstrated . Since complement levels in the human lung are normally low, the role of complement-dependent phagocytosis in the pathogenesis of Legionnaires' disease is doubtful . However, the contribution of other potential phagocytosis-related host cell surface molecules to the phagocytosis of L . pneumophila has never been investigated . We therefore analyzed the role of complement receptors 1 (CD35) and 3 (CD11b/18), the lipopolysaccharide (LPS) receptor (CD14), the beta(1)-integrin chain of the fibronectin receptor (CD29), the intercellular adhesion molecule 1 (ICAM-1, CD54) and the transferrin receptor (CD71) in the complement-independent uptake of L . pneumophila . To exclude any influence of culture conditions onto phagocytosis rates, we compared a fresh clinical isolate with an agar-adapted isolate of L . pneumophila . In addition, we used three different host cell types (MM6, HeLa and Jurkat cells) expressing different rates of complement receptors . We could show that both strains of L . pneumophila were phagocytized by the three host cell lines to the same extent, but intracellular multiplication was only found in MM6 and, although to a much lesser degree, in Jurkat cells . Preincubation of MM6 cells with monoclonal antibodies directed against the above cited phagocytosis-related receptors did not result in inhibition of L . pneumophila uptake . We therefore conclude that typical phagocytosis-related cell surface receptors are not involved in the complement-independent phagocytosis of L . pneumophila.

FEMS Microbiol Lett, 2003 Feb 28, 219(2), 159 - 65
rRNA and nifD phylogeny of Bradyrhizobium from sites across the Pacific Basin; Qian J et al.; Many undomesticated legumes harbor nodule bacteria related to the soybean symbiont Bradyrhizobium elkanii, but little is known about their phylogenetic relationships or geographic distribution . Sequences of ribosomal genes (16S rRNA and partial 23S rRNA) and the nitrogenase alpha-subunit gene (nifD) were analyzed in 22 isolates of this group sampled from diverse legumes in Korea, Japan, the USA, Mexico, Costa Rica and Panama . Some strains from Asia and North America shared identical sequences for both ribosomal genes . However, pairs of strains with closely related nifD sequences were almost never found in different regions . The major exceptions involved North American isolates B . elkanii USDA 76 and USDA 94, which had nifD sequences highly similar to certain Korean strains . However, 16S rRNA sequences of USDA 76 and USDA 94 were closely related to Central American rather than Asian bradyrhizobia, implying that these strains are genetic mosaics combining sequences from distinct ancestral areas . Several other conflicts between rRNA and nifD tree topologies indicated that the genealogical histories of these loci have been influenced by recurrent lateral gene transfer events.

FEMS Microbiol Lett, 2003 Feb 28, 219(2), 151 - 8
The multitalented type III chaperones: all you can do with 15 kDa; Feldman MF et al.; Despite the fact that type III chaperones were discovered approximately 10 years ago, the precise role of most of them is still mysterious . A panoply of functions has been proposed for the members of this family of proteins . Type III chaperones have been suggested to act as anti-aggregation and stabilizing factors . They have also been proposed to keep their substrates in unfolded or partially folded structures, set a hierarchy on secretion, and participate in the regulation of the transcription of the type III substrates . Here, we review this enigmatic family of proteins, and discuss the experimental data supporting the roles proposed for type III chaperones.

Genomics, 2003 Feb, 81(2), 112 - 25
Novel raf kinase protein-protein interactions found by an exhaustive yeast two-hybrid analysis; Yuryev A et al.; We have performed an exhaustive unbiased yeast two-hybrid analysis to identify interaction partners of two human Raf kinase isoforms, A-Raf and C-Raf, using their N-terminal regulatory domain as "bait." A total of 20 different human proteins were found to interact with Raf isoforms . Several of these interactions were novel and an extensive bioinformatics evaluation was performed for each . The novel putative interactions include a signalosome component, TOPK/PBK kinase, and two new putative protein phosphatases . The cysteine-rich zinc-binding domain (CRD) of Raf was found to interact with all 20 proteins and to achieve isoform-specific interactions . Since similar putative CRDs are present in a variety of protein serine-threonine kinases, the data suggest that the CRD may function as a major protein-protein interaction domain of these kinases . We propose possible functional consequences of these novel Raf interactions.

Microb Pathog, 2003 Jan, 34(1), 11 - 6
Differential expression of genes encoding membrane proteins between acute and continuous Chlamydia pneumoniae infections; Hogan RJ et al.; Chlamydia pneumoniae is associated with several chronic human diseases, including chronic obstructive pulmonary disease and atherosclerotic cardiovascular disease . During chronic disease, organisms are believed to exist in a persistent phase that is not well understood at the genetic level . Long-term in vitro continuous infections are spontaneously persistent and are less susceptible than in vitro acute infections to treatment with antibiotics, and are therefore particularly relevant as an in vitro model of in vivo chronic disease . Real-time reverse transcriptase-PCR (r-t RT-PCR) was used to quantitate transcript copy numbers of 13 genes in continuous and acute infections with C . pneumoniae . The set of genes studied encodes proteins with known or predicted functions in the cell membrane, the inclusion membrane, cell division, metabolism, and immunopathology . Significant upregulation was seen for five genes (CPn0483, nlpD, ompA, pmp1 and porB) in continuous cultures . The genes omcB, pmp1, and porB, all of which encode membrane proteins, shared similar patterns of expression over both acute and continuous profiles . These results show that Chlamydia in the long-term continuous model of persistence have a unique transcription profile, adding to our knowledge of regulation of this important stage of chlamydial growth.

Phytochemistry, 2003 Feb, 62(3), 399 - 413
On the origin of family 1 plant glycosyltransferases; Paquette S et al.; The phylogeny of highly divergent multigene families is often difficult to validate but can be substantiated by inclusion of data outside of the phylogeny, such as signature motifs, intron splice site conservation, unique substitutions of conserved residues, similar gene functions, and out groups . The Family 1 Glycosyltransferases (UGTs) comprises such a highly divergent, polyphyletic multigene family . Phylogenetic comparisons of UGTs from plants, animals, fungi, bacteria, and viruses reveal that plant UGTs represent three distinct clades . The majority of the plant sequences appears to be monophyletic and have diverged after the bifurcation of the animal/fungi/plant kingdoms . The two minor clades contain the sterol and lipid glycosyltransferases and each show more homology to non-plant sequences . The lipid glycosyltransferase clade is homologous to bacterial lipid glycosyltransferases and reflects the bacterial origin of chloroplasts . The fully sequenced Arabidopsis thaliana genome contains 120 UGTs including 8 apparent pseudogenes . The phylogeny of plant glycosyltransferases is substantiated with complete phylogenetic analysis of the A . thaliana UGT multigene family, including intron-exon organization and chromosomal localization.

Cell Mol Biol (Noisy-le-grand), 2002 Nov, 48(7), 819 - 21
The use of neutron radiography in agriculture to improve the food quality; de Jesus SP et al.; The report presented in the 7th ONU's Conference (USA, 2001) about climatic changes that took place at the end of 2001 informs that, in less than 50 years, more than 45% of the world population will be suffering from lack of water . This fact occurs by the absence of management on water resources, mainly, in agriculture . As the excess as the lack of humidity in soil can change the harvest quality, causing physiologic anomalies in food and promoting soil diseases incidence caused by bacteria and fungus . In order to establish a larger control in the food quality, a study has been performed, through the neutron radiographic technique, that proposes the optimization of agricultural harvests in relation to the minimum quantity of water necessary for the plant to develop and, also, of the soil compactness . Thus, neutron radiographic images of the system root-soil can be produced so that each root will be evaluated for its ability to penetrate in the soil layers, having the advantage of not interfering in this system what it is not possible through the usual techniques yet . The initial tests using bean roots showed that the soil thickness, which involved the roots, resulted in low contrast images, what impeded their visualization with enough clearness so that their grow could not be observed . For this reason, it was opted to the gadolinium as a contrast agent so that we have been studying its transport through the roots.

Indian J Pediatr, 2003 Jan, 70(1), 33 - 6
Etiology of acute lower respiratory tract infection; Kabra SK et al.; OBJECTIVE: To identify pathogens responsible for acute severe lower respiratory tract infection (ALRTI) in under five children by non-invasive methods . METHOD: 95 children hospitalized with acute severe lower respiratory tract infection were investigated for identification of viruses, bacteria, chlamydia or mycoplasma by nasopharyngeal aspirates, blood culture and serology . RESULT: Etiological agents could be identified in 94% of the patients . Viruses from NP aspirate could be isolated in 36 (38%), bacterial isolates from blood cultures in 15 (16%); mycoplasma was identified in 23 (24%) and chlamydia in 10 (11%) by serological tests; mixed infections were present in 8 (8%) patients . CONCLUSION: Noninvasive methods can be useful in identifying etiological agents in severe ALRTI.

Can J Microbiol, 2002 Dec, 48(12), 1082 - 8
Water activity, temperature, and pH effects on growth of the biocontrol agent Pantoea agglomerans CPA-2; Costa E et al.; The growth response of the biocontrol agent Pantoea agglomerans to changes in water activity (a(w)), temperature, and pH was determined in vitro in nutrient yeast extract-sucrose medium . The minimum temperature at which P . agglomerans was able to grow was 267-272 kelvins (-6 to -1 degrees C), and growth of P . agglomerans did not change at varying pH levels (4.5-8.6) . The minimum a(w) for growth was 0.96 in media modified with glycerol and 0.95 in media modified with NaCl or glucose . Solute used to reduce water activity had a great influence on bacterial growth, especially at unfavourable conditions (e.g., low pH or temperature) . NaCl stimulated bacterial growth under optimum temperatures but inhibited it under unfavourable pH conditions (4.5 or 8.6) . In contrast, the presence of glucose in the medium allowed P . agglomerans to grow over a broad range of temperature (3-42 degrees C) or pH (5-8.6) regimes . This study has defined the range of environmental conditions (a(w), pH, and temperature) over which the bacteria may be developed for biological control of postharvest diseases.

Biosci Biotechnol Biochem, 2003 Jan, 67(1), 192 - 4
Biocatalytic deprotection of a cetraxate ester by Microbacterium sp . strain 7-1W cells; Honda K et al.; Enzymatic deprotection of the terminal ester bond of a cetraxate methyl ester was done with resting cells of Microbacterium sp . strain 7-1W, which produces an esterase catalyzing a regioselective hydrolysis reaction, as the catalyst . When 20 g of cetraxate methyl ester in 50 ml of a reaction mixture was incubated with 5 g of wet cells for 17 h, 96% of the substrate was converted to the desired product, cetraxate, quantitatively.

J Bacteriol, 2003 Mar, 185(6), 1825 - 30
tmRNA in Caulobacter crescentus is cell cycle regulated by temporally controlled transcription and RNA degradation; Keiler KC et al.; SsrA, or tmRNA, is a small RNA found in all bacteria that intervenes in selected translation reactions to target the nascent polypeptide for rapid proteolysis . We have found that the abundance of SsrA RNA in Caulobacter crescentus is regulated with respect to the cell cycle . SsrA RNA abundance increases in late G(1) phase, peaks during the G(1)-S transition, and declines in early S phase, in keeping with the reported role for SsrA in the timing of DNA replication initiation . Cell cycle regulation of SsrA RNA is accomplished by a combination of temporally controlled transcription and regulated RNA degradation . Transcription from the ssrA promoter peaks late in G(1), just before the peak in SsrA RNA abundance . SsrA RNA is stable in G(1)-phase cells and late S-phase cells but is degraded with a half-life of 4 to 5 min at the onset of S phase . This degradation is surprising, since SsrA RNA is both highly structured and highly abundant . This is the first observation of a structural RNA that is cell cycle regulated.

J Biol Chem, 2003 May 9, 278(19), 17291 - 8 Epub 2003 Mar 04.
Homomeric ring assemblies of eukaryotic Sm proteins have affinity for both RNA and DNA . Crystal structure of an oligomeric complex of yeast SmF; Collins BM et al.; Sm and Sm-like proteins are key components of small ribonucleoproteins involved in many RNA and DNA processing pathways . In eukaryotes, these complexes contain seven unique Sm or Sm-like (Lsm) proteins assembled as hetero-heptameric rings, whereas in Archaea and bacteria six or seven-membered rings are made from only a single polypeptide chain . Here we show that single Sm and Lsm proteins from yeast also have the capacity to assemble into homo-oligomeric rings . Formation of homo-oligomers by the spliceosomal small nuclear ribonucleoprotein components SmE and SmF preclude hetero-interactions vital to formation of functional small nuclear RNP complexes in vivo . To better understand these unusual complexes, we have determined the crystal structure of the homomeric assembly of the spliceosomal protein SmF . Like its archaeal/bacterial homologs, the SmF complex forms a homomeric ring but in an entirely novel arrangement whereby two heptameric rings form a co-axially stacked dimer via interactions mediated by the variable loops of the individual SmF protein chains . Furthermore, we demonstrate that the homomeric assemblies of yeast Sm and Lsm proteins are capable of binding not only to oligo(U) RNA but, in the case of SmF, also to oligo(dT) single-stranded DNA.

Chem Biol, 2003 Feb, 10(2), 123 - 30
Selective chemical treatment of cellular microdomains using multiple laminar streams; Takayama S et al.; There are many experiments in which it would be useful to treat a part of the surface or interior of a cell with a biochemical reagent . It is difficult, however, to achieve subcellular specificity, because small molecules diffuse distances equal to the extent of the cell in seconds . This paper demonstrates experimentally, and analyzes theoretically, the use of multiple laminar fluid streams in microfluidic channels to deliver reagents to, and remove them from, cells with subcellular spatial selectivity . The technique made it possible to label different subpopulations of mitochondria fluorescently, to disrupt selected regions of the cytoskeleton chemically, to dislodge limited areas of cell-substrate adhesions enzymatically, and to observe microcompartmental endocytosis within individual cells . This technique does not require microinjection or immobilization of reagents onto nondiffusive objects; it opens a new window into cell biology.

Sex Transm Dis, 2003 Mar, 30(3), 249 - 56
The molecular epidemiology of genital Chlamydia trachomatis in the greater Reykjavik area, Iceland; Jonsdottir K et al.; BACKGROUND: The diversity in MOMP (major outer membrane protein) of Chlamydia trachomatis is thought to be necessary for the bacteria to survive in its environment . The rate of change in the omp1 gene (coding for MOMP) is not known . Iceland offers a good opportunity to study the epidemiology of chlamydial infections because the population is small (280,000) and geographically well defined . GOAL: The goal was to determine the number and distribution of genotypes in a population attending the STD clinic in Reykjavik and to assess changes in omp1 sequences over a period of 2 years . STUDY DESIGN: Three-hundred thirty isolates of C trachomatis collected periodically from January 1999 to January 2001 were omp1 genotyped with nested PCR and sequencing . RESULTS: The serotypes found, in descending order of prevalence, were E, D, J, F, K, G, H, and I . Eighteen distinctive genotypes were found . During the study period no significant changes in frequency of genotypes were noted, and introduction of new or changed genotypes was not observed . CONCLUSION: The results indicate a relatively stable situation of genotypes and suggest an ecological advantage of serotype E.

Plant Cell, 2003 Mar, 15(3), 597 - 611
Alteration of microtubule dynamic instability during preprophase band formation revealed by yellow fluorescent protein-CLIP170 microtubule plus-end labeling; Dhonukshe P et al.; At the onset of mitosis, plant cells form a microtubular preprophase band that defines the plane of cell division, but the mechanism of its formation remains a mystery . Here, we describe the use of mammalian yellow fluorescent protein-tagged CLIP170 to visualize the dynamic plus ends of plant microtubules in transfected cowpea protoplasts and in stably transformed and dividing tobacco Bright Yellow 2 cells . Using plus-end labeling, we observed dynamic instability in different microtubular conformations in live plant cells . The interphase plant microtubules grow at 5 micro m/min, shrink at 20 micro m/min, and display catastrophe and rescue frequencies of 0.02 and 0.08 events/s, respectively, exhibiting faster turnover than their mammalian counterparts . Strikingly, during preprophase band formation, the growth rate and catastrophe frequency of plant microtubules double, whereas the shrinkage rate and rescue frequency remain unchanged, making microtubules shorter and more dynamic . Using these novel insights and four-dimensional time-lapse imaging data, we propose a model that can explain the mechanism by which changes in microtubule dynamic instability drive the dramatic rearrangements of microtubules during preprophase band and spindle formation in plant cells.

Mol Biochem Parasitol, 2003 Feb, 126(2), 239 - 50
Characterization of a unique aspartate-rich protein of the SET/TAF-family in the human malaria parasite, Plasmodium falciparum, which inhibits protein phosphatase 2A; Dobson S et al.; A search for physiological inhibitors of protein phosphatases led to the identification of a Plasmodium falciparum (Pf) cDNA that had the potential to code for an aspartate-rich protein and hence named ARP . The PfARP was virtually identical to its Plasmodium berghei counterpart in gene structure and protein sequence . The PfARP coding sequence contained two introns, and the predicted protein contained 269 amino acid residues . Its primary structure showed significant similarity to eukaryotic proteins of the SET and TAF-family that included two inhibitors of mammalian serine/threonine protein phosphatase 2A (PP2A), namely I1(PP2A) and I2(PP2A) . Like the SET and TAF proteins, it had an extremely acidic tail . The cDNA was confirmed by recombinant expression in bacteria . Native parasitic ARP was purified and was found to be highly thermostable . PfARP specifically inhibited the parasitic PP2A at nanomolar concentrations, with no effect on PP1, PP2B, PP5, or PPJ . Expression of PfARP in HeLa cells led to elevated phosphorylation of c-Jun, and activation of transcription factors AP1 and NF-kappa B . These functional properties are also characteristic of the SET/TAF-family proteins . The ARP mRNA and protein were detectable in all the erythrocytic asexual stages of the parasite, and the protein was located mainly in the parasitic cytoplasm . Thus, PfARP is a unique cytoplasmic member of the SET/TAF-family and a candidate physiological regulator of the Plasmodium PP2A .

Bioorg Med Chem, 2003 Mar 20, 11(6), 811 - 6
A biaryl peptide crosslink in a MetJ peptide model confers cooperative, nonspecific binding to DNA that ablates both repressor binding and in vitro transcription; Yoburn JC et al.; The MetJ repressor is the archetypal example of the beta-ribbon-helix-helix DNA binding motif . A model of the MetJ beta-ribbon (residues 22-28) was prepared by forming a dityrosine crosslinked dimer from the heptapeptide KKYTVSI . Using SPR, the peptide dimer 2 was shown to bind to dsDNA under physiologically relevant conditions, whereas the monomeric peptide did not . The peptide dimer appeared to inhibit binding of the MetJ repressor to natural met operators . Based on the stoichiometry of binding, the binding of peptide dimer 2 seems both highly co-operative and to lack sequence specificity . Peptide binding also appears to prevent transcription in vitro.

Lancet Infect Dis, 2003 Mar, 3(3), 141 - 7
Modern laboratory diagnosis of tuberculosis; Drobniewski FA et al.; One-third of the global population is believed to be infected with bacteria of the Mycobacterium tuberculosis complex, the causative agent of tuberculosis . More than 8 million new cases of tuberculosis occur annually leading to 2 million deaths . Mortality is particularly high in those coinfected with HIV and where the bacteria are multiple-drug-resistant strains--ie, strains resistant to at least isoniazid and rifampicin . Early diagnosis of tuberculosis and drug resistance improves survival and by identifying infectious cases promotes contact tracing, implementation of institutional cross-infection procedures, and other public-health actions . This review addresses significant advances made in the diagnosis of infection, clinical disease, and drug resistance over the past decade . It proposes operational criteria for a modern diagnostic service in the UK (as a model of a low-incidence country) and explores some of the economic issues surrounding the use of these techniques.

Biochemistry, 2003 Mar 11, 42(9), 2625 - 33
Inhibition of nuclear import mediated by the Rev-arginine rich motif by RNA molecules; Fineberg K et al.; The HIV-1 Rev protein plays a pivotal role in viral replication, and therefore, inhibition of its function should block the progression of the virus-induced immune deficiency syndrome (AIDS) . Here, RNA molecules have been shown to inhibit import of the HIV-1 Rev protein into nuclei of permeabilized cells . Nuclear uptake of biotinylated recombinant His-tagged Rev-GFP was assessed in nuclear extracts from digitonin-permeabilized cells by binding to either importin beta-receptors or nickel molecules immobilized on a microtiter plate . Using this method together with fluorescence microscopy, we determined that nuclear import of Rev is inhibited by the addition of a reticulocyte lysate which routinely is used as a source of nuclear import receptors . This inhibition was released by treatment with the RNase enzyme . Also t-RNA molecules and the oligoribonucleotide RRE IIB, namely, the second stem structure of the Rev responsive element (RRE) of the viral RNA, inhibit Rev nuclear import . Similar results were obtained when BSA molecules with covalently attached Rev-arginine rich motif (ARM) peptides were used as a nuclear transport substrate, indicating that the nuclear import inhibition of the Rev protein is due to the presence of the ARM domain . Binding experiments revealed that the RNA molecules inhibit the interaction between the ARM region and importin beta, implying that the RNA prevents the formation of the import complex . The implication of our results for the regulation of the nuclear import of Rev as well as for the use of RNA molecules as antiviral drugs is discussed.

J Mol Evol, 2003 Mar, 56(3), 351 - 61
Alpha/beta hydrolase2, a predicated gene adjacent to mad in Drosophila melanogaster, belongs to a new global multigene family and is associated with obesity; Wisotzkey RG et al.; The experimental validation of genes predicted from genomic sequence and the identification of functions for these genes is an increasingly important task . We report a multidisciplinary analysis of CG3488, a predicted gene adjacent to Mothers against dpp in Drosophila melanogaster . We cloned and sequenced a cDNA corresponding to CG3488 and we show that it is expressed in embryos . A computational analysis shows that CG3488 contains a number of conserved domains present in enzymes capable of lipid hydrolysis . A phylogenetic analysis shows that CG3488 is the homolog of human alpha/beta hydrolase2 and that these genes belong to a novel multigene family with members in animals, plants, fungi, and bacteria . A genetic analysis shows that heterozygosity for a chromosomal deletion that removes CG3488 dominantly enhances the excess lipid phenotype associated with a mutation in adipose, an uncloned obesity gene . Further, overexpression of a CG3488 transgene rescues this obesity phenotype . Overall, the data suggests that CG3488 functions as a lipase and that analyses of its homologs will provide unique insights into lipid metabolism in many species.

Mol Cell Biol, 2003 Mar, 23(6), 1922 - 34
Molecular determinants of glucocorticoid receptor mobility in living cells: the importance of ligand affinity; Schaaf MJ et al.; The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), which is activated upon ligand binding, and can alter the expression of target genes either by transrepression or transactivation . We have applied FRAP (fluorescence recovery after photobleaching) to quantitatively assess the mobility of the yellow fluorescent protein (YFP)-tagged human GR alpha-isoform (hGRalpha) in the nucleus of transiently transfected COS-1 cells and to elucidate determinants of its mobility . Addition of the high-affinity agonist dexamethasone markedly decreases the mobility of the receptor in a concentration-dependent manner, whereas low-affinity ligands like corticosterone decrease the mobility to a much lesser extent . Analysis of other hGRalpha ligands differing in affinity suggests that it is the affinity of the ligand that is a major determinant of the decrease in mobility . Similar results were observed for two hGRalpha antagonists, the low-affinity antagonist ZK98299 and the high-affinity antagonist RU486 . The effect of ligand affinity on mobility was confirmed with the hGRalpha mutant Q642V, which has an altered affinity for triamcinolone acetonide, dexamethasone, and corticosterone . Analysis of hGRalpha deletion mutants indicates that both the DNA-binding domain and the ligand-binding domain of the receptor are required for a maximal ligand-induced decrease in receptor mobility . Interestingly, the mobility of transfected hGRalpha differs among cell types . Finally, the proteasome inhibitor MG132 immobilizes a subpopulation of unliganded receptors, via a mechanism requiring the DNA-binding domain and the N-terminal part of the ligand-binding domain . Ligand binding makes the GR resistant to the immobilizing effect of MG132, and this effect depends on the affinity of the ligand . Our data suggest that ligand binding induces a conformational change of the receptor which is dependent on the affinity of the ligand . This altered conformation decreases the mobility of the receptor, probably by targeting the receptor to relatively immobile nuclear domains with which it transiently associates . In addition, this conformational change blocks immobilization of the receptor by MG132.

Adv Exp Med Biol, 2002, 516, 1 - 25
Molecular mechanisms of TRS instability; Parniewski P et al.; To date several neurodegenerative disorders including myotonic dystrophy, Huntington's disease, Kennedy's disease, fragile X syndrome, spinocerebellar ataxias or Friedreich's ataxia have been linked to the expanding trinucleotide sequences . Although phenotypic features vary among these debilitating diseases, the structural abnormalities of the triplet repeat containing DNA sequences is the primary cause for all of these disorders . Expansions of the CAG repeat within coding regions of miscellaneous genes result in the synthesis of aberrant proteins containing enormously long polyglutamine stretches . Such proteins acquire toxic functions and/or may direct cells into the apoptotic cycle . On the other hand, massive expansions of various triplet repeats (i.e., CTG/CAG, CGG/CCG/, GAA/TTC) inside the noncoding regions lead to the silencing of transcription and therefore affect expression of the adjacent genes . The repetitive character of TRS allows stretches of such tracts to form slipped-stranded structures, self-complementary hairpins, triplexes or more complex configurations called "sticky DNA", which are not equally processed by some cellular mechanisms, as compared to random DNA . It is likely that the instability of the short TRS (below the threshold level) occurs due to the SILC pathway, which is driven by the DNA slippage . Accumulation of the short expansions leads to the disease premutation state where the MLC pathway becomes predominant . Independent of which mechanism is involved in the MLC pathway (replication, transcription, repair or recombination) the process of complementary strand synthesis is crucial for the TRS instability . Generally, dependent on the location of the tract which has higher potential to form secondary DNA structure, further processing of such tract may result in expansions (secondary structure formed at the newly synthesized strand) or deletions (structure present on the template strand) . Analyses of molecular mechanisms of the TRS genetic instability using bacteria, yeast, cell lines and transgenic animals as models allowed the scientists to better understand the role of some major cellular processes in the development of neurodegenerative disorders in humans . However, it is necessary to remember that most of these investigations were focused on the involvement of each particular process separately . Much less of this work though was dedicated to the search for the interactions between such cellular systems that in effect could result in different rate of TRS expansions . Thus, more intensive studies are necessary in order to fully understand the phenomenon ofthe dynamic mutations leading to the human hereditary neurodegenerative diseases.

J Clin Pathol, 2003 Mar, 56(3), 194 - 9
Increased Nox2 expression in human cardiomyocytes after acute myocardial infarction; Krijnen PA et al.; BACKGROUND/AIMS: Recent studies indicate the presence of reactive oxygen species (ROS) producing homologues of the enzymatic subunit (Nox2) of phagocytic NADPH oxidase in non-phagocytic cells . Interestingly, in these cells, ROS produced by the Nox2 homologue(s) was shown to play a role in various regulatory processes, including cell death, proliferation, and aging . The purpose of this study was to investigate whether human cardiomyocytes express Nox2 . METHODS: The expression of Nox2 was studied in human cardiomyocytes using western blot and immunohistochemical analysis . To analyse the putative expression of Nox2 in human heart disease, cardiac samples from patients who had died subsequent to acute myocardial infarction (AMI) were studied . RESULTS: Both in western blot and immunohistochemical studies, Nox2 expression was found in normal human cardiomyocytes . In patients with AMI, a significant increase in Nox2 expression was found both in viable and in jeopardised cardiomyocytes in the infarcted area . In addition, in the "remote from infarction" area, Nox2 expression was present in cardiomyocytes, but was not increased . CONCLUSIONS: Nox2 or its homologue(s) is expressed in normal and jeopardised human cardiomyocytes . This expression is increased in patients with AMI, suggesting a role for this ROS producing Nox2 homologue(s) in the human heart after AMI.

Biophys J, 2003 Mar, 84(3), 2112 - 29
Application of singular value decomposition to the analysis of time-resolved macromolecular x-ray data; Schmidt M et al.; Singular value decomposition (SVD) is a technique commonly used in the analysis of spectroscopic data that both acts as a noise filter and reduces the dimensionality of subsequent least-squares fits . To establish the applicability of SVD to crystallographic data, we applied SVD to calculated difference Fourier maps simulating those to be obtained in a time-resolved crystallographic study of photoactive yellow protein . The atomic structures of one dark state and three intermediates were used in qualitatively different kinetic mechanisms to generate time-dependent difference maps at specific time points . Random noise of varying levels in the difference structure factor amplitudes, different extents of reaction initiation, and different numbers of time points were all employed to simulate a range of realistic experimental conditions . Our results show that SVD allows for an unbiased differentiation between signal and noise; a small subset of singular values and vectors represents the signal well, reducing the random noise in the data . Due to this, phase information of the difference structure factors can be obtained . After identifying and fitting a kinetic mechanism, the time-independent structures of the intermediates could be recovered . This demonstrates that SVD will be a powerful tool in the analysis of experimental time-resolved crystallographic data.

Insect Biochem Mol Biol, 2003 Mar, 33(3), 355 - 69
Mamestra configurata serpin-1 homologues: cloning, localization and developmental regulation; Chamankhah M et al.; A screen of a Mamestra configurata (bertha armyworm) midgut cDNA library identified three types of cDNA clones that resemble the Manduca sexta serpin-1 gene family . Two serpins, 1b and 1c, possess a common conserved serpin amino terminal scaffold domain but bear no similarity to any members of the M . sexta gene family within the reactive centre loop . These serpins differ from one another by only two amino acids in the reactive centre loop (S(363)-->P) and serpin signature (M(369)-->T) regions . The other member, denoted serpin-1a, is closely related to the M . sexta serpin-1Z . M . configurata serpins as a group were expressed in all insect developmental stages including eggs, larvae and adult moths . Within larvae, serpin gene expression was restricted to the early to middle instar developmental phase and mainly in the fat body and hemocytes . Stress imposed by starvation strongly induced expression in fat body and to a lesser degree in alimentary organs, nervous system and Malphigian tubules . Conversely, starvation decreased expression in hemocytes . Wounding or inoculation with bacteria did not induce serpin gene transcription but did lead to the formation of higher and lower molecular weight forms, presumably serpin-protease complexes and resultant truncated serpin, respectively . Two dimensional PAGE and western blotting analysis revealed at least 12 distinct serpins consisting primarily of neutral, but also highly acidic and basic isoforms, as well as additional high and low molecular weight immuno-reactive species . Serpins-1b/1c are the more prominent serpin isoforms and are expressed predominantly in the fat body and subsequently exported to the hemolymph as revealed by western blotting and immunolocalization . The serpin-1b/1c isoform was found only as the fully glycosylated species within the hemolymph . Hemolymph protease activity was comprised mostly of serine proteases whose overall activity increased dramatically at the onset of the molt concomitant with a sharp decline in serpin gene expression.

J Periodontal Res, 2003 Apr, 38(2), 204 - 9
The effect of chronic emotional stress on the humoral immune response to Porphyromonas gingivalis in mice; Houri-Haddad Y et al.; Previous studies have shown that psychological stress plays a significant role in the outcome of infectious diseases, but data related to the effect of stress on periodontal infection is limited . The present study was designed to test the impact of emotional stress on the humoral immune response to the periodontal pathogen Porphyromonas gingivalis in a mouse model of local inflammation . Chambers constructed from titanium wire were implanted in the subcutaneous dorsolumbar region of mice . All mice were immunized with P . gingivalis followed by an intrachamber challenge with the bacteria . One group of mice was used as control, while the other two experienced experimental stress conditions (isolation/restraint stress) . Stress-1 group was stressed during the immunization period, while Stress-2 group was stressed during the local challenge period . Chamber exudates and serum were collected and analyzed for levels of anti-P . gingivalis antibodies (IgG, IgG1 and IgG2a) . The levels of serum antibodies to P . gingivalis were not different between the three tested groups, excluding increased levels of IgG2a in Stress-1 group at baseline . The levels of antibodies in the chamber exudates were significantly lower in the stressed groups at baseline, but higher at d 7 . The IgG1 to IgG2a ratio was significantly higher in the control group compared with the two stressed groups . The findings of the present study suggest that chronic psychological stress had a marked impact on the localized response to P . gingivalis challenge . The lower IgG1/IgG2a ratio observed in the stress groups suggests elevated Th1 response during stress.

J Nat Prod, 2003 Feb, 66(2), 157 - 60
Oxygenated analogues of gorgosterol and ergosterol from the soft coral Capnella lacertiliensis; Wright AD et al.; From the dichloromethane solubles of Capnella lacertiliensis five new sterols were isolated that are highly functionalized with oxygen-containing substituents: 12beta-acetoxy-7alpha-hydroxygorgosterol (1), 12beta-acetoxy-7alpha,19-dihydroxygorgosterol (2), 12beta-acetoxyergost-5-ene-3beta,23-diol (4), 12beta-acetoxyergost-5-ene-3beta,11beta,16-triol (5), and 11beta-acetoxyergost-5-ene-3beta,12beta,16-triol (6) . The structures of all compounds were deduced from interpretation of their spectroscopic data, mainly 1D and 2D NMR spectra and HREIMS . Biological activities of the isolates were assessed, and all were found to be weakly antifungal . Compounds 5 and 6 were also found to have weak tyrosine kinase p56(lck)() (TK) inhibitory activity at the 200 microgram/mL level.

Scand J Gastroenterol, 2003 Jan, 38(1), 73 - 9
Biological activity of Desulfovibrio desulfuricans lipopolysaccharides evaluated via interleukin-8 secretion by Caco-2 cells; Weglarz L et al.; BACKGROUND: Although Desulfovibrio desulfuricans species, besides existing in the natural environment, is also found in the human digestive tract, no information is currently available on its role in the intestinal ecosystem and its activity in regard to the intestinal mucosa . Bacterial products (lipopolysaccharides, LPSs) are generally known for their ability to trigger inflammatory response by stimulating cytokine expression, such as interleukin-8 (IL-8) . METHODS: Colonic Caco-2 cells were exposed to LPSs isolated from the soil type and intestinal wild strains of D . desulfuricans bacteria . The amount of IL-8 secreted was measured by ELISA . The effects of sodium butyrate and cell preincubation with sodium butyrate on the IL-8 secretion in response to LPSs were also analysed . RESULTS: LPSs from D . desulfuricans down-regulated IL-8 secretion by the cells . Incubation of these cells with butyrate alone resulted in a dose-dependent stimulation of IL-8 release . Butyrate also modulated IL-8 secretion by cells stimulated with LPSs . CONCLUSIONS: Our findings suggest the lack of inflammatory response of intestinal mucosa in the presence of LPSs of D . desulfuricans . This response can be conditioned by the natural bacterial product, butyrate, which exerts a stimulatory effect on the IL-8 secretion and modulates its release in response to LPSs.

Vestn Ross Akad Med Nauk, 2003, (1), 30 - 5
{Rickettsiosis: state of the art at the turn of the 21st century}; Lukin EP et al.; Information about changes in the modern taxonomy of intracellular bacteria conditionally united within the nomination of "Rickettsioses" is presented in the paper . Due to a total hobby related with keeping home animals (pets), cats and dogs, apart from the cattle, joined the natural ecological cycles of rickettsioses stimulation . The morbidity of rickettsioses of the acaroid group has been persistently growing; like in case with other pathologies (Lyme's disease) involving the acaroid transfer factor, its obvious "urbanization" is pronounced . Absolute and relative morbidity indices in respect to rickettsioses, which are epidemiologically important for Russia, are presented . The modern knowledge database concerning the rickettsioses makes it possible to control the epidemic process for this infection category . It is noteworthy, that Prowazek's rickettsiosis of its both forms (i.e . the epidemic and relapsing ones), which does not have an independent cycle of circulation of its stimulator in wild nature, and unlike the acaroid group rickettsioses, turned into a socially controllable infection . It will be totally eliminated, during 10-15 years, both in Russia and the CIS countries . The practitioners are well supplied with a variety of drugs of tetracycline and fluorine-quinol groups, which makes it possible to arrest the infection process in patients rapidly and effectively and to prevent the lethal cases . A low incidence rate of rickettsioses, including Q fever, within the general infection morbidity, and taking into consideration the availability of methods for effective therapy and prevention, makes one consider a comprehensive vaccination against the discussed group of infections to be irrational . Obviously, such vaccination must be still applied in respect to a limited number of persons from among high-risk groups and for two or three varieties of rickettsioses only (i.e . Prowazek's rickettsioses, Rocky Mountain spotted fever and Q fever) . Live vaccines, obtained on the basis of attenuated and recombined strains (which have a full-scale set of specific antigens), are recommended.

Nippon Rinsho, 2003 Jan, 61(1), 125 - 8
{The prospects of vaccination against H . pylori infection}; Minoura T et al.; H . pylori causes numerous gastroduodenal diseases including malignancies . Although eradication of H . pylori using antibiotics is clinically performed, it is sometimes unsuccessful because of resistant bacteria and patient non-compliance with treatment . Therefore prophylactic and therapeutic vaccination against H . pylori infection is necessary for clinical use but it is still under study . H . pylori is widely believed to be transmitted in childhood . If long and chronic H . pylori infection causes gastric cancer, vaccination should be targeted at children for the prophylaxis of cancer . Systemic immunization with aluminum hydroxide which has been already proved to be safe for human child, deserve attention.

Biol Reprod, 2003 May, 68(5), 1877 - 87 Epub 2002 Dec 27.
Activin signaling pathways in ovine pituitary and LbetaT2 gonadotrope cells; Dupont J et al.; In the pituitary, activin stimulates the synthesis and release of FSH . However, the activin receptor signaling pathways that mediate these effects are poorly known . We investigated these mechanisms in primary ovine pituitary cells (POP) and in the murine LbetaT2 gonadotrope cell line . POP cells and LbetaT2 cells express the different activin receptors (types IA, IB, IIA, and IIB) and the Smad proteins (Smad-2, -3, -4, and -7) . In both POP and LbetaT2 cells, activin activated several signaling pathways: Smad-2, extracellular regulated kinase-1/2 (ERK1/2), p38, and phosphatidylinositol 3'-kinase (PI3K)/Akt . Phosphorylation of ERK1/2 and p38 were stimulated (3- to 6-fold) rapidly in 5 min, whereas activation of both Smad-2 and Akt (3- to 5-fold) occurred later, in 60 min . Activin also increased the association of activin receptor IIB with PI3K . Using specific inhibitors, we demonstrated that the activation of Smad-2 was partially blocked by the inhibition of PI3K but not by the inhibition of ERK1/2 or p38, suggesting a cross-talk between the Smad and PI3K/Akt pathways . In both POP and LbetaT2 cells, FSH expression and secretion in response to activin were not altered by the inhibition of PI3K/Akt, ERK1/2, or p38 pathways, whereas they were reduced by about 2-fold by expression of a dominant negative of Smad-2 or the natural inhibitory Smad-7 in LbetaT2 cells . These results indicate that activin activates several signaling pathways with different time courses in both POP and LbetaT2 cells, but only the Smad-2 pathway appears to be directly implicated in FSH expression and release in LbetaT2 cells.

Am J Physiol Gastrointest Liver Physiol, 2003 Jul, 285(1), G163 - 76 Epub 2003 Feb 26.
Effects of Helicobacter pylori on intracellular Ca2+ signaling in normal human gastric mucous epithelial cells; Marlink KL et al.; In stomach, Helicobacter pylori (Hp) adheres to gastric mucous epithelial cells (GMEC) and initiates several different signal transduction events . Alteration of intracellular Ca2+ concentration ({Ca2+}i) is an important signaling mechanism in numerous bacteria-host model systems . Changes in {Ca2+}i induced by Hp in normal human GMEC have not yet been described; therefore, we examined effects of Hp on {Ca2+}i in normal human GMEC and a nontransformed GMEC line (HFE-145) . Cultured cells were grown on glass slides, porous filters, or 96-well plates and loaded with fura 2 or fluo 4 . Hp wild-type strain 60190 and vacA-, cagA-, and picB-/cagE- isogenic mutants were incubated with cells . Changes in {Ca2+}i were recorded with a fluorimeter or fluorescence plate reader . Wild-type Hp produced dose-dependent biphasic transient {Ca2+}i peak and plateau changes in both cell lines . Hp vacA- isogenic mutant produced changes in {Ca2+}i similar to those produced by wild type . Compared with wild type, cagA- and picB-/cagE- isogenic mutants produced lower peak changes and did not generate a plateau change . Preloading cultures with intracellular Ca2+ chelator BAPTA blocked all Hp-induced {Ca2+}i changes . Thapsigargin pretreatment of cultures to release Ca2+ from internal stores reduced peak change . Extracellular Ca2+ removal reduced plateau response . Hp-induced peak response was sensitive to G proteins and PLC inhibitors . Hp-induced plateau change was sensitive to G protein inhibitors, src kinases, and PLA2 . These findings are the first to show that H . pylori alters {Ca2+}i in normal GMEC through a Ca2+ release/influx mechanism that depends on expression of cagA and picB/cagE genes.

Environ Int, 2003 Mar, 28(8), 801 - 2
Toxicity assessment of wood preservatives; Lebkowska M et al.; The article describes the influence of wood preservatives on water bioceonosis . The "battery" of toxicology tests was used to cover luminescent bacteria test, algae growth inhibition test, crustacean and fish lethal tests . The test samples used were pure wood preservative and water after the wood preservative leaching from wood sample . It was observed that the wood preservative is leaching from the wood sample in 10% within 1 month of exposition . Achieved results indicate the necessity of toxic assessment of wood preservative after wood element protection.

Mar Pollut Bull, 2003 Mar, 46(3), 346 - 52
Seasonal changes in zooplanktonic alkaline phosphatase activity in Toulon Bay (France): the role of Cypris larvae; Jean N et al.; We studied zooplankton contribution to the total particulate phosphatase activity, the kinetics of this activity, the relation to the different taxonomic groups and the role of particle-bound bacteria . The activity of total particulate material collected from a liter of seawater was more elevated in May, June and August than during the rest of the year . These high activities resulted from a high contribution of the >90 microm fraction which account then for more than 60% of the total particulate activity . Two Michaelian processes with high and low V(max) were disclosed on this fraction . The high V(max) component was responsible for the high summer activities . During these periods, high densities of cirriped Cypris were found which were statistically correlated with this high V(max) component as with its specific activity . Moreover, the contribution of attached bacteria to these high activities was low . In return, this contribution was predominant during the periods of low activity . A simple method was developed to characterise this bacterial activity.

Mol Microbiol, 2003 Mar, 47(5), 1433 - 44
Adaptation of the Lyme disease spirochaete to the mammalian host environment results in enhanced glycosaminoglycan and host cell binding; Parveen N et al.; The Lyme disease spirochaete, Borrelia burgdorferi, is transmitted to mammals by Ixodes ticks and can infect multiple tissues . Host cell attachment may be critical for tissue colonization, and B . burgdorferi cultivated in vitro recognizes heparin- and dermatan sulphate-related glycosaminoglycans (GAGs) on the surface of mammalian cells . To determine whether growth of the spirochaete in the mammalian host alters GAG binding, we assessed the cell attachment activities of B . burgdorferi grown in vitro or in dialysis membrane chambers implanted intraperitoneally in rats . Host-adapted B . burgdorferi exhibited approximately threefold better binding to purified heparin and dermatan sulphate and to GAGs expressed on the surface of cultured endothelial cells . Three B . burgdorferi surface proteins, Bgp, DbpA and DbpB, have been demonstrated previously to bind to GAGs or to GAG-containing molecules, and we show here that recombinant derivatives of each of these proteins were able to bind to purified heparin and dermatan sulphate . Immunofluorescent staining of in vitro-cultivated or host-adapted spirochaetes revealed that DbpA and DbpB were present on the bacterial surface at higher levels after host adaptation . Recombinant Bgp, DbpA and DbpB each partially inhibited attachment of host-adapted B . burgdorferi to cultured mammalian cells, consistent with the hypothesis that these proteins may promote attachment of B . burgdorferi during growth in the mammalian host . Nevertheless, the partial nature of this inhibition suggests that multiple pathways promote mammalian cell attachment by B . burgdorferi in vivo . Given the observed increase in cell attachment activity upon growth in the mammalian host, analysis of host-adapted bacteria will facilitate identification of the cell binding pathways used in vivo.

Mol Microbiol, 2003 Mar, 47(5), 1385 - 93
Conjugal immunity of Streptomyces strains carrying the integrative element pSAM2 is due to the pif gene (pSAM2 immunity factor); Possoz C et al.; Mechanisms of conjugal immunity preventing redundant exchange between two cells harbouring the same conjugative element have been reported in diverse bacteria . Such a system does exist for pSAM2, a conjugative and integrative element of Streptomyces . The apparition of the conjugative free form of pSAM2 in the donor strain during mating can be considered as the initial step of transfer . We analysed the genes involved in transfer inhibition by mating donors harbouring pSAM2 with recipient strains containing different regions of pSAM2 . The conjugal immunity was previously thought to be mediated by the transcriptional repressor KorSA . Although the transfer efficiency is reduced by its presence in the recipient, the initiation of the transfer process is not affected . In contrast, the presence in the recipient strain of a single pSAM2 gene, pif (pSAM2 immunity factor), was sufficient to abolish both transfer and initiation of transfer . Thus, the clustered genes korSA and pif act complementarily to maintain pSAM2 in a 'prophage' state under non-conjugal conditions . KorSA is involved in intracellular signalling, whereas Pif participates in intercellular signalling . The Pif nudix motif is essential for its activity . This is the first protein of the nudix family shown to be involved in bacterial conjugation.

Mol Microbiol, 2003 Mar, 47(5), 1239 - 49
Cytochrome c oxidase genes required for nitrogenase activity and diazotrophic growth in Anabaena sp . PCC 7120; Valladares A et al.; N2 fixation is an O2-sensitive process and some filamentous diazotrophic cyanobacteria that grow performing oxygenic photosynthesis confine their N2 fixation machinery to heterocysts, specialized cells that maintain a reducing environment adequate for N2 fixation . Respiration is thought to contribute to the diazotrophic metabolism of heterocysts and the genome of the heterocyst-forming cyanobacterium Anabaena sp . PCC 7120 bears three gene clusters putatively encoding cytochrome c oxidases . Transcript analysis of these cox gene clusters through RNA/DNA hybridization identified two cox operons, cox2 and cox3, that are induced after nitrogen step-down in an NtcA- and HetR-dependent manner and appear to be expressed specifically in heterocysts . In contrast, cox1 was expressed only in vegetative cells . Expression of cox2 and cox3 occurred at an intermediate stage (about 9 h) during the process of heterocyst development following nitrogen step-down . Inactivation of genes in the two inducible cox operons, but not separately in either of them, strongly reduced nitrogenase activity and prevented diazotrophic growth in aerobic conditions . These results show that the nitrogen-regulated cytochrome c oxidase-type respiratory terminal oxidases Cox2 and Cox3 are essential for heterocyst function in Anabaena sp . PCC 7120.

Helicobacter, 2003 Feb, 8(1), 21 - 8
Helicobacter pylori infection in children and adults: a single pathogen but a different pathology; Gallo N et al.; BACKGROUND: The aims of this retrospective study were to ascertain in large series of children and adults: the relationship of the infecting strain to gastric mucosal lesions; and the relationship of the infecting strain to its duodenal localization . MATERIALS AND METHODS: We studied 307 and 604 consecutive children and adults . In gastric mucosal samples H . pylori was cultured, genotyped and histologically assessed, while inflammation, activity and intestinal metaplasia were graded . In a subset of 171 patients H . pylori ureaseA (ureA) and cagA genes were amplified (PCR) using mucosal biopsies from the duodenum . RESULTS: H . pylori infection was diagnosed in 40 children and 308 adults . cagA was identified in 50% and 65.5% of infected children and adults . Antral activity was associated with the density of infecting bacteria (p <.001) and with cagA (p <.01) . Intestinal metaplasia was correlated with cagA (p <.001) . The ureA gene was found in 56 duodenal samples from 82 H . pylori positive patients . Duodenal H . pylori ureA was significantly more frequent in patients with duodenal diseases than in those without (p <.01), cagA positive strains being mainly involved in the infection of this anatomical area (p <.01) . CONCLUSIONS: A severe H . pylori-associated gastritis is more prevalent when the density of infecting bacteria is high and when cagA positive strains cause the infection . The most virulent cagA positive H . pylori colonizes not only the gastric, but also the duodenal mucosa, which can be directly damaged by the bacteria itself or by its products.

Proteomics, 2003 Feb, 3(2), 224 - 37
Proteome analysis of the plant pathogen Xylella fastidiosa reveals major cellular and extracellular proteins and a peculiar codon bias distribution; Smolka MB et al.; The bacteria Xylella fastidiosa is the causative agent of a number of economically important crop diseases, including citrus variegated chlorosis . Although its complete genome is already sequenced, X . fastidiosa is very poorly characterized by biochemical approaches at the protein level . In an initial effort to characterize protein expression in X . fastidiosa we used one- and two-dimensional gel electrophoresis and mass spectrometry to identify the products of 142 genes present in a whole cell extract and in an extracellular fraction of the citrus isolated strain 9a5c . Of particular interest for the study of pathogenesis are adhesion and secreted proteins . Homologs to proteins from three different adhesion systems (type IV fimbriae, mrk pili and hsf surface fibrils) were found to be coexpressed, the last two being detected only as multimeric complexes in the high molecular weight region of one-dimensional electrophoresis gels . Using a procedure to extract secreted proteins as well as proteins weakly attached to the cell surface we identified 30 different proteins including toxins, adhesion related proteins, antioxidant enzymes, different types of proteases and 16 hypothetical proteins . These data suggest that the intercellular space of X . fastidiosa colonies is a multifunctional microenvironment containing proteins related to in vivo bacterial survival and pathogenesis . A codon usage analysis of the most expressed proteins from the whole cell extract revealed a low biased distribution, which we propose is related to the slow growing nature of X . fastidiosa . A database of the X . fastidiosa proteome was developed and can be accessed via the internet (URL: www.proteome.ibi.unicamp.br).

J Med Virol, 2003 Apr, 69(4), 495 - 502
Heparin-interacting sites of bovine lactoferrin are involved in anti-adenovirus activity; Di Biase AM et al.; Lactoferrin, a member of the transferrin family of approximately 80 kDa, consists of a single polypeptide chain folded in two symmetric, globular lobes (N- and C-lobes), each able to bind one ferric ion . This glycoprotein, found in physiological fluids of mammals, plays an important role in immune regulation and in defense mechanisms against bacteria, fungi, parasites, and viruses . Although the antiviral activity of lactoferrin is one of the major biological functions of such protein, the mechanism of action is still under debate . We have investigated both the role of tryptic fragments of bovine lactoferrin and the mechanism of lactoferrin antiviral effect toward adenovirus infection in HEp-2 cells . The results obtained demonstrated that the anti-adenovirus activity of lactoferrin is mediated by the N-terminal half of the protein as the N-lobe was able to inhibit adenovirus infection, even if at lower extent than undigested lactoferrin, whereas C-lobe was ineffective . The results also showed that the anti-adenovirus action of lactoferrin and of its N-terminal peptide lactoferricin took place on virus attachment to cell membrane, mainly through competition for common glycosaminoglycan receptors . The data provide evidence that the anti-adenovirus activity of lactoferrin is mediated mainly by the cluster of positive charges at the N-terminus of whole molecule and that the N-terminal peptide lactoferricin alone is sufficient to prevent infection .

Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2420 - 5 Epub 2003 Feb 24.
Interaction of mismatch repair protein PMS2 and the p53-related transcription factor p73 in apoptosis response to cisplatin; Shimodaira H et al.; Mismatch repair (MMR) proteins contribute to genome integrity by correcting replication errors . In higher eukaryotes, MMR proteins also regulate the cellular response to DNA lesions such as oxidized, alkylated, or crosslinked bases . Previous studies have linked MMR proteins to the activation of apoptosis through p53-dependent and p53-independent mechanisms . MMR-deficient cells exhibit variable defects in the induction of p53 and its related p73, which are activators of apoptosis . However, the specific role of each MMR protein in the regulation of apoptosis has not been determined . Here, we describe an interaction between PMS2, an MMR protein, and p73 . This interaction causes the stabilization of p73 and the redistribution of PMS2 to the nuclear compartment . Exposure to cisplatin enhances the association between PMS2 and p73 . Moreover, stimulation of the p73 proapoptotic function by cisplatin requires PMS2 . These results suggest that PMS2 contributes to genome integrity not only through DNA repair but also by enhancing DNA damage-induced apoptosis.

Protein Eng, 2002 Dec, 15(12), 987 - 95
Analysis of membrane stereochemistry with homology modeling of sn-glycerol-1-phosphate dehydrogenase; Daiyasu H et al.; Different enantiomeric isomers, sn-glycerol-1-phosphate and sn-glycerol-3-phosphate, are used as the glycerophosphate backbones of phospholipids in the cellular membranes of Archaea and the remaining two kingdoms, respectively . In Archaea, sn-glycerol-1-phosphate dehydrogenase is involved in the generation of sn-glycerol-1-phosphate, while sn-glycerol-3-phosphate dehydrogenase synthesizes the enantiomer in Eukarya and Bacteria . The coordinates of sn-glycerol-3-phosphate dehydrogenase are available, although neither the tertiary structure nor the reaction mechanism of sn-glycerol-1-phosphate dehydrogenase is known . Database searching revealed that the archaeal enzyme shows sequence similarity to glycerol dehydrogenase, dehydroquinate synthase and alcohol dehydrogenase IV . The glycerol dehydrogenase, with coordinates that are available today, is closely related to the archaeal enzyme . Using the structure of glycerol dehydrogenase as the template, we built a model structure of the Methanothermobacter thermautotrophicus sn-glycerol-1-phosphate dehydrogenase, which could explain the chirality of the product . Based on the model structure, we determined the following: (1) the enzyme requires a Zn(2+) ion for its activity; (2) the enzyme selectively uses the pro-R hydrogen of the NAD(P)H; (3) the putative active site and the reaction mechanism were predicted; and (4) the archaeal enzyme does not share its evolutionary origin with sn-glycerol-3-phosphate dehydrogenase.

J Biol Chem, 2003 May 2, 278(18), 16443 - 50 Epub 2003 Feb 24.
Interactions of the cbbII promoter-operator region with CbbR and RegA (PrrA) regulators indicate distinct mechanisms to control expression of the two cbb operons of Rhodobacter sphaeroides; Dubbs JM et al.; In a previous study (Dubbs, J . M., Bird, T . H., Bauer, C . E., and Tabita, F . R . (2000) J . Biol . Chem . 275, 19224-19230), it was demonstrated that the regulators CbbR and RegA (PrrA) interacted with both promoter proximal and promoter distal regions of the form I (cbb(I)) promoter operon specifying genes of the Calvin-Benson-Bassham cycle of Rhodobacter sphaeroides . To determine how these regulators interact with the form II (cbb(II)) promoter, three cbbF(II)::lacZ translational fusion plasmids were constructed containing various lengths of sequence 5' to the cbb(II) operon of R . sphaeroides CAC . Expression of beta-galactosidase was monitored under a variety of growth conditions in both the parental strain and knock-out strains that contain mutations that affect synthesis of CbbR and RegA . The binding sites for both CbbR and RegA were determined by DNase I footprinting . A region of the cbb(II) promoter from +38 to -227 bp contained a CbbR binding site and conferred low level regulated cbb(II) expression . The region from -227 to -1025 bp contained six RegA binding sites and conferred enhanced cbb(II) expression under all growth conditions . Unlike the cbb(I) operon, the region between -227 and -545 bp that contains one RegA binding site, was responsible for the majority of the observed enhancement . Both RegA and CbbR were required for maximal cbb(II) expression . Two potentially novel and specific cbb(II) promoter-binding proteins that did not interact with the cbb(I) promoter region were detected in crude extracts of R . sphaeroides . These results, combined with the observation that chemoautotrophic expression of the cbb(I) operon is RegA independent, indicated that the mechanisms controlling cbb(I) and cbb(II) operon expression during chemoautotrophic growth are quite different.

J Biol Chem, 2003 May 2, 278(18), 15815 - 24 Epub 2003 Feb 24.
Interactions of the non-coding RNA DsrA and RpoS mRNA with the 30 S ribosomal subunit; Worhunsky DJ et al.; Expression of sigma(s), the gene product of rpoS, is controlled translationally in response to many environmental stresses . DsrA, a small 87-nucleotide non-coding RNA molecule, acts to increase translational efficiency of RpoS mRNA under some growth conditions . In this work, we demonstrate that DsrA binds directly to the 30 S ribosomal subunit with an observed equilibrium affinity of 2.8 x 10(7) m(-1) . DsrA does not compete with RpoS mRNA or tRNA(f)(Met) for binding to the 30 S subunit . The 5' end of DsrA binds to 30 S subunits with an observed equilibrium association constant of 2.0 x 10(6) m(-1), indicating that the full affinity of the interaction requires the entire DsrA sequence . In order to investigate translational efficiency of RpoS mRNA, we examined both ribosome-binding site accessibility and the binding of RpoS mRNA to 30 S ribosomal subunits . We find that that ribosome-binding site accessibility is modulated as a function of divalent cation concentration during mRNA renaturation and by the presence of an antisense sequence that binds to nucleotides 1-16 of the RpoS mRNA fragment . The ribosome-binding site accessibility correlates with the amount of RpoS mRNA participating in 30 S-mRNA "pre-initiation" translational complex formation and provides evidence that regulation follows a competitive model of regulation.

Neurobiol Aging, 2003 May-Jun, 24(3), 501 - 9
Glutathione S-transferase isoenzymatic response to aging in rat cerebral cortex and cerebellum; Martinez-Lara E et al.; Aging is associated with increased oxidant generation . One mechanism involved in the defense of oxidative products is the family of glutathione transferases (GST) . We have analyzed the activity, distribution and expression of GSTP1 and GSTA4 isoenzymes in the cerebral cortex and cerebellum of young, adult and aged rats . The total GST activity, measured with the universal substrate 1-chloro-2,4-dinitrobenzene (CDNB), increased only with the maturation process; however GSTA4 activity, using the specific substrate 4-hydroxynonenal (HNE), did show an age-dependent increase in both brain regions . Cellular location of GSTA4 in astrocytes was not changed except for young cerebral cortex and adult/aged cerebellum that also showed immunoreactivity in layer III pyramidal neurons and Bergman radial glia, respectively . Distribution of GSTP1 was similar among groups and only an increased number of positive oligodendrocytes was found in the Purkinje and granular layer of adult/aged cerebellum . The GSTA4 and GSTP1 expression increased from young to adult/aged brain and GSTA4 even augmented in the aged cerebral cortex . These results suggest a GST isoenzymatic response with aging, but above all with the maturation process.

Water Res, 2003 Apr, 37(7), 1583 - 92
Phytoplankton, pelagic community and nutrients in a deep oligotrophic alpine lake: ratios as sensitive indicators of the use of P-resources (DRP:DOP:PP and TN:TP:SRSi); Teubner K; The different use of P-resources between two sites in the deep oligotrophic Traunsee was studied by seasonal and vertical patterns of phytoplankton and nutrients from 12/1997 to 10/1998 . The P-resources were evaluated from the proportion between the P-fractions, the dissolved reactive P (DRP), dissolved non-reactive P (DOP) and particulate organic P (PP) and from the stoichiometry between nutrients, the total N (TN), the total P (TP) and soluble reactive Si (SRSi) . Significant differences between an inshore site impacted by industrial tailings (Ebensee Bay, EB) and an open water reference site (Viechtau, VI) were evident from vertical profiles of both the P-accumulation (%PP of TP) evaluated by DRP:DOP:PP and the distribution of phytoplankton assessed by Si-exhaustion (TN:TP:SRSi), but not from the seasonal patterns of phytoplankton composition, S:V ratios of the algal community or surface layer nutrient dynamics . Low TP and the stable stratification from May to September triggered the relative accumulation of epilimnetic P at VI as it was evident from both the higher portion of particulate P within TP (%PP of TP) and from the shift towards P-enrichment in nutrient stoichiometry of TN:TP:SRSi . The predominance of around 55-52% algal carbon over bacteria at the surface layer to 20m coincided spatially with the lowest Si content relative to N and P . The disturbances at the impacted site was summarised by: up to 11% less P accumulation by organisms at the surface, no stoichiometric shift towards TP in the epilimnion when compared with deeper layers and a reduction of the trophogenic zone to the top 10m . Reasons for this disturbance are seen in the unstable stratification, turbidity, higher TP and the metazoan dominated food chain . Both triple ratios, DRP:DOP:PP and TN:TP:SRSi, were sensitive indicators of the use of P-resources by plankton communities, while inorganic dissolved fractions (DIN:DRP:SRSi, DIN = dissolved inorganic N) provided only insufficient information on nutrient resources in Traunsee .

Cell, 2003 Feb 21, 112(4), 549 - 60
Adenylyl cyclase localization regulates streaming during chemotaxis; Kriebel PW et al.; We studied the role of the adenylyl cyclase ACA in Dictyostelium discoideum chemotaxis and streaming . In this process, cells orient themselves in a head to tail fashion as they are migrating to form aggregates . We show that cells lacking ACA are capable of moving up a chemoattractant gradient, but are unable to stream . Imaging of ACA-YFP reveals plasma membrane labeling highly enriched at the uropod of polarized cells . This localization requires the actin cytoskeleton but is independent of the regulator CRAC and the effector PKA . A constitutively active mutant of ACA shows dramatically reduced uropod enrichment and has severe streaming defects . We propose that the asymmetric distribution of ACA provides a compartment from which cAMP is secreted to locally act as a chemoattractant, thereby providing a unique mechanism to amplify chemical gradients . This could represent a general mechanism that cells use to amplify chemotactic responses.

Cell, 2003 Feb 21, 112(4), 467 - 80
The molecular motor toolbox for intracellular transport; Vale RD; Eukaryotic cells create internal order by using protein motors to transport molecules and organelles along cytoskeletal tracks . Recent genomic and functional studies suggest that five cargo-carrying motors emerged in primitive eukaryotes and have been widely used throughout evolution . The complexity of these "Toolbox" motors expanded in higher eukaryotes through gene duplication, alternative splicing, and the addition of associated subunits, which enabled new cargoes to be transported . Remarkably, fungi, parasites, plants, and animals have distinct subsets of Toolbox motors in their genomes, suggesting an underlying diversity of strategies for intracellular transport.

Hua Xi Yi Ke Da Xue Xue Bao, 2002 Jan, 33(1), 75 - 6, 161
{The effects of fluoride-containing trace element agents on the growth of mutants of S . mutans}; Wu H et al.; OBJECTIVE: To compare the influence of 6 trace element agents containing fluoride versus 6 trace element agents not containing fluoride on the growth of mutants of S . mutans and detect the interaction between fluoride and trace elements . METHODS: Six trace element agents containing fluoride {SnF2, ZnF2, SrF2, LaF2, (NH3)2MoF8, NaF} and 6 trace element agents not containing fluoride {SnCl2, SrCl2, LaCl2, ZnAc2, (NH3)2MoO4, NaCl} were selected . The continuous anaerobic cultivating technique and the absorbency of bacteria liquid and the count of bacteria were used to assess the effects of different agents on the growth of S . mutans . RESULTS: NaF, SnF2, SnCl2, ZnF2, ZnAc2, (NH3)2MoF8 and (NH3)2MoO4 were found to have strong inhibition effects on the growth of mutant of S . mutans(P < 0.01) . SnF2 had more stronger inhibition effects than SnCl2; ZnF2 had more stronger inhibition effects than ZnAc2, no significant differences were seen between (NH3)2MoF8 and (NH3)2MoO4 . SnF2 and ZnF2 had most potent inhibition effects(P < 0.01) . No significant differences were observed between SrF2, SrCl2, LaCl2, LaF2, NaCl and the control groups(P > 0.05) . CONCLUSION: The agents containing fluoride and trace elements, such as stannum and zinc had the effects of prohibiting the growth of mutant of S . mutans, As far as caries prevention is concerned, the agents containing both fluoride and trace elements are more effective than mono-fluoride, the potential mechanism may be the synergistic action between fluoride and trace elements.

J Exp Bot, 2003 Mar, 54(384), 1115 - 6
Isolation of two plastid division ftsZ genes from Chlamydomonas reinhardtii and its evolutionary implication for the role of FtsZ in plastid division; Wang D et al.; In order to elucidate the origin of the plastid division gene ftsZ in green plant lineage, and to understand the significance of this divergence for the function of FtsZ proteins in plants, two full-length cDNAs (accession numbers AF449446 and AB084236) were isolated from Chlamydomonas reinhardtii, a base species of green plant lineage . A phylogenetic analysis based on amino acid sequences of eukaryotic FtsZs reveals that an ancient duplication of the ftsZ gene occurred after the endosymbiotic event . The ancient duplication implies that two ftsZ families might play an indispensable role at the early endosymbiotic stage.

J Med Entomol, 2003 Jan, 40(1), 1 - 5
Molecular phylogeny of Wolbachia endosymbionts in Southeast Asian mosquitoes (Diptera: Culicidae) based on wsp gene sequences; Ruang-Areerate T et al.; Wolbachia are maternally inherited intracellular bacteria that infect a wide range of arthropods and nematodes and are associated with various reproductive abnormalities in their hosts . Insect-associated Wolbachia form a monophyletic clade in the alpha-Proteobacteria and recently have been separated into two supergroups (A and B) and 19 groups . Our recent polymerase chain reaction (PCR) survey using wsp specific primers indicated that various strains of Wolbachia were present in mosquitoes collected from Southeast Asia . Here, we report the phylogenetic relationship of the Wolbachia strains found in these mosquitoes using wsp gene sequences . Our phylogenetic analysis revealed eight new Wolbachia strains, five in the A supergroup and three in the B supergroup . Most of the Wolbachia strains present in Southeast Asian mosquitoes belong to the established Mors, Con, and Pip groups.

J Food Prot, 2003 Feb, 66(2), 335 - 40
Monitoring volatile and nonvolatile amines in dried and salted roes of tuna (Thunnus thynnus L.) during manufacture and storage; Periago MJ et al.; Dried and salted roe, obtained from the reproductive organs of female tuna (Thunnus tynnus L.), is a typical fish-based food in the Mediterranean area of Spain . In the present study, we monitored the formation of volatile amines (trimethyamine nitrogen {TMA-N} and total basic volatile nitrogen {TBVN}) and nonvolatile amines (biogenic amines) in dried and salted tuna roe after processing and storage for 8 weeks at 4, 20, and 30 degrees C . The salting and drying process significantly increased the TBVN, cadaverine, tyramine, phenylethylamine, and tryptamine contents, and bacteria with histamine decarboxylase activity were detected both in raw and in dried and salted tuna roes . During storage of tuna roe, TMA-N and TBVN levels increased significantly after the fourth week of storage at 30 degrees C, whereas biogenic amine contents remained more or less constant . However, samples stored at 30 degrees C showed histamine formation after the first week of storage, with a concentration of < 50 ppm . The volatile and nonvolatile amine concentrations in tuna roe were below the consumer safety limit, with the exception of the total biogenic amine level in roe stored at 30 degrees C, which exceeded the European Community's recommended limit (300 ppm) . These results indicate that in properly stored tuna roe, histamine formation will not represent a serious health risk to consumers unless the tuna roe has previously been mishandled.

Crit Care Nurs Clin North Am, 2003 Mar, 15(1), 13 - 25
The cellular basis of septic shock; Sommers MS; Severe sepsis and septic shock are among the most complex and challenging conditions treated by critical care practitioners . Although the pathophysiology of severe sepsis and septic shock is not fully understood, bacteria and immune responses are known to trigger the release of cytokines . These cytokines initiate a cascade of events that lead to illness behaviors such as fever, anorexia, and sleepiness, as well as a host of physiologic events such as activation of the coagulation cascade, vasodilation, hypotension, and increased vessel permeability . As research advances the understanding of severe sepsis and septic shock, practitioners must become aware of the cellular basis of events so that treatments can be implemented knowledgeably and evaluated.

Curr Top Microbiol Immunol, 2003, 274, 79 - 112
SMC protein complexes and the maintenance of chromosome integrity; Yokomori K; Structural maintenance of chromosomes (SMC) family proteins have attracted much attention for their unique protein structure and critical roles in mitotic chromosome organization . Elegant genetic and biochemical studies in yeast and Xenopus identified two different SMC heterodimers in two conserved multiprotein complexes termed 'condensin' and 'cohesin' . These complexes are required for mitotic chromosome condensation and sister chromatid cohesion, respectively, both of which are prerequisite to accurate segregation of chromosomes . Although structurally similar, the SMC proteins in condensin and cohesin appear to have distinct functions, whose specificity and cell cycle regulation are critically determined by their interactions with unique sets of associated proteins . Recent studies of subcellular localization of SMC proteins and SMC-containing complexes, identification of their interactions with other cellular factors, and discovery of new SMC family members have uncovered unexpected roles for SMC proteins and SMC-containing complexes in different aspects of genome functions and chromosome organization beyond mitosis, all of which are critical for the maintenance of chromosome integrity.

Chemphyschem, 2003 Jan 13, 4(1), 14 - 30
Raman spectroscopy--a prospective tool in the life sciences; Petry R et al.; Although the physics of Raman spectroscopy and its application to purely chemical problems is long established, it offers a noninvasive, nondestructive, and water-insensitive probe to problems in the life sciences . Starting from the principles of Raman spectroscopy, its advantages, and methods for signal enhancement, the bulk of the review highlights recent applications . Structural investigations of a hormone receptor, testing the biocompatibility of dental implants, probing soil components and plant tissue alkaloids, and localization of single bacteria are just four problems in which Raman spectroscopy offers a solution or complements existing methods.

Proteins, 2003 Apr 1, 51(1), 96 - 108
Efficient identification of side-chain patterns using a multidimensional index tree; Hamelryck T; Convergent evolution often produces similar functional sites in nonhomologous proteins . The identification of these sites can make it possible to infer function from structure, to pinpoint the location of a functional site, to identify enzymes with similar enzymatic mechanisms, or to discover putative functional sites . In this article, a novel method is presented that (a) queries a database of protein structures for the occurrence of a given side chain pattern and (b) identifies interesting side-chain patterns in a given structure . For efficiency and to make a robust statistical evaluation of the significance of a similarity possible, patterns of three residues (or triads) are considered . Each triad is encoded as a high-dimensional vector and stored in an SR (Sphere/Rectangle) tree, an efficient multidimensional index tree . Identifying similar triads can then be reformulated as identifying neighboring vectors . The method deals with many features that otherwise complicate the identification of meaningful patterns: shifted backbone positions, conservative substitutions, various atom label ambiguities and mirror imaged geometries . The combined treatment of these features leads to the identification of previously unidentified patterns . In particular, the identification of mirror imaged side-chain patterns is unique to the here-described method . Interesting triads in a given structure can be identified by extracting all triads and comparing them with a database of triads involved in ligand binding . The approach was tested by an all-against-all comparison of unique representatives of all SCOP superfamilies . New findings include mirror imaged metal binding and active sites, and a putative active site in bacterial luciferase .

Proteins, 2003 Apr 1, 51(1), 10 - 20
Dynamic fluorescence studies of beta-glycosidase mutants from Sulfolobus solfataricus: effects of single mutations on protein thermostability; Bismuto E et al.; Multiple sequence alignment on 73 proteins belonging to glycosyl hydrolase family 1 reveals the occurrence of a segment (83-124) in the enzyme sequences from hyperthermophilic archaea bacteria, which is absent in all the mesophilic members of the family . The alignment of the known three-dimensional structures of hyperthermophilic glycosidases with the known ones from mesophilic organisms shows a similar spatial organizations of beta-glycosidases except for this sequence segment whose structure is located on the external surface of each of four identical subunits, where it overlaps two alpha-helices . Site-directed mutagenesis substituting N97 or S101 with a cysteine residue in the sequence of beta-glycosidase from hyperthermophilic archaeon Sulfolobus solfataricus caused some changes in the structural and dynamic properties as observed by circular dichroism in far- and near-UV light, as well as by frequency domain fluorometry, with a simultaneous loss of thermostability . The results led us to hypothesize an important role of the sequence segment present only in hyperthermophilic beta-glycosidases, in the thermal adaptation of archaea beta-glycosidases . The thermostabilization mechanism could occur as a consequence of numerous favorable ionic interactions of the 83-124 sequence with the other part of protein matrix that becomes more rigid and less accessible to the insult of thermal-activated solvent molecules .

Bioessays, 2003 Mar, 25(3), 232 - 42
Phylogenomics of type II DNA topoisomerases; Gadelle D et al.; Type II DNA topoisomerases (Topo II) are essential enzymes implicated in key nuclear processes . The recent discovery of a novel kind of Topo II (DNA topoisomerase VI) in Archaea led to a division of these enzymes into two non-homologous families, (Topo IIA and Topo IIB) and to the identification of the eukaryotic protein that initiates meiotic recombination, Spo11 . In the present report, we have updated the distribution of all Topo II in the three domains of life by a phylogenomic approach . Both families exhibit an atypical distribution by comparison with other informational proteins, with predominance of Topo IIA in Bacteria, Eukarya and viruses, and Topo IIB in Archaea . However, plants and some Archaea contain Topo II from both families . We confront this atypical distribution with current hypotheses on the evolution of the three domains of life and origin of DNA genomes .

Science, 2003 Feb 21, 299(5610), 1214 - 7
Molecular fossil record of elevated methane levels in late Pleistocene coastal waters; Hinrichs KU et al.; Accumulating evidence suggests that methane has been released episodically from hydrates trapped in sea floor sediments during many intervals of rapid climate warming . Here we show that sediments from the Santa Barbara Basin deposited during warm intervals in the last glacial period contain molecular fossils that are diagnostic of aerobic and anaerobic methanotrophs . Sediment intervals with high abundances of these compounds indicate episodes of vigorous methanotrophic activity in methane-laden water masses . Signals for anaerobic methanotrophy in 44,100-year-old sediment are evidence for particularly intense methane emissions and suggest that the basin's methane cycle can profoundly affect oxygen budgets in the water column.

Physiol Genomics, 2003 Apr 16, 13(2), 85 - 95
A splice variant of the G protein beta 3-subunit implicated in disease states does not modulate ion channels; Ruiz-Velasco V et al.; A single-nucleotide polymorphism (C825T) in the GNB3 gene produces an alternative splice variant of the heterotrimeric G protein beta3 subunit (Gbeta3) . Translation of the alternatively spliced mRNA results in a protein product, Gbeta3-s, in which 41 amino acids are deleted from Gbeta3 . Interestingly, previous studies indicate that the C825T allele occurs with a high frequency in patients with certain vascular disorders . However, little information is available regarding the functional role Gbeta3-s might play in ion channel modulation . To examine this aspect, Gbeta3 or Gbeta3-s, along with either Ggamma2 or Ggamma5, were expressed in rat sympathetic neurons by nuclear microinjection of vector encoding the desired protein . In contrast to Gbeta3, expression of Gbeta3-s did not modulate N-type Ca(2+) or G protein-gated inwardly rectifying K(+) channels . In addition, Gbeta3-s did not appear to complex with a pertussis toxin-insensitive mutant of Galpha(i2) or couple to natively expressed alpha(2)-adrenergic receptors . Finally, fluorescence resonance energy transfer (FRET) measurements indicated that enhanced yellow fluorescent protein (EYFP)-labeled Gbeta3-s does not form a Gbetagamma heterodimer when coexpressed with enhanced cyan fluorescent protein (ECFP)-labeled Ggamma2 . Therefore, when expressed in sympathetic neurons, Gbeta3-s appears to lack biological activity--hence pathological conditions in patients carrying the homozygous C825T allele may result from a functional knockout of Gbeta3.

Infect Immun, 2003 Mar, 71(3), 1587 - 9
Interleukin-12 regulates chemokine gene expression during the early immune response to Leishmania major; Zaph C et al.; Following infection with Leishmania major, the chemokines XCL1, CXCL10, and CCL2 were preferentially expressed in draining lymph nodes of resistant mice . Neutralization of interleukin 12 (IL-12) or gamma interferon in resistant mice resulted in decreased chemokine expression, while administration of IL-12 to susceptible mice resulted in an increase in the level of chemokine gene expression.

Infect Immun, 2003 Mar, 71(3), 1225 - 33
Nitric oxide partially controls Coxiella burnetii phase II infection in mouse primary macrophages; Zamboni DS et al.; In most primary or continuous cell cultures infected with the Q-fever agent Coxiella burnetii, bacteria are typically sheltered in phagolysosome-like, large replicative vacuoles (LRVs) . We recently reported that only a small proportion of mouse peritoneal macrophages (PMPhi) infected with a nonvirulent, phase II strain of C . burnetii developed LRVs and that their relative bacterial load increased only slowly . In the majority of infected PMPhi, the bacteria were confined to the small vesicles . We show here that nitric oxide (NO) induced by the bacteria partially accounts for the restricted development of LRVs in primary macrophages . Thus, (i) PMPhi and bone marrow-derived macrophages (BMMPhi) challenged with phase II C . burnetii produced significant amounts of NO; (ii) the NO synthase inhibitors aminoguanidine and N-methyl-L-arginine reduced the production of NO and increased the frequency of LRVs (although the relative bacterial loads of individual LRVs did not change, the estimated loads per well increased appreciably); (iii) gamma interferon (IFN-gamma) or the NO donor sodium nitroprusside, added to BMMPhi prior to or after infection, reduced the development and the relative bacterial loads of LRVs and lowered the yield of viable bacteria recovered from the cultures; and (iv) these effects of IFN-gamma may not be entirely dependent on the production of NO since IFN-gamma also controlled the infection in macrophages from inducible NO synthase knockout mice . It remains to be determined whether NO reduced the development of LRVs by acting directly on the bacteria; by acting on the traffic, fusion, or fission of cell vesicles; or by a combination of these mechanisms.

Infect Immun, 2003 Mar, 71(3), 1134 - 40
Deletion of two-component regulatory systems increases the virulence of Mycobacterium tuberculosis; Parish T et al.; Two-component regulatory signal transduction systems are widely distributed among bacteria and enable the organisms to make coordinated changes in gene expression in response to a variety of environmental stimuli . The genome sequence of Mycobacterium tuberculosis contains 11 complete two-component systems, four isolated homologous regulators, and three isolated homologous sensors . We have constructed defined mutations in six of these genes and measured virulence in a SCID mouse model . Mice infected with four of the mutants (deletions of devR, tcrXY, trcS, and kdpDE) died more rapidly than those infected with wild-type bacteria . The other two mutants (narL and Rv3220c) showed no change compared to the wild-type H37Rv strain . The most hypervirulent mutant (devRdelta) also grew more rapidly in the acute stage of infection in immunocompetent mice and in gamma interferon-activated macrophages . These results define a novel class of genes in this pathogen whose presence slows down its multiplication in vivo or increases its susceptibility to host killing mechanisms . Thus, M . tuberculosis actively maintains a balance between its own survival and that of the host.

J Mol Biol, 2003 Mar 7, 326(5), 1523 - 38
The structure and thermal motion of the B800-850 LH2 complex from Rps.acidophila at 2.0A resolution and 100K: new structural features and functionally relevant motions; Papiz MZ et al.; The structure at 100K of integral membrane light-harvesting complex II (LH2) from Rhodopseudomonas acidophila strain 10050 has been refined to 2.0A resolution . The electron density has been significantly improved, compared to the 2.5A resolution map, by high resolution data, cryo-cooling and translation, libration, screw (TLS) refinement . The electron density reveals a second carotenoid molecule, the last five C-terminal residues of the alpha-chain and a carboxy modified alpha-Met1 which forms the ligand of the B800 bacteriochlorophyll . TLS refinement has enabled the characterisation of displacements between molecules in the complex . B850 bacteriochlorophyll molecules are arranged in a ring of 18 pigments composed of nine approximate dimers . These pigments are strongly coupled and at their equilibrium positions the excited state dipole interaction energies, within and between dimers, are approximately 370cm(-1) and 280cm(-1), respectively . This difference in coupling energy is similar in magnitude to changes in interaction energies arising from the pigment displacements described by TLS tensors . The displacements appear to be non-random in nature and appear to be designed to optimise the modulation of pigment energy interactions . This is the first time that LH2 pigment displacements have been quantified experimentally . The calculated energy changes indicate that there may be significant contributions to inter-pigment energy interactions from molecular displacements and these may be of importance to photosynthetic energy transfer.

Philos Trans R Soc Lond B Biol Sci, 2003 Jan 29, 358(1429), 267 - 74
The redox protein construction kit: pre-last universal common ancestor evolution of energy-conserving enzymes; Baymann F et al.; Genome analyses and the resolution of three-dimensional structures have provided evidence in recent years for hitherto unexpected family relationships between redox proteins of very diverse enzymes involved in bioenergetic electron transport . Many of these enzymes appear in fact to be constructed from only a limited set of building blocks . Phylogenetic analysis of selected units from this "redox enzyme construction kit" indicates an origin for several prominent bioenergetic enzymes that is very early, lying before the divergence of Bacteria and Archaea . Possible scenarios for the early evolution of selected complexes are proposed based on the obtained tree topologies.

Philos Trans R Soc Lond B Biol Sci, 2003 Jan 29, 358(1429), 231 - 43
Type I photosynthetic reaction centres: structure and function; Heathcote P et al.; We review recent advances in the study of the photosystem I reaction centre, following the determination of a spectacular 2.5 A resolution crystal structure for this complex of Synechococcus elongatus . Photosystem I is proving different to type II reaction centres in structure and organization, and the mechanism of transmembrane electron transfer, and is providing insights into the control of function in reaction centres that operate at very low redox potentials . The photosystem I complex of oxygenic organisms has a counterpart in non-oxygenic bacteria, the strictly anaerobic phototrophic green sulphur bacteria and heliobacteria . The most distinctive feature of these type I reaction centres is that they contain two copies of a large core polypeptide (i.e . a homodimer), rather than a heterodimeric arrangement of two related, but different, polypeptides as in the photosystem I complex . To compare the structural organization of the two forms of type I reaction centre, we have modelled the structure of the central region of the reaction centre from green sulphur bacteria, using sequence alignments and the structural coordinates of the S . elongatus Photosystem I complex . The outcome of these modelling studies is described, concentrating on regions of the type I reaction centre where important structure-function relationships have been demonstrated or inferred.

Philos Trans R Soc Lond B Biol Sci, 2003 Jan 29, 358(1429), 165 - 77; discussion 177-9
On the origin of mitochondria: a genomics perspective; Andersson SG et al.; The availability of complete genome sequence data from both bacteria and eukaryotes provides information about the contribution of bacterial genes to the origin and evolution of mitochondria . Phylogenetic analyses based on genes located in the mitochondrial genome indicate that these genes originated from within the alpha-proteobacteria . A number of ancestral bacterial genes have also been transferred from the mitochondrial to the nuclear genome, as evidenced by the presence of orthologous genes in the mitochondrial genome in some species and in the nuclear genome of other species . However, a multitude of mitochondrial proteins encoded in the nucleus display no homology to bacterial proteins, indicating that these originated within the eukaryotic cell subsequent to the acquisition of the endosymbiont . An analysis of the expression patterns of yeast nuclear genes coding for mitochondrial proteins has shown that genes predicted to be of eukaryotic origin are mainly translated on polysomes that are free in the cytosol whereas those of putative bacterial origin are translated on polysomes attached to the mitochondrion . The strong relationship with alpha-proteobacterial genes observed for some mitochondrial genes, combined with the lack of such a relationship for others, indicates that the modern mitochondrial proteome is the product of both reductive and expansive processes.

Philos Trans R Soc Lond B Biol Sci, 2003 Jan 29, 358(1429), 155 - 62; discussion 162-4
Parasite plastids: maintenance and functions; Wilson RJ et al.; Malaria and related parasites retain a vestigial, but biosynthetically active, plastid organelle acquired far back in evolution from a red algal cell . The organelle appears to be essential for parasite transmission from cell to cell and carries the smallest known plastid genome . Why has this genome been retained? The genes it carries seem to be dedicated to the expression of just two "housekeeping" genes . We speculate that one of these, called ycf24 in plants and sufB in bacteria, is tied to an essential "dark" reaction of the organelle--fatty acid biosynthesis . "Ball-park" clues to the function of bacterial suf genes have emerged only recently and point to the areas of iron homeostasis, {Fe-S} cluster formation and oxidative stress . We present experimental evidence for a physical interaction between SufB and its putative partner SufC (ycf16) . In both malaria and plants, SufC is encoded in the nucleus and specifies an ATPase that is imported into the plastid.

Philos Trans R Soc Lond B Biol Sci, 2003 Jan 29, 358(1429), 99 - 106; discussion 106-7
Evolution of the chloroplast genome; Howe CJ et al.; We discuss the suggestion that differences in the nucleotide composition between plastid and nuclear genomes may provide a selective advantage in the transposition of genes from plastid to nucleus . We show that in the adenine, thymine (AT)-rich genome of Borrelia burgdorferi several genes have an AT-content lower than the average for the genome as a whole . However, genes whose plant homologues have moved from plastid to nucleus are no less AT-rich than genes whose plant homologues have remained in the plastid, indicating that both classes of gene are able to support a high AT-content . We describe the anomalous organization of dinoflagellate plastid genes . These are located on small circles of 2-3 kbp, in contrast to the usual plastid genome organization of a single large circle of 100-200 kbp . Most circles contain a single gene . Some circles contain two genes and some contain none . Dinoflagellate plastids have retained far fewer genes than other plastids . We discuss a similarity between the dinoflagellate minicircles and the bacterial integron system.

Surg Infect (Larchmt), 2000 Autumn, 1(3), 239 - 48
Can one use biologic modifiers to prevent multiple organ dysfunction syndrome after abdominal infections?
Dellinger EP.
Postoperative or trauma-induced intra-abdominal infections can result in sepsis and multiple organ dysfunction syndrome (MODS) . Enteric bacteria and endotoxin released from the gut into the peritoneal cavity in response to injury can directly stimulate the inflammatory cascade responsible for the development of systemic inflammation and subsequent MODS . Therapeutic strategies, such as biologic modifiers that are aimed at blocking or enhancing specific mediators of the inflammatory response, have been developed and tested in animal models with varying efficacy in preventing mortality . Specific therapies that have shown beneficial effects in animal models have not proved successful in prospective, randomized human studies, and it is as yet unclear whether cytokine-based therapies will ultimately have a role in preventing MODS . Testing novel therapies in appropriate animal models that closely simulate human intra-abdominal infection is crucial in developing drugs that will be beneficial in preventing sepsis-induced mortality in critically ill patients.

Surg Infect (Larchmt), 2000 Autumn, 1(3), 217 - 25
Pathogenesis of multiple organ dysfunction syndrome: gut origin, protection, and decontamination; Rotstein OD; Clinical and experimental studies performed over the past several decades have implicated bacterial and endotoxin translocation (BET) from the gut to distant organs in the pathogenesis of multiple organ dysfunction syndrome (MODS) . Experimental studies in animals directed at maintaining the integrity of the intestinal mucosa have shown efficacy in preventing BET and the induction of distant inflammatory processes, suggesting that the egress of bacteria and their surface endotoxins might be pivotal in inducing MODS . However, clinical studies have failed to convincingly recapitulate these beneficial effects . Selective digestive decontamination, although it effectively decreases rates of respiratory infection, has failed to reduce MODS in critically ill patients and, except in certain patient subsets, has had no demonstrable effect on mortality . Nevertheless, the gut is an immunologically active organ that, irrespective of BET occurrence, appears to contribute significantly to the development of distant organ dysfunction following ischemia/reperfusion injury . Resuscitation strategies aimed at minimizing the inflammatory effects of gut-derived mediators, such as toxic oxygen species, appear promising in preventing the development of distant organ injury in the critically ill patient.

Surg Infect (Larchmt), 2001 Summer, 2(2), 163 - 8; discussion 168-70
Peritoneal host defenses: modulation by carbon dioxide insufflation; Rotstein OD; BACKGROUND: The host response to inflammatory stimuli in the peritoneal cavity consists of coordinated interactions of physical factors, cells, and soluble mediator molecules . Following infection, this is characterized by rapid eradication of large numbers of bacteria via the diaphragmatic lymphatics, clearance of bacteria through phagocytosis and intracellular killing, and, finally, sequestration of residual bacteria by inflammatory fibrinous exudate . Increasingly, surgical infections and major operations with the potential for postoperative infection are being managed laparoscopically, with CO(2) insufflation . The effects of CO(2) on intraperitoneal host defenses are still being defined . METHODS: Review of the pertinent literature and results from our laboratory . RESULTS: Exposure of peritoneal macrophages in vitro to CO(2) causes intracellular acidification, which in turn suppresses production of tumor necrosis factor (TNF) in response to exposure to lipopolysaccharide (LPS) . In comparison, neither room air nor helium had any effect on cytosolic pH or the peritoneal macrophage response to LPS . Insufflation of CO(2) into the preperitoneal space in vivo caused similar peritoneal lining acidosis and reduced TNF production compared with insufflation of helium . CONCLUSION: CO(2) pneumoperitoneum created during laparoscopy appears to exert immunosuppressive effects through its ability to acidify the intracellular compartment of inflammatory cells . This may contribute to the improved recovery in patients undergoing laparoscopic surgery.

Ann N Y Acad Sci, 2002 Dec, 980, 23 - 31
Achieving congruency of phylogenetic trees generated by W-curves of genomic sequences; Cork DJ et al.; Comparative genomic analysis at its most fundamental level involves alignment and analysis of linear strings of DNA . Many useful and powerful tools, such as BlastN and ClustalW are able to respectively, search for, and align similar strings of DNA from a variety of species . However, interesting genomic patterns cannot be immediately visualized within the information contact embedded in long genomic strings without extensive a priori knowledge . More problematic is the question of whether we will be able to crystallize long genomic sequences and analyze their true secondary and tertiary structures . It is, of course, these putative motifs that are binding to the three-dimensional structures of proteins and inducing replication and transcription events . The W-curve is a numerical mapping algorithm that allows one to geometrically visualize the information content of genomic motifs . Patterns of ALU, LINES, SINEs, and duplication sequences may be easily visualized with the W-curve . It is our hope that this pattern recognition algorithm will lead to visualization tools to track the evolutionary history of motif patterns . The combinatorics of DNA motif crossover-recombination events will be more easily followed as we continue to sequence more and more genomes . In our laboratory we are currently collaborating with mathematicians and computer scientists to develop and test tools, such as the W-curve, for analyzing patterns of long genomic sequences . In this paper, we examine the limitations of using the W-curve to infer the phylogenetic history of species.

FEMS Microbiol Lett, 2003 Feb 14, 219(1), 53 - 62
Effect of root exudates on the exopolysaccharide composition and the lipopolysaccharide profile of Azospirillum brasilense Cd under saline stress; Fischer SE et al.; The effect of wheat root exudates on the exopolysaccharide (EPS) composition and the lipopolysaccharide (LPS) profile of Azospirillum brasilense Cd under saline stress was studied . EPS of A . brasilense Cd was composed of glucose (47%), mannose (3%), xylose (4%), fucose (28%), rhamnose (6%), arabinose (1%) and galactose (11%) . Under saline stress, A . brasilense produced a totally different EPS, composed mainly of galactose . Root exudates induced changes in A . brasilense EPS composition only under normal conditions, consisting of higher amounts of arabinose and xylose compared with EPS of bacteria grown without root exudates . No changes were induced by root exudates when A . brasilense was grown under saline stress . Additionally, root exudates induced changes in the LPS profile, both under normal and stress conditions.

Biochem J, 2003 May 15, 372(Pt 1), 77 - 86
Mannose metabolism is required for mycobacterial growth; Patterson JH et al.; Mycobacteria are the causative agents of tuberculosis and several other significant diseases in humans . All species of mycobacteria synthesize abundant cell-wall mannolipids (phosphatidylinositol mannosides, lipoarabinomannan), a cytoplasmic methylmannose polysaccharide and O-mannosylated glycoproteins . To investigate whether these molecules are essential for mycobacterial growth, we have generated a Mycobacterium smegmatis mannose auxotroph by targeted deletion of the gene encoding phosphomannose isomerase (PMI) . The PMI deletion mutant displayed a mild hyperseptation phenotype, but grew normally in media containing an exogenous source of mannose . When this mutant was suspended in media without mannose, ongoing synthesis of both the mannolipids and methylmannose polysaccharides was halted and the hyperseptation phenotype became more pronounced . These changes preceded a dramatic loss of viability after 10 h in mannose-free media . Mannose starvation did not lead to detectable changes in cell-wall ultrastructure or permeability to hydrophobic drugs, or to changes in the rate of biosynthesis of other plasma-membrane or wall-associated phospholipids . These results show that mannose metabolism is required for growth of M . smegmatis and that one or more mannose-containing molecules may play a role in regulating septation and cell division in these bacteria.

J Periodontol, 2003 Jan, 74(1), 38 - 43
Human monoclonal antibody inhibits Porphyromonas gingivalis hemagglutinin activity; Kaizuka K et al.; BACKGROUND: Porphyromonas gingivalis has been implicated as an important pathogen in the development of chronic periodontitis, and its colonization of subgingival sites is critical in the pathogenic process . One potential virulence factor, hemagglutinin, may mediate bacteria attachment onto and penetration into host cells, as well as agglutinate and lyse erythrocytes to intake heme, an absolute requirement for growth . We previously cloned the gene encoding the 130 kDa hemagglutinin domain (130k HMGD) and identified its functional domain . The construction of a human monoclonal antibody that is capable of inhibiting the hemagglutinating ability is significant and important toward the development of passive immunotherapy . METHODS: Human lymphocytes isolated from a donor, who had high antibody titer against the recombinant 130k HMGD (r130k HMGD), were immortalized by Epstein-Barr virus, and specific antibody-producing B cells were established by panning using the r130k HMGD . RESULTS: The constructed HuMAb-HMGD1, IgG subclass, recognized the r130k HMGD as well as the 43 and 49 kDa major bands in P . gingivalis cells and vesicles . The HuMAb-HMGD1 significantly inhibited hemagglutinating activity of P . gingivalis vesicles in a dose-dependent manner . Furthermore, the HuMAb-HMGD1 recognized the synthetic peptide, EGSNEFAPVQNLTGSSVG, which contains the functional domain of 130k HMGD . CONCLUSION: The newly constructed HuMAb-HMGD1 may prove to be useful for the development of passive immunization against periodontal diseases caused by P . gingivalis infection, pending the results of fertility study in disease mode.

Lijec Vjesn, 2002 Sep, 124 Suppl 1, 33 - 6
{The rapid urease test}; Filipec T et al.; Today there are many methods in diagnostics of Helicobacter pylori infection . They are divided in two major groups based on using of endoscopy (invasive and non-invasive methods) . Helicobacter pylori bacteria are specific because of having very big amounts of urease enzyme that divides urea on NH3 and CO2 which enables environment suitable for survival . Rapid ureas test is based on detecting of the enzyme activity . Because of its simplicity and high sensitivity and specificity it belongs to methods that are used in every day practice in endoscopic laboratories.

Lijec Vjesn, 2002 Sep, 124 Suppl 1, 28 - 33
{The urea breath test}; Filipec T et al.; Helicobacter pylori is infective cause of peptic ulcer and a risk factor for gastric carcinoma . The discovery of the bacterial ed to importance of finding a new reliable and inexpensive diagnostic method for detection of infection before and after eradication therapy . Urea breath test is isotope based test . It has become the most specific and sensitive method in detection of Helicobacter pylori infection, therefore many other isotope based tests become popular in diagnostic of gastrointestinal diseases . Because of its simplicity and no need in using endoscopy in the procedure it is very popular in primary diagnosis and in controlling eradication . It is successfully used in diagnostics of Helicobacter pylori and bacteria eradication success testing, where it is one of most reliable diagnostic methods.

Lijec Vjesn, 2002 Sep, 124 Suppl 1, 5 - 9
{Epidemiology of Helicobacter pylori infection}; Strnad M et al.; About 50% of adults in the developed and 80-90% in the developing countries are estimated to be infected by Helicobacter pylori . Being 68% nationally, this rate is higher in the northern continental parts of Croatia, which also have higher gastric cancer rates . Low socio-economic status, poor living conditions in childhood (the age when Helicobacter pylori is typically acquired), and exposure to the stomach content of an infected person are risk factors for Helicobacter pylori . Most of the infected are symptomless, with 10 to 20% subsequently developing the disease, and this mainly from peptic ulcer, asymptomatic chronic gastritis and chronic dyspepsia . Less than 5/10,000 become affected with adenocarcinoma, MALT lymphoma or primary non-Hodgkin's gastric lymphoma . Helicobacter pylori is under intensive study for possible association with other diseases . As transmission route of the infection is still unclear, any mechanism allowing the bacteria entry into a non-infected individual's stomach is probably a possibility . In addition to improved socio-economic status, eradication or vaccination may be contributors to the reduction in the number of the infected.

J Allergy Clin Immunol, 2003 Feb, 111(2 Suppl), S677 - 86
22 . Immune responses to malignancies; Whiteside TL; Immune responses to tumor-associated antigens exist in tumor-bearing hosts but are usually not successful in eliminating malignant cells or preventing the development of metastases . Patients with cancer generate robust immune responses to infectious agents (bacteria and viruses) perceived as a "danger signal" but only ineffective, weak responses to tumor-associated antigens, which are considered as "self." This fundamental difference in responses to self versus non-self is further magnified by the ability of tumors to subvert the host immune system . Tumors induce dysfunction, as well as apoptosis in CD8(+) antitumor effector cells . The escape of tumors from immune cells is mediated by several distinct molecular mechanisms . Insights into these mechanisms and more effective control of tumor-orchestrated immune dysfunction are needed . Novel strategies for immunotherapy of cancer must address protection and survival of antitumor effector cells in the tumor microenvironment.

J Allergy Clin Immunol, 2003 Feb, 111(2 Suppl), S476 - 85
3 . Lymphocytes; Alam R et al.; The fundamental task of the immune system is to defend "self" from "nonself." Lymphocytes are the primary cells of the immune system that developed one of the most sophisticated and comprehensive defense mechanisms in the biological system . T cells play a central role in orchestrating the immune response . Further, they are instrumental in eliminating intracellular pathogens (viruses, some bacteria) through the generation of cytotoxic T cells . B cells defend against extracellular pathogens by producing antibodies . Natural killer cells are an important component of innate immunity . Dendritic cells play a key role in initiating the immune response by presenting foreign antigens to T cells . The interaction among T cells, B cells, dendritic cells, and natural killer cells constitute the fundamental defense network of the host . The failure of any of these components severely jeopardizes the integrity of the immune system and its ability to mount the most appropriate immune response.

J Exp Med, 2003 Feb 17, 197(4), 403 - 11
Regulatory T cells selectively express toll-like receptors and are activated by lipopolysaccharide; Caramalho I et al.; Regulatory CD4 T cells (Treg) control inflammatory reactions to commensal bacteria and opportunist pathogens . Activation of Treg functions during these processes might be mediated by host-derived proinflammatory molecules or directly by bacterial products . We tested the hypothesis that engagement of germline-encoded receptors expressed by Treg participate in the triggering of their function . We report that the subset of CD4 cells known to exert regulatory functions in vivo (CD45RB(low) CD25(+)) selectively express Toll-like receptors (TLR)-4, -5, -7, and -8 . Exposure of CD4(+) CD25(+) cells to the TLR-4 ligand lipopolysaccharide (LPS) induces up-regulation of several activation markers and enhances their survival/proliferation . This proliferative response does not require antigen-presenting cells and is augmented by T cell receptor triggering and interleukin 2 stimulation . Most importantly, LPS treatment increases CD4(+) CD25(+) cell suppressor efficiency by 10-fold and reveals suppressive activity in the CD4(+) CD45RB(low) CD25(-) subset that when tested ex-vivo, scores negative . Moreover, LPS-activated Treg efficiently control naive CD4 T cell-dependent wasting disease . These findings provide the first evidence that Treg respond directly to proinflammatory bacterial products, a mechanism that likely contributes to the control of inflammatory responses.

J Bacteriol, 2003 Mar, 185(5), 1712 - 8
Structural model for 12-helix transporters belonging to the major facilitator superfamily; Hirai T et al.; The major facilitator superfamily includes a large collection of evolutionarily related proteins that have been implicated in the transport of a variety of solutes and metabolites across the membranes of organisms ranging from bacteria to humans . We have recently reported the three-dimensional structure, at 6.5 A resolution, of the oxalate transporter, OxlT, a representative member of this superfamily . In the oxalate-bound state, 12 helices surround a central cavity to form a remarkably symmetrical structure that displays a well-defined pseudo twofold axis perpendicular to the plane of the membrane as well as two less pronounced, mutually perpendicular pseudo twofold axes in the plane of the membrane . Here, we combined this structural information with sequence information from other members of this protein family to arrive at models for the arrangement of helices in this superfamily of transport proteins . Our analysis narrows down the number of helix arrangements from about a billion starting possibilities to a single probable model for the relative spatial arrangement for the 12 helices, consistent both with our structural findings and with the majority of previous biochemical studies on members of this superfamily.

J Bacteriol, 2003 Mar, 185(5), 1624 - 33
Role of the cytoplasmic C terminus of the FliF motor protein in flagellar assembly and rotation; Grunenfelder B et al.; Twenty-six FliF monomers assemble into the MS ring, a central motor component of the bacterial flagellum that anchors the structure in the inner membrane . Approximately 100 amino acids at the C terminus of FliF are exposed to the cytoplasm and, through the interaction with the FliG switch protein, a component of the flagellar C ring, are essential for the assembly of the motor . In this study, we have dissected the entire cytoplasmic C terminus of the Caulobacter crescentus FliF protein by high-resolution mutational analysis and studied the mutant forms with regard to the assembly, checkpoint control, and function of the flagellum . Only nine amino acids at the very C terminus of FliF are essential for flagellar assembly . Deletion or substitution of about 10 amino acids preceding the very C terminus of FliF resulted in assembly-competent but nonfunctional flagella, making these the first fliF mutations described so far with a Fla(+) but Mot(-) phenotype . Removal of about 20 amino acids further upstream resulted in functional flagella, but cells carrying these mutations were not able to spread efficiently on semisolid agar plates . At least 61 amino acids located between the functionally relevant C terminus and the second membrane-spanning domain of FliF were not required for flagellar assembly and performance . A strict correlation was found between the ability of FliF mutant versions to assemble into a flagellum, flagellar class III gene expression, and a block in cell division . Motile suppressors could be isolated for nonmotile mutants but not for mutants lacking a flagellum . Several of these suppressor mutations were localized to the 5' region of the fliG gene . These results provide genetic support for a model in which only a short stretch of amino acids at the immediate C terminus of FliF is required for flagellar assembly through stable interaction with the FliG switch protein.

J Bacteriol, 2003 Mar, 185(5), 1543 - 54
A two-component regulator of universal stress protein expression and adaptation to oxygen starvation in Mycobacterium smegmatis; O'Toole R et al.; We identified a response regulator in Mycobacterium smegmatis which plays an important role in adaptation to oxygen-starved stationary phase . The regulator exhibits strong sequence similarity to DevR/Rv3133c of M . tuberculosis . The structural gene is present on a multigene locus, which also encodes a sensor kinase . A devR mutant of M . smegmatis was adept at surviving growth arrest initiated by either carbon or nitrogen starvation . However, its culturability decreased several orders of magnitude below that of the wild type under oxygen-starved stationary-phase conditions . Two-dimensional gel analysis revealed that a number of oxygen starvation-inducible proteins were not expressed in the devR mutant . Three of these proteins are universal stress proteins, one of which is encoded directly upstream of devR . Another protein closely resembles a proposed nitroreductase, while a fifth protein corresponds to the alpha-crystallin (HspX) orthologue of M . smegmatis . None of the three universal stress proteins or nitroreductase, and a considerably lower amount of HspX was detected in carbon-starved wild-type cultures . A fusion of the hspX promoter to gfp demonstrated that DevR directs gene expression when M . smegmatis enters stationary phase brought about, in particular, by oxygen starvation . To our knowledge, this is the first time a role for a two-component response regulator in the control of universal stress protein expression has been shown . Notably, the devR mutant was 10(4)-fold more sensitive than wild type to heat stress . We conclude that DevR is a stationary-phase regulator required for adaptation to oxygen starvation and resistance to heat stress in M . smegmatis.

Math Biosci, 2003 Apr, 182(2), 151 - 66
Multiple limit cycles in the chemostat with variable yield; Pilyugin SS et al.; The global asymptotic behavior of solutions of the variable yield model is determined . The model generalizes the classical Monod model and it assumes that the yield is an increasing function of the nutrient concentration . In contrast to the Monod model, it is demonstrated that the variable yield model exhibits sustained oscillations . Moreover, it is shown that the variable yield model may undergo a subcritical Hopf bifurcation and feature at least two distinct limit cycles . Implications for the coexistence of competing populations are discussed.

Biochemistry, 2003 Feb 25, 42(7), 2062 - 71
Dynamics of protein and chromophore structural changes in the photocycle of photoactive yellow protein monitored by time-resolved optical rotatory dispersion; Chen E et al.; The dynamics of the PYP photocycle have been studied using time-resolved optical rotatory dispersion (TRORD) spectroscopy in the visible and far-UV spectral regions to probe the changes in the chromophore configuration and the protein secondary structure, respectively . The changes in the secondary structure in PYP upon photoisomerization of the chromophore can be described by two exponential lifetimes of 2 +/- 0.8 and 650 +/- 100 ms that correspond to unfolding and refolding processes, respectively . The TRORD experiments that follow the dynamics of the chromophore report three exponential components, with lifetimes of 10 +/- 3 micros, 1.5 +/- 0.5 ms, and 515 +/- 110 ms . A comparison of the kinetic behaviors of the chromophore and protein shows that during the decay of pR(465) an initial relaxation that is localized to the chromophore hydrophobic pocket precedes the formation of the chromophore and protein structures found in pB(355) . In contrast, the protein and chromophore processes occur with similar time constants during inactivation of the signaling state.

Biochem Biophys Res Commun, 2003 Feb 21, 301(4), 899 - 906
Differential control of the NIMA-related kinases, Nek6 and Nek7, by serum stimulation; Minoguchi S et al.; Neks (NIMA-related kinases) are mammalian serine/threonine (Ser/Thr) protein kinases structurally related to Aspergillus NIMA (Never in Mitosis, gene A), which plays essential roles in mitotic signaling . Among these kinases, Nek6 and Nek7 are structurally related and constitute a subfamily in the NIMA/Nek family, although their functions still remain almost elusive . In this report, we studied the enzymatic regulation of Nek6 and Nek7 to gain an insight into their cellular functions . Recombinant Nek7 produced in bacteria was active comparably to Nek6; however, the Nek7 activity in mammalian cells was found to be significantly lower than Nek6 . Since Nek6 previously has been reported to in vitro phosphorylate p70 ribosomal S6 kinase at Thr412, we examined if Nek6 and Nek7 activities were controlled by the amino acid supplement, which is known to affect the phosphorylation at Thr412, and did not observe any significant effect . However, we unexpectedly found that Nek7 kinase activity was rapidly and efficiently increased by serum deprivation, while Nek6 activity was decreased . This is well consistent with the lower activity of Nek7 in cells under normal growth conditions . In addition, it was suggested that Nek7 activity would be regulated in a cell cycle-dependent manner, although Nek6 was not . These clear differences in enzymatic control between the highly similar kinases, Nek6 and Nek7, suggest their distinct signaling functions in mammalian cells.

Ann Pharm Fr, 2003 Jan, 61(1), 30 - 50
Peripheral benzodiazepine receptor: structure and function in health and disease; Papadopoulo V; In vitro studies using biochemical, pharmacological and molecular approaches demonstrated that the peripheral-type benzodiazepine receptor (PBR) is a mitochondrial protein, involved in the regulation of cholesterol transport from the outer to the inner mitochondrial membrane, the rate-determining step in steroid biosynthesis . In vivo animal models and ontogeny studies validated the role of PBR in steroidogenesis . Targeted disruption of the PBR gene in Leydig cells resulted in the arrest of cholesterol transport into mitochondria and steroid formation . Molecular modeling of PBR suggested that it might function as a channel for cholesterol . Indeed, cholesterol uptake and transport by bacteria cells was induced upon PBR expression . Amino acid deletion and site-directed mutagenesis studies identified a cholesterol recognition/interaction amino acid consensus sequence in the cytoplasmic carboxy-terminus of the receptor . In vitro reconstitution experiments demonstrated that the 18 kDa PBR protein binds with high affinity both drug ligands and cholesterol, suggesting that this protein might serve numerous functions considering the critical role of cholesterol in membrane biogenesis and human pathology . In this context, PBR expression correlated with the quality of kidney preservation, indicating that it might serve as an index of kidney and mitochondrial viability during ischemia-reperfusion injury . PBR overexpression was also found to be a prognostic indicator of the aggressive phenotype in breast, colorectal and prostate cancers . Moreover, in Alzheimer's disease brain specimens, PBR levels were increased and paralleled the elevated neurosteroid synthesis observed in specific brain areas . The role for PBR in these pathological conditions remains to be elucidated . paralleled the elevated neurosteroid synthesis observed in specific brain areas . The role for PBR in these pathological conditions remains to be elucidated.

Mol Biol Cell, 2003 Feb, 14(2), 396 - 406
Actin filament polymerization regulates gliding motility by apicomplexan parasites; Wetzel DM et al.; Host cell entry by Toxoplasma gondii depends critically on actin filaments in the parasite, yet paradoxically, its actin is almost exclusively monomeric . In contrast to the absence of stable filaments in conventional samples, rapid-freeze electron microscopy revealed that actin filaments were formed beneath the plasma membrane of gliding parasites . To investigate the role of actin filaments in motility, we treated parasites with the filament-stabilizing drug jasplakinolide (JAS) and monitored the distribution of actin in live and fixed cells using yellow fluorescent protein (YFP)-actin . JAS treatment caused YFP-actin to redistribute to the apical and posterior ends, where filaments formed a spiral pattern subtending the plasma membrane . Although previous studies have suggested that JAS induces rigor, videomicroscopy demonstrated that JAS treatment increased the rate of parasite gliding by approximately threefold, indicating that filaments are rate limiting for motility . However, JAS also frequently reversed the normal direction of motility, disrupting forward migration and cell entry . Consistent with this alteration, subcortical filaments in JAS-treated parasites occurred in tangled plaques as opposed to the straight, roughly parallel orientation observed in control cells . These studies reveal that precisely controlled polymerization of actin filaments imparts the correct timing, duration, and directionality of gliding motility in the Apicomplexa.

Mol Cell Biol, 2003 Mar, 23(5), 1623 - 32
Collaborative competition mechanism for gene activation in vivo; Miller JA et al.; The mechanism by which gene regulatory proteins gain access to their DNA target sites is not known . In vitro, binding is inherently cooperative between arbitrary DNA binding proteins whose target sites are located within the same nucleosome . We refer to such competition-based cooperativity as collaborative competition . Here we show that arbitrarily chosen foreign DNA binding proteins, LexA and Tet repressor, cooperate with an adjacently binding endogenous activator protein, Gcn4, to coactivate expression of chromosomal reporter genes in Saccharomyces cerevisiae . Coactivation requires that the cooperating target sites be within a nucleosome-length distance; it leads to increased occupancy by Gcn4 at its binding site; and it requires both Gcn5 and Swi/Snf which, at an endogenous Gcn4-dependent promoter, act subsequent to Gcn4 binding . These results imply that collaborative competition contributes to gene regulation in vivo . They further imply that, even in the presence of the cell's full wild-type complement of chromatin remodeling factors, competition of regulatory proteins with histone octamer for access to regulatory target sites remains a quantitative determinant of gene expression levels . We speculate that initial target site recognition and binding may occur via spontaneous nucleosomal site exposure, with remodeling factor action required downstream to lock in higher levels of regulatory protein occupancy.

Pathol Int, 2002 Dec, 52(12), 755 - 63
Chlamydia pneumoniae and multiple infections in the aorta contribute to atherosclerosis; Shi Y et al.; Our previous study on herpesvirus infection including Herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV), cytomegalovirus (CMV) and atherosclerosis revealed that the prevalence of herpesvirus is higher in atherosclerotic aorta than in non-atherosclerotic aorta . Infections with two or three forms of the virus have been found only in atherosclerotic aorta . In our current study, we examined both Chlamydia pneumoniae and Chlamydia trachomatis in herpesvirus-infected aortic tissues, by means of immunohistochemistry, polymerase chain reaction, Southern hybridization, in situ hybridization, electron microscopy and electron-microscopic immunohistochemistry . In particular, the bacteria were found in atherosclerotic lesions . In atherosclerotic aorta, 40% of tissues examined were positive for C . pneumoniae in contrast to absence of this bacteria in non-atherosclerotic aorta . Elementary bodies of C . pneumoniae were found in macrophage-like cells in the intima of atherosclerotic aorta by electron microscopy . Chlamydia trachomatis was not found in both atherosclerotic and non-atherosclerotic aorta . Our findings suggest that multiple infections in aortic tissue contribute to the development of atherosclerosis . Furthermore, the absence of C . pneumoniae compared to herpesviruses in normal arterial tissue suggests that C . pneumoniae is specific for atherosclerotic lesions . In contrast to 'abortive infection' of viruses in arteries, C . pneumoniae infection was demonstrated in macrophages by electron microscopy and electron-microscopic immunohistochemistry in atherosclerotic lesion.Chlamydia pneumoniae may be the most important pathogen related to the development of atherosclerosis.

Biologist (London), 2003 Feb, 50(1), 15 - 9
Sinking methane; Reay DS; Concentrations of the powerful greenhouse gas, methane, in our atmosphere have doubled since the beginning of the industrial age . Reducing these levels is a vital part of global efforts to combat global warming . Could we make use of the Earth's own methane sinks?

Genetics, 2003 Jan, 163(1), 203 - 15
Analysis of Ras-induced overproliferation in Drosophila hemocytes; Asha H et al.; We use the Drosophila melanogaster larval hematopoietic system as an in vivo model for the genetic and functional genomic analysis of oncogenic cell overproliferation . Ras regulates cell proliferation and differentiation in multicellular eukaryotes . To further elucidate the role of activated Ras in cell overproliferation, we generated a collagen promoter-Gal4 strain to overexpress Ras(V12) in Drosophila hemocytes . Activated Ras causes a dramatic increase in the number of circulating larval hemocytes (blood cells), which is caused by cellular overproliferation . This phenotype is mediated by the Raf/MAPK pathway . The mutant hemocytes retain the ability to phagocytose bacteria as well as to differentiate into lamellocytes . Microarray analysis of hemocytes overexpressing Ras(V12) vs . Ras(+) identified 279 transcripts that are differentially expressed threefold or more in hemocytes expressing activated Ras . This work demonstrates that it will be feasible to combine genetic and functional genomic approaches in the Drosophila hematopoietic system to systematically identify oncogene-specific downstream targets.

Vet Immunol Immunopathol, 2003 Feb 10, 91(3-4), 183 - 97
Characterisation of porcine monocyte-derived dendritic cells according to their cytokine profile; Johansson E et al.; The influence of interferon (IFN)-alpha on the in vitro differentiation of myeloid porcine dendritic cells (DC) was evaluated as the ability of the DC to stimulate to cell proliferation in a mixed leukocyte reaction (MLR), and as their ability to produce cytokines at exposure to bacterial and viral preparations . Porcine monocytes were enriched from purified peripheral blood mononuclear cells (PBMC) by plastic adherence and cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 or in GM-CSF, IL-4 and IFN-alpha . After 5 days of culture, the cells developed a dendritic morphology and the proportion of cells expressing MHC class II and B7 molecules was increased as determined by flow cytometry . Dendritic cells, differentiated for 5 days in GM-CSF, IL-4 and IFN-alpha, were able to stimulate both allogeneic and syngeneic PBMC to proliferation in an MLR . The DC produced the Th1 associated cytokines IFN-alpha at Sendai virus stimulation, and IL-12 at stimulation with plasmid DNA (pre-incubated in the presence of lipofectin), heat-inactivated Actinobacillus pleuropneumoniae, UV-inactivated Aujeszky's disease virus and live Sendai virus . The heat-inactivated bacteria and Sendai virus also induced production of the Th2 associated cytokines IL-10 and IL-6 . The addition of IFN-alpha during differentiation of DC in GM-CSF and IL-4 enhanced their ability to stimulate allogeneic and syngeneic MLR, but did not alter their ability to produce cytokines .

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