Microorganisms

J Gen Virol, 2001 Jul, 82(Pt 7), 1581 - 7
Epstein-Barr virus nuclear antigen 5 interacts with HAX-1, a possible component of the B-cell receptor signalling pathway; Dufva M et al.; Using a yeast two-hybrid screen of a B-cell cDNA library with an Epstein-Barr nuclear antigen 5 (EBNA5) molecule containing seven repeats of the W(1)W(2) domain as bait, we have isolated the EBNA5-interacting protein HAX-1 . HAX-1 has previously been shown to associate with HS1, a protein specifically expressed in cells of the haematopoietic lineage, and is thought to be involved in signal transduction in B-cells . Immunofluorescence experiments showed that HAX-1 co-localized with the hsp60 protein that is associated with the mitochondria in the cell cytoplasm . Pull down experiments with a fusion protein between glutathione S-transferase and the seven copy repeat EBNA5 synthesized in bacteria and in yeast cells confirmed that HAX-1 can interact with EBNA5 in vitro . Conventionally, EBNA5 is regarded as a nuclear protein . However, we show here that the smallest EBNA5 species, composed of the unique Y domain and only one copy of the W(1)W(2) repeat domain, like HAX-1, co-localizes with the mitochondrial hsp60 protein in the B-cell cytoplasm . Furthermore, immunoprecipitation experiments demonstrate that the single repeat EBNA5 associates with HAX-1 in transfected B-lymphoblastoid cells.

J Exp Bot, 2001 Apr, 52(357), 747 - 60
Further observations on the interaction between sugar cane and Gluconacetobacter diazotrophicus under laboratory and greenhouse conditions; James EK et al.; Sugar cane (Saccharum spp.) variety SP 70-1143 was inoculated with Gluconacetobacter diazotrophicus strain PAL5 (ATCC 49037) in two experiments . In experiment 1 the bacteria were inoculated into a modified, low sucrose MS medium within which micropropagated plantlets were rooted . After 10 d there was extensive anatomical evidence of endophytic colonization by G . diazotrophicus, particularly in lower stems, where high numbers of bacteria were visible within some of the xylem vessels . The identity of the bacteria was confirmed by immunogold labelling with an antibody raised against G . diazotrophicus . On the lower stems there were breaks caused by the separation of the plantlets into individuals, and at these 'wounds' bacteria were seen colonizing the xylem and intercellular spaces . Bacteria were also occasionally seen entering leaves via damaged stomata, and subsequently colonizing sub-stomatal cavities and intercellular spaces . A localized host defence response in the form of fibrillar material surrounding the bacteria was associated with both the stem and leaf invasion . In experiment 2, stems of 5-week-old greenhouse-grown plants were inoculated by injection with a suspension of G . diazotrophicus containing 10(8) bacteria ml(-1) . No hypersensitive response (HR) was observed, and no symptoms were visible on the leaves and stems for the duration of the experiment (7 d) . Close to the point of inoculation, G . diazotrophicus cells were observed within the protoxylem and the xylem parenchyma, where they were surrounded by fibrillar material that stained light-green with toluidine blue . In leaf samples taken up to 4 cm from the inoculation points, G . diazotrophicus cells were mainly found within the metaxylem, where they were surrounded by a light green-staining material . The bacteria were growing in relatively low numbers adjacent to the xylem cell walls, and they were separated from the host-derived material by electron-transparent 'haloes' that contained material that reacted with the G . diazotrophicus antibody.

Gut, 2001 Jul, 49(1), 18 - 22
Helicobacter pylori induced transactivation of SRE and AP-1 through the ERK signalling pathway in gastric cancer cells; Mitsuno Y et al.; BACKGROUND AND AIMS: Helicobacter pylori infection induces expression of proinflammatory cytokines such as interleukin (IL)-8 and tumour necrosis factor alpha (TNF-alpha) in gastric mucosa, and their genes have AP-1 binding sites in the promoter region . c-Fos is important for transactivation of AP-1 which has SRE in the promoter region . We conducted this study to confirm H pylori induced transactivation of these binding sites . METHODS: Transactivation of SRE and AP-1 was evaluated in human gastric cancer cells TMK1 and MKN45 by luciferase reporter assay in transient transfection . We compared the effects of coculture with four H pylori strains, a cag pathogenicity island (PAI) positive strain TN2, its isogenic vacA negative (TN2-DeltavacA) or cagE negative (TN2-DeltacagE) mutants, and a cag PAI negative clinical isolate T68 . Phosphorylation of ERK1/2, JNK, and c-Jun was measured by immunoblot, induction of IL-8 secretion by ELISA, and the effects of MEK by inhibitor U0126 . RESULTS: Both SRE and AP-1 were transactivated by coculture with TN2 . Although TN2-DeltavacA induced comparable transactivation, TN2-DeltacagE and T68 showed decreased transactivation of SRE (65% and 51%) and AP-1 (71% and 54%, respectively, of TN2) . Heat killed TN2 or indirect contact using a permeable membrane inhibited transactivation . Levels of phosphorylated ERK1/2, JNK, and c-Jun were increased by coculture with TN2 . MEK inhibitor U0126 reduced TN2 induced transactivation of SRE and AP1, as well as secretion of IL-8, by 83%, 87%, and 53%, respectively, of TN2 . CONCLUSIONS: Transactivation of SRE and AP-1, through ERK/MAPK and JNK/SAPK cascades, respectively, was found in gastric cancer cells cocultured with H pylori . Direct contact with viable bacteria possessing intact cag PAI is a prerequisite for the onset of intracellular signalling leading to AP-1 transactivation.

Curr Biol, 2001 Apr 3, 11(7), R278 - 80
Recombination: homologous recombination branches out; Hiom K; Homologous recombination can be divided into three key steps: strand exchange, branch migration and resolution . The identification of a protein complex that catalyses branch migration and Holliday junction resolution argues that the mechanism of homologous recombination is conserved from bacteria to man.

FEBS Lett, 2001 Jun 8, 498(2-3), 168 - 71
Rho proteins, PI 3-kinases, and monocyte/macrophage motility; Ridley AJ; Rho proteins and phosphatidylinositide 3-kinases (PI 3-kinases) have been widely implicated in regulating cell motility both in cultured cells and in animal models . Monocytes are recruited from the bloodstream in response to inflammatory signals, and migrate across the endothelial barrier into the tissues, where they differentiate into macrophages and phagocytose bacteria and cells . Studies of monocytes and macrophages have revealed that different Rho family members and PI 3-kinases are not functionally redundant but play unique and distinct roles in motile responses.

FEBS Lett, 2001 Jun 8, 498(2-3), 135 - 9
Mutualists and parasites: how to paint yourself into a (metabolic) corner; Tamas I et al.; Eukaryotes have developed an elaborate series of interactions with bacteria that enter their bodies and/or cells . Genome evolution of symbiotic and parasitic bacteria multiplying inside eukaryotic cells results in both convergent and divergent changes . The genome sequences of the symbiotic bacteria of aphids, Buchnera aphidicola, and the parasitic bacteria of body louse and humans, Rickettsia prowazekii, provide insights into these processes . Convergent genome characteristics include reduction in genome sizes and lowered G+C content values . Divergent evolution was recorded for amino acid and cell wall biosynthetic genes . The presence of pseudogenes in both genomes provides examples of recent gene inactivation events and offers clues to the process of genome deterioration and host-cell adaptation.

Clin Chim Acta, 2001 Jun, 308(1-2), 179 - 81
Simultaneous amplification of DNA and RNA virus using multiplex PCR system; Singh VK et al.; A large number of disease-causing bacteria and viruses are being sequenced and PCR is increasingly used for the diagnosis of the diseases . We have designed a multiplex PCR system for hepatitis B virus (HBV), a DNA virus, and hepatitis E virus (HEV), an RNA virus . A modified technique has been standardized for simultaneous extraction of DNA and RNA, followed by a one-step RT-PCR/PCR.

J Environ Sci Health B, 2001 May, 36(3), 355 - 64
Methodology for management of endosulfan contaminated eluent; Sudhakar Y et al.; Management of Endosulfan contaminated eluent (24 mg/l) resulting from a treatment process to remove Endosulfan from water with wood charcoal, was attempted using various methods viz . volatilisation, hydrolysis and sorption by viable cell bacteria with and without acclimatisation . Volatilisation failed in giving better result, as Endosulfan was not considerably volatile . It could achieve a removal efficiency of 1.4-2% . Hydrolysis resulted in 28.4% and 17.9% removal of Endosulfan in acidic and alkaline media, respectively . Viable cell bacteria (aerobic) without prior acclimatization showed efficiency of 89.7% and after prior acclimatisation showed 96% removal efficiency . Sorption by the acclimatized biomass was found a suitable method for the removal of Endosulfan at a concentration of 24 mg/l.

Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 891 - 9
Proposal to transfer some members of the genera Haemobartonella and Eperythrozoon to the genus Mycoplasma with descriptions of 'Candidatus Mycoplasma haemofelis', 'Candidatus Mycoplasma haemomuris', 'Candidatus Mycoplasma haemosuis' and 'Candidatus Mycoplasma wenyonii'; Neimark H et al.; Cell-wall-less uncultivated parasitic bacteria that attach to the surface of host erythrocytes currently are classified in the order Rickettsiales, family Anaplasmataceae, in the genera Haemobartonella and Eperythrozoon . Recently 16S rRNA gene sequences have been determined for four of these species: Haemobartonella felis and Haemobartonella muris and Eperythrozoon suis and Eperythrozoon wenyonii . Phylogenetic analysis of these sequence data shows that these haemotrophic bacteria are closely related to species in the genus Mycoplasma (class Mollicutes) . These haemotrophic bacteria form a new phylogenetic cluster within the so-called pneumoniae group of Mycoplasma and share properties with one another as well as with other members of the pneumoniae group . These studies clearly indicate that the classification of these taxa should be changed to reflect their phylogenetic affiliation and the following is proposed: (i) that Haemobartonella felis and Haemobartonella muris should be transferred to the genus Mycoplasma as 'Candidatus Mycoplasma haemofelis' and 'Candidatus Mycoplasma haemomuris' and (ii) that Eperythrozoon suis and Eperythrozoon wenyonii should be transferred to the genus Mycoplasma as 'Candidatus Mycoplasma haemosuis' and 'Candidatus Mycoplasma wenyonii' . The former Haemobartonella and Eperythrozoon species described here represent a new group of parasitic mycoplasmas that possess a pathogenic capacity previously unrecognized among the mollicutes . These haemotrophic mycoplasmas have been given the trivial name haemoplasmas . These results call into question the affiliation of the remaining officially named species of Haemobartonella and Eperythrozoon which should be considered species of uncertain affiliation pending the resolution of their phylogenetic status.

Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 815 - 7
'Candidatus Mycoplasma haemominutum', a low-virulence epierythrocytic parasite of cats; Foley JE et al.; The phylogenetic position and some taxonomically relevant characteristics of a small, low-virulence bacterial parasite of cats are described . A 16S rDNA analysis revealed that the organism was in the Mycoplasma clade and was most closely related to a parasite of pigs previously designated Eperythrozoon suis . As the organism has not been cultured in vitro and is maintained in serial passage in cats in vivo, Candidatus status is proposed for this novel taxon as 'Candidatus Mycoplasma haemominutum'.

J Biomed Mater Res, 2001, 58(4), 427 - 35
Physicochemical, mechanical, and biological properties of commercial membranes for GTR; Milella E et al.; Barrier membranes for guided tissue regeneration (GTR) to treat bone defects have to satisfy criteria of biocompatibility, cell-occlusiveness, spacemaking, tissue integration, and clinical manageability . In this study, the morphological and mechanical properties of two commercial biodegradable membranes (Resolut LT and Biofix) as a function of the incubation time have been compared . Moreover, their permeability to both fluids and epithelial cells as well as the bacteria adhesion have been evaluated . The membranes are asymmetric and composed of a dense polymeric layer coupled with nonwoven (Resolut LT) or woven (Biofix) fibers . Both of the membranes, when incubated in complete culture medium, completely lose the structural and mechanical properties within 30 days . Moreover the results of solute permeability show that Resolut LT and Biofix membranes cannot be considered selective membranes to the solute crossing . On the contrary, they act as a barrier to the passage of the gingivial cells and to S . mutans bacteria .

Nucleic Acids Res, 2001 Jun 15, 29(12), 2654 - 60
Massive parallel analysis of the binding specificity of histone-like protein HU to single- and double-stranded DNA with generic oligodeoxyribonucleotide microchips; Krylov AS et al.; A generic hexadeoxyribonucleotide microchip has been applied to test the DNA-binding properties of HU histone-like bacterial protein, which is known to have a low sequence specificity . All 4096 hexamers flanked within 8mers by degenerate bases at both the 3'- and 5'-ends were immobilized within the 100 x 100 x 20 mm polyacrylamide gel pads of the microchip . Single-stranded immobilized oligonucleotides were converted in some experiments to the double-stranded form by hybridization with a specified mixture of 8mers . The DNA interaction with HU was characterized by three type of measurements: (i) binding of FITC-labeled HU to microchip oligonucleotides; (ii) melting curves of complexes of labeled HU with single-stranded microchip oligonucleotides; (iii) the effect of HU binding on melting curves of microchip double-stranded DNA labeled with another fluorescent dye, Texas Red . Large numbers of measurements of these parameters were carried out in parallel for all or many generic microchip elements in real time with a multi-wavelength fluorescence microscope . Statistical analysis of these data suggests some preference for HU binding to G/C-rich single-stranded oligonucleotides . HU complexes with double-stranded microchip 8mers can be divided into two groups in which HU binding either increased the melting temperature (T(m)) of duplexes or decreased it . The stabilized duplexes showed some preference for presence of the sequence motifs AAG, AGA and AAGA . In the second type of complex, enriched with A/T base pairs, the destabilization effect was higher for longer stretches of A/T duplexes . Binding of HU to labeled duplexes in the second type of complex caused some decrease in fluorescence . This decrease also correlates with the higher A/T content and lower T(m) . The results demonstrate that generic microchips could be an efficient approach in analysis of sequence specificity of proteins.

Am J Bot, 2001 Jun, 88(6), 975 - 979
Effect of aquatic weeds on methane emission from submerged paddy soil; Inubushi K et al.; Paddy fields are one of the dominant anthropogenic sources of methane emission to the atmosphere, and the main passageway of methane from paddy soil is through the rice plant . However, the effect of aquatic weeds on methane emission from rice paddies has not been properly evaluated yet . Methane emission from weeded pots and unweeded ones with anaerobic paddy soil was measured throughout the period of rice growth . More than double the amount of methane was emitted from weeded pots compared with unweeded ones . Peroxidase activity of rice root was not different between weeded and unweeded pots . However, methanogenic bacteria populations were higher in weeded pots than in unweeded ones, while methane oxidation activity, measured by the propylene oxidation technique, was higher in unweeded pots than in weeded ones . Methane oxidation activity of roots from three typical aquatic weeds in paddy fields, Lipocarpha sp., Rotala indica, and Ludwigia epilobioides, was higher than that of rice plants, while lower stems of these aquatic plants showed similar or lower activity compared with the same areas of rice plants . These results indicate that the role of aquatic weeds in paddy soil in methane emission should not be overlooked in evaluating mitigation options for reducing methane emission from paddy fields.

Gene, 2001 Jun 13, 271(1), 13 - 20
Degenerate oligonucleotide gene shuffling (DOGS): a method for enhancing the frequency of recombination with family shuffling; Gibbs MD et al.; Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling . Shuffling techniques can be used on a collection of mutants of the same gene, or related families of genes can be shuffled to produce mutants encoding chimeric gene products . One difficulty with current shuffling procedures is the predominance of unshuffled ("parental") molecules in the pool of mutants . We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled and reduces the regeneration of unshuffled parental genes . This procedure has the advantage of avoiding the use of endonucleases for gene fragmentation prior to shuffling and allows the use of random mutagenesis of selected segments of the gene as part of the procedure . We illustrate the use of the technique with a diverse family of beta-xylanase genes that possess widely different G+C contents.

FEMS Microbiol Lett, 2001 Jun 12, 200(1), 67 - 72
Phylogenetic analysis of archaeal 16S rRNA libraries from the rumen suggests the existence of a novel group of archaea not associated with known methanogens; Tajima K et al.; Molecular diversity of rumen archaea was analyzed by PCR amplification and sequencing of two 16S rRNA clone libraries prepared from the bovine rumen fluid using two different archaea-specific primer sets . The first library of 19 clones which was generated with primers D30 and D33, produced essentially two groups of sequences, one affiliated with Methanomicrobium mobile (21% of clones) and the other -- with the uncultured archaeal sequences from anaerobic digester, which are distantly associated with Thermoplasma (79% of clones) . The second library of 25 clones, which was generated with primers 0025e Forward and 1492 Reverse, produced a higher degree of diversity: in addition to the previous two groups, with the M . mobile- (56%) and Thermoplasma-associated sequences (20%), four clones (16%) were identified as Methanobrevibacter spp . The remaining two sequences were associated with unidentified archaeal sequences from the rumen and swine waste . Phylogenetic placement of eight almost complete 16S rRNA sequences revealed the existence of a novel cluster of the rumen Euryarchaeota, which is not affiliated with the known methanogenic archaea.

FEMS Microbiol Lett, 2001 Jun 12, 200(1), 1 - 7
Molecular phylogeny of the genus Bartonella: what is the current knowledge?
Houpikian P, Raoult D.
Species of the genus Bartonella are involved in an increasing variety of human diseases . In addition to the 14 currently recognized species, several Bartonella strains have been recovered from a wide range of wild and domestic mammals in Europe and America . Such a high diversity of geographic distributions, animal reservoirs, arthropod vectors and pathogenic properties makes clarification of our knowledge about the phylogeny of Bartonella species necessary . Phylogenetic data have been inferred mainly from 16S rDNA, 16S--23S rRNA intergenic spacer, citrate synthase and 60 kDa heat-shock protein gene sequences, which are available in GenBank . Comparison of phylogenetic organizations obtained from various genes allowed six statistically significant evolutionary clusters to be identified . Bartonella bacilliformis and Bartonella clarridgeiae appear to be divergent species . Bartonella henselae, Bartonella koehlerae and Bartonella quintana cluster together, as well as Bartonella vinsonii subsp . vinsonii and B . vinsonii subsp . berkhoffii . The fifth group includes bacteria isolated from various rodents that belong to native species from the New World and in the sixth, Bartonella tribocorum, Bartonella elizabethae and Bartonella grahamii are grouped with several strains associated with Old World indigenous rodents . The position of the other species could not be consistently determined . As some cat- or rodent-associated Bartonella appeared to cluster together, it has been suggested that these bacteria and their reservoir hosts may co-evolve . Lack of host specificity, however, seems to be frequent and may reflect the influence of vector specificity . Host or vector specificity may also explain the current geographic distribution of Bartonella species.

Biochim Biophys Acta, 2001 Jun 11, 1547(2), 329 - 38
Impact of the tryptophan residues of Humicola lanuginosa lipase on its thermal stability; Zhu K et al.; Thermal stability of wild type Humicola lanuginosa lipase (wt HLL) and its two mutants, W89L and the single Trp mutant W89m (W117F, W221H, and W260H), were compared . Differential scanning calorimetry revealed unfolding of HLL at T(d)=74.4 degrees C whereas for W89L and W89m this endotherm was decreased to 68.6 and 62 degrees C, respectively, demonstrating significant contribution of the above Trp residues to the structural stability of HLL . Fluorescence emission spectra revealed the average microenvironment of Trps of wt HLL and W89L to become more hydrophilic at elevated temperatures whereas the opposite was true for W89m . These changes in steady-state emission were sharp, with midpoints (T(m)) at approx . 70.5, 61.0, and 65.5 degrees C for wt HLL, W89L, and W89m, respectively . Both steady-state and time resolved fluorescence spectroscopy further indicated that upon increasing temperature, the local movements of tryptophan(s) in these lipases were first attenuated . However, faster mobilities became evident when the unfolding temperatures (T(m)) were exceeded, and the lipases became less compact as indicated by the increased hydrodynamic radii . Even at high temperatures (up to 85 degrees C) a significant extent of tertiary and secondary structure was revealed by circular dichroism . Activity measurements are in agreement with increased amplitudes of conformational fluctuations of HLL with temperature . Our results also indicate that the thermal unfolding of these lipases is not a two-state process but involves intermediate states . Interestingly, a heating and cooling cycle enhanced the activity of the lipases, suggesting the protein to be trapped in an intermediate, higher energy state . The present data show that the mutations, especially W89L in the lid, contribute significantly to the stability, structure and activity of HLL.

Immunol Lett, 2001 Jul 2, 77(3), 151 - 8
Differentiation by in vitro treatment of lidocaine-epinephrine and prilocaine-felypressine in neutrophils; Azuma Y et al.; Neutrophils are often the first cells of the immune system to encounter an invader, such as bacteria and fungi . Lidocaine-epinephrine induced transient potentiation of the production of superoxide anion, while prilocaine-felypressine induced persistent inhibition of the production in neutrophils . Moreover, lidocaine-epinephrine inhibited the production of hydrogen peroxide in spite that it potentiated the production of superoxide anion, while prilocaine-felypressine inhibited the production of hydrogen peroxide as well as superoxide anion . By contrast, lidocaine-epinephrine and prilocaine-felypressine are both effective in significantly inhibiting adhesion and phagocytosis . Using flow cytometric analysis, both local anesthetics were found to be effective in inhibiting the expression of Mac-1 (CD11b/CD18) in neutrophils . These results suggest that lidocaine-epinephrine and prilocaine-felypressine differentially modulate the production of superoxide anion, and could similarly inhibit adhesion, phagocytosis, and the production of hydrogen peroxide by neutrophils.

EMBO J, 2001 Jun 15, 20(12), 3229 - 37
Telomere resolution in the Lyme disease spirochete; Chaconas G et al.; The genus Borrelia includes the causative agents of Lyme disease and relapsing fever . An unusual feature of these bacteria is a genome that includes linear DNA molecules with covalently closed hairpin ends referred to as telomeres . We have investigated the mechanism by which the hairpin telomeres are processed during replication . A synthetic 140 bp sequence having the predicted structure of a replicated telomere was shown to function as a viable substrate for telomere resolution in vivo, and was sufficient to convert a circular replicon to a linear form . Our results suggest that the final step in the replication of linear Borrelia replicons is a site-specific DNA breakage and reunion event to regenerate covalently closed hairpin ends . The telomere substrate described here will be valuable both for in vivo manipulation of linear DNA in Borrelia and for in vitro studies to identify and characterize the telomere resolvase.

EMBO J, 2001 Jun 15, 20(12), 3029 - 35
High-resolution AFM topographs of Rubrivivax gelatinosus light-harvesting complex LH2; Scheuring S et al.; Light-harvesting complexes 2 (LH2) are the accessory antenna proteins in the bacterial photosynthetic apparatus and are built up of alphabeta-heterodimers containing three bacteriochlorophylls and one carotenoid each . We have used atomic force microscopy (AFM) to investigate reconstituted LH2 from Rubrivivax gelatinosus, which has a C-terminal hydrophobic extension of 21 amino acids on the alpha-subunit . High-resolution topographs revealed a nonameric organization of the regularly packed cylindrical complexes incorporated into the membrane in both orientations . Native LH2 showed one surface which protruded by approximately 6 A and one that protruded by approximately 14 A from the membrane . Topographs of samples reconstituted with thermolysin-digested LH2 revealed a height reduction of the strongly protruding surface to approximately 9 A, and a change of its surface appearance . These results suggested that the alpha-subunit of R.gelatinosus comprises a single transmembrane helix and an extrinsic C-terminus, and allowed the periplasmic surface to be assigned . Occasionally, large rings ( approximately 120 A diameter) surrounded by LH2 rings were observed . Their diameter and appearance suggest the large rings to be LH1 complexes.

Biochim Biophys Acta, 2001 May 28, 1519(1-2), 143 - 6
Cloning and characterization of the major groESL operon from a nitrogen-fixing cyanobacterium Anabaena sp . strain L-31; Rajaram H et al.; The major heat-shock-responsive operon groESL has been cloned from the cyanobacterial diazotroph Anabaena . The bicistronic operon harbors an upstream negative regulatory CIRCE element and is transcriptionally activated upon temperature upshift . The deduced amino acid sequence displays strong identity/similarity with other cyanobacterial GroES and GroEL proteins.

Cochrane Database Syst Rev . 2001;(2):CD002996.
Inhaled hyperosmolar agents for bronchiectasis; Wills P et al.; BACKGROUND: Mucus retention in the lungs is a prominent feature of bronchiectasis . The stagnant mucus becomes chronically colonised with bacteria, which elicit a host neutrophilic response . This fails to eliminate the bacteria, and the large concentration of host-derived protease may contribute to the airway damage . The sensation of retained mucus is itself a cause of suffering, and the failure to maintain airway sterility probably contributes to the frequent respiratory infections experienced by many patients . Hypertonic saline inhalation is known to accelerate tracheobronchial clearance in many conditions, probably by inducing a liquid flux into the airway surface, which alters mucus rheology in a way favourable to mucociliary clearance . Inhaled dry powder mannitol has a similar effect . Such agents are an attractive approach to the problem of mucostasis, and deserve further clinical evaluation . OBJECTIVES: To determine whether inhaled hyperosmolar substances are efficacious in the treatment of bronchiectasis SEARCH STRATEGY: MEDLINE and Cochrane databases were searched, and leaders in the field contacted . SELECTION CRITERIA: Any trial using hyperosmolar inhalation in patients with bronchiectasis not caused by cystic fibrosis . DATA COLLECTION AND ANALYSIS: One reference were identified by the searches conducted . MAIN RESULTS: Only one trial was identified, a crossover study of 11 patients with bronchiectasis . The outcome measure was tracheobronchial clearance of a particulate radioaerosol after inhalation of dry mannitol on a single occasion, with appropriate controls . Airway clearance doubled in the central and intermediate regions of the lung, but not in the peripheral region, after mannitol administration . No side effects were observed, but two patients were premedicated with nedocromil to prevent bronchospasm . REVIEWER'S CONCLUSIONS: Dry powder mannitol has been shown to improve tracheobronchial clearance in bronchiectasis, as well as cystic fibrosis, asthmatics, and normal subjects . It is not yet available for clinical use . Hypertonic saline has not been specifically tested in bronchiectasis, but improve clearance in these other conditions and in chronic bronchitis . Longer term randomised controlled studies of mannitol and hypertonic saline with clinical endpoints are now needed.

Biochemistry (Mosc), 2001 May, 66(5), 541 - 7
Studies of electron transport dynamics in photosynthetic reaction centers using fast temperature changes; Chamorovsky SK et al.; Rates of thermoinduced conformational transitions of reaction center (RC) complexes providing effective electron transport were studied in chromatophores and isolated RC preparations of various photosynthesizing purple bacteria using methods of fast freezing and laser-induced temperature jump . Reactions of electron transfer from the primary to secondary quinone acceptors and from the multiheme cytochrome c subunit to photoactive bacteriochlorophyll dimer were used as probes of electron transport efficiency . The thermoinduced transition of the acceptor complex to the conformational state facilitating electron transfer to the secondary quinone acceptor was studied . It was shown that neither the characteristic time of the thermoinduced transition within the temperature range 233-253 K nor the characteristic time of spontaneous decay of this state at 253 K exceeded several tens of milliseconds . In contrast to the quinone complex, the thermoinduced transition of the macromolecular RC complex to the state providing effective electron transport from the multiheme cytochrome c to the photoactive bacteriochlorophyll dimer within the temperature range 220-280 K accounts for tens of seconds . This transition is thought to be mediated by large-scale conformational dynamics of the macromolecular RC complex.

Mol Genet Genomics, 2001 May, 265(3), 445 - 54
Organization, expression, and function of Caulobacter crescentus genes needed for assembly and function of the flagellar hook; Mullin DA et al.; This paper reports on the organization, expression, and function of the divergently transcribed flbG and flaN operons in the hook gene cluster of Caulobacter crescentus . The transcription initiation site of flbG was determined previously, and in this work the transcription map was completed by locating the 3' end of the mRNA using nuclease S1 protection assays . A previous genetic study had suggested that the flbG operon is comprised of four genes; however, the nucleotide sequence revealed three tandemly arranged ORFs that correspond to 5'-flbG, flbH, and flgE . FlbG is similar to FliK proteins which are required for termination of hook synthesis, FlbH is similar to FlgD proteins which are essential scaffolding proteins that cap the hook during its assembly, and FlgE corresponds to the hook structural protein . The divergently transcribed flaN gene codes for a hook associated protein I homolog based on its inferred amino acid sequence similarity to FlgK proteins . Based on the amino acid sequence similarities and phenotypes of mutants, flbG, flbH, and flaN have been renamed fliK, flgD, and flgK, FlgD, FlgE, and FlgK proteins, with apparent molecular masses of 23, 68, and 41 kDa, respectively, were expressed from plasmids in a cell-free coupled transcription-translation system, and a protein corresponding to FliK was identified as part of a 190-kDa FliK-LacZ fusion protein . We present evidence showing that, in addition to its role in termination of hook synthesis, FliK is also required for initiation of hook assembly.

Clin Lab, 2001, 47(5-6), 279 - 88
The determination of free and protein-bound haemoglobin in plasma using a combination of HPLC and absorption spectrometry; Wood WG et al.; The aim of the study was to develop a method for the determination of haemoglobin in plasma suitable for use to set target values for external quality assessment schemes for this analyte using commercially available test kits and equipment . In the early phase of the method development it became clear that the use of a single method, namely HPLC, would not be possible . However, by combining HPLC and absorption spectrophotometry, both qualitative and quantitative rapid determinations of protein-bound and free haemoglobin were able to be performed on equipment present in most routine clinical chemistry laboratories . The separation of protein-bound and free haemoglobin could be carried out using commercial HPLC equipment for the determination of haemoglobin A1c (HbA1c) without modification of the conditions used . Instead of haemolysed blood, the same volume of plasma (10 microl) was injected . The eluate was not discarded, but collected in 1-minute fractions so that the void volume (protein-bound Hb) and the haemoglobin peaks (free Hb) were available for the colorimetric determination of haemoglobin using the pseudoperoxidase activity of the haem moiety on hydrogen peroxide and a chromogen (3,3',5,5'-tetramethylbenzidine) in concentrated acetic acid and optimal determination at 600 nm . (In this publication at 578 nm due to the use of a spectrophotometer with Hg-discharge lamp and filter) . The appearance of a blue colour in the reaction tube or cuvette indicated the presence of haemoglobin . The use of the above chromogen, with its absorption maximum around 600 nm excluded interference from serum components such as bilirubin, which may interfere in the conventional method often used to determine plasma haemoglobin . The method can be used quantitatively by including an aqueous human haemoglobin standard in the run . This elutes from the HPLC column only as free haemoglobin in the concentration range from 0.1 to 10 g/l . Addition of human haemoglobin to haemoglobin-free plasma resulted in the binding of all Hb to plasma proteins up to a concentration between 2 and 3 g/l (void-volume fraction) . At higher concentrations free Hb appeared in the 3-5 minute fractions . These observations agree with published data on the scavenging capacity of plasma for Hb released from erythrocytes . The method is rapid, (HPLC-run maximally 6 min, quantitative colorimetric results 5-10 min) precise (inter-assay coefficients of variation < 8%) and suitable for answering the question as to whether the protein-binding (scavenging) system which prevents the nephro- and cerebrotoxic effects of haemoglobin has been saturated or not, an important question in patients with acute haemolysis problems . A qualitative result is obtainable within 10 minutes of injecting the sample into the HPLC-system . The use of this assay in controlling blood transfusion and haemolytic events arising from surgery, intravascular haemolytic bacteria or artificial heart valves can help in rapid corrective action, if needed.

Prim Dent Care, 2000 Jul, 7(3), 105 - 7
Advances in endodontics; Gulabivala K; Progress in diagnosis, prevention of diseases, treatment of exposed pulp, root canal treatment, retreatment and surgery is reported . Contemporary biological research techniques could set the foundation for a more rational approach to treatment . Pulp inflammation may be prevented by inhibiting bacteria from colonising dentine surfaces . Specific factors could be synthesised to stimulate normal dentine deposition over pulp exposures . Significant improvements have been made in instruments and techniques available for root canal treatment and surgery but there is no clinical evidence that they improve periapical healing.

Proc Natl Acad Sci U S A, 2001 Jun 19, 98(13), 7540 - 5 Epub 2001 Jun 12.
Light regulation of type IV pilus-dependent motility by chemosensor-like elements in Synechocystis PCC6803; Bhaya D et al.; To optimize photosynthesis, cyanobacteria move toward or away from a light source by a process known as phototaxis . Phototactic movement of the cyanobacterium Synechocystis PCC6803 is a surface-dependent phenomenon that requires type IV pili, cellular appendages implicated in twitching and social motility in a range of bacteria . To elucidate regulation of cyanobacterial motility, we generated transposon-tagged mutants with aberrant phototaxis; mutants were either nonmotile or exhibited an "inverted motility response" (negative phototaxis) relative to wild-type cells . Several mutants contained transposons in genes similar to those involved in bacterial chemotaxis . Synechocystis PCC6803 has three loci with chemotaxis-like genes, of which two, Tax1 and Tax3, are involved in phototaxis . Transposons interrupting the Tax1 locus yielded mutants that exhibited an inverted motility response, suggesting that this locus is involved in controlling positive phototaxis . However, a strain null for taxAY1 was nonmotile and hyperpiliated . Interestingly, whereas the C-terminal region of the TaxD1 polypeptide is similar to the signaling domain of enteric methyl-accepting chemoreceptor proteins, the N terminus has two domains resembling chromophore-binding domains of phytochrome, a photoreceptor in plants . Hence, TaxD1 may play a role in perceiving the light stimulus . Mutants in the Tax3 locus are nonmotile and do not make type IV pili . These findings establish links between chemotaxis-like regulatory elements and type IV pilus-mediated phototaxis.

Mol Ther, 2001 Jun, 3(6), 882 - 91
Targeting of adenovirus to endothelial cells by a bispecific single-chain diabody directed against the adenovirus fiber knob domain and human endoglin (CD105); Nettelbeck DM et al.; The use of adenoviruses for antivascular cancer gene therapy is limited by their low transduction efficiency for endothelial cells . We have developed a recombinant bispecific antibody as a molecular bridge, linking the adenovirus capsid to the endothelial cell surface protein endoglin, for vascular targeting of adenoviruses . Endoglin (CD105), a component of the transforming growth factor beta receptor complex, represents a promising target for antivascular cancer therapy . Endoglin is expressed predominantly on endothelial cells and is upregulated in angiogenic areas of tumors . We isolated single-chain Fv fragments directed against human endoglin from a human semisynthetic antibody library . One of the isolated scFv fragments (scFv C4) bound specifically to various proliferating primary endothelial cells or cell lines including HUVEC, HDMEC, HMVEC, and HMEC . ScFv C4 was therefore used to construct a bispecific single-chain diabody directed against endoglin and the adenovirus fiber knob domain (scDb EDG-Ad) . This bispecific molecule mediated enhanced and selective adenovirus transduction of HUVECs, which was independent from binding to the coxsackievirus and adenovirus receptor (CAR) and alpha(v)-integrins . Thus, adenovirus infection was redirected to a new cellular receptor (CD105) and cell entry pathway . These results demonstrate the utility of bispecific single-chain diabodies, which can be produced in large quantities in bacteria, for the retargeting of adenoviruses in cancer gene therapy.

Adv Microb Physiol, 2001, 44, 141 - 81
Environmental sensing mechanisms in Bordetella; Coote JG; The success of a bacterial pathogen may depend on its ability to sense and respond to different environments . This is particularly true of those pathogens whose survival depends on adaptation to different niches both within and outside the host . Members of the genus Bordetella cause infections in humans, other animals and birds . Two closely related species, B . pertussis and B . bronchiseptica, cause respiratory disease and express a similar range of virulence factors during infection, but exhibit different host ranges and responses to environmental change . B . pertussis has no known reservoir other than humans and is assumed to be transmitted directly via aerosol droplets between hosts . B . bronchiseptica, on the other hand, has the potential to survive and grow in the natural environment . Comparison of the manner in which these two organisms respond to external signals has provided important insights into the co-ordinate regulation of gene expression as a response to a changing environment . During infection, both species produce a range of virulence factors whose expression is co-ordinated by two members of the two-component family of signal transduction proteins, the bvg (bordetella virulence gene) and ris (regulator of intracellular stress response) loci . When active, the bvg locus directs the activity of a number of virulence determinants in both species whose products, such as adhesins and toxins, establish colonization of the host by the bacteria, although each organism has evolved a slightly different strategy during pathogenesis . B . pertussis, the causative agent of whooping cough, promotes an acute disease and tends to be more virulent than B . bronchiseptica which generally causes chronic and persistent asymptomatic colonization of the respiratory tract . The recently identified ris locus appears to control the expression of factors important for intracellular survival of B . bronchiseptica, but a role for this regulatory locus in B . pertussis infection has not been established . Expression of the virulence determinants controlled by the bvg and ris loci is subject to modulation by different environmental signals, such as low temperature, which act through these two-component systems . Evidence indicates that, for B . bronchiseptica, bvg-controlled determinants expressed under modulating conditions, such as motility, facilitate adaptation and survival in environments outside the host . With B . pertussis, however, there is no apparent requirement for prolonged survival outside the host and this difference is reflected in the expression of different, as yet uncharacterized, determinants as a response to modulating signals . The nature of the gene products involved and their assumed role in the life cycle of B . pertussis remains to be determined . Thus, comparative analysis of these species provides an excellent model for understanding the genetic requirements for pathogenesis of respiratory infection and adaptation to changing environments, both within and outside the host.

Endocr Rev, 2001 Jun, 22(3), 319 - 41
Environmental signaling: what embryos and evolution teach us about endocrine disrupting chemicals; McLachlan JA; The term "endocrine disrupting chemicals" is commonly used to describe environmental agents that alter the endocrine system . Laboratories working in this emerging field-environmental endocrine research-have looked at chemicals that mimic or block endogenous vertebrate steroid hormones by interacting with the hormone's receptor . Environmental chemicals known to do this do so most often with receptors derived from the steroid/thyroid/retinoid gene family . They include ubiquitous and persistent organochlorines, as well as plasticizers, pharmaceuticals, and natural hormones . These chemicals function as estrogens, antiestrogens, and antiandrogens but have few, if any, structural similarities . Therefore, receptor-based or functional assays have the best chance of detecting putative biological activity of environmental chemicals . Three nuclear estrogen receptor forms-alpha, beta, and gamma-as well as multiple membrane forms and a possible mitochondrial form have been reported, suggesting a previously unknown diversity of signaling pathways available to estrogenic chemicals . Examples of environmental or ambient estrogenization occur in laboratory experiments, zoo animals, domestic animals, wildlife, and humans . Environmentally estrogenized phenotypes may differ depending upon the time of exposure-i.e., whether the exposure occurred at a developmental (organizational and irreversible) or postdevelopmental (activational and reversible) stage . The term "estrogen" must be defined in each case, since steroidal estrogens differ among themselves and from synthetic or plant-derived chemicals . An "estrogen-like function" seems to be an evolutionarily ancient signal that has been retained in a number of chemicals, some of which are vertebrate hormones . Signaling, required for symbiosis between plants and bacteria, may be viewed, therefore, as an early example of hormone cross-talk . Developmental feminization at the structural or functional level is an emerging theme in species exposed, during embryonic or fetal life, to estrogenic compounds . Human experience as well as studies in experimental animals with the potent estrogen diethylstilbestrol provide informative models . Advances in the molecular genetics of sex differentiation in vertebrates facilitate mechanistic understanding . Experiments addressing the concept of gene imprinting or induction of epigenetic memory by estrogen or other hormones suggest a link to persistent, heritable phenotypic changes seen after developmental estrogenization, independent of mutagenesis . Environmental endocrine science provides a new context in which to examine the informational content of ecosystem-wide communication networks . As common features come to light, this research may allow us to predict environmentally induced alterations in internal signaling systems of vertebrates and some invertebrates and eventually to explicate environmental contributions to human reproductive and developmental health.

ILAR J, 1998 Sep, 39(4), 322 - 330
Deer Mice As Laboratory Animals; Joyner CP et al.; Although laboratory mice (Mus) and rats (Rattus) are the most widely used research rodents, deer mice (Peromyscus maniculatus) and their congeneric species are favored as nontraditional alternatives for some purposes . Mice of the native genus Peromyscus are the most abundant and widely distributed rodents in North America . They occur in a great diversity of habitats and play a significant role in natural ecosystems . Because of their abundance, peromyscines are commonly hosts for larva of ticks that transmit Lyme disease bacteria, and they are implicated in several other vector-borne diseases . Deer mice also are the principal carriers of the virus that causes hantaviral pulmonary syndrome, or "Four Corners disease." Deer mice are useful as laboratory models for a variety of other types of pure and applied research . They are easily maintained and bred in captivity using the husbandry protocols developed for other small laboratory rodent species . The Peromyscus Genetic Stock Center at the University of South Carolina maintains more than 50 laboratory-bred, well-characterized stocks of deer mice and other peromyscine species for research and educational use.

Hong Kong Med J, 2001 Mar, 7(1), 67 - 72
Infections of the nervous system: an update on recent developments; Kay R et al.; The past decade has seen major changes in the field of infectious diseases . In particular, many new infections of the nervous system have been recognised, including the lethal infections of Enterovirus 71, and the Nipah and West Nile viruses . Increased interest in prion diseases has occurred, following the recognition of animal-to-human transmission in Europe . Familiar bacteria such as the pneumococcus continue to cause problems due to increasing resistance to multiple antibiotics . Furthermore, human immunodeficiency virus-infected and other immunocompromised patients are under the constant threat of opportunistic infections, many of which are targeted towards the brain and spinal cord . This paper reviews the changing world of nervous system infections, highlighting some of the most significant recent developments.

Int J Parasitol, 2001 Jun, 31(8), 798 - 809
Role of oligosaccharides in the immune response of sheep vaccinated with Lucilia cuprina larval glycoprotein, peritrophin-95; Tellam RL et al.; The larvae of the fly Lucilia cuprina cause a cutaneous myiasis in mammalian hosts, particularly sheep . The glycoprotein, peritrophin-95, isolated from Lucilia cuprina larval peritrophic matrix, is a candidate vaccine antigen . This protein induced an immune response in vaccinated sheep that inhibited larval growth . Recombinant forms of peritrophin-95 were produced in bacteria and baculovirus-infected insect cells . The bacterial protein was not glycosylated and incorrectly folded whereas the insect cell-expressed protein was glycosylated and probably correctly folded . Sheep immunised with purified native peritrophin-95 generated strong larval growth inhibitory activity in their sera, whereas sheep immunised with either recombinant form of peritrophin-95 generated only relatively weak inhibitory activity . Ingested ovine antibodies to native peritrophin-95 mediated the anti-larval growth activity and this was independent of the presence of ovine complement . The activity was associated with IgG(1) and IgG(2) but not IgM . There were strong antibody responses to both the correctly folded native peritrophin-95 polypeptide and the oligosaccharides present on this glycoprotein . Immuno-affinity isolation of antibody to the peritrophin-95 polypeptide and antibody to peritrophin-95 oligosaccharides demonstrated that the larval growth inhibitory activity resided with both antibodies . Lectin blots and ELISA data showed substantial differences between the oligosaccharides attached to native peritrophin-95 and insect cell-expressed recombinant peritrophin-95 . It was concluded that the oligosaccharides attached to native peritrophin-95 and its unique polypeptide structure are essential for the induction of larval growth inhibitory activity in the sera of sheep vaccinated with this antigen.

Best Pract Res Clin Gastroenterol, 2001 Jun, 15(3), 511 - 21
Occurrence and significance of gastric colonization during acid-inhibitory therapy; Williams C; There are now a wide variety of drugs available that are able profoundly to reduce the production of gastric acid . These drugs are currently widely prescribed for the treatment of peptic ulceration and gastro-oesophageal reflux disease . One of the main functions of gastric acid is to kill ingested bacteria . Colonization of the gastric lumen occurs in patients on anti-secretory medication, the degree of bacterial overgrowth depending upon the degree of elevation of the pH . There have been concerns that these bacteria may produce carcinogenic nitrosamines and increase the risk of gastric cancer, but there is at present no definitive evidence in support of this . A profound suppression of gastric acid may also facilitate the colonization of the upper small intestine, leading to deconjugation of the bile salts and malabsorption . There is some evidence that profound gastric acid suppression may decrease the number of ingested pathogens required to produce enteric disease . This chapter discusses these potential bacterial complications of therapeutic acid suppression and the evidence for them.

Syst Appl Microbiol, 2001 Apr, 24(1), 83 - 97
Design and application of new 16S rRNA-targeted oligonucleotide probes for the Azospirillum-Skermanella-Rhodocista-cluster; Stoffels M et al.; The genera Azospirillum, Skermanella and Rhodocista form a phylogenetic subgroup within the alfa subclass of Proteobacteria . Based on comparative 16S rRNA sequence analysis a nested set of new oligonucleotide probes was designed . It comprises probes for the whole genus cluster Azospirillum-Skermanella-Rhodocista, for the Azospirilli subcluster I including A . lipoferum, A . doebereinerae, A . largimobile, A . brasilense and A . halopraeferens, for the Azospirilli subcluster II including A . amazonense, A . irakense and the genus Skermanella, for the genus Rhodocista as well as for all Azospirilli species or species cluster . The new probes allow a fast and reliable in situ identification of bacteria belonging to the Azospirillum-Skermanella-Rhodocista-cluster at different phylogenetic levels . The specificity of the new probes was tested with 56 strains of the Azospirillum-Rhodocista-Skermanella-cluster and selected reference cells from other genera by hybridising with the complete probe set . In addition, applications of the fluorescently labelled probes for in situ identification of isolates and for the in situ localisation of A . brasilense on maize roots were demonstrated using confocal laser scanning microscopy.

Syst Appl Microbiol, 2001 Apr, 24(1), 63 - 6
Genetic characterization of a Chlamydophila pneumoniae isolate from an African frog and comparison to currently accepted biovars; Hotzel H et al.; The amphibian isolate DE177 identified as Chlamydophila (C.) pneumoniae was sequenced in five genomic regions: 16S ribosomal RNA gene, 16-23S intergenic spacer, ompA, ompB, and groESL genes . Comparison with corresponding sequences of the currently accepted equine, human and koala biovars of C . pneumoniae revealed that koala strains represented the most closely related taxon, although sequence dissimilarities in the ompA (VD4) and ompB gene regions were noted . In this respect, the present isolate is distinct from a previously described frog isolate (Berger et al., 1999) whose sequence analysis yielded identity to the koala biovar . As three of the nucleotide substitutions in ompA (VD4) of DE177 will be translated into two altered amino acids the possible existence of another biovar is discussed.

Arch Intern Med, 2001 May 28, 161(10), 1289 - 94
Clinician attributions for symptoms and treatment of Gulf War-related health concerns; Richardson RD et al.; BACKGROUND: Several clinical syndromes are defined solely on the basis of symptoms, absent an identifiable medical etiology . When evaluating and treating individuals with these syndromes, clinicians' beliefs might shape decisions regarding referral, diagnostic testing, and treatment . To assess clinician beliefs about the etiology and treatment of "Gulf War illness," we surveyed a sample of general internal medicine clinicians (GIMCs) and mental health clinicians (MHCs) . METHODS: Clinicians (77 GIMCs and 214 MHCs) at the Veterans Affairs Puget Sound Health Care System, Seattle, Wash, and the Veterans Affairs Medical Center in Portland, Ore, responded to a mailed survey of their beliefs about Gulf War illness . RESULTS: Compared with GIMCs, MHCs were more likely to believe that Gulf War illness was the result of a "physical disorder" and that symptoms resulted from viruses or bacteria, immunizations, exposure to toxins, chemical weapons, or a combination of toxins and stress (P <.05) . Conversely, GIMCs were more likely than MHCs to believe that Gulf War illness was a "mental disorder" and that symptoms were due to stress or posttraumatic stress disorder (P <.05) . In addition, MHCs were more likely to endorse biological interventions to treat Gulf War illness (P <.01), whereas GIMCs were more likely to endorse psychological interventions . CONCLUSIONS: Clinicians' beliefs about the etiology and effective treatment of Gulf War illness vary and thus might contribute to the multiple referrals often reported by Gulf War veterans . Health care models for Gulf War veterans and others with symptom-based disorders necessitate collaborative interdisciplinary approaches.

J Biol Chem, 2001 Aug 31, 276(35), 33181 - 95 Epub 2001 Jun 11.
Proteomic analysis of the mammalian mitochondrial ribosome . Identification of protein components in the 28 S small subunit; Suzuki T et al.; The mammalian mitochondrial ribosome (mitoribosome) has a highly protein-rich composition with a small sedimentation coefficient of 55 S, consisting of 39 S large and 28 S small subunits . In the previous study, we analyzed 39 S large subunit proteins from bovine mitoribosome (Suzuki, T., Terasaki, M., Takemoto-Hori, C., Hanada, T., Ueda, T., Wada, A., and Watanabe, K . (2001) J . Biol . Chem . 276, 21724-21736) . The results suggested structural compensation for the rRNA deficit through proteins of increased molecular mass in the mitoribosome . We report here the identification of 28 S small subunit proteins . Each protein was separated by radical-free high-reducing two-dimensional polyacrylamide gel electrophoresis and analyzed by liquid chromatography/mass spectrometry/mass spectrometry using electrospray ionization/ion trap mass spectrometer to identify cDNA sequence by expressed sequence tag data base searches in silico . Twenty one proteins from the small subunit were identified, including 11 new proteins along with their complete cDNA sequences from human and mouse . In addition to these proteins, three new proteins were also identified in the 55 S mitoribosome . We have clearly identified a mitochondrial homologue of S12, which is a key regulatory protein of translation fidelity and a candidate for the autosomal dominant deafness gene, DFNA4 . The apoptosis-related protein DAP3 was found to be a component of the small subunit, indicating a new function for the mitoribosome in programmed cell death . In summary, we have mapped a total of 55 proteins from the 55 S mitoribosome on the two-dimensional polyacrylamide gels.

Infect Immun, 2001 Jul, 69(7), 4654 - 6
Normal IncA expression and fusogenicity of inclusions in Chlamydia trachomatis isolates with the incA I47T mutation; Pannekoek Y et al.; To investigate the correlation between the incA I47T mutation in Chlamydia trachomatis and the nonfusogenic phenotype, the incA genes of 25 isolates were sequenced . Four major sequence types were identified . Seven isolates (28%) had the I47T mutation . Isolates representing the four sequence types expressed IncA in the membrane of one large single inclusion . In conclusion, the incA I47T mutation is not associated with the nonfusogenic phenotype.

Infect Immun, 2001 Jul, 69(7), 4479 - 85
Evidence of recombination in Porphyromonas gingivalis and random distribution of putative virulence markers; Frandsen EV et al.; The association of Porphyromonas gingivalis to periodontal disease is not clearly understood . Similar proportions of P . gingivalis may be cultivated from both inactive and actively degrading periodontal pockets . Differences in virulence among strains of P . gingivalis exist, but the molecular reason for this remains unknown . We examined the population structure of P . gingivalis to obtain a framework in which to study pathogenicity in relation to evolution . Phylogenetic trees derived from the sequencing of fragments of four housekeeping genes, ahp, thy, rmlB, and infB, in 57 strains were completely different with no correlation between clustering of strains in the four dendrograms . Combining the various alleles of the four gene fragments sequenced resulted in 41 different sequence types . The index of association, I(A), based on a single representative of each sequence type was 0.143 +/- 0.202, indicating a population at linkage equilibrium . Inclusion of all isolates for the calculation of I(A) resulted in a value of 0.206 +/- 0.171 . This suggests an epidemic population structure supported by the finding of genetically identical strains in different parts of the world . We observed a random distribution of two virulence-associated mobile genetic elements, the ragB locus and the insertion sequence IS1598, among 132 strains tested . In conclusion, P . gingivalis has a nonclonal population structure characterized by frequent recombination . Our study suggests that particular genotypes, possibly with increased pathogenic potential, may spread successfully in the human population.

Front Biosci, 2001 Jun 01, 6, D737 - 47
Entry mechanisms of mycobacteria; El-Etr SH et al.; Since many mycobacteria are facultative intracellular pathogens, their ability to cause disease involves entry, survival and replication within host cells . Despite the fact that mycobacteria were first associated with disease more than 125 years ago, the first step in the production of an infection, entry into host cells, is not well understood . Mycobacteria have the ability to enter a number of different cell types, but the primary cell type that they are thought to replicate within during human disease is macrophages . Since macrophages have a large number of receptors that are designed for relatively non-specific uptake of foreign particles, there are multiple routes by which nearly any bacteria can be taken up . The outcome of mycobacterial entry into macrophages via different mechanisms is unclear . Although it is thought that mycobacteria may enter macrophages by a mechanism that allows them to avoid lysosomal fusion, it remains possible that mycobacteria enter by more than one mechanism, yet remain viable and replicate intracellularly through modification of the phagosome . In the current discussion we will review mycobacterial research specifically relating to the mechanisms of entry into host cells . Although much progress has been made in our understanding of entry by mycobacteria, we anticipate that clarification of the role of entry in pathogenesis will require further application of newly developed molecular tools to dissect each of the proposed mechanisms.

Mol Microbiol, 2001 Jun, 40(5), 1201 - 14
RpoS co-operates with other factors to induce Legionella pneumophila virulence in the stationary phase; Bachman MA et al.; Legionella pneumophila replicates within amoebae and macrophages and causes the severe pneumonia Legionnaires' disease . When broth cultures enter the post-exponential growth (PE) phase or experience amino acid limitation, L . pneumophila accumulates the stringent response signal (p)ppGpp and expresses traits likely to promote transmission to a new phagocyte . The hypothesis that a stringent response mechanism regulates L . pneumophila virulence was bolstered by our finding that the avirulent mutant Lp120 contains an internal deletion in the gene encoding the stationary phase sigma factor RpoS . To test directly whether RpoS co-ordinates virulence with stationary phase, isogenic wild-type, rpoS-120 and rpoS null mutant strains were constructed and analysed . PE phase L . pneumophila became cytotoxic by an RpoS-independent pathway, but their sodium sensitivity and maximal expression of flagellin required RpoS . Likewise, full induction of sodium sensitivity by experimentally induced (p)ppGpp synthesis required RpoS . To replicate efficiently in macrophages, L . pneumophila used both RpoS-dependent and -independent pathways . Like those containing the dotA type IV secretory apparatus mutant, phagosomes harbouring either rpoS or dotA rpoS mutants rapidly acquired the late endosomal protein LAMP-1, but not the lysosomal marker Texas red-ovalbumin . Together, the data support a model in which RpoS co-operates with other regulators to induce L . pneumophila virulence in the PE phase.

Mol Microbiol, 2001 May, 40(4), 1037 - 44
Variations in the surface proteins and restriction enzyme systems of Mycoplasma pulmonis in the respiratory tract of infected rats; Gumulak-Smith J et al.; Restriction and modification (R-M) systems are generally thought to protect bacteria from invasion by foreign DNA . This paper proposes the existence of an alternative role for the phase-variable R-M systems encoded by the hsd loci of Mycoplasma pulmonis . Populations of M . pulmonis cells that arose during growth in different environments were compared with respect to R-M activity and surface antigen production . When M . pulmonis strain X1048 was propagated in laboratory culture medium, > 95% of colony-forming units (cfu) lacked R-M activity and produced the variable surface protein VsaA . Mycoplasmas isolated from the nose of experimentally infected rats also lacked R-M activity and produced VsaA . In contrast, the cell population of mycoplasmas isolated from the lower respiratory tract of the infected rats was more complex . The most dramatic results were obtained for mycoplasmas isolated from the trachea . At 14 days postinfection, 38% of mycoplasma isolates produced a Vsa protein other than VsaA, and 34% of isolates had active restriction systems . These data suggest that differences in selection pressures in animal tissues affect the surface proteins and the R-M activity of the mycoplasmal cell population . We propose that variations in the production of R-M activity and cell surface proteins are important for the survival of the mycoplasma within the host.

Mol Microbiol, 2001 May, 40(4), 991 - 9
Mutational analysis of the high-affinity BvgA binding site in the fha promoter of Bordetella pertussis; Boucher PE et al.; In order to define a consensus binding sequence for the response regulator BvgA, we have undertaken a systematic analysis of contributions made by each nucleotide within the heptad half-sites that are present in an inverted orientation at the promoter for the fha operon . Using in vitro binding assays, we examined the full complement of 21 single point mutations symmetrically arranged in this heptad repeat . Both gel shift and nitrocellulose filter-binding assays provided evidence that nucleotides at positions 3 (thymidine), 4 (cytosine) and 7 (adenine) in the binding heptad contribute substantially to sequence-specific recognition by BvgA . Furthermore, a T to A conversion at position 6 reduced binding . Selected binding site mutations were introduced into a modified fha promoter and examined for their effects on BvgA activation of promoter activity in vivo . Only those substitutions most severely affecting binding in vitro affected promoter activity in vivo . The in vivo effects of substitutions that had a significant effect on binding in vitro but did not severely affect in vivo promoter activity under standard culture conditions could be detected in vivo either in combination with additional substitutions or from their effect on the sensitivity of the mutant promoters to modulation by magnesium sulphate.

Mol Microbiol, 2001 May, 40(4), 941 - 50
The role of HetN in maintenance of the heterocyst pattern in Anabaena sp . PCC 7120; Callahan SM et al.; The gene hetN encodes a putative oxidoreductase that is known to suppress heterocyst differentiation when present on a multicopy plasmid in Anabaena sp . PCC 7120 . To mimic the hetN null phenotype and to examine where HetN acts in the regulatory cascade that controls heterocyst differentiation, we replaced the native chromosomal hetN promoter with the copper-inducible petE promoter . In the presence of copper, heterocyst formation was suppressed in undifferentiated filaments . When hetN expression was turned off by transferring cells to media lacking copper, the filaments initially displayed the wild-type pattern of single heterocysts but, 48 h after the induction of heterocyst formation, a pattern of multiple contiguous heterocysts predominated . Suppression of heterocyst formation by HetN appears to occur both upstream and downstream of the positive regulator HetR: overexpression of hetN in undifferentiated filaments prevents the wild-type pattern of hetR expression as well as the multiheterocyst phenotype normally observed when hetR is expressed from an inducible promoter . Green fluorescent protein fusions show that the expression of hetN in wild-type filaments normally occurs primarily in heterocysts . We propose that HetN is normally involved in the maintenance of heterocyst spacing after the initial heterocyst pattern has been established, but ectopic expression of hetN can also block the initial establishment of the pattern.

Mol Microbiol, 2001 May, 40(4), 900 - 8
Heterologous expression of archaeal selenoprotein genes directed by the SECIS element located in the 3' non-translated region; Rother M et al.; Previous in silico analysis of selenoprotein genes in Archaea revealed that the selenocysteine insertion (SECIS) motif necessary to recode UGA with selenocysteine was not adjacent to the UGA codon as is found in Bacteria . Rather, paralogous stem-loop structures are located in the 3' untranslated region (3' UTR), reminiscent of the situation in Eukarya . To assess the function of such putative SECIS elements, the Methanococcus jannaschii MJ0029 (fruA, which encodes the A subunit of the coenzyme F420-reducing hydrogenase) mRNA was mapped in vivo and probed enzymatically in vitro . It was shown that the SECIS element is indeed transcribed as part of the respective mRNA and that its secondary structure corresponds to that predicted by RNA folding programs . Its ability to direct selenocysteine insertion in vivo was demonstrated by the heterologous expression of MJ0029 in Methanococcus maripaludis, resulting in the synthesis of an additional selenoprotein, as analysed by 75Se labelling . The selective advantage of moving the SECIS element in the untranslated region may confer the ability to insert more than one selenocysteine into a single polypeptide . Evidence for this assumption was provided by the finding that the M . maripaludis genome contains an open reading frame with two in frame TGA codons, followed by a stem-loop structure in the 3' UTR of the mRNA that corresponds to the archaeal SECIS element.

Mol Microbiol, 2001 May, 40(4), 879 - 89
Regulation of catalase-peroxidase (KatG) expression, isoniazid sensitivity and virulence by furA of Mycobacterium tuberculosis; Pym AS et al.; Mycobacterium tuberculosis has two genes for ferric uptake regulator orthologues, one of which, furA, is situated immediately upstream of katG encoding catalase-peroxidase, a major virulence factor that also activates the prodrug isoniazid . This association suggested that furA might regulate katG and other genes involved in pathogenesis . Transcript mapping showed katG to be expressed from a strong promoter, with consensus -10 and -35 elements, preceding furA . No promoter activity was demonstrated downstream of the furA start codon, using different gene reporter systems, indicating that furA and katG are co-transcribed from a common regulatory region . The respective roles of these two genes in the isoniazid susceptibility and virulence of M . tuberculosis were assessed by combinatorial complementation of a Delta(furA-katG) strain that is heavily attenuated in a mouse model of tuberculosis . In the absence of furA, katG was upregulated, cells became hypersensitive to isoniazid, and full virulence was restored, indicating that furA regulates the transcription of both genes . When furA alone was introduced into the Delta(furA-katG) mutant, survival in mouse lungs was moderately increased, suggesting that FurA could regulate genes, other than katG, that are involved in pathogenesis . These do not include the oxidative stress genes ahpC and sodA, or those for siderophore production.

Biochemistry, 2001 Jun 19, 40(24), 7149 - 57
Carbon monoxide adducts of KatG and KatG(S315T) as probes of the heme site and isoniazid binding; Lukat-Rodgers GS et al.; KatG, the catalase peroxidase from Mycobacterium tuberculosis, is important in the activation of the antitubercular drug, isoniazid . About 50% of isoniazid-resistant clinical isolates contain a mutation in KatG wherein the serine at position 315 is substituted with threonine, KatG(S315T) . The heme pockets of KatG and KatG(S315T) and their interactions with isoniazid are probed using resonance Raman (rR) spectroscopy to characterize their ferrous CO complexes . Three vibrational modes, C-O and Fe-C stretching and Fe-CO bending, are assigned using 12CO and 13CO isotope shifts . Two conformers are observed for KatG-CO and KatG(S315T)-CO . Resonance Raman features assigned to form I are consistent with it having a neutral proximal histidine ligand and the Fe-C-O moiety hydrogen bonded to a distal residue . The nu(C-O) band for form I is sharp, consistent with a conformationally homogeneous Fe-CO unit . Form II also has a neutral proximal histidine ligand but is not hydrogen bonded . This appears to result in a conformationally disordered Fe-CO unit, as evidenced by a comparatively broad C-O stretching band . The 13CO-sensitive bands assigned to form II are predominant in the KatG(S315T)-CO rR spectrum . Isoniazid binding is apparent from the resonance Raman signatures of both WT KatG-CO and KatG(S315T)-CO . Moreover, isoniazid binding elicits an increase in the form I population of wild-type KatG-CO while having little, if any, effect on the already low population of form I of KatG(S315T)-CO . Since oxyKatG (compound III) also contains a low-spin diatomic ligand-heme adduct (heme-O2), it is reasonable to suggest that it too would exist as a mixture of conformers . Because the small form I population of KatG(S315T)-CO correlates with its inability to activate INH, we hypothesize that form I plays a role in INH activation.

Ann Hematol, 2001 Apr, 80(4), 189 - 94
Thrombin converts singlet oxygen (1O2)-oxidized fibrinogen into a soluble t-PA cofactor . A new method for preparing a stimulator for functional t-PA assays; Stief TW et al.; Activated phagocytes, particularly polymorphonuclear leukocytes (neutrophils), by means of oxidative photonic burst, i.e., the combined activation of NADPH-oxidase and myeloperoxidase, generate large amounts of oxidants of the hypochlorite/chloramine type that are an important physiologic source for the nonradical, photon-emitting oxidant singlet oxygen (1O2), which (in the dark blood stream) is both a signal and an agent of defense against bacteria or fibrin . 1O2-oxidized fibrinogen or oxidized fibrin monomer has previously been shown to be unpolymerizable, and methionine to methionine sulfoxide-oxidized fibrinogen occurs in circulating blood . The present study demonstrates that thrombin converts oxidized fibrinogen into a soluble stimulator of tissue-type plasminogen activator (t-PA) . After addition of 0.1 IU thrombin to 25 microl oxidized normal human plasma and an incubation time of 10 min (room temperature), t-PA activity increases about 20-fold when compared with oxidized plasma without the addition of thrombin . Thus, since oxidized fibrin monomer is a t-PA cofactor, thrombin-degraded oxidized fibrinogen can be used as a stimulator in functional t-PA assays.

Eur Respir J, 2001 Apr, 17(4), 623 - 7
Serum procalcitonin in pneumococcal pneumonia in children; Korppi M et al.; Serum procalcitonin (PCT), a marker of bacterial infection, was measured in children with pneumonia to examine whether PCT can be used to screen pneumococcal (PNC) from viral pneumonia . The number of patients was 132; mean age 3.0 yrs, and 64% were males . In all cases, pneumonia was radiologically confirmed, being alveolar in 46 and interstitial in 86 cases . The aetiology of infection was studied by a panel of serological tests for PNC, for five other respiratory bacteria and for seven common respiratory viruses . PNC infection was found in 25, mixed viral-PNC infections in 13 and viral infection in 17 cases . In general, serum PCT was not associated with the type or aetiology of pneumonia . PCT values were >1.0 mg.L(-1) in 40% of PNC cases, as compared to 12-15% in viral or mixed cases, respectively (p<0.05) . PCT values were significantly higher in >2 yrs old children than in younger ones . The cut-off limits of 0.5 ng.mL(-1), 1.0 ng.mL(-1) and 2.0 ng.mL(-1) were tested for screening between PNC and viral pneumonia . The highest sensitivity of 55% was found at the 0.5 ng.mL(-1) cut-off level, whereas the highest specificity of 88% was reached at the level of 1.0 ng.mL(-1) . The likelihood ratios, however, were far from optimal for both the positive and negative results . Although marginally higher in pneumococcal pneumonia than in viral pneumonia, serum procalcitonin cannot be used to discriminate between these two types of pneumonia.

Eur Cytokine Netw, 2001 Apr-Jun, 12(2), 280 - 9
Inflammatory cytokine production in interferon-gamma-primed mice, challenged with lipopolysaccharide . Inhibition by SK&F 86002 and interleukin-1 beta-converting enzyme inhibitor; Spinelle-Jaegle S et al.; Mice challenged with lipopolysaccharide (LPS) produce variable serum levels of pro-inflammatory cytokines, and particularly low levels of interleukin-1 beta (IL-1 beta) . Interferon-gamma (IFN-gamma) has been shown to be an important mediator of bacteria-induced hypersensitivity to LPS in mice . In the present study, we show that mice pretreated with IFN-gamma exhibit an enhanced capacity to produce serum IL-1 beta, IL-1 alpha, tumour necrosis factor (TNF-alpha) as well as IL-6 in response to LPS . Priming with intraperitoneal (i.p.) injection of 15 mg rat recombinant IFN-gamma, 18 hours prior to the i.p . LPS (300 mg) challenge resulted in a 4-fold increase in the LPS-stimulated release of IL-1 beta and a 2- to 7-fold increase in the release of IL-1 alpha, TNF-alpha, as well as IL-6 into the serum . LPS induced a concentration-dependent increase in the release of IL-1 beta in isolated peritoneal macrophages from IFN-gamma-primed mice whereas macrophages from unprimed mice released minute amounts of IL-1 beta . In addition, nigericin markedly enhanced the release of IL-1 beta in unprimed mice but not in macrophages from IFN-gamma primed mice . The cytokine synthesis inhibitor SK&F 86002, administered per os (100 mg/kg), 1 hour prior to LPS challenge, strongly inhibited the rise in serum levels of the four cytokines . Furthermore, treatment with the IL-1 beta converting enzyme (ICE) specific reversible inhibitor YVAD-CHO resulted in a sharp dose- and time-dependent inhibition of IL-1 beta secretion in the serum, whereas the other cytokines were not affected . In conclusion, IFN-gamma priming strongly potentiates the release of proinflammatory cytokines in the serum of mice as compared to LPS stimulation alone, and provides therefore a useful way to test the in vivo potency and selectivity of cytokine synthesis inhibitors.

J Mol Biol, 2001 Jun 22, 309(5), 1007 - 15
Sfl1 functions via the co-repressor Ssn6-Tup1 and the cAMP-dependent protein kinase Tpk2; Conlan RS et al.; Ssn6 (Cyc8) is a component of the yeast general corepressor Ssn6-Tup1 that inhibits the transcription of many diversely regulated genes . The corepressor does not interact directly with DNA but is recruited to different promoters through interactions with distinct pathway-specific, DNA-binding repressor proteins . Using yeast two-hybrid and GST chromatography interaction experiments, we have determined that Sfl1, a novel repressor protein, interacts directly with Ssn6, and in vivo repression data suggest that Sfl1 inhibits transcription by recruiting Ssn6-Tup1 via a specific domain in the Sfl1 protein . Sin4 and Srb10, components of specific RNA polymerase II sub-complexes that are required for Ssn6-Tup1 repression activity, are found to be required for Sfl1 repression function . These results indicate a possible mechanism for Sfl1-mediated repression via Ssn6-Tup1 and specific subunits of the RNA polymerase II holoenzyme . Electrophoretic mobility shift and chromatin immuno-precipitation assays demonstrate that Sfl1 is present at the promoters of three Ssn6-Tup1-repressible genes; namely, FLO11, HSP26, and SUC2 . Sfl1 is known to interact with Tpk2, a cAMP-dependent protein kinase that negatively regulates Sfl1 function . Consistently, we show that phosphorylation by protein kinase A inhibits Sfl1 DNA binding in vitro, and that a tpk2Delta mutation increases the levels of Sfl1 protein associated with specific promoter elements in vivo . These data indicate a possible mechanism for regulating Sfl1-mediated repression through modulation of DNA binding by cAMP-dependent protein kinase-dependent phosphorylation . Taken together with previous data, these new observations suggest a link between cAMP signaling and Ssn6-Tup1-mediated transcriptional repression .

Vet Ophthalmol, 2000, 3(2-3), 111 - 119
Evaluation of tear film proteinases in horses with ulcerative keratitis; Strubbe DT et al.; Ulcerative keratitis is a common and potentially blinding ocular disease of horses, capable of progressing to corneal perforation in as little as 24 h . This rapid stromal degeneration is mediated in part by exogenous and endogenous proteinases . We measured and compared the concentrations of two matrix metalloproteinases (MMP-2 and MMP-9) and a serine proteinase (neutrophil elastase) present in the precorneal tear film of normal horses and horses with rapidly progressing ulcerative keratitis . Precorneal tear film samples were collected from 23 ulcerated and 21 unaffected eyes of 23 horses with unilateral ulcerative keratitis, and from 33 normal eyes of 17 control horses . MMP-2, MMP-9, and neutrophil elastase were identified by casein and gelatin zymography and quantified by computerized image analysis . Median MMP-9 levels were significantly higher in the precorneal tear film of young control horses vs . older control horses (P = 0.005) . Median MMP-2, MMP-9, and neutrophil elastase levels were significantly higher in the precorneal tear film of ulcerated eyes when compared to age-matched normal controls (P = 0.004, P = 0.001, and P = 0.012, respectively) . Median MMP-2 levels were also significantly higher in the precorneal tear film of contralateral eyes of affected horses when compared to age-matched normal controls (P = 0.004) . No significant differences in median proteinase levels were detected between 'sterile' ulcers and those from which bacteria or mixed infections (bacteria and fungi) were isolated . However, median MMP-2 and neutrophil elastase levels were significantly higher in the precorneal tear film of eyes with 'sterile' ulcers when compared with ulcerated eyes from which fungi were isolated (P < 0.05) . The results of this study support the use of topical antiproteinase therapy which targets both MMPs and serine proteinases in progressive equine ulcerative keratitis.

Vet Ophthalmol, 1999, 2(4), 207 - 211
Corneal stromal abscess in a horse; Andrew SE; A thoroughbred yearling presented with a focal, yellow, midstromal corneal opacity with concurrent iridocyclitis which was consistent with a corneal stromal abscess . When continued, appropriate, medical therapy failed to improve the patient's condition, penetrating keratoplasty was performed for diagnosis and therapy . Histopathology showed that the deep corneal stroma and Descemet's membrane were severely infiltrated with necrotic neutrophils and numerous, intralesional fungal hyphae . Culture was negative . Bacteria were not isolated, consistent with a fungal corneal stromal abscess . The resultant corneal scar did not interfere with the horse's racing career.

Vet Ophthalmol, 1998, 1(1), 31 - 39
Anterior chamber to frontal sinus shunt for the diversion of aqueous humor: a pilot study in four normal dogs; Cullen CL et al.; Silicone tubing was used to divert aqueous humor from the anterior chamber of the right eye to the rostral compartment of the right frontal sinus in four clinically normal mixed-breed dogs . Biomicroscopic examination, and pneumoapplanation tonometry and tonography, completed for up to 18 weeks postoperatively, confirmed gonioimplant function in all four cases . The dogs were euthanized at 6, 8, 16 and 18 weeks postoperatively . Gonioimplant patency was further confirmed by postmortem examination of the globes, implants and frontal sinuses . Gross and light microscopic examinations revealed iridal attachments to the implant (n = 4), mild anterior uveitis (n = 3), anterior subcapsular cataracts (n = 4), and focal corneal (n = 3) and scleral (n = 3) scarring in the operated globes . Light microscopic examination of frontal sinus specimens revealed mild lymphocytic proliferation and fibrosis immediately adjacent to the implant entrance site . There were no bacteria detected on aerobic or anaerobic cultures of the frontal sinuses or light microscopic examination of the globes or frontal sinuses . Results indicate that the frontal sinus shunting of aqueous humor is a safe and effective means of extraorbital aqueous diversion with potential applicability in the management of glaucoma.

J Mol Biol, 2001 Jun 8, 309(3), 589 - 603
The basal transcription factors TBP and TFB from the mesophilic archaeon Methanosarcina mazeii: structure and conformational changes upon interaction with stress-gene promoters; Thomsen J et al.; Transcription of archaeal non-stress genes involves the basal factors TBP and TFB, homologs of the eucaryal TATA-binding protein and transcription factor IIB, respectively . No comparable information exists for the archaeal molecular-chaperone, stress genes hsp70(dnaK), hsp40(dnaJ), and grpE . These do not occur in some archaeal species, but are present in others possibly due to lateral transfer from bacteria, which provides a unique opportunity to study regulation of stress-inducible bacterial genes in organisms with eukaryotic-like transcription machinery . Among the Archaea with the genes, those from the mesophilic methanogen Methanosarcina mazeii are the only ones whose basal (constitutive) and stress-induced transcription patterns have been determined . To continue this work, tbp and tfb were cloned from M . mazeii, sequenced, and the encoded recombinant proteins characterized in solution, separately and in complex with each other and with DNA . M . mazeii TBP ranks among the shortest within Archaea and, contrary to other archaeal TBPs, it lacks tryptophan or an acidic tail at the C terminus and has a basic N-terminal third . M . mazeii TFB is similar in length to archaeal and eucaryal homologs and all have a zinc finger and HTH motifs . Phylogenetically, the archaeal and eucaryal proteins form separate clusters and the M . mazeii molecules are closer to the homologs from Archaeoglobus fulgidus than to any other . Antigenically, M . mazeii TBP and TFB are close to archaeal homologs within each factor family, but the two families are unrelated . The purified recombinant factors were functionally active in a cell-free in vitro transcription system, and were interchangeable with the homologs from Methanococcus thermolithotrophicus . The M . mazeii factors have a similar secondary structure by circular dichroism (CD) . The CD spectra changed upon binding to the promoters of the stress genes grpE, dnaK, and dnaJ, with the changes being distinctive for each promoter; in contrast, no effect was produced by the promoter of a non-stress-gene . Factor(s)-DNA modeling predicted that modifications of H bonds are caused by TBP binding, and that these modifications are distinctive for each promoter . It also showed which amino acid residues would contact an extended TATA box with a B recognition element, and evolutionary conservation of the TBP-TFB-DNA complex orientation between two archaeal organisms with widely different optimal temperature for growth (37 and 100 degrees C) .

Arch Biochem Biophys, 2001 Jun 15, 390(2), 149 - 57
Structure and function of sulfotransferases; Negishi M et al.; Sulfotransferases (STs) catalyze the transfer reaction of the sulfate group from the ubiquitous donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to an acceptor group of numerous substrates . This reaction, often referred to as sulfuryl transfer, sulfation, or sulfonation, is widely observed from bacteria to humans and plays a key role in various biological processes such as cell communication, growth and development, and defense . The cytosolic STs sulfate small molecules such as steroids, bioamines, and therapeutic drugs, while the Golgi-membrane counterparts sulfate large molecules including glucosaminylglycans and proteins . We have now solved the X-ray crystal structures of four cytosolic and one membrane ST . All five STs are globular proteins composed of a single alpha/beta domain with the characteristic five-stranded beta-sheet . The beta-sheet constitutes the core of the Paps-binding and catalytic sites . Structural analysis of the PAPS-, PAP-, substrate-, and/or orthovanadate (VO(3-)(4))-bound enzymes has also revealed the common molecular mechanism of the transfer reaction catalyzed by sulfotransferses . The X-ray crystal structures have opened a new era for the study of sulfotransferases .

Endoscopy, 2001 May, 33(5), 443 - 7
Chronology of histological changes after band ligation of esophageal varices in humans; Polski JM et al.; BACKGROUND AND STUDY AIMS: While the histological effects of endoscopic sclerotherapy in humans have been extensively described, the effects of endoscopic ligation have been reported in only two cases . The purpose of this study was to reconstruct the chronological sequence of histological changes after ligation of esophageal varices . PATIENTS AND METHODS: Autopsy specimens from six patients who received ligation of varices from nine hours to 22 months ante-mortem were evaluated for gross and microscopic changes . RESULTS: Early after ligation, the appearance was that of a polyp with its base compressed by the band . Variceal thrombosis was seen on day 2 . Varying degrees of ischemic necrosis of the polyp were present on days 0-5 . If the bands did not remain in situ for two days (premature loss), necrosis of the polyp and dilated variceal vessels were seen . On day 22, superficial ulcers were observed . After complete healing, fibrosis was seen in the submucosa . CONCLUSIONS: The changes seen in the present study are similar to those described in animals . The delay in ulcer healing, compared with the gross changes reported during follow-up endoscopic examinations, may be related to the severity of the underlying illness and the compromised immune status of patients in the present series.

Mol Membr Biol, 2001 Jan-Mar, 18(1), 65 - 72
Recent molecular advances in studies of the concentrative Na+-dependent nucleoside transporter (CNT) family: identification and characterization of novel human and mouse proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib); Ritzel MW et al.; The human concentrative (Na+-linked) plasma membrane transport proteins hCNT1 and hCNT2, found primarily in specialized epithelia, are selective for pyrimidine nucleosides (system cit) and purine nucleosides (system cif), respectively . Both have orthologs in other mammalian species and belong to a gene family (CNT) that also includes members in lower vertebrates, insects, nematodes, pathogenic yeast and bacteria . The CNT transporter family also includes a newly identified human and mouse CNT3 transporter isoform . This paper reviews the studies of CNT transport proteins that led to the identification of hCNT3 and mCNT3, and gives an overview of the structural and functional properties of these latest CNT family members . hCNT3 and mCNT3 have primary structures that place them in a CNT subfamily separate from CNT1/2, transport a wide range of physiological pyrimidine and purine nucleosides and antineoplastic and antiviral nucleoside drugs (system cib), and exhibit a Na+:uridine coupling ratio of at least 2:1 (cf 1:1 for hCNT1/2) . Cells and tissues containing hCNT3 transcripts include mammary gland, differentiated HL-60 cells, pancreas, bone marrow, trachea, liver, prostrate and regions of intestine, brain and heart . In HL-60 cells, hCNT3 is transcriptionally regulated by phorbol myristate (PMA) . The hCNT3 gene, which contains an upstream PMA response element, mapped to 9q22.2 (cf chromosome 15 for hCNT1 and hCNT2).

J Biol Chem, 2001 Aug 10, 276(32), 30514 - 20 Epub 2001 Jun 06.
Functional groups required for the stability of yeast RNA triphosphatase in vitro and in vivo; Bisaillon M et al.; Cet1, the RNA triphosphatase component of the yeast mRNA capping apparatus, catalyzes metal-dependent gamma-phosphate hydrolysis within the hydrophilic interior of an eight-strand beta barrel (the "triphosphate tunnel"), which rests upon a globular protein core (the "pedestal") . We performed a structure-guided alanine scan of 17 residues located in the tunnel (Ser(373), Thr(375), Gln(405), His(411), Ser(429), Glu(488), Thr(490)), on the tunnel's outer surface (Ser(378), Ser(487), Thr(489), His(491)), at the tunnel-pedestal interface (Ile(304), Met(308)) and in the pedestal (Asp(315), Lys(317), Arg(321), Asp(425)) . Alanine mutations at 14 positions had no significant effect on Cet1 phosphohydrolase activity in vitro and had no effect on Cet1 function in vivo . Two of the mutations (R321A and D425A) elicited a thermosensitive (ts) yeast growth phenotype . The R321A and D425A proteins had full phosphohydrolase activity in vitro, but were profoundly thermolabile . Arg(321) and Asp(425) interact to form a salt bridge within the pedestal that tethers two of the strands of the tunnel . Mutations R321Q and D411N resulted in ts defects in vivo and in vitro, as did the double-mutant R321A-D435A, whereas the R321K protein was fully stable in vivo and in vitro . These results highlight the critical role of the buried salt bridge in Cet1 stability . Replacement of Ser(429) by alanine or valine elicited a cold-sensitive (cs) yeast growth phenotype . The S429A and S429V proteins were fully active when produced in bacteria at 37 degrees C, but were inactive when produced at 17 degrees C . Replacement of Ser(429) by threonine partially suppressed the cold sensitivity of the Cet1 phosphohydrolase, but did not suppress the cs growth defect in yeast.

J Bacteriol, 2001 Jul, 183(13), 3875 - 84
Promoters of the CATG-specific methyltransferase gene hpyIM differ between iceA1 and iceA2 Helicobacter pylori strains; Xu Q et al.; Helicobacter pylori strains can be divided into two groups, based on the presence of two unrelated genes, iceA1 and iceA2, that occupy the same genomic locus . hpyIM, located immediately downstream of either gene, encodes a functional CATG-specific methyltransferase . Despite the strong conservation of the hpyIM open reading frame (ORF) among all H . pylori strains, the sequences upstream of the ORF in iceA1 and iceA2 strains are substantially different . To explore the roles of these upstream sequences in hpyIM regulation, promoter analysis of hpyIM was performed . Both deletion mutation and primer extension analyses demonstrate that the hpyIM promoters differ between H . pylori strains 60190 (iceA1) and J188 (iceA2) . In strain 60190, hpyIM has two promoters, P(a) or P(I), which may function independently, whereas only one hpyIM promoter, P(c), was found in strain J188 . The XylE assay showed that the hpyIM transcription level was much higher in strain 60190 than in strain J188, indicating that regulation of hpyIM transcription differs between the H . pylori iceA1 strain (60190) and iceA2 strains (J188) . Since the iceA1 and iceA2 sequences are highly conserved within iceA1 or iceA2 strains, we conclude that promoters of the CATG-specific methylase gene hpyIM differ between iceA1 and iceA2 strains, which leads to differences in regulation of hpyIM transcription.

J Periodontol, 2001 May, 72(5), 626 - 33
Adhesion of Porphyromonas gingivalis strains to cultured epithelial cells from patients with a history of chronic adult periodontitis or from patients less susceptible to periodontitis; Quirynen M et al.; BACKGROUND: The present study aimed to explain the interindividual variation in periodontitis susceptibility by differences in the initial adhesion rate of Porphyromonas gingivalis to the pocket epithelium of these individuals, and/or by inter-P . gingivalis strain differences in association capacity (adhesion and internalization) . METHODS: Adhesion assays were performed on epithelial monolayers (cultured in vitro from pocket epithelium belonging to patients who were less or more susceptible to chronic adult periodontitis) using 11 genetically different clinical strains of P . gingivalis . RESULTS: Both the disease category (less susceptible versus susceptible) and the interstrain variation were found to have a significant effect (both P <0.05) on the initial bacterial association . The chronic adult periodontitis group showed significantly more association of P . gingivalis when compared to less susceptible patients (4.2 x 10(6) versus 3.5 x 10(6)) . Also, the interstrain variation was significant, with strains Pg 4 and 5 representing the least and best associating bacteria (1.8 x 10(6) colony forming units for Pg 4, 9 x 10(6) for Pg 5) . CONCLUSIONS: These results indicate that periodontitis susceptibility is influenced by both the interindividual differences in pocket epithelium (allowing more adhesion of P . gingivalis) or by the strain type by which the patient is infected (intra-species differences in adhesion capacity).

J Periodontol, 2001 May, 72(5), 590 - 7
Expression of cytokines and inducible nitric oxide synthase in inflamed gingival tissue; Hirose M et al.; BACKGROUND: Periodontopathic bacteria induce inflammation of periodontal tissues . The cytokines and nitric oxide released in periodontal lesions have been reported to play a protective role in bacterial infection and to relate to the process of inflammation . To clarify the relationship between colonization of periodontopathic bacteria and cytokines, we evaluated profiles of inflammatory cytokines, chemokine, anti-inflammatory cytokines, and inducible nitric oxide synthase (iNOS) and colonization by Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, which are major pathogens of periodontitis . METHODS: mRNA expression of cytokines and iNOS in inflamed and healthy gingival tissue was determined using reverse transcription-polymerase chain reaction (RT-PCR), and the relationship between their profiles and the detection of specific bacteria was analyzed . RESULTS: The relative expression of interleukin (IL)-6 and iNOS mRNAs in periodontal lesions was significantly higher than those in healthy individuals . IL-6 mRNA expression was also significantly higher at bleeding on probing (BOP)-positive sites than at BOP-negative sites . The expressions of IL-1alpha and IL-8 increased, but IL-10 expression decreased at sites where A . actinomycetemcomitans was detected . We found no correlation between the expression of cytokine and iNOS mRNA and infection by P . gingivalis . CONCLUSIONS: The expression of IL-6 may reflect inflammation in gingival tissue, and iNOS may be involved in the inflammatory process in periodontitis . The presence of A . actinomycetemcomitans or P . gingivalis might relate to the different cytokine profiles of IL-1alpha, IL-8, and IL-10.

Indoor Air, 2001 Jun, 11(2), 127 - 33
Irritants and allergens at school in relation to furnishings and cleaning; Smedje G et al.; In order to study the influence of furnishings and cleaning on the indoor air quality at school, 181 randomly chosen classrooms were investigated . The amounts of open shelves, textiles and other fittings were noted, data were gathered on cleaning routines, and a number of pollutants were measured in the classrooms . In classrooms with more fabrics there was more settled dust and the concentration of formaldehyde was higher . Classrooms with more open shelves had more formaldehyde, and more pet allergens in settled dust, and classrooms with a white board, instead of a chalk board, were less dusty . Classrooms mainly cleaned through wet mopping had more airborne viable bacteria but less settled dust than classrooms mainly cleaned by dry methods . In rooms where the desks and curtains were more often cleaned, the concentrations of cat and dog allergen in settled dust were lower . It is concluded that furnishings and textiles in the classroom act as significant reservoirs of irritants and allergens and have an impact on the indoor air quality at school.

Environ Sci Technol, 2001 May 15, 35(10), 2022 - 5
Spectrophotometric assay of POE nonionic surfactants and its application to surfactant sorption isotherms; Brown DG et al.; The operational range and suitability toward environmental samples of an iodine-iodide (I-I) assay for nonionic surfactants were assessed . The I-I assay provides a rapid and repeatable method for determining aqueous nonionic surfactant concentrations . Through a systematic examination of surfactant structure, the operational range of the assay was shown to be on the order of 10(-6) to 10(-3) MEO, where the concentration unit MEO is defined as the molar surfactant concentration multiplied by the number of ethylene oxide units in the surfactant molecule . For environmental samples, it was shown that the I-I assay can be applied to measurement of surfactant sorption isotherms to aquifer sands and bacteria cultures . A potential limitation of the I-I assay is interference with humic acids, with the magnitude of the interference dependent on the concentration of humic acids present . The main benefit of the I-I assay is that its high accuracy and ease of application allows measurement of low levels of surfactant sorption . Surfactant sorption to aquifer sand could be measured down to the range of 10(-9) mol/g.

Cancer Gene Ther, 2001 Apr, 8(4), 252 - 8
Preclinical and therapeutic utility of HVJ liposomes as a gene transfer vector for hepatocellular carcinoma using herpes simplex virus thymidine kinase; Hasegawa H et al.; Although gene therapy has been suggested to be a novel strategy to treat hepatocellular carcinoma (HCC), no study showing the clinical feasibility of vectors to treat HCC has been reported . In this preclinical study, we show evidence indicating that hemagglutinating virus of Japan (HVJ) liposomes are a feasible vector to treat HCC in a clinical setting using ganciclovir (GCV) and herpes simplex virus thymidine kinase (HSV-tk), which is driven by the cytomegalovirus immediate early enhancer/promoter (plasmid pcDNA3/HSV-tk) . In in vitro experiments, almost complete tumor cell regression was achieved with the optimal GCV concentration (100 microg/mL) and more than 1/3 regression was seen even with a 20% transduction ratio using HuH7 HCC cells stably transformed by HSV-tk . HVJ liposomes showed a 19.7% (mean) transduction rate of the lacZ gene in a relatively large mass of more than 300 mm3 in vivo, which is a clinically detectable size, implanted into SCID mice . Moreover, a single HSV-tk injection of HVJ liposomes followed by GCV treatment inhibited tumor growth at least within a week, and repeat administration was more effective . Furthermore, subcutaneous injection of an HVJ liposomes vehicle induced no apparent inflammatory response in C3H/HeN mice, whereas lacZ gene transfection resulted in inflammatory pathology, suggesting a lower immunogenicity of the HVJ envelope protein than those of bacteria-derived plasmid DNA or the beta-galactosidase gene product . From these findings, we conclude that HVJ liposomes are a clinically safe and effective gene transfer vector to treat HCC.

J Formos Med Assoc, 2001 Mar, 100(3), 192 - 7
Cadaveric study of blood supply to the lower intraorbital fat: etiologic relevance to the complication of anaerobic cellulitis in orbital floor fracture; Chien HF et al.; BACKGROUND AND PURPOSE: Although orbital fractures are common, orbital cellulitis rarely develops following orbital fracture . We hypothesized that compromise of the blood supply to the intraorbital fat during orbital floor fracture is responsible for this condition . The purpose of this study was to determine whether or not the lower intraorbital fat is supplied by a branch of the infraorbital artery along the orbital groove or canal on the orbital floor . MATERIALS AND METHODS: We dissected 14 orbits from seven fixed human cadavers and 12 orbits from six fresh cadaver heads following dye injection into the maxillary artery . The sites of dye-filled vessels branching from the infraorbital artery supplying the lower intraorbital fat were measured and plotted on a two-dimensional orbital floor graph . RESULTS: A main branch of the infraorbital artery rose through the medial orbital floor to supply the lower intraorbital fat in all of the cadaver orbits . The sites of the branching point of the vessel ranged from 0 to 5 mm (mean, 2.2 mm; n = 14) medial to the line connecting the infraorbital foramen and the infraorbital groove . The shortest distance measured from the branching point to the orbital rim ranged from 3 to 20 mm (mean, 14.1 mm; n = 14) . This suggests that if orbital fracture were to occur around the infraorbital groove or canal, this vascular pedicle would be in danger of being incarcerated by bone fragments . CONCLUSION: Our cadaveric investigation revealed that the lower intraorbital fat is supplied by a branch of the infraorbital artery along the infraorbital groove or canal on the orbital floor . This finding suggests that compromised blood supply to the intraorbital fat may cause anaerobic cellulitis or enophthalmos.

Curr Microbiol, 2001 Aug, 43(2), 79 - 82
A rapid method for determining the tuberculocidal activity of liquid chemical germicides; Erickson BD et al.; A rapid, quantitative method has been developed for determining the tuberculocidal activity of liquid chemical germicides . In this method, a test strain of Mycobacterium bovis that carries the firefly luciferase gene is exposed to a germicide, and the surviving bacteria are detected by bioluminescence . The tuberculocidal activities of five commercially available glutaraldehyde-based disinfectants were tested, and all reduced the number of surviving mycobacteria by greater than five orders of magnitude . In contrast, a phenol-based disinfectant with tuberculocidal claims gave less than one order of magnitude reduction of the test organism . With this method for determining tuberculocidal activity, results can be obtained in less than one day, compared with weeks or months for the standard tuberculocidal assays.

News Physiol Sci, 1998 Jun, 13, 137 - 142
The Dichotomy of MIP Family Suggests Two Separate Origins of Water Channels; Ishibashi K et al.; MIP family proteins can be divided into two groups according to their primary sequences . The CHIP group is predominant in the plant and animal kingdoms and functions primarily as water channels . The GLP group is a minor group with limited prevalence and functions primarily as glycerol transporters . Both prototypes are present in bacteria and may have evolved separately.

Microbiology, 2001 Jun, 147(Pt 6), 1451 - 60
Identification of lipopolysaccharide O antigen synthesis genes required for attachment of the S-layer of Caulobacter crescentus; Awram P et al.; The outer surface of Caulobacter crescentus consists of a two-dimensional crystalline protein lattice layer (S-layer) . A fraction of the LPS has an O antigen polymer attached to the core to form a 'smooth' LPS (S-LPS), which is required for attachment of the protein S-layer to the outer-membrane surface . A method to screen for strains defective in LPS production, based on loss of S-layer attachment, was developed and applied to libraries of transposon-generated mutants . Eighteen distinct insertions were found with transposon interruptions in genes affecting S-LPS production, 12 of which were located near the S-layer subunit protein gene, rsaA, and its transporter genes . Sequence adjacent to transposon insertion points was determined and used to search a C . crescentus genome database . Twelve ORFs likely to be involved in S-LPS synthesis were identified . Seven of the predicted ORFs were linked to rsaA . Six of the putative genes had identity with proteins involved in synthesis of sugar residues, including five predicted to make perosamine . The remaining six ORFs were similar to glycosyltransferases involved in forming linkages between sugar residues in the O antigen, while one may be a transcription repressor . Other chemical and preliminary proton NMR studies of the S-LPS O antigen indicate that it contains an N-acetylated 4,6-dideoxy-4-aminohexose, but is not assembled as a simple, uniform homopolymer, consisting of several different linkages between sugar residues . The ORFs described here include homologues of all the enzymes involved in the synthesis of N-acetylperosamine, a 4,6-dideoxy-4-aminohexose . Overall, the data are consistent with the hypothesis that the O antigen of C . crescentus S-LPS consists primarily of N-acetylperosamine residues polymerized with multiple anomeric linkages.

Microbiology, 2001 Jun, 147(Pt 6), 1425 - 36
Visualization of Borrelia burgdorferi sensu lato by fluorescence in situ hybridization (FISH) on whole-body sections of Ixodes ricinus ticks and gerbil skin biopsies; Hammer B et al.; The objective of this study was to visualize borreliae directly in whole-body sections of Ixodes ricinus by fluorescence in situ hybridization (FISH) . Borrelia afzelii mono-infected or Borrelia burgdorferi sensu stricto (ss)/B . afzelii double-infected nymphs were fixed, embedded in cold polymerizing resin and sectioned . The same sample processing was applied to skin biopsies taken from a Mongolian gerbil after an infectious tick-bite . FISH was carried out using 16S-rRNA-directed, fluorescence-labelled oligonucleotide probes specific for the genus Borrelia and specific within the group of Lyme borreliosis-associated genospecies B . afzelii, B . burgdorferi ss, Borrelia garinii and Borrelia valaisiana . Sensitivity and specificity of the newly designed probes were evaluated using PCR, dot-blot hybridizations and FISH . Despite significant autofluorescence of certain tick tissues (which allowed good histological orientation within the sections), borreliae showing the typical spirochaetal morphotype were clearly visible in five out of six putatively infected ticks . These findings were confirmed by electron microscopy of ticks from the same infected batch as used for FISH . Attempts to produce ticks infected by two different Borrelia genospecies were not successful . FISH on whole-body sections of resin-embedded ticks offers the possibility of visualizing and identifying borreliae within tick tissues . This technique has great potential for the investigation of the transmission of bacteria or other micro-organisms by arthropod vectors . Furthermore, clear visualization of single spirochaetes distributed along subcutaneous fat cell membranes in gerbil skin biopsies suggests that FISH might also be suitable for the detection of borreliae in clinical tissue specimens.

J Immunol, 2001 Jun 15, 166(12), 7381 - 8
Fc receptor-mediated phagocytosis makes a significant contribution to clearance of influenza virus infections; Huber VC et al.; Fc receptors for IgG expressed on macrophages and NK cells are important mediators of opsonophagocytosis and Ab-dependent cell-mediated cytotoxicity . Phagocyte-mediated opsonophagocytosis is pivotal for protection against bacteria, but its importance in recovery from infection with intracellular pathogens is unclear . We have now investigated the role of opsonophagocytosis in protection against lethal influenza virus infection by using FcR gamma(-/-) mice . Absence of the FcR gamma-chain did not affect the expression of IFN-gamma and IL-10 in the lungs and spleens after intranasal immunization with an influenza subunit vaccine . Titers of serum and respiratory Abs of the IgM, IgG1, IgG2a, and IgA isotypes in FcR gamma(-/-) mice were similar to levels seen in FcR gamma(+/+) mice . Nevertheless, FcR gamma(-/-) mice were highly susceptible to influenza infection, even in the presence of anti-influenza Abs from immune FcR gamma(+/+) mice . NK cells were not necessary for the observed Ab-mediated viral clearance, but macrophages were found to be capable of actively ingesting opsonized virus particles . We conclude that Fc receptor-mediated phagocytosis plays a pivotal role in clearance of respiratory virus infections.

Biochem Pharmacol, 2001 Jul 15, 62(2), 255 - 9
Oxidation of hydrogen sulfide and methanethiol to thiosulfate by rat tissues: a specialized function of the colonic mucosa; Furne J et al.; Colonic bacteria release large quantities of the highly toxic thiols hydrogen sulfide (H(2)S) and methanethiol (CH(3)SH) . These gases rapidly permeate the colonic mucosa, and tissue damage would be expected if the mucosa could not detoxify these compounds rapidly . We previously showed that rat cecal mucosa metabolizes these thiols via conversion to thiosulfate . The purpose of the present study in rats was to determine if this conversion of thiols to thiosulfate is (a) a generalized function of many tissues, or (b) a specialized function of the colonic mucosa . The tissues studied were mucosa from the cecum, right colon, mid-colon, ileum, and stomach; liver; muscle; erythrocytes; and plasma . The metabolic rate was determined by incubating homogenates of the various tissues with H(2)(35)S and CH(3)(35)SH and measuring the rate of incorporation of (35)S into thiosulfate and sulfate . The detoxification activity of H(2)S (expressed as nmol/mg per min) that resulted in thiosulfate production was at least eight times greater for cecal and right colonic mucosa than for the non-colonic tissues . Thiosulfate production from CH(3)SH was at least five times more rapid for cecal and right colonic mucosa than for the non-colonic tissues . We conclude that colonic mucosa possesses a specialized detoxification system that allows this tissue to rapidly metabolize H(2)S and CH(3)SH to thiosulfate . Presumably, this highly developed system protects the colon from what otherwise might be injurious concentrations of H(2)S and CH(3)SH . Defects in this detoxification pathway possibly could play a role in the pathogenesis of various forms of colitis.

J Antimicrob Chemother, 2001 Jun, 47(6), 829 - 35
Chemotherapy of Mycobacterium tuberculosis infections in mice with a combination of isoniazid and rifampicin entrapped in Poly (DL-lactide-co-glycolide) microparticles; Dutt M et al.; Strategies to improve patient compliance in tuberculosis chemotherapy include the use of sustained release drug delivery systems . In this study, Poly (DL-lactide-co-glycolide) (PLG) microparticles containing a combination of isoniazid and rifampicin were developed as sustained release carrier systems . A single dose of PLG microparticles exhibited a sustained release of isoniazid and rifampicin in vivo up to 7 and 6 weeks, respectively . Free drugs (in combination) injected in the same doses were detectable in vivo up to 24 h only . One dose of PLG microparticles cleared bacteria more effectively from lungs and liver in an experimental murine model of tuberculosis after low-dose PLG combination drug therapy and in liver after high-dose PLG combination drug therapy as compared with a daily administration of the free drugs . These results suggest that PLG microparticles offer an improvement for tuberculosis chemotherapy over the conventional treatment.

Hum Reprod, 2001 Jun, 16(6), 1151 - 4
Distribution of a spermicide containing Nonoxynol-9 in the vaginal canal and the upper female reproductive tract; Barnhart KT et al.; Topical, intravaginal microbicides and spermicides are greatly needed to prevent transmission of sexually transmitted diseases and/or unwanted pregnancies . The development of such compounds is a high research priority . The presumed method of action of existing, or novel, microbicides/spermicides is to provide a chemical barrier to the vaginal epithelium preventing exposure to micro-organisms . Other intravaginal products are used to treat vaginal bacteria of fungal infections . Little is known, however, about the actual or optimal initial distribution and subsequent spread of medications placed in the vagina . We describe a sensitive new technique to quantify the spread of a gel placed in the vagina using magnetic resonance imaging (MRI) . Five millilitres of an over-the-counter spermicide containing Nonoxynol-9 was mixed with Gadolinium . MRI was used to quantify spread of the mixture 10 min after insertion with a standard applicator . We demonstrated contiguous spread of gel throughout the vagina . The coverage of material was thicker in the upper vagina than in the lower vagina . We also demonstrated, for the first time, that spermicidal compounds may migrate from the vaginal canal into the endocervix within 10 min of insertion . This finding suggests that topical microbicides/spermicides may act both in the vaginal canal and in the upper female genital tract.

Hum Reprod, 2001 Jun, 16(6), 1110 - 4
Semen sample collection in medium enhances the implantation rate following ICSI in patients with severe oligoasthenoteratozoospermia; Zollner U et al.; Ejaculation in medium increases the proportion of antibody-free spermatozoa in semen samples containing anti-sperm antibodies and thereby enhances the fertilization rate in vitro . The aim of this study was to investigate whether this technique is also beneficial in semen samples with severe oligoasthenoteratozoospermia (OAT) where bacteria and detritus are often present . A prospective randomized controlled trial was carried out to study the results of sperm preparation and fertilization and pregnancy rates after intracytoplasmic sperm injection (ICSI) for OAT . Of the 114 couples (one cycle per couple) studied between 1998 and 2000, 55 men were randomized to have semen collection into sterile dry pots (group A) and the remaining 59 had samples collected into 20 ml HEPES buffered Ham's F-10 medium with 10% human serum albumin (group B) . In group B the ejaculates were incubated for 30 min and mixed gently . The samples were then processed by mild centrifugation and washing followed by a mini-swim-up technique . The ejaculates in group A were prepared by the swim-up procedure only . The overall fertilization rate was 71.8% and was similar in groups A (fertilization rate = 66.7%) and B (fertilization rate = 64.3%) . In group A, 10/55 clinical pregnancies were recorded (pregnancy rate 18%), with an implantation rate (IR) of 6.9% per embryo . In group B, 16 of 59 patients conceived leading to significantly higher implantation (9.9%, P < 0.001) and clinical pregnancy rates (27%, P < 0.001) . It is postulated that the addition of medium before liquefaction could inhibit the binding of bacteria and detritus to the sperm surface and may diminish DNA damage caused by reactive oxygen species, leading to improved efficiency of fertilization . The results demonstrate that the addition of HEPES buffered Ham's F-10 medium to sample collection pots significantly improves the pregnancy rate after ICSI in patients with severe OAT.

FASEB J, 2001 Jun, 15(8), 1398 - 403
Hypothesis: inappropriate colonization of the premature intestine can cause neonatal necrotizing enterocolitis; Claud EC et al.; Neonatal necrotizing enterocolitis (NEC) is a major cause of morbidity in preterm infants . We hypothesize that the intestinal injury in this disease is a consequence of synergy among three of the major risk factors for NEC: prematurity, enteral feeding, and bacterial colonization . Together these factors result in an exaggerated inflammatory response, leading to ischemic bowel necrosis . Human milk may decrease the incidence of NEC by decreasing pathogenic bacterial colonization, promoting growth of nonpathogenic flora, promoting maturation of the intestinal barrier, and ameliorating the proinflammatory response.

EMBO J, 2001 Jun 1, 20(11), 2931 - 42
Determinants for hairpin formation in Tn10 transposition; Allingham JS et al.; Tn10 transposition involves the formation of a hairpin intermediate at the transposon termini . Here we show that hairpin formation exhibits more stringent DNA sequence requirements at the terminal two base pairs than either transpososome assembly or first strand nicking . We also observe a significant DNA distortion at the terminal base pairs upon transpososome assembly by chemical nuclease footprinting . Interestingly, mutations at these positions do not necessarily inhibit the formation of the distortion . However, it remains a possibility that the inhibitory effect of these mutations is due to a defect in protein-DNA interactions subsequent to this deformation . Terminal base pair mutations also inhibited strand transfer, providing evidence that transposase interactions with the terminal residues on both 'transferred' and 'non-transferred' strands are important for hairpin formation . We also demonstrate that mutation of a highly conserved tyrosine residue that is a component of the YREK motif, Y285, results in a phenotype comparable to that of the terminal base pair mutations . In contrast, a mutation at another conserved position, W265, is shown to relax the specificity of the hairpin formation reaction.

EMBO J, 2001 Jun 1, 20(11), 2923 - 30
Tipping the balance between replicative and simple transposition; Tavakoli NP et al.; The bacterial insertion sequence IS903 has the unusual ability to transpose both replicatively and non-replicatively . The majority of products are simple insertions, while co-integrates, the product of replicative transposition, occur at a low frequency (<0.1% of simple insertions) . In order to define the critical steps that determine the outcome of IS903 transposition, we have isolated mutants that specifically increase the rate of replicative transposition . Here we show that the nucleotide immediately flanking the transposon influences both overall transposition frequency and co-integrate formation . In particular, when the 3'-flanking nucleotide is A, co-integrates are increased 500-fold compared with a 3' C . In addition, we have isolated five transposase mutants that increase replicative transposition . These residues are close to the catalytic residues and are thus likely to be part of the active site . These are the first transposase mutations described that affect the product of transposition . Our results are consistent with the hypothesis that a delay in cleavage of the 5'-flanking DNA will increase the effective half-life of the 3'-nicked transposon intermediate and consequently enhance co-integrate formation.

Parasitology, 2000, 121 Suppl, S97 - 111
Animal models of intestinal nematode infections of humans; Boes J et al.; In this paper we discuss several established and potential animal models for human parasitic infection, with a focus on rodent, pig and primate models and the nematodes Ascaris, Trichuris and Toxocara spp . Firstly, we discuss the relevance of choosing a suitable animal host to fit the particular study hypothesis, and the interaction between mathematical modelling and animal models . Secondly, we review the use of animal models for the study of nutrition-parasite interaction, evaluation of treatment and control strategies, and bacteria-parasite interactions . We show that rodent, pig and primate models are all very useful in parasitological research, and that each model has its limitations . However, based on recent experience with the pig-Ascaris and pig-Trichuris models, a more extensive use of the pig-parasite model is advocated, especially for the study of the interaction between human malnutrition and helminth infection, and congenital helminth infection.

Antonie Van Leeuwenhoek, 2000 Dec, 78(3-4), 253 - 61
Glucose kinase of Streptomyces coelicolor A3(2): large-scale purification and biochemical analysis; Mahr K et al.; Glucose kinase of Streptomyces coelicolor A3(2) is essential for glucose utilisation and is required for carbon catabolite repression (CCR) exerted through glucose and other carbon sources . The protein belongs to the ROK-family, which comprises bacterial sugar kinases and regulators . To better understand glucose kinase function, we have monitored the cellular activity and demonstrated that the choice of carbon sources did not significantly change the synthesis and activity of the enzyme . The DNA sequence of the Streptomyces lividans glucose kinase gene glkA was determined . The predicted gene product of 317 amino acids was found to be identical to S . coelicolor glucose kinase, suggesting a similar role for this protein in both organisms . A procedure was developed to produce pure histidine-tagged glucose kinase with a yield of approximately 10 mg/l culture . The protein was stable for several weeks and was used to raise polyclonal antibodies . Purified glucose kinase was used to explore protein-protein interaction by surface plasmon resonance . The experiments revealed the existence of a binding activity present in S . coelicolor cell extracts . This indicated that glucose kinase may interact with (an)other factor(s), most likely of protein nature . A possible cross-talk with proteins of the phosphotransferase system, which are involved in carbon catabolite repression in other bacteria, was investigated.

Antonie Van Leeuwenhoek, 2000 Dec, 78(3-4), 243 - 51
Sugar uptake and utilisation in Streptomyces coelicolor: a PTS view to the genome; Parche S et al.; Our research group is studying the phosphotransferase system (PTS) of Streptomyces coelicolor, which, in other bacteria, is centrally involved in carbon source uptake and regulation . We have surveyed the public available S . coelicolor genome sequence produced by the ongoing genome sequencing project for pts gene homologues . Three genes encoding homologues of the general PTS components enzyme I (ptsI), HPr (ptsH), and enzyme IIA(Crr) (crr; IIA(Glc)-homologue) and six genes encoding homologues of sugar-specific PTS components were identified . The deduced primary sequences of the sugar-specific components shared significant similarities to PTS permeases of the mannitol/fructose family and of the glucose/sucrose family . A model is presented, in which possible functions of the novel described PTS homologues are discussed.

Water Sci Technol, 2000, 41(3), 33 - 41
Anaerobic batch degradation of solid poultry slaughterhouse waste; Salminen E et al.; We studied anaerobic batch degradation of solid poultry slaughterhouse wastes with different initial waste and inoculum concentrations and waste-to-inoculum ratios and simulated the dynamics of the process with a new generation <METHANE> model . Our modelling results suggest that inhibited propionate degradation by long-chain fatty acids (LCFA) and inhibited hydrolysis by a high propionate concentration constituted the rate-limiting step in the waste degradation . Palmitate was the most abundant LCFA in the assays . Within 27 days of incubation, up to 0.55 to 0.67 m3 of methane (STP)/kg VS added was produced under the studied conditions . Lower waste-to-inoculum ratios exhibited a faster onset and rate of specific methane production . In all the assays, ammonification occurred within 3 to 6 days and accounted for 50 to 60% of total nitrogen.

Mikrobiologiia, 2001 Mar-Apr, 70(2), 215 - 25
{Biogeochemical processes of methane cycle in the soils, swamps and lakes of Western Siberia}; Gal'chenko VF et al.; The biogeochemical processes of methane production and oxidation were studied in the upper horizons of tundra and taiga soils and of raised bogs and lake bottom sediments nearby the Tarkosalinsk gas field in western Siberia . Both in dry and water-logged soils, the total methane concentration (in soil particles and gaseous phase) was an order of magnitude higher than in the soil gaseous phase alone (22 and 1.1 nl/cm3, respectively) . In bogs and lake bottom sediments, methane concentration was as high as 11 microliters/cm3 . Acetate was the major precursor of the newly formed methane . The rate of aceticlastic methanogenesis reached 55 ng C/(cm3 day), whereas that of autotrophic methanogenesis was an order of magnitude lower . The most active methane production and oxidation were observed in bogs and lake sediments where the delta 13C values of CO2 were inversely related to the intensity of bacterial methane oxidation . Methane diffusing from bogs and lake bottom sediments showed delta 13C values ranging from -78 to -47@1000, whereas the delta 13C value of carbon dioxide ranged from -18 to -6@1000 . In these ecosystems, methane emission comprised from 3 to 206 mg CH4/(m2 day) . Conversely, the dry and water-logged soils of tundra and taiga took up atmospheric methane at a rate varying from 0.3 to 5.3 mg CH4/(m2 day) . Methane consumption in soils was of biological rather than of adsorptive nature . This was confirmed by the radioisotopic method and chamber experiments, in which weighting of methane carbon was observed (the delta 13C value changed from -51 to -41@1000).

Eur J Immunol, 2001 Jun, 31(6), 1674 - 84
Immune evasion of Borrelia burgdorferi by acquisition of human complement regulators FHL-1/reconectin and Factor H; Kraiczy P et al.; To understand immune evasion mechanisms of Borrelia burgdorferi we compared serum-resistant B . afzelii and serum-sensitive B . garinii isolates for their capacity toacquire human complement regulators . Here we demonstrate that the two borrelial genospecies show different binding of the two important human complement regulators, FHL-1/reconectin and Factor H . All serum-resistant B . afzelii isolates bound FHL-1/reconectin and also Factor H, and all analyzed serum-sensitive B . garinii isolates showed no or a significantly lower binding activity . Using recombinant deletion mutants, the binding domains were localized to the C terminus of FHL-1/reconectin to short consensus repeats 5-7 . The borrelial binding proteins were located in the surface of the bacteria as demonstrated by immunofluorescence staining of intact, serum-exposed bacteria and by enrichment of outer membrane proteins . The surface-attached complement regulators maintained complement regulatory activity as demonstrated in a cofactor assay . By ligand blotting two different borrelial binding proteins were identified that were responsible for the surface attachment of FHL-1/reconectin and Factor H . These borrelial complement regulators acquiring surface proteins (CRASP) were further characterized as either CRASP-1, a 27.5-kDa molecule which preferentially binds FHL-1/reconectin and which was present in all serum-resistant borreliae, or CRASP-2, a 20/21-kDa protein which interacts preferentially with Factor H and the expression of which was more restricted, being detected in four of the six isolates analyzed . In summary, we describe a new immune evasion mechanism of B . burgdorferi, as these bacteria acquire human complement regulators to control complement activation on their surface and to prevent formation of toxic activation products.

Environ Pollut, 2001, 113(2), 155 - 62
Monitoring laboratory-scale bioventing using synchronous scan fluorescence spectroscopy: analysis of the vapor phase; Bachman J et al.; Bioventing is an improved method of soil remediation that is being used with increasing frequency . In this paper, we refine techniques to measure the progress of petroleum hydrocarbon decomposition by monitoring vapor phase composition with synchronous scan fluorescence spectroscopy (SSFS) . Analysis of the vapor phase has advantages compared to standard extraction techniques that require extensive sample handling and clean up . For comparison, hydrocarbon contamination in the soil was measured by analysis of Soxhlet extractions with gas chromatography-mass spectrometry (GC-MS) . Comparison of the GC-MS and SSFS data showed that changes in hydrocarbon composition measured in the vapor phase provide an accurate measure of decomposition reactions taking place in the soil.

Shokuhin Eiseigaku Zasshi, 2001 Feb, 42(1), 7 - 12
Flavor production from a non-stick oil by moulds; Fujikawa H et al.; Natural flavor was accidentally produced from rice cake products in Japan . A non-stick oil had been sprayed on the products during the production process . It was found that a Penicillium corylophilum strain, a contaminant of the oil, produced the flavor from the oil . The ingredients of the flavor were four volatile substances, 2-heptanone, 2-nonanone, 2-heptanol, and 2-nonanol . Challenge tests with the mould strain in a rice cake system were performed under various conditions . The volatile substances were produced in the largest amounts at 25 degrees C, followed by 20 or 30 degrees C then 10 degrees C . 2-Heptanone was produced most remarkably at 25 degrees C, followed by 2-nonanone, 2-heptanol, and 2-nonanol . The growth patterns of the mould were similar between 20-30 degrees C, and the growth at 10 degrees C was delayed . The non-stick oil itself had neither flavor nor volatile substance . The flavor was also produced from coconut oil, which was one of the materials of the non-stick oil . No bacteria or yeasts tested produced any flavor from the non-stick oil, whereas most of the moulds tested produced flavor components.

Int J Radiat Biol, 2001 May, 77(5), 617 - 23
Mechanism of protection against radiation-induced DNA damage in plasmid pBR322 by caffeine; Kumar SS et al.; PURPOSE: Caffeine (1,3,7-trimethyl xanthine), a dietary component, has been shown to have widely varying effects on DNA damage induced by UV and ionizing radiation, depending upon pre- or post-irradiation administration and its concentration . Caffeine administered post-UV irradiation is known to inhibit enzymatic repair of DNA lesions, leading to potentiation of damage, whereas its presence before or during irradiation elicits protection in a wide range of test systems: bacteria, cultured human cells, plant seeds and mouse . The purpose of this study is to test whether caffeine present during gamma-irradiation of plasmid DNA, a system devoid of replication and repair, could elicit protection by scavenging free radicals . MATERIALS AND METHODS: Plasmid pBR322 DNA was exposed to gamma-radiation in the presence or absence of caffeine at a dose-rate of 1.20 Gy min(-1) and damage measured as single-strand breaks . To understand the mechanisms of the observed protection, especially under oxic conditions, reaction of caffeine with superoxide radical (O(2)(-)), hydrogen peroxide (H(2)O(2)) and the deoxyribose peroxyl radical (ROO(*)) were studied . RESULTS: Irradiation of pBR322 was observed to induce a dose-dependent increase in single-strand breaks . Caffeine itself did not induce strand breaks but reduced radiation-induced strand breaks at micromolar to millimolar concentrations . Caffeine has been shown to react with the radiation-derived oxidants . The reaction rate constants observed were 7.5x10(1) M(-1) s(-1) with O(2)(-) 1.05x10(8) M(-1) s(-1) with ROO(*) and 8.8x10(1) M(-1) s(-1) with H(2)O(2) . CONCLUSIONS: Caffeine effectively protects DNA against ionizing radiation in a system devoid of repair and replication machinery . Thus, DNA protection shown by caffeine is possibly due to the scavenging of radiation-derived primary as well as secondary reactive oxygen species, and this physicochemical protective pathway possibly pre-empts any subsequent inhibitory effect of caffeine on the enzymatic repair of DNA.

Hautarzt, 2001 Apr, 52(4), 316 - 21
{What remains of the skin after 2000 years in a bog?}; Stucker M et al.; BACKGROUND AND OBJECTIVE: Mummies have an important place in the study of archaeology and paleopathology because they are so well preserved . For the first time skin samples of six 2300-1600 year old bog bodies from North Germany were examined by histology, transmission electron microscopy and immunohistology . METHODS: For histology the samples were stained with H&E and van Gieson elastic stain . Fixation and embedding in epoxy followed for the transmission electron microscopy . Specific antibodies directed to type IV collagen and S-100 were used . RESULTS: Histologically it was possible to observe collagen bundles in the dermis, with a density similar to recently stained samples . Epidermis was not preserved . The electron microscopy showed collagen fibrils with a diameter of 45-110 nm and the characteristic axial periodicity . Throughout the dermis, a number of spores of bacteria with a diameter of 0.83 +/- 0.051 micron and an electron dense core were found . No activity against the used antibodies could be detected . CONCLUSIONS: Histology and electron microscopy demonstrate the excellent conservation of the dermal collagen in the bog . In contrast to ice mummies like "Otzi" and mummies from Egypt, no cellular elements could be found in the skin of bog bodies.

Water Sci Technol, 2000, 41(3), 93 - 100
Bench-scale anaerobic bioconversion of newsprint and office paper; Clarkson WW et al.; Anaerobic bioconversion of newsprint and waste office paper was performed in bench-scale reactors with three inocula sources: landfill, rumen, and anaerobic digester . Office paper bioconversion was nearly complete within 20 days but continued for about 165 days with methane yield efficiencies ranging from 71-85% of potential chemical oxygen demand (COD) conversion . Average newsprint methane conversion efficiencies ranged from 32-41% of total COD under strictly anaerobic conditions for 300 days . Mass balance calculations revealed that more than 80% of newsprint cellulose was biodegraded . The apparent limiting factor for anaerobic bioconversion of newsprint was the physical association between lignin and cellulose . After proper acclimation, the three inocula tested equally well for methane production under strictly anaerobic conditions . Testing of ground, shredded strips, and whole paper pieces showed no effects of feedstock size on bioconversion rate or extent . Alkali pretreatment with NaOH concentration up to 10% significantly improved newsprint biodegradability . Treatment for longer duration or at elevated temperatures increased the solubilization of lignin, but did not improve bioconversion of newsprint to methane . Neutralizing treated samples with carbon dioxide gave higher methane yields compared to sulfuric or hydrochloric acids, suggesting that digester neutralization could be combined with biogas scrubbing.

Water Sci Technol, 2000, 41(3), 299 - 304
Full scale experience with the BIOCEL process; ten Brummeler E; The BIOCEL process is a mesophilic dry anaerobic batch digestion system for solid organic wastes . In the BIOCEL process organic solid wastes, such as source separated organic fraction of MSW (biowaste) is converted into enriched compost and biogas . In the process net energy production is achieved by converting the biogas to heat and power with a heat-electric power production unit . In September 1997 the first full scale plant is started-up in Lelystad, The Netherlands . This plant is processing 50,000 tons of biowaste (organic fraction of MSW from source separation) per year . The plant has a net energy production and therefore contributes to prevention of CO2 emissions from fossil fuels . In the BIOCEL-system the several compost fractions are produced with a "wet" separation process . During the wet separation sand and contaminants are removed . An important aspect of compost quality is the absence of several types of pathogens . It appears that anaerobic digestion with the BIOCEL-process results in complete inactivation of several important groups of plant and animal pathogens . The mechanism that causes the inactivation is not yet fully understood, but the relatively high Volatile Fatty Acids concentration during the first two weeks of the digestion process might presumably be the key factor.

Water Sci Technol, 2000, 41(3), 239 - 46
Anaerobic treatment of landfill leachate by sulfate reduction; Henry JG et al.; The present study was conducted to investigate the effectiveness of the sulphate-reduction pathway in the anaerobic treatment of landfill leachate . The effects of several COD/SO4 ratios (keeping COD constant) and loadings on anaerobic filter performance were studied and compared with the results from anaerobic filters which followed the methanogenic pathway . Results indicated that the treatability of leachate by sulphate reducing bacteria (SRB) was dependent upon the leachate strength . With high strength leachate (COD = 15,000 mg/L) from the Keele Valley Landfill, it was found that at lower COD/SO4 ratios (< or = 1.6) toxic conditions developed in the system that were more inhibitory to the SRB than to the methane producing bacteria (MPB) . As the COD/SO4 ratio increased, methanogenesis predominated . No predominance of SRB occurred at any COD/SO4 ratio with high strength leachate . The highest COD removal achieved was about 70% of which 20% was accomplished by the SRB at a COD/SO4 ratio of 1.6 and an organic loading rate (OLR) of 4 kg COD/m3.d . With low strength leachate (COD = 1500-3300 mg/L) from the Brock West Landfill, and a COD/SO4 ratio < or = 1, SRB became predominant . In these anaerobic filters in which SRB were predominant, the SRB reduced the COD as well as the MPB could . Sulphide inhibition did not take place at any loading in units treating low strength leachate . Consequently, both SRB and MPB should function at COD/SO4 ratios between 1 and 3 . About 60% COD removal was achieved at a loading of 2.8 kg COD/m3.d and a COD/SO4 ratio of 1.0 . However at a loading of 6 kg COD/m3.d only 27% COD removal was achieved, all of it through the sulphate-reduction pathway . These OLR values are comparable to those applied in systems where methanogenesis was dominant . It was also observed that once the methanogens were established in the units, it was not possible to displace them completely . However, where methanogenesis had not been previously established, it was found that sulphate-reduction could be the sole pathway for COD removal . From this study, it can be concluded that there is no advantage to the sulphate-reduction pathway in the anaerobic treatment of landfill leachate . The other options for increasing the loadings, i.e . the use of high surface/volume filter media (to achieve higher biomass concentrations) or high rate systems are likely to be more successful.

Water Sci Technol, 2000, 41(3), 223 - 30
Characterization and anaerobic treatment of the sanitary landfill leachate in Istanbul; Inanc B et al.; In this study, characterization and anaerobic treatability of leachate from Komurcuoda Sanitary Landfill located on the Asian part of Istanbul were investigated . Time based fluctuations in characteristics of leachate were monitored for an 8 month period . Samples were taken from a 200 m3 holding tank located at the lowest elevation of the landfill . COD concentrations have ranged between 18,800 and 47,800 mg/l while BOD5 between 6820 and 38,500 mg/L . COD and BOD5 values were higher in summer and lower in winter due to dilution by precipitation . On the other hand, it was quite interesting that such a dilution effect was not observed for ammonia . The highest ammonia concentration, 2690 mg/L was in November 1998 . BOD5/COD ratio was larger than 0.7 for most samples indicating high biodegradability, and acidic phase of decomposition in the landfill . For anaerobic treatability, three different reactors, namely an upflow anaerobic sludge bed reactor, an anaerobic upflow filter and a hybrid bed reactor, were used . The anaerobic reactors were operated for more than 230 days and were continuing operation when this paper was prepared . Organic loading was increased gradually from 1.3 kg COD/m3.day to 8.2 kg COD/m3.day while hydraulic retention time was reduced from 2.4 days to 2.0 days . All the reactors showed similar performances against organic loadings with efficiencies between 80% and 90% . However the reactors have experienced high ammonia concentrations several times throughout the experimental period, and showed different inhibition levels . Anaerobic filter was the least affected reactor while UASB was the most . Hybrid bed reactor has exhibited a similar performance to anaerobic filter although not to the same degree.

Water Sci Technol, 2000, 41(3), 171 - 9
Effects of disintegration on anaerobic degradation of sewage excess sludge in downflow stationary fixed film digesters; Engelhart M et al.; The effects of mechanical disintegration on anaerobic digestibility of sewage excess sludge in downflow stationary fixed film (DSFF) digesters were investigated on laboratory scale . Mechanical pretreatment using a high pressure homogenizer led to significantly enhanced concentrations of soluble proteins and carbohydrates in the feed sludge . Using DSFF digesters with two different tubular plastic media as support material it was shown that a stable digestion process could be achieved at hydraulic retention times (HRT) down to 5 days . Compared to conventional digesters at 10 d and 15 d HRT respectively, the degradation of volatile solids was enhanced up to 25%, also resulting in a higher specific biogas production . Further investigations on degradation of soluble proteins and carbohydrates showed that a slowly degradable fraction of carbohydrates was released via disintegration . Using the distribution of chain length and the concentrations of volatile fatty acids as process parameters, the dependability on the HRT and the degree of disintegration (the release of soluble COD) predominated the effects of specific surface area of the support media.

Water Sci Technol, 2000, 41(3), 17 - 24
Anaerobic hydrolysis kinetics of particulate substrates; Sanders WT et al.; A mathematical description of the surface related hydrolysis kinetics for spherical particles in a batch digestion is presented as well as a verification of this model with particulate starch as a substrate . Three substrates containing starch with different particle size distributions (PSD) were used . Two were obtained from fresh potatoes by wet sieving and for the third substrate a commercially available starch was used . The substrates were batch digested at 30 degrees C with granular sludge as inoculum and the hydrolysis efficiency was measured and fitted with the model . The results revealed that the hydrolysis rates for the three substrates were equal, viz . 0.4 +/- 0.1 g starch/m2/hour . Moreover, for the commercial starch not only the hydrolysis efficiency but also the changes within the PSD of the starch was determined several times with the use of light microscopy and image analysis . The obtained experimental PSD showed good similarity with the theoretical PSD from model calculations . This shows that the surface of the particulate substrate is the key factor for the hydrolysis process.

Water Sci Technol, 2000, 41(3), 155 - 62
Influence of the size reduction of organic waste on their anaerobic digestion; Palmowski LM et al.; The rate-limiting step in anaerobic digestion of organic solid waste is generally their hydrolysis . A size reduction of the particles and the resulting enlargement of the available specific surface can support the biological process in two ways . Firstly, in case of substrates with a high content of fibres and a low xegradability, their comminution yields to an improved digester gas production . This leads to a decreased amount of residues to be disposed of and to an increased quantity of useful digester gas . The second effect of the particle size reduction observed with all the substrates but particularly with those of low degradability is a reduction of the technical digestion time . Furthermore, the particle size of organic waste has an influence on the dewaterability after codigestion with sewage sludge . The presence of organic waste residues improves the dewaterability measured as specific resistance to filtration but this positive effect is attenuated if the particle size of the solids is reduced.

Water Sci Technol, 2000, 41(3), 145 - 53
Increase of anaerobic degradation of particulate organic matter in full-scale biogas plants by mechanical maceration; Hartmann H et al.; Different concepts of implementation of mechanical pretreatment for enhancing the biogas potential from fibers in manure feedstock were evaluated by sampling before and after macerators at different biogas plants and from a fiber separation unit . An increase of the biogas potential of up to 25% by pretreatment of the whole feed in the macerator before the reactor was observed . Implementation concepts with a treatment of the fibers alone after separation from the manure showed to be not efficient due to a low recovery of organic matter in the fibers by the separation unit . The low operational costs of a macerator make it attractive to use this pretreatment method for a more complete degradation of particulate organic matter . Investigation of the size distribution of the fibers showed that a change in biogas potential was not correlated to a smaller size of the fibers . Results from the macerators indicate that the biodegradability of the fibers is rather enhanced by shearing which is not necessarily reflected by a change in fiber size.

Water Sci Technol, 2000, 41(3), 119 - 27
Effect of moisture content on anaerobic digestion of dewatered sludge: ammonia inhibition to carbohydrate removal and methane production; Fujishima S et al.; The purpose of this study is to investigate the effect of moisture content on anaerobic digestion of dewatered sewage sludge under mesophilic condition . The moisture contents of sludge fed to reactors were 97.0%, 94.6%, 92.9%, 91.1% and 89.0% . The VS removal efficiency changed from 45.6% to 33.8%, as the moisture content of sludge fed to digester decreased from 97.0% to 89.0% . The carbohydrate removal efficiency also decreased from 71.1% to 27.8% . Methane production decreased when the moisture content of sludge was lower than 91.1% . The number of glucose consuming acidogenic bacteria was decreased from 3.1 x 10(6) to 3.1 x 10(8) (MPN/mL) as the moisture content decreased from 91.1% to 89.0% . The numbers of hydrogenotrophic and acetoclastic methanogenic bacteria decreased by one order of magnitude when the moisture content was lower than 91.1% . The decrease in numbers of glucose consuming acidogenic bacteria and methanogenic bacteria was found to correspond to the decrease in the carbohydrate removal efficiency and the accumulation of propionic acid . Batch experiments showed that acetoclastic methanogenic bacteria were acclimated to high ammonia concentration, on the other hand, glucose consuming acidogenic bacteria were inhibited.

Water Sci Technol, 2000, 41(3), 101 - 10
Pilot-scale gasification of municipal solid wastes by high-rate and two-phase anaerobic digestion (TPAD); Ghosh S et al.; Bioconversion of municipal solid waste-sludge blend by conventional high-rate and two-phase anaerobic digestion was studied . RDF (refused-derived fuel)-quality feed produced in a Madison, Wisconsin, USA, MRF (materials-recovery facility) was used . High-rate digestion experiments were conducted with bench-scale digesters under target operating conditions developed from an economic feasibility study . The effects of digestion temperature, RDF content of digester feed, HRT, loading rate, RDF particle size, and RDF pretreatment with cellulase or dilute solutions of NaOH or lime on digester performance were studied . A pilot-scale two-phase digestion plant was operated with 80:20 (weight ratio) RDF-sludge blends to show that this process exhibited a higher methane yield, and produced a higher methane-content digester gas than those obtained by single-stage, high-rate anaerobic digestion.

J Mol Graph Model, 2001, 19(1), 26 - 59
Intrinsically disordered protein; Dunker AK et al.; Proteins can exist in a trinity of structures: the ordered state, the molten globule, and the random coil . The five following examples suggest that native protein structure can correspond to any of the three states (not just the ordered state) and that protein function can arise from any of the three states and their transitions . (1) In a process that likely mimics infection, fd phage converts from the ordered into the disordered molten globular state . (2) Nucleosome hyperacetylation is crucial to DNA replication and transcription; this chemical modification greatly increases the net negative charge of the nucleosome core particle . We propose that the increased charge imbalance promotes its conversion to a much less rigid form . (3) Clusterin contains an ordered domain and also a native molten globular region . The molten globular domain likely functions as a proteinaceous detergent for cell remodeling and removal of apoptotic debris . (4) In a critical signaling event, a helix in calcineurin becomes bound and surrounded by calmodulin, thereby turning on calcineurin's serine/threonine phosphatase activity . Locating the calcineurin helix within a region of disorder is essential for enabling calmodulin to surround its target upon binding . (5) Calsequestrin regulates calcium levels in the sarcoplasmic reticulum by binding approximately 50 ions/molecule . Disordered polyanion tails at the carboxy terminus bind many of these calcium ions, perhaps without adopting a unique structure . In addition to these examples, we will discuss 16 more proteins with native disorder . These disordered regions include molecular recognition domains, protein folding inhibitors, flexible linkers, entropic springs, entropic clocks, and entropic bristles . Motivated by such examples of intrinsic disorder, we are studying the relationships between amino acid sequence and order/disorder, and from this information we are predicting intrinsic order/disorder from amino acid sequence . The sequence-structure relationships indicate that disorder is an encoded property, and the predictions strongly suggest that proteins in nature are much richer in intrinsic disorder than are those in the Protein Data Bank . Recent predictions on 29 genomes indicate that proteins from eucaryotes apparently have more intrinsic disorder than those from either bacteria or archaea, with typically > 30% of eucaryotic proteins having disordered regions of length > or = 50 consecutive residues.

Genome Res, 2001 Jun, 11(6), 981 - 93
Genome evolution at the genus level: comparison of three complete genomes of hyperthermophilic archaea; Lecompte O et al.; We have compared three complete genomes of closely related hyperthermophilic species of Archaea belonging to the Pyrococcus genus: Pyrococcus abyssi, Pyrococcus horikoshii, and Pyrococcus furiosus . At the genomic level, the comparison reveals a differential conservation among four regions of the Pyrococcus chromosomes correlated with the location of genetic elements mediating DNA reorganization . This discloses the relative contribution of the major mechanisms that promote genomic plasticity in these Archaea, namely rearrangements linked to the replication terminus, insertion sequence-mediated recombinations, and DNA integration within tRNA genes . The combination of these mechanisms leads to a high level of genomic plasticity in these hyperthermophilic Archaea, at least comparable to the plasticity observed between closely related bacteria . At the proteomic level, the comparison of the three Pyrococcus species sheds light on specific selection pressures acting both on their coding capacities and evolutionary rates . Indeed, thanks to two independent methods, the "reciprocal best hits" approach and a new distance ratio analysis, we detect the false orthology relationships within the Pyrococcus lineage . This reveals a high amount of differential gains and losses of genes since the divergence of the three closely related species . The resulting polymorphism is probably linked to an adaptation of these free-living organisms to differential environmental constraints . As a corollary, we delineate the set of orthologous genes shared by the three species, that is, the genes that may characterize the Pyrococcus genus . In this conserved core, the amino acid substitution rate is equal between P . abyssi and P . horikoshii for most of their shared proteins, even for fast-evolving ones . In contrast, strong discrepancies exist among the substitution rates observed in P . furiosus relative to the two other species, which is in disagreement with the molecular clock hypothesis.

Heredity, 2001 Feb, 86(Pt 2), 161 - 6
Two male-killing Wolbachia strains coexist within a population of the butterfly Acraea encedon; Jiggins FM et al.; Inherited bacteria that kill male hosts early in their development are known from five insect orders . We ask to what extent the incidence of male-killers might be restricted by the rate at which new host-parasite interactions arise, by testing whether multiple male-killers have invaded a single host species . In Uganda, the butterflies Acraea encedon and A . encedana are both infected by the same strain of male-killing Wolbachia and there was no evidence of variation within the population . In Tanzanian A . encedon however, two phylogenetically distinct strains of male-killing Wolbachia were found within the same population . If this pattern of male-killer polymorphism is found to be general across infected species, it suggests that new male-killing infections arise frequently on an evolutionary time scale . Whether this polymorphism is stable, and what forces may be maintaining it, are unknown.

Water Sci Technol, 2001, 43(2), 213 - 20
Heterogeneous catalytic ozonation of 2-chlorophenol aqueous solution with alumina as a catalyst; Ni CH et al.; Heterogenous catalytic ozonation of 2-chlorophenol (2-CP) in the presence of gamma-alumina as a solid catalyst has been investigated in this research . It showed that the rate for degradation of TOC could increase from 21% to 43% . The pseudo-first reaction constants of 2-CP could increase from 0.8688 min-1 to 0.1270, increasing by approximately 40% . At the same time, the consumption of ozone was only half that of ozone alone . This research also explored the effects of the catalyst dosage, pH values and removal kinetics of 2-CP . In addition, three consecutive running with the same catalyst revealed insignificant reduction of the activity . Furthermore, the elimination of toxicity was evaluated by Microtox analysis . The detoxification was more stable and with good results.

J Dent Res, 2001 Mar, 80(3), 914 - 8
Increased frequency of FcgammaRIIIb-NA1 allele in periodontitis-resistant subjects in an elderly Japanese population; Sugita N et al.; Many elderly people show minimum periodontal tissue destruction, which might be partly due to genetic advantages in host immune response against periodontopathic bacteria . The human IgG Fc receptor IIIb on neutrophils bears a NA1-NA2 polymorphism . The FcgammaRIIIb-NA1 displays a more efficient interaction with IgG1- and IgG3-opsonized bacteria, compared with the FcgammaRIIIb-NA2 . We investigated a 70-year-old Japanese population (n = 599) to determine whether the FcgammaRIIIb polymorphism was associated with resistance to periodontitis . Among subjects with > or = 20 teeth present, periodontitis-resistant (n = 46) and periodontitis-susceptible groups (n = 73) were selected based on the percentage of sites with > or = 4 mm probing attachment loss in the entire dentition . The FcgammaRIIIb-NA1 allotype was overrepresented in the periodontitis-resistant group, compared with the periodontitis-susceptible group (chi2 = 4.89, p = 0.03, odds ratio = 1.87, 95% CI, 1.07 to 3.28) . This suggests that FcgammaRIIIb-NA1 may be associated with resistance to periodontitis.

Water Sci Technol, 2001, 43(5), 51 - 60
River Water Quality Model no . 1 (RWQM1): case study II . Oxygen and nitrogen conversion processes in the River Glatt (Switzerland); Reichert P; Various simplifications of the river water quality model no . 1 are applied to data sets from the river Glatt in Switzerland . In a first application, the biomass responsible for nitrogen and oxygen conversion processes is quantified based on known reaeration rates, measured concentrations of ammonia, nitrite and oxygen and assumed growth parameters of algae and bacteria . In a second application, the model is extended to calculate chemical equilibria of inorganic carbon compounds dissolved in the water and daily variations in pH . The influence of partially unknown inflow concentrations and of calcite precipitation on fluctuations in electrical conductivity and pH are discussed . In the last model, the processes of growth of sessile algae and bacteria, detachment of algae, and grazing by benthic organisms are introduced . Due to lack of data for quantifying these processes, this last model application is speculative . Nevertheless, it is interesting because it shows a direction to which river water quality modelling would have to proceed in order to increase its predictive capabilities.

Water Sci Technol, 2001, 43(5), 365 - 72
Complete remediation of PCE contaminated unsaturated soils by sequential anaerobic-aerobic bioventing; Mihopoulos PG et al.; Bioventing principles have been applied to completely dechlorinate tetrachloroethylene vapors in the unsaturated zone in a sequential anaerobic-aerobic pattern . The aerobic step yields trans-DCE and VC as PCE reductive dechlorination byproducts, while TCE and cis-DCE are observed as intermediates . The aerobic step results in rapid oxidation of the VC and trans-DCE to carbon dioxide . Hydrogen was delivered in the gas phase as a reducing agent for the anaerobic step at levels of 1%, and oxygen at 4.2% was used as an electron acceptor in the aerobic step . PCE and VC half lives in the anaerobic and aerobic steps respectively, where less than 10 min.

Water Sci Technol, 2001, 43(5), 175 - 82
Water quality response to riparian restoration in an agricultural watershed in Vermont, USA; Meals DW; Achievement of management goals for Lake Champlain (Vermont/New York, USA and Quebec, Canada) will require reduction of agricultural phosphorus loads, the dominant nonpoint source in the Basin . Cost-effective phosphorus reduction strategies need reliable treatment techniques beyond basic cropland and waste management practices . The Lake Champlain Basin Agricultural Watersheds National Monitoring Program (NMP) Project evaluates the effectiveness of livestock exclusion, streambank protection, and riparian restoration practices in reducing concentrations and loads of nutrients, sediment, and bacteria in surface waters . Treatment and control watersheds in northwestern Vermont have been monitored since 1994 according to a paired-watershed design . Monitoring consists of continuous stream discharge recording, flow-proportional sampling for total P, total Kjeldahl N, and total suspended solids, grab sampling for indicator bacterial, and land use/agricultural monitoring . Strong statistical calibration between the control and treatment watersheds has been achieved . Installation of riparian fencing, protected stream crossings, and streambank bioengineering was completed in 1997 . Early post-treatment data suggest significant reduction in P concentrations and loads and in bacteria counts in the treated watershed . Monitoring is scheduled to continue through 2000.

Water Sci Technol, 2001, 43(1), 27 - 34
Performance characterization of anaerobic sequencing batch reactor process for digestion of night soil; Lee JG et al.; Laboratory experiments were conducted to investigate the performance of an anaerobic sequencing batch reactor (ASBR) process for night soil treatment . Performances of the reactors were evaluated at an equivalent hydraulic retention time (HRT) of 10 days with an equivalent loading rate of 2.6 kgVS/m3/d (3.1 kgCOD/m3/day) at 35 degrees C . Digestion of a night soil was possible using the ASBR at an HRT of 10 days in spite of high concentration of ammonia nitrogen and settleable solids . Solids were accumulated rapidly in the ASBRs, and their concentrations were 2.3-2.4 times higher than that in a completely mixed control reactor . Remarkable increases in gas production were observed in the ASBRs compared with the control reactor . Average increases in equivalent daily gas production from the ASBRs were 205-220% compared with that from the control run . The ASBR with reaction period/thickening period ratio (R/T ratio) of 1 showed a little higher gas production and organic removal efficiency than that with R/T ratio of 3 . Volatile solids removals based on supernatant of the ASBRs were 12-14% higher than that of the control reactor . Thus, the ASBR was a stable and effective process for the treatment of night soil having high concentration of settleable organics and ammonia nitrogen.

Water Sci Technol, 2001, 43(1), 19 - 26
Mechanical disintegration of sewage sludge; Lehne G et al.; Mechanical disintegration can be used for an accelerated and improved anaerobic digestion of excess sludge . The hydrolysis is the limiting step of this process . Mechanical disintegration can be used to disrupt the cell walls and to cause the release of the organic material from the cells . Particle size analysis describes the size reduction but is not suitable for characterising the release of the organic material and the cell disruption . Two biochemical methods were developed for these phenomena . One of the parameters provides information about the disruption of micro-organisms, the other one gives information about the release of organic material . Different ultrasonic homogenizers, a high pressure homogenizer and stirred ball mills were used for disintegration experiments using various parameters . The influences of a mechanical disintegration on the particle size and of the energy intensity on the disintegration were investigated . Further investigations had to detect the influence of the solid content on the disintegration results . For sludge with a higher solid content better results in terms of energy consumption could be achieved . An optimum of the bead diameter and the stress intensity in stirred ball mills could be detected . A comparison of the results of different methods of sludge disintegration shows that the investigated ultrasonic homogenizers are inferior to a high pressure homogenizer and a stirred ball mill in terms of energy consumption.

Eur J Hum Genet, 2001 May, 9(5), 347 - 54
Genetic polymorphism of MUC7: allele frequencies and association with asthma; Kirkbride HJ et al.; MUC7 encodes a small salivary mucin, previously called MG2, a glycoprotein with a putative role in facilitating the clearance of oral bacteria . The central domain of this glycoprotein was previously shown to comprise five or six tandemly repeated units of 23 amino-acids which carry most of the O-linked glycans . The polymorphism of these two allelic forms (MUC7*5 or MUC7*6) has been confirmed in this study in which we have analysed a large cohort of subjects (n = 375) of various ethnic origins . We have also identified a novel rare allele with eight tandem repeats (MUC7*8) . MUC7*6 was the most common allele (0.78-0.95) in all the populations tested . The tandem repeat arrays of 22 MUC7*5 alleles and 34 MUC7*6 alleles were sequenced . No sequence differences were detected in any of the MUC7*6 alleles . Twenty-one MUC7*5 alleles sequenced lacked the 4th tandem repeat (structure TR12356), while one showed the structure TR12127 . The structure of the MUC7*8 allele was TR12343456 . Because of the known role of MUC7 in bacterial binding, and thus its potential involvement in susceptibility to chest disease we also tested MUC7 in our previously described series of Northern European atopic individuals with and without associated asthma . The MUC7*5 allele was rarer in the atopic asthmatics than in the atopic non-asthmatics (P = 0.014, OR for no asthma in atopic individuals 3.13, CI 1.01-6.10), and the difference in frequency between all asthmatics and all non-asthmatics was statistically significant (P = 0.009) while there was no difference between atopy and non-atopy (P = 0.199) . In this study we also report the electrophoretic analysis of the MUC7 glycoprotein in saliva from individuals of different MUC7 genotype.

Minerva Stomatol, 2001 Mar-Apr, 50(3-4), 63 - 9
{SEM study of morphologyc and incidence of accessory canals in the furcation region of permanent molars}; Luglie PF et al.; BACKGROUND: The lateral canals in the molar furcation region could represent a connection, well separated from the apical region, between dental pulp and periodontal tissues and may be used by bacteria or toxins both in an endodontic-periodontal direction and vice versa . A SEM study was carried out on the furcation region of permanent molars to evaluate the morphology and incidence of lateral canals . METHODS: A total of 53 permanent molars were selected . The microinfiltration test was performed on each sample using methylene blue 1% before being sectioned and examined using a Zeiss Opmi 9-FC stereomicroscope and SEM ISI DS 130 . RESULTS: Openings of lateral canals were found in the furcation region of 50.94% of the samples examined . The mean diameter of the orifices was 130 mm . The apertures were elliptical and flared . The holes occasionally appeared to be the end of probable single or double canals, but were not always patent . CONCLUSIONS: In the light of these findings, the authors advise disinfecting and filling the dental pulp cavity during root treatment and, in the event of severe periodontal disease, evaluating the vitality of dental elements before surgery, thus ensuring a correct therapeutic approach by not overlooking the possibility of combined lesions.

Curr Opin Microbiol, 2001 Jun, 4(3), 274 - 9
Host-pathogen interactions promoting inflammatory Lyme arthritis: use of mouse models for dissection of disease processes; Wooten RM et al.; Recent studies have confirmed the infectious and inflammatory nature of arthritis induced by Borrelia burgdorferi, or Lyme arthritis . This arthritis is directed by the presence of the bacteria in joint tissue, and is mediated through activation of the Toll-like receptor 2 (TLR2) signaling pathways by borrelial lipoproteins . Several host genes regulate the severity of arthritis, possibly by regulating the balance of pro- and anti-inflammatory responses.

Curr Opin Genet Dev, 2001 Jun, 11(3), 247 - 57
Computational analysis of human disease-associated genes and their protein products; Sreekumar KR et al.; The complete genome sequences for human, Drosophila melanogaster and Arabidopsis thaliana have been reported recently . With the availability of complete sequences for many bacteria and archaea, and five eukaryotes, comparative genomics and sequence analysis are enabling us to identify counterparts of many human disease genes in model organisms, which in turn should accelerate the pace of research and drug development to combat human diseases . Continuous improvement of specialized protein databases, together with sensitive computational tools, have enhanced the power and reliability of computational prediction of protein function.

Structure (Camb), 2001 May 9, 9(5), 377 - 87
Activation of the redox-regulated molecular chaperone Hsp33--a two-step mechanism; Graumann J et al.; BACKGROUND: Hsp33 is a novel redox-regulated molecular chaperone . Hsp33 is present in the reducing environment of the cytosol and is, under normal conditions, inactive . The four highly conserved cysteines found in Hsp33 constitute a novel zinc binding motif . Upon exposure to oxidative stress, Hsp33's chaperone activity is turned on . This activation process is initiated by the formation of two intramolecular disulfide bonds . Recently, the 2.2 A crystal structure of Hsp33 has been solved, revealing that Hsp33 is present as a dimer in the structure (Vijayalakshmi et al., this issue, 367-375 {1}) . RESULTS: We show here that oxidized, highly active Hsp33 is a dimer in solution . In contrast, reduced and inactive Hsp33 is monomeric . The incubation of reduced Hsp33 in H(2)O(2) leads to the simultaneous formation of two intramolecular disulfide bonds and the concomitant release of zinc . This concentration-independent step is followed by a concentration-dependent association reaction . The dimerization of Hsp33 requires highly temperature-sensitive structural rearrangements . This allows Hsp33's activation process to be greatly accelerated at heat shock temperatures . CONCLUSIONS: The regulation of Hsp33's chaperone function is highly sophisticated . On a transcriptional level, Hsp33 is under heat shock control . This increases the concentration of Hsp33 under heat and oxidative stress, a process that favors dimerization, a critical step in Hsp33's activation reaction . On a posttranslational level, Hsp33 is redox regulated . Dimerization of disulfide-bonded Hsp33 monomers leads to the formation of two extended, putative substrate binding sites . These sites might explain Hsp33's high and promiscuous affinity for unstructured protein folding intermediates.

Toxicol In Vitro, 2001 Jun, 15(3), 191 - 8
Inhibitory actions of luteolin on the growth and arylamine N-acetyltransferase activity in strains of Helicobacter pylori from ulcer patients; Chung JG et al.; Helicobacter pylori is now recognized as an important cause of type B gastritis, which is strongly associated with gastric and duodenal ulcer disease . H . pylori may be a causative factor in patients with gastric cancer . The growth inhibition and N-acetylation of 2-Aminofluorene (AF) or P-aminobenzoic acid (PABA) by arylamine N-acetyltransferase (NAT) in H . pylori were inhibited by luteolin, a component in herbal medicine . The growth inhibition was based on the changes of optical density (OD) by using a spectrophotometer . The N-acetylation of AF or PABA by NAT from H . pylori were assayed by the amounts of acetylated and non-acetylated AF or PABA in cytosols and intact bacteria of H . pylori by using HPLC . An inhibition of growth on H . pylori demonstrated that luteolin elicited a dose-dependent growth inhibition in the H . pylori cultures . Cytosols and suspensions of H . pylori with or without specific concentrations of luteolin co-treatment showed different percentages of AF or PABA acetylation . The data indicated that there was decreased NAT activity associated with increased levels of luteolin in H . pylori cytosols and suspensions . Using standard steady-state kinetic analysis, it was demonstrated that luteolin was a possible uncompetitive inhibitor to NAT enzyme in H . pylori . This report is the first demonstration to show that luteolin can inhibit H . pylori growth and NAT activity.

Prog Biophys Mol Biol, 2001, 75(3), 121 - 64
Determining the structure of biological macromolecules by transmission electron microscopy, single particle analysis and 3D reconstruction; Ruprecht J et al.; Single particle analysis and 3D reconstruction of molecules imaged by transmission electron microscopy have provided a wealth of medium to low resolution structures of biological molecules and macromolecular complexes, such as the ribosome, viruses, molecular chaperones and photosystem II . In this review, the principles of these techniques are introduced in a non-mathematical way, and single particle analysis is compared to other methods used for structural studies . In particular, the recent X-ray structures of the ribosome and of ribosomal subunits allow a critical comparison of single particle analysis and X-ray crystallography . This has emphasised the rapidity with which single particle analysis can produce medium resolution structures of complexes that are difficult to crystallise . Once crystals are available, X-ray crystallography can produce structures at a much higher resolution . The great similarities now seen between the structures obtained by the two techniques reinforce confidence in the use of single particle analysis and 3D reconstruction, and show that for electron cryo-microscopy structure distortion during sample preparation and imaging has not been a significant problem . The ability to analyse conformational flexibility and the ease with which time-resolved studies can be performed are significant advantages for single particle analysis . Future improvements in single particle analysis and electron microscopy should increase the attainable resolution . Combining single particle analysis of macromolecular complexes and electron tomography of subcellular structures with high-resolution X-ray structures may enable us to realise the ultimate dream of structural biology-a complete description of the macromolecular complexes of the cell in their different functional states.

FEBS Lett, 2001 May 18, 497(1), 26 - 30
The role of the N-terminal domain of photoactive yellow protein in the transient partial unfolding during signalling state formation; van der Horst MA et al.; It is shown that the N-terminal domain of photoactive yellow protein (PYP), which appears relatively independently folded in the ground state of the protein, plays a key role in the transient unfolding during signalling state formation: genetic truncation of the N-terminal domain of PYP significantly decreases the extent of cooperativity of the titration curve that describes chromophore protonation in the ground state of PYP, which is in agreement with the notion that the N-terminal domain is linked through a hydrogen-bonding network with the chromophore-containing domain of the protein . Furthermore, deletion of the N-terminal domain completely abolishes the non-linearity of the Arrhenius plot of the rate of ground state recovery.

Nucleic Acids Res, 2001 Jun 1, 29(11), 2401 - 8
Association of an RNA kissing complex analyzed using 2-aminopurine fluorescence; Rist M et al.; The fluorescent probe, 2-aminopurine-2'-O-methyl riboside (2-AP) has been selectively incorporated at adenosine positions in stem-loops (so called R1inv and R2inv), derived from the ColE1 plasmid encoded RNA I and RNA II transcripts, that interact to form stable loop-loop kissing complexes and bind the RNA one modulator (Rom) protein, such that fluorescence-detected stopped-flow and equilibrium methods could be used to study the detailed mechanism of this RNA-RNA interaction . Formation of loop-loop kissing complexes between R1inv and R2inv hairpins, substituted with 2-AP at positions in the complementary loops, results in a 5-10-fold fluorescence emission decrease (F(max) = 370 nm), which provides a sensitive measure for the binding reaction . The 2-AP substituted complexes are found to have equilibrium binding properties (average K(D) = 2.6 +/- 1.7 nM) and affinity for Rom (average K(D) = 60 +/- 24 nM) that are similar to complexes formed with equivalent unlabeled hairpins . Using stopped-flow experiments, it was found that the 2-AP probes experienced at least three different microenvironments during association of the RNA complex, thus suggesting a kinetic intermediate in the kissing pathway . In contrast, dissociation of the complex was found to fit a single exponential decay (average k(off) = 8.9 x 10(-5) s(-1)) . Consistent with these observations, a two-step mechanism for RNA loop-loop complex association is proposed in which the complementary loops of R1inv and R2inv first base pair to form the loop-loop helix (average k(1) = 0.13 microM(-1)s(-1)) in the initial encounter reaction, and subsequently isomerize to the final tertiary fold in a second slower step (average k(2) = 0.09 s(-1)), where the helical stacking around the junctions is optimized.

J Clin Microbiol, 2001 Jun, 39(6), 2261 - 6
Identification of Aspergillus fumigatus and related species by nested PCR targeting ribosomal DNA internal transcribed spacer regions; Zhao J et al.; Aspergillus fumigatus is the most common species that causes invasive aspergillosis . In order to identify A . fumigatus, partial ribosomal DNA (rDNA) from two to six strains of five different Aspergillus species was sequenced . By comparing sequence data from GenBank, we designed specific primer pairs targeting rDNA internal transcribed spacer (ITS) regions of A . fumigatus . A nested PCR method for identification of other A . fumigatus-related species was established by using the primers . To evaluate the specificities and sensitivities of those primers, 24 isolates of A . fumigatus and variants, 8 isolates of Aspergillus nidulans, 7 isolates of Aspergillus flavus and variants, 8 isolates of Aspergillus terreus, 9 isolates of Aspergillus niger, 1 isolate each of Aspergillus parasiticus, Aspergillus penicilloides, Aspergillus versicolor, Aspergillus wangduanlii, Aspergillus qizutongii, Aspergillus beijingensis, and Exophiala dermatitidis, 4 isolates of Candida, 4 isolates of bacteria, and human DNA were used . The nested PCR method specifically identified the A . fumigatus isolates and closely related species and showed a high degree of sensitivity . Additionally, four A . fumigatus strains that were recently isolated from our clinic were correctly identified by this method . Our results demonstrate that these primers are useful for the identification of A . fumigatus and closely related species in culture and suggest further studies for the identification of Aspergillus fumigatus species in clinical specimens.

J Clin Microbiol, 2001 Jun, 39(6), 2079 - 82
Three days of incubation may be sufficient for routine blood cultures with BacT/Alert FAN blood culture bottles; Bourbeau PP et al.; BacT/Alert FAN blood culture bottles have been shown to enhance the recovery of bacteria and yeast from blood compared with standard BacT/Alert bottles . It is well established that standard BacT/Alert blood culture bottles require no more than 5 days of incubation for the detection of routine bacteria and yeast . It is less clear, however, whether FAN bottles also routinely require 5 days of incubation . To address this question, we recently reviewed the results of 17,887 blood culture sets collected in FAN blood culture bottles at Geisinger Medical Center . Of these cultures, 1,780 were positive for bacteria or yeast, yielding a total of 1,242 clinically significant isolates . The numbers of isolates recovered on days 1, 2, 3, 4, and 5 were as follows: (values in parentheses are percentages of total significant isolates): 877 (71%), 269 (22%), 65 (5%), 18 (1%) and, 13 (1%), respectively . In total, 97.5% of all clinically significant isolates were detected in the first 3 days of incubation . Of the 31 significant isolates detected on day 4 or 5 of incubation, 17 were detected in concurrent blood cultures within the first 3 days of incubation . Chart reviews were conducted for the 13 patients with the remaining 14 isolates detected on day 4 or 5 to determine whether therapy was changed due to this blood culture result . Therapy was changed for only 1 patient . These results suggest that it may not be necessary to routinely incubate FAN blood culture bottles for more than 3 days.

EMBO Rep, 2001 May, 2(5), 382 - 7
The renaissance of aminoacyl-tRNA synthesis; Ibba M et al.; The role of tRNA as the adaptor in protein synthesis has held an enduring fascination for molecular biologists . Over four decades of study, taking in numerous milestones in molecular biology, led to what was widely held to be a fairly complete picture of how tRNAs and amino acids are paired prior to protein synthesis . However, recent developments in genomics and structural biology have revealed an unexpected array of new enzymes, pathways and mechanisms involved in aminoacyl-tRNA synthesis . As a more complete picture of aminoacyl-tRNA synthesis now begins to emerge, the high degree of evolutionary diversity in this universal and essential process is becoming clearer.

Appl Environ Microbiol, 2001 Jun, 67(6), 2641 - 8
The Sinorhizobium meliloti nutrient-deprivation-induced tyrosine degradation gene hmgA is controlled by a novel member of the arsR family of regulatory genes; Milcamps A et al.; The regulation of the nutrient-deprivation-induced Sinorhizobium meliloti homogentisate dioxygenase (hmgA) gene, involved in tyrosine degradation, was examined . hmgA expression was found to be independent of the canonical nitrogen regulation (ntr) system . To identify regulators of hmgA, secondary mutagenesis of an S . meliloti strain harboring a hmgA-luxAB reporter gene fusion (N4) was carried out using transposon Tn1721 . Two independent Tn1721 insertions were found to be located in a positive regulatory gene (nitR), encoding a protein sharing amino acid sequence similarity with proteins of the ArsR family of regulators . NitR was found to be a regulator of S . meliloti hmgA expression under nitrogen deprivation conditions, suggesting the presence of a ntr-independent nitrogen deprivation regulatory system . nitR insertion mutations were shown not to affect bacterial growth, nodulation of Medicago sativa (alfalfa) plants, or symbiotic nitrogen fixation under the physiological conditions examined . Further analysis of the nitR locus revealed the presence of open reading frames encoding proteins sharing amino acid sequence similarities with an ATP-binding phosphonate transport protein (PhnN), as well as transmembrane efflux proteins.

Appl Environ Microbiol, 2001 Jun, 67(6), 2515 - 25
Two distinct hemolytic activities in Xenorhabdus nematophila are active against immunocompetent insect cells; Brillard J et al.; Xenorhabdus spp . and Photorhabdus spp . are major insect bacterial pathogens symbiotically associated with nematodes . These bacteria are transported by their nematode hosts into the hemocoel of the insect prey, where they proliferate within hemolymph . In this work we report that wild strains belonging to different species of both genera are able to produce hemolysin activity on blood agar plates . Using a hemocyte monolayer bioassay, cytolytic activity against immunocompetent cells from the hemolymph of Spodoptera littoralis (Lepidoptera: Noctuidae) was found only in supernatants of Xenorhabdus; none was detected in supernatants of various strains of Photorhabdus . During in vitro bacterial growth of Xenorhabdus nematophila F1, two successive bursts of cytolytic activity were detected . The first extracellular cytolytic activity occurred when bacterial cells reached the stationary phase . It also displayed a hemolytic activity on sheep red blood cells, and it was heat labile . Among insect hemocyte types, granulocytes were the preferred target . Lysis of hemocytes by necrosis was preceded by a dramatic vacuolization of the cells . In contrast the second burst of cytolytic activity occurred late during stationary phase and caused hemolysis of rabbit red blood cells, and insect plasmatocytes were the preferred target . This second activity is heat resistant and produced shrinkage and necrosis of hemocytes . Insertional inactivation of flhD gene in X . nematophila leads to the loss of hemolysis activity on sheep red blood cells and an attenuated virulence phenotype in S . littoralis (A . Givaudan and A . Lanois, J . Bacteriol . 182:107-115, 2000) . This mutant was unable to produce the early cytolytic activity, but it always displayed the late cytolytic effect, preferably active on plasmatocytes . Thus, X . nematophila produced two independent cytolytic activities against different insect cell targets known for their major role in cellular immunity.

Proc R Soc Lond B Biol Sci, 2001 Jun 7, 268(1472), 1123 - 6
How many species are infected with Wolbachia? Cryptic sex ratio distorters revealed to be common by intensive sampling; Jiggins FM et al.; Inherited bacterial symbionts from the genus Wolbachia have attracted much attention by virtue of their ability to manipulate the reproduction of their arthropod hosts . The potential importance of these bacteria has been underlined by surveys, which have estimated that 17% of insect species are infected . We examined whether these surveys have systematically underestimated the proportion of infected species through failing to detect the low-prevalence infections that are expected when Wolbachia distorts the sex ratio . We estimated the proportion of species infected with Wolbachia within Acraea butterflies by testing large collections of each species for infection . Seven out of 24 species of Acraea were infected with Wolbachia . Four of these were infected with Wolbachia at high prevalence, a figure compatible with previous broad-scale surveys, whilst three carried low-prevalence infections that would have had a very low likelihood of being detected by previous sampling methods . This led us to conclude that sex-ratio-distorting Wolbachia may be common in insects that have an ecology and/or genetics that permit the invasion of these parasites and that previous surveys may have seriously underestimated the proportion of species that are infected.

Philos Trans R Soc Lond B Biol Sci, 2001 May 29, 356(1409), 681 - 9
Treatment with immunotoxin; Knechtle SJ; T-cell depletion prior to or beginning at the time of transplantation has been shown to be a valuable adjunct to the induction of immunological unresponsiveness . Both total lymphoid irradiation and anti-lymphocyte globulin have been used for this purpose in experimental models of transplantation as well as in human organ transplant recipients . However, these methods of T-cell depletion are limited in their ability to deplete T cells selectively due to non-specific targeting and limited efficacy . A new anti-CD3 immunotoxin has been developed with a far more potent ability to deplete T cells selectively as measured by flow cytometry analysis of peripheral blood T lymphocytes as well as lymph node lymphocytes . This immunotoxin is well tolerated by rhesus monkeys when administered in vivo . When administered as a single immunosuppressive agent pretransplant, it substantially promotes allograft survival, inducing tolerance in at least one-third of recipients as measured by subsequent acceptance of donor skin grafts and rejection of third-party skin grafts . When administered on the day of transplant in combination with steroid pretreatment and a brief course of deoxyspergualin or mycophenolate mofetil (4 to 14 days), long-term unresponsiveness is also produced and in a more reliable manner than using immunotoxin alone . A new immunotoxin directed at the human CD3epsilon has been developed with excellent potency in T-cell killing and lacking the Fc portion of the CD3 antibody . This construct may be useful for T-cell depletion in humans and has a potential application in tolerance induction in human organ transplantation . Lessons learned from anti-CD3 immunotoxin in the non-human primate model to date include (i) profound (2-3 log) depletion of T-cells can be accomplished safely without inducing lymphoma or infection, (ii) such depletion is a useful adjunct for tolerance induction to allogeneic organ transplants, and (iii) tolerance to both allogeneic renal transplants and xenogeneic islet transplants has been accomplished using such strategies to date in non-human primates and in pigs . Immunotoxin may be useful for the induction of chimerism using strategies that include donor bone marrow infusion . Successful strategies for tolerance induction have also been developed using immunotoxin without the adjunct of donor bone marrow or stem cell infusion . Clinical application of immunotoxin will use a newly engineered construct with the potential for causing cytokine release, less susceptibility to neutralization by anti-diphtheria antibody and not dependent on chemical conjugation of an antibody and toxin . The usefulness of immunotoxin is directly related to its tremendous potency for depleting T cells . Based on results in nonhuman primates, it is anticipated that it will become a useful agent in tolerance induction in humans.

J Nat Prod, 2001 May, 64(5), 668 - 70
Radiosumin B, an unusual dipeptide from the cyanobacterium Microcystis aeruginosa; Coleman JE et al.; Radiosumin B (1), an N-methyl dipeptide containing two unusual amino acid residues, was isolated from the cyanobacterium Microcystis aeruginosa Kutzing . The structure and stereochemical details were elucidated on the basis of 1D and 2D NMR data, MS data, and chemical degradation.

J Nat Prod, 2001 May, 64(5), 651 - 2
Hortein, a new natural product from the fungus Hortaea werneckii associated with the sponge Aplysina aerophoba; Brauers G et al.; The fungus Hortaea werneckii isolated from the Mediterranean sponge Aplysina aerophoba, collected at Banyuls-sur-Mer in southern France, yielded a new compound named hortein (1), which possesses a unique ring system hitherto unknown for natural products . The structure of 1 was established on the basis of 1D and 2D NMR spectroscopic and mass spectrometric (ESI, CI, FAB, EI) data.

Langenbecks Arch Surg, 2001 Mar, 386(2), 75 - 81
Incidence and pathophysiology of peptic ulcer bleeding; Arlt GD et al.; Peptic ulcer accounts for about 50% of all cases of upper gastrointestinal bleeding . Acute mortality may be as high as 14% . Infection with Helicobacter plyori (Hp) and the use of nonsteroidal anti-inflammatory drugs (NSAIDs) are the predominant risk factors . While the prevalence of Hp in ulcer bleeding is still debated, there is strong evidence that eradication of bacteria reduce the risk of re-bleeding significantly . The use of NSAIDs increases the frequency of ulcer bleeding about four- to sixfold on average . Additional factors such as advanced age, concomitant use of corticosteroids or anticoagulants, prior ulcer complications and co-morbid diseases may further increase the risk of bleeding . Whether or not Hp infection also represents an additive risk factor in NSAID-related bleeding remains to be clarified . The pathophysiologic action of both Hp and NSAIDs is quite complex . Hp promotes the aggressive factor acid and damages several mucosal defence mechanisms by liberating lipopolysaccharide, urease and vacuolating cytotoxin . In NSAID toxicity the cyclo-oxygenase enzymes (COX) have been studied intensively . With the advent of COX-2-selective NSAIDs, the clinical problem of NSAID-induced ulcer bleeding may be markedly reduced or abolished completely.

Nat Struct Biol, 2001 Jun, 8(6), 505 - 9
Supramolecular assembly and acid resistance of Helicobacter pylori urease; Ha NC et al.; Helicobacter pylori, an etiologic agent in a variety of gastroduodenal diseases, produces a large amount of urease, which is believed to neutralize gastric acid by producing ammonia for the survival of the bacteria . Up to 30% of the enzyme associates with the surface of intact cells upon lysis of neighboring bacteria . The role of the enzyme at the extracellular location has been a subject of controversy because the purified enzyme is irreversibly inactivated below pH 5 . We have determined the crystal structure of H . pylori urease, which has a 1.1 MDa spherical assembly of 12 catalytic units with an outer diameter of approximately 160 A . Under physiologically relevant conditions, the activity of the enzyme remains unaffected down to pH 3 . Activity assays under different conditions indicated that the cluster of the 12 active sites on the supramolecular assembly may be critical for the survival of the enzyme at low pH . The structure provides a novel example of a molecular assembly adapted for acid resistance that, together with the low Km value of the enzyme, is likely to enable the organism to inhabit the hostile niche.

Microb Pathog, 2001 May, 30(5), 289 - 97
Molecular analysis of Mycobacterium tuberculosis phosphate specific transport system in Mycobacterium smegmatis . Characterization of recombinant 38 kDa (PstS-1); Torres A et al.; The functionality of the putative Mycobacterium tuberculosis phosphate transport operon was studied by operon- lacZ promoterless fusions in Mycobacterium smegmatis . The expression of the operon genes was evaluated in transformed M . smegmatis growing in medium with low and high phosphate concentration . Although the gene fusions expressed beta-galactosidase in medium with phosphate, a higher activity was detected in bacteria growing in medium with low phosphate . In contrast, alkaline phosphatase activity from M . smegmatis was detected only in bacteria growing in medium with low phosphate . The expression of the operon genes was driven by a promoter located 5' upstream from the start codon of the pstB gene . A second putative internal promoter 5' upstream of the pstS-1 gene was also detected . Furthermore, comparative analysis between the native and recombinant PstS-1 proteins showed that they were very similar . Like the native protein, the recombinant protein was also secreted to the culture medium as a glycosylated band . The results show that M . smegmatis recognized phosphate regulatory signals of the M . tuberculosis phosphate transport operon genes, and open the possibility to study gene phosphate regulation in mycobacteria .

J Med Entomol, 2001 May, 38(3), 382 - 7
Wolbachia-induced cytoplasmic incompatibility in single- and superinfected Aedes albopictus (Diptera: Culicidae); Dobson SL et al.; Maternally inherited bacteria of the genus Volbachia can cause cytoplasmic incompatibility resulting in the developmental arrest of early embryos . Previous studies have shown that both single- and superinfections of Wolbachia naturally occur in populations of Aedes albopictus (Skuse) . Here, we report crossing experiments using three infection types occurring in Ae . albopictus: uninfected, single-infected, and superinfected individuals . Crosses were monitored over the lifetime of adults to detect possible effects of host age on cytoplasmic incompatibility levels and infection virulence . Both single- and superinfections induced high levels of cytoplasmic incompatibility throughout the lifetime of Ae . albopictus, demonstrating that both the single- and superinfections are well adapted for invasion of Ae . albopictus populations . Superinfected females were the longest lived and had the highest oviposition rates, whereas in males, uninfected individuals were the longest lived . These latter results demonstrate the need for additional experiments to better elucidate Wolbachia effects on host fitness in addition to cytoplasmic incompatibility.

Neurol Med Chir (Tokyo), 2001 Mar, 41(3), 121 - 6
Discrimination of brain abscess and cystic tumor by in vivo proton magnetic resonance spectroscopy; Kadota O et al.; Proton magnetic resonance (MR) spectroscopy was evaluated for the differentiation of brain abscesses and cystic brain tumors . Proton MR spectroscopy was performed in vivo in two patients with brain abscess and eight patients with various cystic brain tumors (anaplastic astrocytoma, glioblastoma, and metastatic brain tumor) . MR imaging with contrast medium demonstrated ring-like enhanced mass lesions in all patients . The various resonance peaks in proton MR spectra were assigned to metabolites according to chemical shifts . Treatment of the cystic brain lesions was based on the information from proton MR spectroscopy . Aspirated pus from one patient with brain abscess was examined using ex vivo proton MR spectroscopy . The in vivo spectra of brain abscess contained resonance peaks attributed to acetate, lactate, alanine, amino acids, and lipids in both cases, and an additional peak of succinate in one case . In vivo spectra of the neoplasms contained resonance peaks corresponding to lactate, lipids, choline, creatine, and N-acetyl aspartate . Proton MR spectroscopy is useful for discriminating brain abscess from cystic tumors with similar neuroimaging appearance, which is very important for determining the treatment strategy.

J Bacteriol, 2001 Jun, 183(12), 3597 - 605
Ralstonia solanacearum needs motility for invasive virulence on tomato; Tans-Kersten J et al.; Ralstonia solanacearum, a widely distributed and economically important plant pathogen, invades the roots of diverse plant hosts from the soil and aggressively colonizes the xylem vessels, causing a lethal wilting known as bacterial wilt disease . By examining bacteria from the xylem vessels of infected plants, we found that R . solanacearum is essentially nonmotile in planta, although it can be highly motile in culture . To determine the role of pathogen motility in this disease, we cloned, characterized, and mutated two genes in the R . solanacearum flagellar biosynthetic pathway . The genes for flagellin, the subunit of the flagellar filament (fliC), and for the flagellar motor switch protein (fliM) were isolated based on their resemblance to these proteins in other bacteria . As is typical for flagellins, the predicted FliC protein had well-conserved N- and C-terminal regions, separated by a divergent central domain . The predicted R . solanacearum FliM closely resembled motor switch proteins from other proteobacteria . Chromosomal mutants lacking fliC or fliM were created by replacing the genes with marked interrupted constructs . Since fliM is embedded in the fliLMNOPQR operon, the aphA cassette was used to make a nonpolar fliM mutation . Both mutants were completely nonmotile on soft agar plates, in minimal broth, and in tomato plants . The fliC mutant lacked flagella altogether; moreover, sheared-cell protein preparations from the fliC mutant lacked a 30-kDa band corresponding to flagellin . The fliM mutant was usually aflagellate, but about 10% of cells had abnormal truncated flagella . In a biologically representative soil-soak inoculation virulence assay, both nonmotile mutants were significantly reduced in the ability to cause disease on tomato plants . However, the fliC mutant had wild-type virulence when it was inoculated directly onto cut tomato petioles, an inoculation method that did not require bacteria to enter the intact host from the soil . These results suggest that swimming motility makes its most important contribution to bacterial wilt virulence in the early stages of host plant invasion and colonization.

Biophys J, 2001 Jun, 80(6), 2843 - 55
Energy transfer in the peridinin chlorophyll-a protein of Amphidinium carterae studied by polarized transient absorption and target analysis; Krueger BP et al.; The peridinin chlorophyll-a protein (PCP) of dinoflagellates differs from the well-studied light-harvesting complexes of purple bacteria and green plants in its large (4:1) carotenoid to chlorophyll ratio and the unusual properties of its primary pigment, the carotenoid peridinin . We utilized ultrafast polarized transient absorption spectroscopy to examine the flow of energy in PCP after initial excitation into the strongly allowed peridinin S2 state . Global and target analysis of the isotropic and anisotropic decays reveals that significant excitation (25-50%) is transferred to chlorophyll-a directly from the peridinin S2 state . Because of overlapping positive and negative features, this pathway was unseen in earlier single-wavelength experiments . In addition, the anisotropy remains constant and high in the peridinin population, indicating that energy transfer from peridinin to peridinin represents a minor or negligible pathway . The carotenoids are also coupled directly to chlorophyll-a via a low-lying singlet state S1 or the recently identified SCT . We model this energy transfer time scale as 2.3 +/- 0.2 ps, driven by a coupling of approximately 47 cm(-1) . This coupling strength allows us to estimate that the peridinin S1/SCT donor state transition moment is approximately 3 D.

In Vitro Cell Dev Biol Anim, 2001 Mar, 37(3), 127 - 40
Permanent phenotypic and genotypic changes of prostate cancer cells cultured in a three-dimensional rotating-wall vessel; Rhee HW et al.; A three-dimensional (3D) integrated rotating-wall vessel cell-culture system was used to evaluate the interaction between a human prostate cancer cell line, LNCaP, and microcarrier beads alone, or microcarrier beads previously seeded with either prostate or bone stromal cells . Upon coculture of LNCaP cells with microcarrier beads either in the presence or in the absence of prostate or bone stromal cells, 3D prostate organoids were formed with the expected hormonal responsiveness to androgen, increased cell growth, and prostate-specific antigen production . In this communication, we define permanent phenotypic and genotypic changes of LNCaP cells upon coculture with microcarrier beads alone, or with microcarrier beads previously seeded with either prostate or bone stromal cells . Most notably, we observed selective genetic changes, i.e., chromosomal losses or gains, as evaluated by both conventional cytogenetic and comparative genomic hybridization, in LNCaP sublines derived from the prostate organoids . Moreover, the derivative LNCaP cells appear to have altered growth profiles, and exhibit permanent and stable changes in response to androgen, estrogen, and growth factors . The derivative LNCaP sublines showed increased anchorage-independent growth rate, and enhanced tumorigenicity and metastatic potential when inoculated orthotopically in castrated athymic mice . Our results support the hypothesis that further nonrandom genetic and phenotypic changes in prostate cancer epithelial cells can occur through an event that resembles "adaptive mutation" such as has been described in bacteria subjected to nutritional starvation . The occurrence of such permanent changes may be highly contact dependent, and appears to be driven by specific microenvironmental factors surrounding the tumor cell epithelium grown as 3D prostate organoids.

Med Klin (Munich), 2001 Apr 15, 96(4), 234 - 7
{A rare cause of hepatic coma}; Adamek MU et al.; CASE REPORT: A 65-year-old female patient was admitted to the hospital in somnolent state . During physical examination, she had an increase in blood pressure and a positive bilateral Babinski's sign . Laboratory findings showed elevated liver enzymes, metabolic alkalosis and slightly elevated kidney values . 3 h hours later she lapsed into a hepatic coma (ammonia > 400 micrograms/dl) . Subsequently, indirect anamnesis revealed the following affections: ureterosigmoidostomy in 1942, cystectomy in 1943, and right-sided nephrectomy in 1950 . DISCUSSION: This case presents with an uncommon origin of the hepatic encephalopathy: in the operated colon, bacterial overgrowth (bacteriogenic ureapoiesis) developed, which led to a hyperammonemia . Furthermore, in mild alcoholic liver disease--as in this case--ammonia cannot be metabolized to urea and leads to hepatic encephalopathy . We considered making this part of the colon poor in bacteria (by consequent administration of antibiotics), thus reducing the formation of ammonia.

J Exp Med, 2001 May 21, 193(10), 1213 - 20
Induction of M3-restricted cytotoxic T lymphocyte responses by N-formylated peptides derived from Mycobacterium tuberculosis; Chun T et al.; Major histocompatibility complex (MHC) class I-restricted CD8(+) T cells play a critical role in the protective immunity against Mycobacterium tuberculosis (Mtb) . However, only a few Mtb peptides recognized by MHC class Ia-restricted CD8(+) T cells have been identified . Information on epitopes recognized by class Ib-restricted T cells is even more limited . M3 is an MHC class Ib molecule that preferentially presents N-formylated peptides to CD8(+) T cells . Because bacteria initiate protein synthesis with N-formyl methionine, the unique binding specificity of M3 makes it especially suitable for presenting these particular bacterial epitopes . We have scanned the full sequence of the Mtb genome for NH2-terminal peptides that share features with other M3-binding peptides . Synthetic peptides corresponding to these sequences were tested for their ability to bind to M3 in an immunofluorescence-based peptide-binding assay . Four of the N-formylated Mtb peptides were able to elicit cytotoxic T lymphocytes (CTLs) from mice immunized with peptide-coated splenocytes . The Mtb peptide-specific, M3-restricted CTLs lysed the Mtb-infected macrophages effectively, suggesting that these N-formylated Mtb peptides are presented as the naturally processed epitopes by Mtb-infected cells . Furthermore, T cells from Mtb-infected lungs, spleen, and lymph nodes responded to N-formylated Mtb peptides in an M3-restricted manner . Taken together, our data suggest that M3-restricted T cells may participate in the immune response to Mtb.

Microbes Infect, 2001 Apr, 3(5), 417 - 24
The molecular puzzle of two-component signaling cascades; Foussard M et al.; Two-component systems constitute prevalent signaling pathways in bacteria and mediate a large variety of adaptative cellular responses . Signaling proceeds through His-Asp phosphorelay cascades that involve two central partners, the histidine protein kinase and the response regulator protein . Structural studies have provided insights into some design principles and activation mechanisms of these multi-domain proteins implicated in the control of virulence gene expression in several pathogens.

Arch Biochem Biophys, 2001 Apr 15, 388(2), 253 - 60
Mutational analyses of Aquifex pyrophilus DNA ligase define essential domains for self-adenylation and DNA binding activity; Lim JH et al.; We constructed nine deletion mutants of NAD+-dependent DNA ligase from Aquifex pyrophilus to characterize the functional domains . All of DNA ligase deletion mutants were analyzed in biochemical assays for NAD+-dependent self-adenylation, DNA binding, and nick-closing activity . Although the mutant lsub1 (91-362) included the active site lysine (KxDG), self-adenylation was not shown . However, the mutants lsub6 (1-362), lsub7 (1-516), and lsub9 (1-635) showed the same adenylation activity as that of wild type . The lsub5 (91-719), which has the C-terminal domain (487-719) as to lsub4 (91-486), showed minimal adenylation activity . These results suggest that the presence of N-terminal 90 residues is essential for the formation of an enzyme-AMP complex, while C-terminal domain (487-719) appears to play a minimal role in adenylation . It was found that the presence of C-terminal domain (487-719) is indispensable for DNA binding activity of lsub5 (91-719) . The mutant lsub9 (1-635) showed reduced DNA binding activity compared to that of wild type, suggesting the contribution of the domain (636-719) for the DNA binding activity . Thus, we concluded that the N-terminal 90 residues and C-terminal domain (487-719) of NAD+-dependent DNA ligase from A . pyrophilus are mutually indispensable for binding of DNA substrate.

Sheng Li Xue Bao, 1998 Oct, 50(5), 557 - 62
{Effects of glutamic acid and acetylcholine on induction of heat shock proteins 70 mRNA in PC12 cells}; Wu BY et al.; The heat shock response has been found in many strains of bacteria to human beings . Besides heat stimuli, many kinds of factors could also induce the synthesis of heat shock proteins (hsp) . It is still unknown whether neurotransmitter could induce the increase of hsp expression in mammalian cells . In the present study, the effects of glutamic acid and acetylcholine (ACh) on the induction of hsp70 mRNA in PC12 cells were studied by Northern blot method . The probe used is specific for inducible hsp70 mRNA . Our results showed that the glutamic acid under limited conditions (such as at 50-500 mumol/L and action time 5-30 min) could induce the expression of hsp70 mRNA, which was partly mediated by NMDA receptors . On the other hand, ACh (0.1-1,000 mumol/L) could not induce the expression of hsp 70 mRNA.

Photochem Photobiol, 2001 May, 73(5), 551 - 5
Expression of a ketolase gene mediates the synthesis of canthaxanthin in Synechococcus leading to tolerance against photoinhibition, pigment degradation and UV-B sensitivity of photosynthesis; Albrecht M et al.; The potential of ketocarotenoids to protect the photosynthetic apparatus from damage caused by excess light and UV-B radiation was assessed . Therefore, the cyanobacterium Synechococcus was transformed with a foreign beta-carotene ketolase gene under a strong promoter leading to the accumulation of canthaxanthin . This diketo carotenoid is absent in the original strain . Most of the newly formed canthaxanthin was located in the thylakoid membranes . The endogenous beta-carotene hydroxylase was unable to interact with the ketolase . Therefore, only traces of astaxanthin were found . The transformant was treated with strong light (500 or 1200 mumol m-2 s-1) and with UV-B radiation . In contrast to a nontransformed strain the overall photosynthesis, measured as oxygen evolution, was protected from inhibition by light of 500 mumol m-2 s-1 and UV-B radiation of 6.8 W m-2 . Furthermore, degradation in the light of chlorophyll and carotenoids at an irradiance of 1200 mumol m-2 s-1, which was substantial in the nontransformed control, was prevented . These results indicate that in situ canthaxanthin, which is formed at the expense of zeaxanthin and replaces this hydroxy carotenoid within the photosynthetic apparatus, is a better protectant against solar radiation . These findings are discussed on the basis of the in vitro properties such as inactivating peroxyl radicals, quenching of singlet oxygen and oxidation stability of these different carotenoid structures.

Photochem Photobiol, 2001 May, 73(5), 537 - 44
The effect of UV irradiation on infection of mice with Borrelia burgdorferi; Brown EL et al.; These studies addressed the hypothesis that UV radiation (UVR) could affect immune responses in mice infected with Borrelia burgdorferi . Immunity against the Lyme spirochete B . burgdorferi was studied in a murine model of UV-induced immune suppression . Borrelia-specific cellular and humoral responses were examined following immunosuppressive doses of UVR . Low-passage Borrelia were injected intradermally at the base of the tail following irradiation . At various time points after infection the blood was cultured for the presence of Borrelia and the serum analyzed for Borrelia-specific antibodies . Two weeks after infection one hind-limb joint was cultured for the presence of spirochetes and the contralateral joint was examined histologically for arthritis formation . The results demonstrated that UV irradiation, administered at the site of infection or at a distant site, suppressed Borrelia-specific cellular and humoral responses in infected mice . Suppression of delayed-type hypersensitivity and antibody responses to UV was abrogated by administration of anti-interleukin (IL)-10 after UV irradiation . In addition, UV irradiation altered the dissemination pattern of the bacteria from the skin into the blood and exacerbated arthritis when compared with unirradiated controls . From these studies we concluded that UV irradiation can modulate the immune response to Borrelia and exacerbate the subsequent arthritic component of Lyme disease in mice . Furthermore, our studies suggest that IL-10 is in part responsible for the suppression of both cellular and humoral responses in addition to playing a role in the development of Lyme arthritis.

J Med Genet, 2001 May, 38(5), 310 - 1
The mannose binding lectin gene influences the severity of chronic liver disease in cystic fibrosis; Gabolde M et al.; Chronic liver disease is a major complication of cystic fibrosis . Its incidence and severity show marked heterogeneity, even among the homogeneous group of homozygous DeltaF508 patients, suggesting that environmental or genetic factors other than the deletion DeltaF508 may influence the development of cystic fibrosis related liver disease . We investigated whether the allelic variants of mannose binding lectin, an important protein of the immune system, could be associated with the presence of cirrhosis in a population of 216 homogeneous homozygous DeltaF508 patients . Analysis of the data shows that the presence of cirrhosis in cystic fibrosis patients is significantly associated with a mutated mannose binding lectin genotype (homozygous or compound heterozygous for mannose binding lectin variants) . The modulating role of mannose binding lectin in the occurrence of cirrhosis in cystic fibrosis could be explained by the fact that hepatotoxic damage from viruses or bacteria might be increased by the immunodeficiency associated with mannose binding lectin variants and might facilitate the degradation of liver status . These data highlight the crucial role of mannose binding lectin in the clinical outcome of cystic fibrosis, as it has recently been shown that the mannose binding lectin gene is a modulating gene of the respiratory involvement in cystic fibrosis patients.

Posit Living, 1999 Nov, 8(10), 14, 19 - 20
Smoking: it doesn't make living with HIV any easier; Wongvipat N; AIDS: Smoking is a well-recognized risk to health, particularly for people with HIV . HIV-positive smokers are more likely to develop Pneumocystis carinii pneumonia (PCP), thrush, and oral hairy leukoplakia . The bacteria that causes Mycobacterium avium Complex (MAC), a life-threatening infection found in many people with HIV, has been found in tobacco products and can survive the smoking process . Suggestions to help people with HIV quit smoking include choosing the right approach, using smoking cessation programs and methods, and obtaining information . Contact information is provided for organizations such as the American Lung Association and the American Heart Association, which have smoking-cessation information and programs .

AIDS Treat News . 1998 Feb 6;(No 288):5.
NTZ submitted to FDA for cryptosporidiosis . Food and Drug Administration; James JS; AIDS: Unimed Pharmaceuticals has applied for Food and Drug Administration (FDA) approval of NTZ (nitaxozanide) for treating cryptosporidial diarrhea in people with AIDS . The drug is already approved in Mexico . NTZ, a broad spectrum drug, is active against many bacteria and parasites . The PWA Health Group, the oldest AIDS buyers club in New York, carries the drug .

AIDS Alert, 1998 Jan, 13(1), suppl 1 - 2
Unpasteurized juice may pose danger; Controlling diarrhea; AIDS: Diarrhea, a common problem in people living with HIV, has a number of causes and steps to control it . It is important to diagnose the cause(s) of diarrhea as soon as possible, followed by appropriate treatment and replenishment of body fluids . Causes can include diet, lactose intolerance, or the antiviral regimen itself . Treatments discussed include the use of medications, bulking agents, specific types of fibrous diets, and consumption of certain bacteria and yeast . Anti-diarrhea medications, while helpful, should be used cautiously so that infection-causing agents are not prevented from being purged from the body .

Crit Path AIDS Proj, 1997 Summer, (No 32), 25 - 7
A reportback from the Third National Conference on Women and HIV; Russell A; AIDS: The Third National Conference on Women and HIV addressed a number of important issues related to infection rates, treatments, and research specific to women . While overall death rates for infected men decreased 15 percent in 1996, the rate for women increased 3 percent . The men's decrease is widely attributed to potent combination therapy, but women are penalized by persistent gaps in treatment . Antivirals were approved without sufficient data on how they act in women's bodies . Women are less likely than men to be prescribed protease inhibitors or have had viral load testing, and most drug trials either include small numbers of women or have no gender-specific tests . There has also been little research into the role of vaginal bacteria that appear to suppress HIV transmission .

AIDS Alert . 1997 Feb;12(2):suppl 2.
Don't have a MAC attack . How to fight AIDS-related MAC . National Institutes of Health; NIAID international AIDS research . National Institute of Allergy and Infectious Diseases; AIDS: With the hope of developing a greater understanding of HIV transmission in the developing world, the National Institute of Allergy and Infectious Diseases (NIAID) is supporting its first program of international HIV prevention trials . Investigators will test HIV prevention strategies in Southeast Asia, sub-Saharan Africa, and the West Indies . Epidemiologic studies have found that, unlike the United States, most HIV infections in the developing world occur as a result of heterosexual contact . In addition, people with current sexually transmitted disease (STD) infections have a greatly increased risk for HIV infection . Therefore, NIAID-supported researchers have developed international trials that are targeted specifically to the needs of the international community . One trial is looking at the possibility of prevention of HIV through the use of topical microbicides, and bacteria- and virus-killing agents applied intravaginally prior to sex . Another study, undertaken in Uganda, will explore whether preventive treatment for STDs can prevent HIV infection . NIAID is working to develop less cumbersome and less costly strategies than those used in the United States for preventing perinatal HIV transmission . Finally, NIAID has achieved some level of success already with a behavioral study in Thailand where military recruits received training in safe sex practices .

AIDS Alert, 1995 Jul, 10(7), suppl 1 - 2
Moderate, regular exercise seen best for immune system; Carotenoid biosynthesis and biotechnological application; Botanisches Institut, J . W . Goethe Universitat Frankfurt, GermanyA survey is given on the carotenoid biosynthetic pathway leading to beta-carotene and its oxidation products in bacteria and plants . This includes the synthesis of prenyl pyrophosphates via the mevalonate or the 1-deoxyxylulose-5-phosphate pathways as well as the reaction sequences of carotenoid formation and interconversion together with the properties of the enzymes involved . Biotechnological application of this knowledge resulted in the development of heterologous carotenoid production systems using bacteria and fungi with metabolic engineered precursor supply and crop plants with manipulated carotenoid biosynthesis . The recent developments in engineering crops with increased carotenoid contents are covered.

J Immunol, 2001 Jun 1, 166(11), 6625 - 32
Clustering of peptide-loaded MHC class I molecules for endoplasmic reticulum export imaged by fluorescence resonance energy transfer; Pentcheva T et al.; Fluorescence resonance energy transfer between cyan fluorescent protein- and yellow fluorescent protein-tagged MHC class I molecules reports on their spatial organization during assembly and export from the endoplasmic reticulum (ER) . A fraction of MHC class I molecules is clustered in the ER at steady state . Contrary to expectations from biochemical models, this fraction is not bound to the TAP . Instead, it appears that MHC class I molecules cluster after peptide loading . This clustering points toward a novel step involved in the selective export of peptide-loaded MHC class I molecules from the ER . Consistent with this model, we detected clusters of wild-type HLA-A2 molecules and of mutant A2-T134K molecules that cannot bind TAP, but HLA-A2 did not detectably cluster with A2-T134K at steady state . Lactacystin treatment disrupted the HLA-A2 clusters, but had no effect on the A2-T134K clusters . However, when cells were fed peptides with high affinity for HLA-A2, mixed clusters containing both HLA-A2 and A2-T134K were detected.

Mol Microbiol, 2001 May, 40(3), 691 - 9
Heparin-binding outer membrane protein of chlamydiae; Stephens RS et al.; As an intracellular pathogen, the mechanism by which Chlamydia invade eukaryotic cells represents a cornerstone to understanding chlamydial biology . The ability of chlamydiae specifically to bind heparan sulphate or heparin and the association of this ability to bind and enter mammalian host cells was approached by searching experimentally for chlamydial outer membrane proteins that bind heparin . The 60 000 molecular weight cysteine-rich outer membrane complex protein, OmcB, bound heparin . The ability of OmcB to bind heparin was supported by mapping the region of the protein with heparin-binding capacity and demonstrating that an OmcB synthetic 20-mer peptide from this region specifically bound heparin . Surface localization of OmcB was shown using monospecific antisera specific to the 20-mer OmcB peptide that bound the surfaces of elementary bodies (EB) and by heparin-binding peptide cross-linking of EB surface proteins.

Microbes Infect, 2001 Mar, 3(3), 171 - 9
Characterization of four members of a multigene family encoding outer membrane proteins of Helicobacter pylori and their potential for vaccination; Peck B et al.; In search of protective antigens which can be used in a vaccine to prevent Helicobacter pylori infection, we report on the identification of four genes, hopV, hopW, hopX and hopY, and the characterization of the corresponding proteins which belong to the H . pylori outer membrane protein (Hop) family containing 32 homologous members, some of which were shown to function as porins . Sequence analysis of 16 different H . pylori strains revealed that the proteins HopV, HopW, HopX and HopY are highly conserved . Localization of HopV, HopW, HopX and HopY at the surface of the bacteria was investigated by immunofluorescence . Using a planar lipid bilayer system the proteins HopV and HopX were shown to form pores with single-channel conductances of 1.4 and 3.0 nS, respectively.

J, Exp . Mar . Biol . Ecol. . 2001 May 31, 260(1), 71 - 91
Seasonal utilization of different seston carbon sources by the ribbed mussel, Geukensia demissa (Dillwyn) in a mid-Atlantic salt marsh; Kreeger DA et al.; Seston in salt marshes contains a temporally and spatially complex mixture of natural microparticulate organic material, including phytoplankton, vascular plant detritus, bacteria, heterotrophic nanoflagellates and benthic diatoms . Quantitative information is available concerning how suspension-feeding consumers, such as the ribbed mussel, Geukensia demissa (Dillwyn), utilize some of these components to satisfy their carbon demands . Despite this information there is still a limited understanding of how the relative nutritive contribution of these different dietary items may shift during the year associated with variations in both seston composition and the mussel's physiological condition . To investigate if the mussel's ability to use specific constituents of natural seston varies seasonally, we ran a series of pulse-chase 14C feeding experiments under ambient conditions in March, May, August and November 1996 . Phytoplankton, cellulosic detritus, bacteria, heterotrophic nanoflagellates and benthic diatoms were radiolabeled and supplemented in small amounts to natural marsh water for feeding to mussels . The fate of 14C in mussel tissues, feces, respiration and excretion was quantified and contrasted among the different diet types and seasons . Microcapsules containing radiolabeled carbohydrate and protein were used as standards to differentiate possible between-experiment variations in seston composition from seasonal changes in the mussel's feeding and digestive physiology . Mussel clearance rates for all diets were highest in summer and autumn and lowest in winter and spring . In contrast, seasonal shifts in digestive physiology were only found for certain diets . The seasonal range of assimilation efficiencies for microcapsule standards (18-29%) and field-collected microheterotrophs (bacteria 76-93% and heterotrophic nanoflagellates 87-94%) did not differ significantly during the year, whereas summer and autumn assimilation efficiencies for cellulosic detritus (22-24%), phytoplankton (71-79%) and benthic diatoms (89-93%) were up to twofold greater than those in winter and spring (13%, 40-59% and 45-81%, respectively) . We conclude that the digestive physiology (e.g., digestive enzyme production) of mussels responds to shifts in dietary components during the year.

Oral Microbiol Immunol, 2001 Jun, 16(3), 185 - 7
Effects of the oral spirochete Treponema denticola on interleukin-8 expression from epithelial cells; Deng QD et al.; This communication demonstrates that the interaction of the oral spirochete Treponema denticola 35405 with KB epithelial cells does not lead to the induction of interleukin-8 production as occurs with a variety of other bacteria . Utilizing the dentilisin protease mutant K1 of T . denticola, this property was demonstrated to be primarily a function of the expression of the protease by strain 35405.

Oral Microbiol Immunol, 2001 Jun, 16(3), 178 - 81
Morphological analysis of Helicobacter pylori from gastric biopsies and dental plaque by scanning electron microscopy; Young KA et al.; Helicobacter pylori is rarely cultured from sites other than the gastric mucosa . The morphology of H . pylori in the stomach and dental plaque of adult dyspeptic patients was investigated to determine whether a difference in morphology at these sites could explain the inability to culture the organism from the oral cavity . Five adult patients attending for an upper gastrointestinal endoscopy were investigated . Dental plaque and gastric antral biopsy samples were analysed by culture and polymerase chain reaction (PCR) both before and after immunomagnetic separation using polyclonal rabbit anti-H . pylori IgG . Bead:bacteria aggregates were then examined by scanning electron microscopy (SEM) . Rod and coccoid forms of H . pylori were seen by SEM in all oral and gastric samples which were H . pylori PCR positive . Although rod and coccoid forms have previously been shown to be associated with the gastric mucosa, this is the first time H . pylori cells have been visualized in dental plaque.

Electrophoresis, 2001 Apr, 22(6), 1204 - 23
Proteome analysis of the Chlamydia pneumoniae elementary body; Vandahl BB et al.; Chlamydia pneumoniae is an obligate intracellular human pathogen that causes acute and chronic respiratory tract diseases and that has been implicated as a possible risk factor in the development of atherosclerotic heart disease . C . pneumoniae cultivated in Hep-2 cells were 35S-labeled and infectious elementary bodies (EB) were purified . The EB proteins were separated by two-dimensional gel electrophoresis . Excised protein spots were in-gel digested with trypsin and peptides were concentrated on reverse-phase chromatographic beads for identification analysis by matrix-assisted laser desorption/ionization-mass spectrometry . In the pH range from 3-11, 263 C . pneumoniae protein spots encoded from 167 genes were identified . These genes constitute 15% of the genome . The identified proteins include 31 hypothetical proteins . It has recently been suggested that EB should be able to synthesize ATP . This view may be strengthened by the identification of several proteins involved in energy metabolism . Furthermore, proteins have been found which are involved in the type III secretion apparatus important for pathogenesis of intracellular bacteria . Proteome maps and a table of all identified proteins have been made available on the world wide web at www.gram.au.dk.

Biochem Biophys Res Commun, 2001 May 25, 283(5), 1054 - 60
Application of the fluorescence resonance energy transfer method for studying the dynamics of caspase-3 activation during UV-induced apoptosis in living HeLa cells; Luo KQ et al.; Activation of caspase-3 is a central event in apoptosis . We have developed a GFP-based FRET (fluorescence resonance energy transfer) probe that is highly sensitive to the activation of caspase-3 in intact living cells . This probe was constructed by fusing a CFP (cyan fluorescent protein) and a YFP (yellow fluorescent protein) with a specialized linker containing the caspase-3 cleavage sequence: DEVD . The linker design was optimized to produce a large FRET effect . Using purified protein, we observed a fivefold change in the fluorescence emission ratio when the probe was cleaved by caspase-3 . To demonstrate the usefulness of this method, we introduced this FRET probe into HeLa cells by both transient and stable transfection . We observed that during UV-induced apoptosis, the activation of caspase-3 varied significantly between different cells; but once the caspase was activated, the enzyme within the cell became fully active within a few minutes . This technique will be highly useful for correlating the caspase-3 activation with other apoptotic events and for rapid-screening of potential drugs that may target the apoptotic process .

Vet Pathol, 2001 May, 38(3), 343 - 6
Granulomatous enteritis and lymphadenitis in Iberian pigs naturally infected with Lawsonia intracellularis; Segales J et al.; Intestinal samples and/or lymph nodes of two Iberian pigs from two different farms were submitted for histopathologic examination . Both pigs had proliferation of ileal and/or cecal crypts with almost complete absence of goblet cells . Infection by Lawsonia intracellularis was demonstrated by immunohistochemistry and polymerase chain reaction assay . The mesenteric lymph node of one pig had moderate lymphocyte depletion with granulomatous inflammation of the lymph node parenchyma . Histiocytes and multinucleated giant cells from the lymph node of one pig contained L . intracellularis antigen within the cytoplasm . This pig had also porcine circovirus type 2 (PCV-2) infection, but nucleic acid and antigen of this virus were not demonstrated in the lymph node . The second pig had lymphocyte depletion and marked granulomatous inflammation in Peyer's patches . Histiocytes and multinucleated giant cells in areas of granulomatous inflammation contained L . intracellularis antigen; no PCV-2 nucleic acid or antigen was detected in the tissues of this pig . This is the first description of granulomatous ileitis and lymphadenitis associated with L . intracellularis infection.

Indian J Biochem Biophys, 2000 Dec, 37(6), 447 - 52
Analysis of the activity of promoters from two photosynthesis-related genes psaF and petH of spinach in a monocot plant, rice; Mohanty A et al.; The subunit III of photosystem I and ferredoxin-NADP(+)-oxidoreductase are encoded by nuclear genes, namely psaF and petH . The activity of their promoters from spinach has been evaluated in transgenic tobacco earlier . Evaluation of the activity of these Dicotyledoneae-specific promoters has been carried out in a monocot system (i.e . rice) by transient gene expression system, based on electroporation-mediated gene delivery into protoplasts from leaves and roots . It has been found that various promoter deletions show higher activity in leaf protoplasts and elements for quantitative response are widely distributed . Transgenic rice has also been produced with a petH promoter and gus reporter gene construct . Although petH promoter is a weak promoter in comparison to the 35S promoter, it expresses well in green tissues and could be useful for plant genetic engineering.

Postepy Hig Med Dosw, 2001, 55(1), 177 - 88
{Granules of neutrophils}; Wysocka J et al.; The exocytosis of cytoplasmic granules plays a most important role in the regulation of many neutrophils functions: adhesion, phagocytosis, killing of bacteria and interaction with endothelial cells . Neutrophils contain following types of granules: azurophilic (primary) granules, specific (secondary) granules, gelatinase-rich tertiary granules and secretory vesicles . Neutrophil granules may be classified on the basis of their size, morphology, density or with reference to a given protein.

Biotechniques, 2001 May, 30(5), 1028 - 34
Four-color flow cytometric detection of retrovirally expressed red, yellow, green, and cyan fluorescent proteins; Hawley TS et al.; Flow cytometric procedures are described to detect a "humanized" version of a new red fluorescent protein (DsRed) from the coral Discosoma sp . in conjunction with various combinations of three Aequorea victoria green fluorescent protein (GFP) variants--EYFP, EGFP, and ECFP . In spite of overlapping emission spectra, the combination of DsRed with EYFP, EGFP, and ECFP generated fluorescence signals that could be electronically compensated in real time using dual-laser excitation at 458 and 568 nm . Resolution of fluorescence signals from DsRed, EYFP, and EGFP was also readily achieved by single-laser excitation at 488 nm . Since many flow cytometers are equipped with an argon-ion laser that can be tuned to 488 nm, the DsRed/EYFP/EGFP combination is expected to have broad utility for facile monitoring of gene transfer and expression in mammalian cells . The dual-laser technique is applicable for use on flow cytometers equipped with tunable multiline argon-ion and krypton-ion lasers, providing the framework for studies requiring simultaneous analysis of four fluorescent gene products within living cells.

N Z Dent J, 2001 Mar, 97(427), 9 - 14
Plaque mineral induction and inhibition properties in the formation of supragingival calculus; Pearce EI et al.; Several individual species of dental plaque bacteria have the ability to initiate the precipitation of calcium phosphate minerals in vitro; other plaque components have been shown to inhibit mineralisation . We have examined subjects' overall plaque mineralisation promoter and inhibitor properties, and have attempted to correlate them with supragingival calculus development over 6 months . Three-day-old plaque was collected from 22 adult subjects at the start and end of the study . To detect promoter activity, the plaque was placed in a suspension of brushite, the liquid phase of which was supersaturated with respect to hydroxyapatite . The extent of mineralisation was determined by the rise in phosphate concentration over 4 days . To detect inhibitor activity, plaque was placed in a similar suspension, which also contained hydroxyapatite . Promoter activity was compared with that hydroxyapatite, and inhibitor activity was compared with polyaspartate . The subjects' teeth were scaled at the start of the study, and calculus deposition was measured at the end using the Volpe Manhold method . Most plaque samples showed some promoter or inhibitor activity, or both, but no significant correlation existed between these activities and a subject's development of calculus . A significant inverse correlation existed between plaque mineralisation promoter activity and its inhibitor activity at the start of the study . Our results suggest that the nucleating and mineralisation inhibitory properties of young plaque will probably not be a useful target for a practical preventive methodology for supragingival calculus.

Environ Sci Technol, 2001 May 1, 35(9), 1830 - 9
Assessment of indigenous reductive dechlorinating potential at a TCE-contaminated site using microcosms, polymerase chain reaction analysis, and site data; Fennell DE et al.; A combination of microcosm studies, polymerase chain reaction (PCR) analysis, and site data was used to assess the indigenous reductive dechlorinating potential in a trichloroethene (TCE)-contaminated aquifer at Cape Canaveral Air Station, Florida . Sediment and groundwater were obtained from two distinct locations approximately 10 m apart . Microcosm studies were performed to assess dechlorinating activity under a variety of nutrient and electron donor amendment conditions . Most live microcosms constructed using material from the first location, near well 9 (W09), were negative for dechlorination . All live microcosms constructed using material from the second location (W06) exhibited dechlorination of TCE to vinyl chloride (VC) and ethene (ETH) . DNA encoding 16S ribosomal RNA (rDNA) with a sequence nearly identical with that from Dehalococcoides ethenogenes strain 195 was detected in the active microcosms and in the sediment from W06 with polymerase chain reaction (PCR) using primers targeted to unique regions of Dehalococcoides 16S rDNA . Dehalococcoides was not detected in the autoclaved microcosms from W06, nor in sediment and most microcosms from W09 . The results of the microcosm studies and PCR analysis were supported by field data, which indicated significant accumulation of cis-1,2-dichloroethene (cisDCE) and VC at W06, but not at W09 . The different microcosm results obtained for the two locations and the spatial variation of positive PCR results indicates heterogeneous distribution of dechlorinating activity and a specific dechlorinating organism, Dehalococcoides, at the site . As both Dehalococcoides and dechlorination activity were similarly, heterogeneously distributed, this suggests that molecular-probing (which could and should be extended in the future to include virtually all known dechlorinators and/or dehalogenases) can provide a relatively quick and facile method for investigating spatial distributions of dechlorinators on-site.

Oral Dis, 2001 Jan, 7(1), 41 - 6
Lipid peroxidation caused by oxygen radicals from Fusobacterium-stimulated neutrophils as a possible model for the emergence of periodontitis; Sheikhi M et al.; OBJECTIVE: The possible contribution of bacteria and polymorphonuclear neutrophils (PMN) to the disease process of periodontitis was evaluated . DESIGN: Fusobacterium nucleatum has been associated with chronic adult periodontitis . Intracellular production and extracellular release of reactive oxygen species (ROS) by PMN stimulated by fusobacteria were evaluated . To estimate the potential extracellular damage that might be caused by the ROS, the lipid peroxidation (LPO) of an exogenous phospholipid, Intralipid, was assayed . METHODS: The ROS production of PMN was studied by the nitroblue tetrazolium and chemiluminescence tests . The levels of malonaldehyde (MDA) and 4-hydroxyalkenals were used to indicate LPO . RESULTS: Fusobacterium nucleatum strains stimulated neutrophils to produce a large amount of ROS, independently of plasma complement factors . The two strains tested induced considerable intracellular, but no extracellular chemiluminescence responses during the first hour, indicating that ROS were released into phagosomes . However an incubation period of 4 h, in the presence of the extracellular lipid resulted in a high degree of LPO, presumably caused by ROS release from the Fusobacterium-stimulated PMN . ROS production and lipid peroxidation could be counteracted by vitamin E . CONCLUSION: In periodontitis local bacteria might stimulate PMN to release ROS, which cause inflammation and destruction.

Amino Acids, 2001, 20(3), 225 - 41
Does phosphoenolpyruvate carboxykinase have a role in both amino acid and carbohydrate metabolism?
Lea PJ, Chen ZH, Leegood RC, Walker RP.
Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the reversible decarboxylation of oxaloacetate to yield phosphoenolpyruvate and CO2 . The role of the enzyme in gluconeogenesis and anaplerotic reactions in a range of organisms is discussed, along with the important function in C4 and CAM photosynthesis in higher plants . In addition, new data are presented indicating that PEPCK may play a key role in amino acid metabolism . It is proposed that PEPCK is involved in the conversion of the carbon skeleton of asparagine/aspartate (oxaloacetate) to that of glutamate/glutamine (2-oxoglutarate) . This metabolism is particularly important in the transport system, seeds and fruits of higher plants.

Rev Neurol, 2001 Mar 16-31, 32(6), 501 - 5
{The presence of anti-Chlamydia pneumoniae antibodies in peripheral vascular and neurological disorders}; Gutierrez-Fernandez J et al.; OBJECTIVES: To make a retrospective analysis of the synthesis of antibodies to the MOMPS and LPS antigens of Chlamydia pneumoniae in patients with occlusive disease of the peripheral arteries (ODPA) and multiple sclerosis (MS) . PATIENTS AND METHODS: We studied 190 samples of plasma from patients included in the following groups: group 1:66 samples from 66 patients with ODPA; group 2:74 samples from 31 patients with MS (20 remittent-relapsing and 11 secondarily progressive), followed over time; and group 3:50 samples from persons acting as controls . In all cases determinations were made using ELISA, of the IgG anti-MOMP and the IgG and IgA anti-LPS . Comparison of the continuous variables was made using the Mann-Whitney U Test . Discrete variables were analysed using the exact bilateral Fisher Test . The Wilcoxon Test over ranges was used to compare the evolution of antibodies in the patients with MS . RESULTS: The percentage of positive results in groups 1 to 3 for anti-LPS IgG were: 24.6%, 18.9% and 20.8%, respectively, with no differences between patients and controls; nor were there any differences with IgA (29%, 29.7% and 25%, respectively) . However differences were seen in the anti-MOMP IgG between patients and controls (group 1:80.3%, group 2:37.8% and group 3: 33.3%) . In patients with MS the results of the evolution of the antibodies did not reflect a uniform tendency of the levels of the different antibodies . CONCLUSION: A higher level of IgG anti-MOMP was seen in ODPA and MS, although this did not occur with anti-LPS or IgA.

Proc Natl Acad Sci U S A, 2001 May 22, 98(11), 6003 - 8 Epub 2001 May 15.
Conformational coupling in the chemotaxis response regulator CheY; Schuster M et al.; CheY, a response regulator protein in bacterial chemotaxis, serves as a prototype for the analysis of response regulator function in two-component signal transduction . Phosphorylation of a conserved aspartate at the active site mediates a conformational change at a distal signaling surface that modulates interactions with the flagellar motor component FliM, the sensor kinase CheA, and the phosphatase CheZ . The objective of this study was to probe the conformational coupling between the phosphorylation site and the signaling surface of CheY in the reverse direction by quantifying phosphorylation activity in the presence and absence of peptides of CheA, CheZ, and FliM that specifically interact with CheY . Binding of these peptides dramatically impacted autophosphorylation of CheY by small molecule phosphodonors, which is indicative of reverse signal propagation in CheY . Autodephosphorylation and substrate affinity, however, were not significantly affected . Kinetic characterization of several CheY mutants suggested that conserved residues Thr-87, Tyr-106, and Lys-109, implicated in the activation mechanism, are not essential for conformational coupling . These findings provide structural and conceptual insights into the mechanism of CheY activation . Our results are consistent with a multistate thermodynamic model of response regulator activation.

Biochemistry, 2001 May 22, 40(20), 6047 - 52
Primary photoreaction of photoactive yellow protein studied by subpicosecond-nanosecond spectroscopy; Imamoto Y et al.; The primary photochemical event of photoactive yellow protein (PYP) was studied by laser flash photolysis experiments on a subpicosecond-nanosecond time scale . PYP was excited by a 390-nm pulse, and the transient difference absorption spectra were recorded by a multichannel spectrometer for a more reliable spectral analysis than previously possible . Just after excitation, an absorbance decrease due to the stimulated emission at 500 nm and photoconversion of PYP at 450 nm were observed . The stimulated emission gradually shifted to 520 nm and was retained up to 4 ps . Then, the formation of a red-shifted intermediate with a broad absorption spectrum was observed from 20 ps to 1 ns . Another red-shifted intermediate with a narrow absorption spectrum was formed after 2 ns and was stable for at least 5 ns . The latter is therefore believed to correspond to I1 (PYP(L)), which has been detected on a nanosecond time scale or trapped at -80 degrees C . Singular value decomposition analysis demonstrated that the spectral shifts observed from 0.5 ps to 5 ns could be explained by two-component decay of excited state(s) and conversion from PYP(B) to PYP(L) . The amount of PYP(L) at 5 ns was less than that of photoconverted PYP, suggesting the formation of another intermediate, PYP(H) . In addition, the absorption spectra of these intermediates were calculated based on the proposed reaction scheme . Together, these results indicate that the photocycle of PYP at room temperature has a branched pathway in the early stage and is essentially similar to that observed under low-temperature spectroscopy.

Biochemistry, 2001 May 22, 40(20), 5854 - 60
The C2B domain of synaptotagmin I is a Ca2+-binding module; Ubach J et al.; Synaptotagmin I is a synaptic vesicle protein that contains two C(2) domains and acts as a Ca(2+) sensor in neurotransmitter release . The Ca(2+)-binding properties of the synaptotagmin I C(2)A domain have been well characterized, but those of the C(2)B domain are unclear . The C(2)B domain was previously found to pull down synaptotagmin I from brain homogenates in a Ca(2+)-dependent manner, leading to an attractive model whereby Ca(2+)-dependent multimerization of synaptotagmin I via the C(2)B domain participates in fusion pore formation . However, contradictory results have been described in studies of Ca(2+)-dependent C(2)B domain dimerization, as well as in analyses of other C(2)B domain interactions . To shed light on these issues, the C(2)B domain has now been studied using biophysical techniques . The recombinant C(2)B domain expressed as a GST fusion protein and isolated by affinity chromatography contains tightly bound bacterial contaminants despite being electrophoretically pure . The contaminants bind to a polybasic sequence that has been previously implicated in several C(2)B domain interactions, including Ca(2+)-dependent dimerization . NMR experiments show that the pure recombinant C(2)B domain binds Ca(2+) directly but does not dimerize upon Ca(2+) binding . In contrast, a cytoplasmic fragment of native synaptotagmin I from brain homogenates, which includes the C(2)A and C(2)B domains, participates in a high molecular weight complex as a function of Ca(2+) . These results show that the recombinant C(2)B domain of synaptotagmin I is a monomeric, autonomously folded Ca(2+)-binding module and suggest that a potential function of synaptotagmin I multimerization in fusion pore formation does not involve a direct interaction between C(2)B domains or requires a posttranslational modification.

J Mol Biol, 2001 May 18, 308(5), 1063 - 79
Two crystal structures of the cytoplasmic molybdate-binding protein ModG suggest a novel cooperative binding mechanism and provide insights into ligand-binding specificity; Delarbre L et al.; The X-ray structures of the cytoplasmic molybdate-binding protein ModG from Azotobacter vinelandii in two different crystal forms have been determined . For such a small protein it is remarkably complex . Each 14.3 kDa subunit contains two small beta-barrel domains, which display an OB-fold motif, also seen in the related structure of ModE, a molybdenum-dependent transcriptional regulator, and very recently in the Mop protein that, like ModG, has been implicated in molybdenum homeostasis within the cell . In contrast to earlier speculation, the functional unit of ModG is actually not a dimer (as in ModE), but a trimer capable of binding a total of eight molybdate molecules that are distributed between two disparate types of site . All the binding sites are located at subunit interfaces, with one type lying on a crystallographic 3-fold axis, whilst the other lies between pairs of subunits . The two types of site are linked by short hydrogen bond networks that may suggest a cooperative binding mechanism . A superposition of two subunits of the ModG trimer on the apo-ModE dimer allows the probable locations of the molybdate-binding sites of the latter to be assigned . Through structural comparisons with other oxyanion-binding proteins, including Mop and ModE, it is possible to speculate about ligand-binding affinities, selectivity and evolution . Copyright 12001 Academic Press.

Plant Mol Biol, 2001 Mar, 45(4), 461 - 8
Plant mitochondria contain proteolytic and regulatory subunits of the ATP-dependent Clp protease; Halperin T et al.; The proteolytic machinery of plant organelles is largely unknown, although indications so far point to several proteases of bacterial origin . In this study an Arabidopsis thaliana cDNA was isolated that encodes a homologue of bacterial ClpX, a molecular chaperone and regulatory subunit of the ATP-dependent, serine-type Clp protease . Computer analysis of the predicted plant ClpX revealed a putative mitochondrial transit peptide at the N-terminus, as well as overall sequence similarity to other eukaryotic ClpX homologues . Specific polyclonal antibodies were made to the Arabidopsis ClpX protein and used to confirm its localization in plant mitochondria . In addition to ClpX, a ClpP protein located in mitochondria was also identified from the numerous ClpP isomers in Arabidopsis . Localization of this nuclear-encoded protein, termed ClpP2, was determined first by its close sequence similarity to mitochondrial ClpP human, and later experimentally using ClpP2-specific antibodies with isolated plant organellar fractions . In Arabidopsis, transcripts for both clpX and clpP2 genes were detected in various tissues and under different growth conditions, with no significant variation in mRNA level (i.e . 2-fold) for each gene between samples . Using beta-casein as a substrate, plant mitochondria were found to possess an ATP-stimulated, serine-type proteolytic activity that could be strongly inhibited by antibodies specific for ClpX or ClpP2, suggesting an active ClpXP protease . The recent discovery of homologous mitochondrial ClpX and ClpP proteins in mammals suggests that this type of protease may be common to multicellular eukaryotes.

J Dairy Sci, 2001 Apr, 84(4), 818 - 23
Bioluminescence as a technique to evaluate udder preparation; Finger R et al.; Food production processes are increasingly influenced by quality and safety concerns . For dairy production, one of the food quality outcomes is a low level of bacteria in unprocessed milk . A putative on-farm control point for low levels of bacteria is teat and udder cleaning before milking . Currently there are no appropriate on-farm schemes to monitor the effectiveness of different processes used to prepare cows for milking . The purpose of this project was to compare levels of teat skin bioluminescence with direct bacterial culture as a tool to evaluate teat cleanliness of dairy cows . Bioluminescence demonstrated average changes in cow cleanliness as animals proceeded through the premilking sanitation steps and, in that manner, could be used as a tool to demonstrate the effectiveness of the process.

Science, 2001 May 11, 292(5519), 1109 - 12
Population biology of multihost pathogens; Woolhouse ME et al.; The majority of pathogens, including many of medical and veterinary importance, can infect more than one species of host . Population biology has yet to explain why perceived evolutionary advantages of pathogen specialization are, in practice, outweighed by those of generalization . Factors that predispose pathogens to generalism include high levels of genetic diversity and abundant opportunities for cross-species transmission, and the taxonomic distributions of generalists and specialists appear to reflect these factors . Generalism also has consequences for the evolution of virulence and for pathogen epidemiology, making both much less predictable . The evolutionary advantages and disadvantages of generalism are so finely balanced that even closely related pathogens can have very different host range sizes.

Science, 2001 May 11, 292(5519), 1099 - 102
The ecology of genetically diverse infections; Read AF et al.; Microparasite infections often consist of genetically distinct clonal lineages . Ecological interactions between these lineages within hosts can influence disease severity, epidemiology, and evolution . Many medical and veterinary interventions have an impact on genetic diversity within infections, but there is little understanding of the long-term consequences of such interventions for public and animal health . Indeed, much of the theory in this area is based on assumptions contradicted by the available data.

Environ Sci Technol, 2001 Feb 1, 35(3), 516 - 21
Reductive dechlorination of cis-1,2-dichloroethene and vinyl chloride by "Dehalococcoides ethenogenes"; Maymo-Gatell X et al.; cis-Dichloroethene (DCE) and vinyl chloride (VC) often accumulate in contaminated aquifers in which tetrachloroethene (PCE) or trichloroethene (TCE) undergo reductive dechlorination . "Dehalococcoides ethenogenes" strain 195 is the first isolate capable of dechlorinating chloroethenes past cis-DCE . Strain 195 could utilize commercially synthesized cis-DCE as an electron acceptor, but doses greater than 0.2 mmol/L were inhibitory, especially to PCE utilization . To test whether the cis-DCE itself was toxic, or whether the toxicity was due to impurities in the commercial preparation (97% nominal purity), we produced cis-DCE biologically from PCE using a Desulfitobacterium sp . culture . The biogenic cis-DCE was readily utilized at high concentrations by strain 195 indicating that cis-DCE was not intrinsically inhibitory . Analysis of the commercially synthesized cis-DCE by GC/mass spectrometry indicated the presence of approximately 0.4% mol/mol chloroform . Chloroform was found to be inhibitory to chloroethene utilization by strain 195 and at least partially accounts for the inhibitory activity of the synthetic cis-DCE . VC, a human carcinogen that accumulates to a large extent in cultures of strain 195, was not utilized as a growth substrate, and cultures inoculated into medium with VC required a growth substrate, such as PCE, for substantial VC dechlorination . However, high concentrations of PCE or TCE inhibited VC dechlorination . Use of a hexadecane phase to keep the aqueous PCE concentration low in cultures allowed simultaneous utilization of PCE and VC . At contaminated sites in which "D . ethenogenes" or similar organisms are present, biogenic cis-DCE should be readily dechlorinated, chloroform as a co-contaminant may be inhibitory, and concentrations of PCE and TCE, except perhaps those near the source zone, should allow substantial VC dechlorination.

Ann Rheum Dis, 2001 Jun, 60(6), 605 - 11
Male sex predominance in Chlamydia trachomatis sexually acquired reactive arthritis: are women more protected by anti-chlamydia antibodies?
Bas S, Scieux C, Vischer TL.
OBJECTIVE: To determine whether the humoral anti-chlamydia antibody response might be related to the ineffective bacterial elimination seen in patients with Chlamydia trachomatis reactive arthritis, particularly in men, who have a higher prevalence of the disease than women . METHODS: The number and specificity of the antibody responses to 27 different C trachomatis antigens were determined by western blots in serum samples from patients with C trachomatis urogenital infection, with and without reactive arthritis, with a special regard to the sex of the patients . RESULTS: Patients with reactive arthritis had antibodies to significantly fewer chlamydia antigens than those with urethritis only . Antibodies from men recognised significantly fewer antigens than antibodies from women . The IgA class antibodies were slightly more relevant than those of the IgG class for differentiation of patients with reactive arthritis from those with uncomplicated genitourinary infection . CONCLUSIONS: In patients with acute C trachomatis infection the development of reactive arthritis may be related, particularly in men, to a deficient humoral response, to antigens which perhaps play a part in the clearance of the bacteria . Men who cannot generate antibodies to a large number of antigens may be less able to contain the local infection, allowing a wide systemic dissemination of the organisms to the joints.

Traffic, 2001 May, 2(5), 345 - 57
Trafficking of yellow-fluorescent-protein-tagged mu1 subunit of clathrin adaptor AP-1 complex in living cells; Huang F et al.; Clathrin adaptor protein AP-1 complex is thought to function in forming clathrin-coated vesicles at the trans-Golgi network (TGN) and mediating transport of cargo between the TGN and endosomes . To study trafficking of AP-1 in living cells, yellow fluorescent protein (YFP) was inserted in the middle of mu1 A subunit of AP-1 . When expressed in a tetracycline-dependent manner in HeLa cells, YFP-mu1 was efficiently incorporated into the AP-1 complex, replacing endogenous mu1 in most of cellular AP-1 . Time-lapse imaging revealed that YFP-mu1/AP-1 departs from TGN as isolated vesicles and spherical structures, or varicosities, associated with fine tubular processes . Typically, several vesicles or varicosities were seen moving sequentially along the same 'tracks' from TGN to cell periphery . These data suggest that AP-1 may function after formation of Golgi transport intermediates in facilitating their intracellular movement . Mutagenesis of YFP-mu1 determined that the structural requirements for its binding to tyrosine-containing sequence motifs are similar to those previously defined in mu2 subunit of AP-2 . Moreover, the carboxyl-terminal half of mu2 could replace the corresponding fragment of mu1 without loss of the ability of the resulting mu1-YFP-mu2 chimeric protein to incorporate into AP-1 and bind tyrosine-containing motifs . Mutations that abolish binding capacity for tyrosine motifs did not mistarget AP-1 in the cell, suggesting that AP-1 interactions with this type of sorting signals are not essential for membrane docking of AP-1 at the TGN . Altogether, this study demonstrates that YFP-tagged mu1 protein can serve as a useful tool for visualizing the dynamics of AP-1 in living cells and for the structure-function analysis of mu1-cargo interactions.

J Gastroenterol Hepatol, 2001 May, 16(5), 513 - 8
Endoscopic duodenitis, gastric metaplasia and Helicobacter pylori; Urakami Y et al.; BACKGROUND AND AIMS: The purpose of this study was to investigate the relationship between gastric metaplasia and Helicobacter pylori in patients with endoscopic duodenitis . METHODS: The subjects were 57 patients with endoscopic duodentitis with or without H . pylori-associated gastritis . Biopsy specimens were obtained from the stomach and duodenal bulb to assess the histological findings and H . pylori infection . Gastric metaplasia was divided into three types: complete, intermediate and incomplete, according to the amount of mucus in the metaplastic cells . In 10 H . pylori-positive patients, endoscopic and histological findings of duodenitis were compared before and after eradication of the bacteria . RESULTS: There was no significant difference in the extent of gastric metaplasia or the appearance and severity of endoscopic duodenitis between H . pylori-positive and -negative groups . The complete type of gastric metaplasia was frequently detected in the H . pylori-negative group, whereas the incomplete type was frequently observed in the H . pylori-positive group . After eradication of H . pylori, the incomplete type changed to the complete type with a decrease of histological inflammation . CONCLUSIONS: The complete type of gastric metaplasia occurred frequently without H . pylori infection, whereas the incomplete type was frequently associated with H . pylori infection.

J Clin Periodontol, 2001 May, 28(5), 419 - 24
Flow-cytometric analysis of lymphocyte subsets and mCD14 expression in patients with various periodontitis categories; Buduneli N et al.; BACKGROUND: Membrane-bound CD14 (mCD14) is expressed mainly on circulating monocytes and tissue macrophages . It is one of the receptors, which act at the recognition of lipopolysaccharides by host cells . Periodontopathic bacteria result in activation of cellular and humoral immune responses . AIM: The aim of the present study was to analyze the peripheral blood mCD14 concentrations as well as cell surface markers of lymphocyte subsets in periodontitis patients of various categories . MATERIALS AND METHODS: Peripheral blood samples were obtained from 22 early onset periodontitis (EOP), 10 adult periodontitis (AP) patients and 13 systemically and periodontally healthy control subjects . Three-color flow cytometry and a panel of relevant monoclonal antibodies were used to determine the percent expression of various cell surface markers on peripheral blood mononuclear cells (PBMCs) . The results were tested statistically by one-way variance analysis and Newman Keuls test . RESULTS: No significant difference was observed between the study groups with regard to the relative counts of B-cells, T-cells, T-helper, T-cytotoxic/suppressor, activated T-cells and natural killer cells . EOP patients expressed significantly lower level of interleukin-2 receptor (IL-2R) when compared with AP patients (6.08% and 19.3% respectively) (p<0.05) . The level of mCD14 in EOP patients (7.18%) was lower than that of AP patients (9.3%) and the control subjects (9.2%), but the differences were not statistically significant . CONCLUSIONS: The low level of IL-2R in the EOP group may be interpreted as an insufficient responsiveness to the periodontopathogens, which may be ultimately related with the more severe tissue destruction . Though not significant, the reduced expression of mCD14 in EOP group may also be related with the immune system deficiencies in these patients.

Environ Toxicol Chem, 2001 Mar, 20(3), 498 - 501
Enhanced mineralization of benzo{a}pyrene in the presence of nonaqueous phase liquids; Kanaly RA et al.; Bacterial mineralization of {7-14C}benzo{a}pyrene (BaP) to 14CO2 was enhanced by the presence of nonaqueous phase liquids (NAPLs) . Mineralization of BaP was affected differently by different NAPLs, and the mode of enhancement of mineralization by a NAPL most likely occurred by a combination of cometabolic and physical effects . Mineralization was enhanced to the greatest extent when BaP was dissolved in a high-boiling distillation product of diesel fuel.

Transplantation, 2001 Apr 15, 71(7), 841 - 50
Modulation of immune responses after portal venous injection of antigen; Wrenshall LE et al.; BACKGROUND: How the localization of antigen to the liver, through means such as oral ingestion, induces tolerance is poorly understood . METHODS: To elucidate potential mechanisms we used an adoptive transfer system wherein ova-specific T cells were infused into a syngeneic host, and antigen-specific T-cell responses after delivery of soluble antigen into the liver were monitored . RESULTS: After infusion of antigen into the portal vein, the frequency of antigen-specific T cells in lymph nodes draining the liver was lower than the frequency in peripheral lymph nodes . These findings were the reverse of what is typically observed after subcutaneous injection of antigen with adjuvant . Infusion of antigen with adjuvant into the portal vein did not alter this pattern of antigen-specific T-cell localization; however, an increased frequency of T cells, compared with antigen alone, was observed in peripheral lymph nodes and spleen . After exposure to antigen via the portal vein, T cells isolated from lymph nodes draining the liver and challenged with antigen in vitro exhibited a diminished proliferative response compared with T cells isolated from nondraining lymph nodes . This hyporesponsiveness was not observed when the antigen was administered with adjuvant . CONCLUSIONS: Our findings suggest that the influence of the liver on immune responses might reflect two processes: (1) loss of antigen-specific T cells after primary antigen injection, and (2) hyporesponsiveness on reexposure to antigen . These mechanisms may contribute to the prevention of undesirable immune responses to foods and enteric bacteria in the gastrointestinal tract . Furthermore, these results underscore the importance of minimizing inflammation in circumstances such as islet transplantation, if endogenous mechanisms of tolerance induction are to be maximized.

Environ Technol, 2001 Feb, 22(2), 157 - 64
River and groundwater nitrogen contamination caused by livestock production; Maekawa T et al.; Water quality of rivers in Japanese domestic dairy and pig raising regions, as well as the groundwater in these regions, was investigated . Regarding the method of disposing livestock excreta, interview results from the livestock production farmers and the results of water quality analysis were evaluated . It is concluded that the rivers and the groundwater were contaminated due to inappropriate disposal methods of the livestock excreta . The concentrations of ammonium nitrogen and nitrate nitrogen in the rivers and groundwater were high . The sludge from the bottom of the rivers was also investigated and bacteria which are characteristic of excreta of dairy cattle and pigs were detected . The above pollutants were, therefore, considered to be of livestock origin.

Infect Immun, 2001 Jun, 69(6), 4168 - 73
gamma-Glutamyltransferase is a Helicobacter pylori virulence factor but is not essential for colonization; McGovern KJ et al.; The contribution of glutamyl transpeptidase (GGT) (gamma-glutamyltransferase {EC 2 . 3 . 2 . 2}) to Helicobacter pylori virulence was investigated in piglets and mice using GGT-deficient isogenic strains . All animals became colonized . However, the bacterial load was significantly lower for mutant bacteria than for parent strains . These results suggest that GGT activity provides an advantage to H . pylori in colonization.

Infect Immun, 2001 Jun, 69(6), 4146 - 53
Distinct regulatory pathways control expression of Borrelia burgdorferi infection-associated OspC and Erp surface proteins; Babb K et al.; Deciphering the mechanisms by which Borrelia burgdorferi controls the synthesis of proteins associated with mammalian infection will be an important step toward understanding the pathogenic properties of Lyme disease-causing bacteria . We present results of studies indicating that B . burgdorferi senses a wide variety of environmental stimuli, including soluble chemicals, which enables it to independently control synthesis of the Erp and OspC proteins . Regulation of OspC and Erp expression appears to occur at the level of transcription . In this regard, we observed that one or more DNA-binding proteins interact specifically with erp promoter DNA but not with the ospC promoter.

Infect Immun, 2001 Jun, 69(6), 4120 - 4
Vaccination with Bordetella pertussis-pulsed autologous or heterologous dendritic cells induces a mucosal antibody response in vivo and protects against infection; George-Chandy A et al.; This study demonstrates for the first time that vaccination with either autologous or heterologous dendritic cells (DC) pulsed with specific antigen induces protective immune responses against noninvasive bacteria, namely Bordetella pertussis . The DC-mediated protection is associated with strong B . pertussis-specific immunoglobulin G (IgG) and IgA responses in the lung.

Infect Immun, 2001 Jun, 69(6), 3995 - 4006
Intracellular survival of Brucella spp . in human monocytes involves conventional uptake but special phagosomes; Rittig MG et al.; Brucella spp . are facultative intracellular parasites of various mammals, including humans, typically infecting lymphoid as well as reproductive organs . We have investigated how B . suis and B . melitensis enter human monocytes and in which compartment they survive . Peripheral blood monocytes readily internalized nonopsonized brucellae and killed most of them within 12 to 18 h . The presence of Brucella-specific antibodies (but not complement) increased the uptake of bacteria without increasing their intracellular survival, whereas adherence of the monocytes or incubation in Ca(2+)- and Mg(2+)-free medium reduced the uptake . Engulfment of all Brucella organisms (regardless of bacterial viability or virulence) initially resulted in phagosomes with tightly apposed walls (TP) . Most TP were fully fusiogenic and matured to spacious phagolysosomes containing degraded bacteria, whereas some TP (more in monocyte-derived macrophages, HeLa cells, and CHO cells than in monocytes) remained tightly apposed to intact bacteria . Immediate treatment of infected host cells with the lysosomotropic base ammonium chloride caused a swelling of all phagosomes and a rise in the intraphagosomal pH, abolishing the intracellular survival of Brucella . These results indicate that (i) human monocytes readily internalize Brucella in a conventional way using various phagocytosis-promoting receptors, (ii) the maturation of some Brucella phagosomes is passively arrested between the steps of acidification and phagosome-lysosome fusion, (iii) brucellae are killed in maturing but not in arrested phagosomes, and (iv) survival of internalized Brucella depends on an acidic intraphagosomal pH and/or close contact with the phagosomal wall.

Infect Immun, 2001 Jun, 69(6), 3772 - 81
CD14 is expressed and released as soluble CD14 by human intestinal epithelial cells in vitro: lipopolysaccharide activation of epithelial cells revisited; Funda DP et al.; Human endothelial as well as epithelial cells were shown to respond to lipopolysaccharides (LPSs) . However, the expression and release of CD14 by these so-called CD14-negative cells have not been studied in detail . We investigated three human intestinal epithelial cell lines (ECLs), SW-480, HT-29, and Caco-2, for their expression of CD14 and CD11c/CD18 as well as their responsiveness to endotoxins . Fluorescence-activated cell sorter analysis revealed no expression of CD11c/CD18, but there was low expression of membrane-bound CD14 on HT-29, Caco-2, and SW-480 ECLs . Both Western blotting and reverse transcription-PCR confirmed the CD14 positivity of all three intestinal ECLs . No substantial modulation of CD14 expression was achieved after 6, 8, 18, 24, and 48 h of cultivation with 10-fold serial dilutions of LPS ranging from 0.01 ng/ml to 100 microg/ml . Interestingly, soluble CD14 was found in the tissue culture supernatants of all three ECLs . Finally, only HT-29 and SW-480, and not Caco-2, cells responded to LPS exposure (range, 0.01 ng/ml to 100 microg/ml) by interleukin 8 release . Thus, we show that HT-29, SW-480, and Caco-2 human intestinal ECLs express membrane-bound CD14 . As Caco-2 cells did not respond to LPS, these cell lines might be an interesting model for studying the receptor complex for LPS . The fact that human intestinal epithelial cells are capable not only of expression but also of release of soluble CD14 may have important implications in vivo, e.g., in shaping the interaction between the mucosal immune system and bacteria in the gut and/or in the pathogenesis of endotoxin shock.

Infect Immun, 2001 Jun, 69(6), 3550 - 5
Tumor necrosis factor-dependent adhesions as a major protective mechanism early in septic peritonitis in mice; Echtenacher B et al.; The occurrence of peritoneal adhesions in surgical patients is positively correlated with tumor necrosis factor (TNF) levels . In a model of septic peritonitis-cecal ligation and puncture-TNF neutralization prevented formation of peritoneal adhesions and increased mortality, most likely because localization of the septic focus was prevented . To discriminate between the coagulation-independent protective TNF effect and a potential protective procoagulant TNF effect, formation of peritoneal adhesions after CLP was inhibited with heparin, hirudin, or urokinase . Each treatment increased mortality and increased the number of bacteria in the peritoneal lavage fluid, kidney, and liver to various degrees . Under these experimental conditions, antibiotics prevented death . In coagulation-compromised mice, lethality was further enhanced by additional TNF neutralization . These findings demonstrate that peritoneal adhesions early in septic peritonitis are an important mechanism of innate immunity that prevents increased spread of bacteria and reduces mortality.

Infect Immun, 2001 Jun, 69(6), 3536 - 41
Purification and characterization of Borrelia burgdorferi from feeding nymphal ticks (Ixodes scapularis); Rathinavelu S et al.; Here we describe a protocol for purifying Borrelia burgdorferi from feeding ticks by velocity centrifugation and Percoll density gradient centrifugation . The purified spirochetes were motile and 10- to 20-fold purer than the bacteria in crude tick homogenates . The purified bacteria were present in sufficient quantity for protein and gene expression studies . In comparison to culture-grown bacteria, tick-borne spirochetes had several proteins that were upregulated and a few that were downregulated . When the levels of B . burgdorferi outer surface proteins OspA and OspC were measured, OspC protein and mRNA levels were lower in cultured bacteria than in bacteria purified from ticks . Although differences in OspA mRNA levels were observed between cultured and tick-borne bacteria, no differences were observed at the protein level . These experiments demonstrate that tick-transmitted borreliae display a gene expression and antigen profile different from that of spirochetes cultured in vitro.

J Microbiol Methods, 2001 Jul, 45(3), 167 - 70
Comparison of usefulness of three types of artificial substrata (glass, wood and plastic) when studying settlement patterns of periphyton in lakes of different trophic status; Danilov RA et al.; Usefulness of three types of artificial substrata (glass, wood and plastic) was tested when studying settlement patterns of periphyton in lakes of different trophic status . Strictly eu-, meso- and oligotrophic lakes in central Sweden were chosen as objects of the study . Glass slides, glass tubes, pieces of plastic (PVC) and pieces of wood of similar dimensions were placed for 9 weeks in July-August vertically 3 cm above bottom at a total depth of ca . 30 cm . Substrata were located at well-illuminated places without any other submerged objects (like macrophytes and stones), which could potentially affect colonisation patterns by algae . Periphyton communities, which colonised both the glass tubes and the pieces of wood tested, were specific enough to enable a clear classification of the lakes studied in eu-, meso- and oligotrophic . Glass tubes turned out to be the most favourable substratum when investigating settlement patterns of periphyton in this study . Although also colonised by periphytic species, wood did not support the same diversity and abundance of species as glass did . No algae were detected on the plastics studied . The plastics were covered entirely by a slime layer of bacteria . It is discussed if the nature of plastics could have some inhibitory effects on algal growth or the slime layer itself may have prevented settlement of algal spores.

Folia Microbiol (Praha), 2000, 45(5), 457 - 63
Interferon-gamma- and lipopolysaccharide-induced tumor necrosis factor-alpha is required for nitric oxide production: tumor necrosis factor-alpha and nitric oxide are independently involved in the killing of Mycobacterium microti in interferon-gamma- and lipopolysaccharide-treated J774A.1 cells; Majumdar S et al.; A comparative study was done using J774A.1 and J774A.1-derived transfected cells (J774A.1 C.1) containing antisense tumor necrosis factor alpha (TNF-alpha) plasmid to determine the role of endogenous TNF-alpha on nitric oxide production as well as on the growth of Mycobacterium microti in interferon gamma (IFN-gamma)- and lipopolysaccharide (LPS)-treated cells . On stimulation with IFN-gamma and LPS a higher level of NO was observed in J774A.1 cells compared to J774A.1 C.1 which indicated that endogenous TNF-alpha is required for the production of NO . Comparing the effect of IFN-gamma and LPS on the intracellular growth of M . microti, the growth-reducing activity was higher in J774A.1 cells than in J774A.1 C.1 cells and was not completely abrogated in the presence of the nitric oxide inhibitor NG-methyl-L-arginine (L-NMA) . J774A.1 C.1 cells infected with M . microti produced a significant amount of NO when exogenous TNF-alpha was added along with IFN-gamma and LPS and the concentration of intracellular bacteria decreased almost to that in IFN-gamma and LPS treated parental J774A.1 cells . Addition of exogenous TNF-alpha even in the presence of L-NMA in J774A.1 C.1 cells could also partially restore intracellular growth inhibition of M . microti caused by IFN-gamma and LPS . TNF-alpha is probably required for the production of NO in J774A.1 cells by IFN-gamma and LPS but TNF-alpha and NO are independently involved in the killing of intracellular M . microti with IFN-gamma and LPS.

Folia Microbiol (Praha), 2000, 45(4), 343 - 7
Performance and persistence of phosphate solubilizing Azotobacter chroococcum in wheat rhizosphere; Kumar V et al.; Survival and establishment of inoculant strains of Azotobacter chroococcum NV 11 and its mutant NV 43 were assessed in sterilized soil and rhizosphere soil of wheat plants at 7 and 15 d interval, respectively, after sowing, i.e., up to 45 d under pot-house conditions . There was an apparent decrease in population of both strains in bulk soil but a steady increase was observed in root zones of the wheat plant . 10(3) to 10(4) introduced bacteria per g root were found sticking to roots . Further studies indicated that inoculant strains of Azotobacter could survive, proliferate and establish well in root zones.

Transfusion, 2001 May, 41(5), 643 - 51
Performance characteristics of the COBAS AmpliScreen HIV-1 test, version 1.5, an assay designed for screening plasma mini-pools; Yang Y et al.; BACKGROUND: The COBAS AmpliScreen HIV-1 test, version 1.5 (v1.5) (Roche Molecular Systems), is designed for screening pools composed of samples from 24 individual units of blood or plasma . A specimen-processing procedure (Multiprep) simultaneously concentrates and extracts HIV-1, HCV, and HBV particles from plasma and incorporates an HIV-1 internal control (IC) RNA . Processed samples are amplified by RT-PCR using HIV-1-specific primers and detected by hybridization of the amplified products to HIV-1- and IC-specific oligonucleotide probes . STUDY DESIGN AND METHODS: Plasma samples containing known quantities of HIV-1 were used to evaluate analytical sensitivity and precision and to validate a pool testing algorithm . Analytical specificity was evaluated by adding various viruses and bacteria to HIV-1-negative plasma . Seroconversion panels were tested to estimate the window-period reduction achieved by RNA testing . RESULTS: The analytical sensitivity of the test (concentration that yields > or = 95% positive results in a set of replicate tests) was 25 copies of HIV-1 RNA per mL of pooled plasma . Representative strains from all HIV-1 group M subtypes were reproducibly detected (> 95% positive results) at concentrations of 20 to 200 viral particles per mL . The test did not cross-react with a set of 31 viral and 5 bacterial isolates, and it yielded negative results on a panel of 500 blood samples from HIV-1-seronegative donors . Plasma samples containing abnormally high levels of Hb, albumin, triglycerides, or bilirubin did not interfere with the test . HIV-1 RNA was detected 2 to 14 days before HIV-1 antibody and 0 to 28 days before p24 antigen . The test specifically detected pools containing a single positive unit with 2400 HIV-1 RNA copies per mL and correctly identified the positive unit . CONCLUSION: The COBAS AmpliScreen HIV-1 test, v1.5, has sufficient sensitivity to detect a single infected unit containing 600 copies of HIV-1 per mL in a pool with 23 uninfected units and should reduce the window period between infection and seroconversion by at least 2 to 14 days.

Med Mycol, 2001 Apr, 39(2), 215 - 9
A specific PCR assay for the dermatophyte fungus Microsporum canis; Liu D et al.; A DNA fragment of approximately 1.2 kb, generated from the common dermatophyte Microsporum canis by arbitrarily primed polymerase chain reaction (PCR) using random primer OPU13, was cloned and sequenced . Based on the resulting sequencing data, a forward primer (MC1F) and a reverse primer (MC1R) have been designed and assessed by PCR for their usefulness in the improved identification of M . canis . The results obtained suggest that these primers are specific for M . canis, as a band of 900 bp was amplified in PCR with genomic DNA from M . canis only, and not from any of the other dermatophyte species or varieties, other fungi or common bacteria examined . Combining this PCR technique with a rapid mini-preparation method for fungal DNA, a definitive diagnosis of M . canis can be achieved within a day from the primary cultures . Future refinement of a DNA purification protocol from clinical specimens would further enhance the potential of the PCR based test for improved detection and identification of M . canis.

J Toxicol Environ Health A, 2001 May 11, 63(1), 53 - 65
Exposure to flaxseed and its purified lignan reduces bone strength in young but not older male rats; Ward WE et al.; Flaxseed is the richest source of the plant lignan secoisolariciresinol diglycoside (SDG), which is converted to the two major mammalian lignans, enterodiol (ED) and enterolactone (EL), by colonic bacteria . Because both ED and EL can produce biological effects similar to estrogen, exposure to lignans during early stages of development may adversely alter the normal development of bone in males since bone is a hormone-sensitive tissue . To determine whether early exposure to flaxseed or its lignan compromised the acquisition of bone mass or reduced bone strength, male offspring were exposed to one of three diets during lactation only (birth through postnatal day {PND} 21) via mother's milk or continuously from the start of lactation through to adolescence (PND 50) or young adulthood (PND 132) . The diets were a basal diet (BD) that was devoid of phytoestrogens, BD containing 10% flaxseed, or BD containing the equivalent quantity of SDG present in a 10% flaxseed diet . To assess bone quantity, the bone mineral content (BMC) and bone mineral density (BMD) of femurs were assessed by dual-energy x-ray absorptiometry . Since the biomechanical properties of bone are indicators of the microarchitecture and thus bone quality, the biomechanical strength of femurs was assessed by three-point bending . At PND 50, ultimate bending stress and Young's modulus, measures of bone strength, were reduced among rats that received the 10% flaxseed diet from PND 0 through PND 50, while there were no marked differences in bone size, BMC, or BMD among groups . Interestingly, this effect does not appear to be due to the lignan in flaxseed, as continuous exposure to the diet containing the equivalent quantity of lignan (10 S diet) did not alter any measures of bone strength . In contrast to PND 50, bone strength did not differ among groups at PND 132, indicating that the compromise in bone strength was not sustained into early adulthood . Bone size, BMC, and BMD continued to be similar among treatment groups at PND 132 . In conclusion, exposing male rats to a diet containing 10% flaxseed or an equivalent quantity of lignan either during lactation only or through to early adulthood is safe with respect to bone health, as measures of bone mass and strength were similar to control rats.

Sci Total Environ, 2001 Apr 23, 271(1-3), 49 - 59
Toxicity assessment of total dissolved solids in effluent of Alaskan mines using 22-h chronic Microtox and Selenastrum capricornutum assays; LeBlond JB et al.; In order to overcome limitations associated with the Daphnia assay, we have explored two alternative assays, the 22-h chronic Microtox test and the 3-day S . capricornutum test, as substitutes . During this study, we compared the two assays using both a simple TDS standard solution and field water samples from two Alaskan mines . Using EC20 values, our results suggest that simple TDS standard solutions are not representative of environmental water samples of equivalent TDS concentrations . When comparing assays, our results showed that the 22-h Microtox assay was more reproducible and sensitive to effluent waters than the algal assay . Principle component analysis indicated that the 22-h Microtox test was generally more sensitive to nickel, ammonia and chloride while the S . capricornutum growth test appeared sensitive to cadmium levels.

Clin Experiment Ophthalmol, 2000 Feb, 28(1), 38 - 40
Intravitreal pefloxacin therapy in postoperative endophthalmitis; Kumar A et al.; PURPOSE: To study the efficacy of intravitreal pefloxacin in the management of suspected bacterial endophthalmitis . METHODS: Twenty eyes with suspected postoperative bacterial endophthalmitis were given an intravitreal injection of pefloxacin (200 microg in 0.1 mL) . If required the injection was repeated after 24 h . The main parameters evaluated were visual acuity, response to intravitreal therapy and any complications due to intravitreal pefloxacin . RESULTS: Fourteen eyes (70%) responded to intravitreal pefloxacin therapy alone, while an additional pars plana vitrectomy was required in six eyes (30%) . Nineteen eyes retained a visual acuity of 6/60 or better at 3 months after the initiation of therapy . A retinal detachment developed in one of the eyes which received intravitreal therapy . CONCLUSIONS: Intravitreal pefloxacin may be a useful aternative therapy in bacteria endophthalmitis.

Proc R Soc Lond B Biol Sci, 2001 Apr 22, 268(1469), 855 - 9
Transfection of Wolbachia in Lepidoptera: the feminizer of the adzuki bean borer Ostrinia scapulalis causes male killing in the Mediterranean flour moth Ephestia kuehniella; Fujii Y et al.; Two species of Lepidoptera, Ostrinia scapulalis and Ephestia kuehniella, harbour Wolbachia, which are maternally transmitted intracellular bacteria that often cause reproductive abnormalities in arthropods . While the infection in O . scapulalis causes conversion of genetic males into functional females (feminization), that in E . kuehniella induces cytoplasmic incompatibility . In the present study, we investigated the relative importance of host and Wolbachia factors in the differential expression of reproductive alterations in these insects . We transferred the Wolbachia harboured by O . scapulalis to E . kuehniella in which the original infection had been cured by tetracycline treatment . The transfected strain of E . kuehniella expressed a maternally inherited, female-biased sex ratio . Unexpectedly, two lines of evidence suggested that the sex ratio distortion was due to male killing . First, higher mortality of young larvae was observed . Second, the removal of the transferred Wolbachia resulted in the recovery of a 1:1 sex ratio, whereas the removal of a feminizer should result in a male-biased sex ratio among offspring . To the authors' knowledge, this is the first report that a single Wolbachia strain can cause two distinct sexual abnormalities in different hosts . Our observations highlighted the importance of host-Wolbachia interactions in determining the phenotype of reproductive alterations.

Am J Rhinol, 2001 Mar-Apr, 15(2), 91 - 4
Scanning electron microscopy of hydroxylated polyvinyl acetal and conventional gauze strip nasal packing materials; Uslu SS et al.; The application of nasal packing is one of the most commonly performed procedures in rhinology . Various materials have been used as nasal packing, including conventional gauze strips and hydroxylated polyvinyl acetal . Complications related to nasal packing may cause problems that lead to increased morbidity . Among those complications, infectious ones range from localized infection in the nasal cavity to toxic shock syndrome . The purpose of this study was to evaluate conventional gauze strips and hydroxylated polyvinyl acetal nasal packing materials by scanning electron microscopy, to reveal their surface characteristics that would promote or prevent the development of infectious complications . The two types of materials were examined before and after application into the nasal cavity . Scanning electron microscopy demonstrated that hydroxylated polyvinyl acetal material had a smooth surface, whereas conventional gauze strips had an irregular surface with fibers projecting, thus increasing the surface area for bacterial adherence and allowing possible evasion of bacteria from the elements of the immune system within this fibrillar structure.

Bull Soc Belge Ophtalmol, 2001, (279), 61 - 5
{Chronic endophthalmitis}; Libert J; Chronic endophthalmitis are intraocular infections, characterized by late onset and prolonged evolution . They are either of exogenous or endogenous origin and may be related to the penetration of "fastidious bacteria", mycoses or viruses . Diagnosis is based on specific techniques, including direct examination peroperatively, prolonged cultures on special medium and electron microscopic analysis . Treatment often combines diagnostic/therapeutic vitrectomy and intraocular injection of antibiotics . Their evolution is usually made of successive reactivations and explanting of the artificial lens is often necessary for a definitive remission in pseudophakic patients.

Bull Soc Belge Ophtalmol, 2001, (279), 35 - 8
Patterns of intraocular inflammation in children; Benezra D et al.; AIM: To report on the causes of uveitis in children and young adults and their effects on visual functions . MATERIALS AND METHODS: Two hundred and seventy six patients, 18 years old or younger, with uveitis were included in this study . The intraocular inflammation (uveitis) was classified according to anatomical site of ocular involvement and the most probable etiological factor . The final diagnosis was based on clinical manifestations and the results of specific laboratory investigations . RESULTS: Bilateral intraocular inflammation was observed in 70.3% of the cases and 29.7% had either the left or the right eye involved . The symptomatology was relatively mild in most cases despite the fact that the visual acuity was markedly affected . An associated systemic disease was detected in 40.2% of the cases classified as non-infectious . Of this group, juvenile rheumatoid arthritis was the most common single systemic associated cause detected in 41 children . In 110 children (59.8%), the uveitis was strictly confined to the eyes with 70 of these (25.4% of the total group) classified as idiopathic . Parasites were the most common infectious-associated cause for the uveitis followed by viruses and bacteria . CONCLUSION: Uveitis is highly prevalent among children . In children, symptomatology of the intraocular inflammation may be very mild . However, visual acuity is markedly reduced leading to amblyopia in the young children . Early detection and treatment is therefore of utmost importance.

Vnitr Lek, 2000 Oct, 46(10), 677 - 80
{Monitoring phagocyte activity and free radicals in Helicobacter pylori infections}; Juraskova B et al.; The presence of infection in organism induces a lot of Immunological reactions accompanied by creating free Radicals and reactive oxygen species (ROS) . These play an important role in elimination of bacteria but also in tissue injury in surrounding . The aim of presented study was to focus on monitoring of ROS and phagocyte activation in subjects infected by Helicobacter pylori (HP) and thus to contribute to our knowledge of the etiopathogenic role of HP in inflammatory gastrointestinal diseases . The results report some differences, which interpretation opens a lot of questions showing how important of role ROS in HP infection may play.

Lasers Surg Med, 2001, 28(4), 371 - 4
Microleakage of composite fillings in Er,Cr:YSGG laser-prepared class II cavities; Gutknecht N et al.; BACKGROUND AND OBJECTIVE: If there is insufficient bonding to the enamel, the shrinkage of composites that occurs during polymerization can result in a gap between the filling material and the cavity wall . This gap permits the passage of bacteria or their metabolic products and also of various molecules and ions . This leads to hypersensitivity or secondary caries and is thus one of the causes of the failures encountered in composite restorations . The aim of this study was to examine the quality of the margins of composite fillings in Er,Cr:YSGG laser-prepared cavities by means of dye penetration . The results were compared with those of restoration in conventionally prepared and conditioned cavities . STUDY DESIGN/MATERIALS AND METHODS: To this end, 45 class II cavities in extracted molars were prepared . The teeth were divided into three groups . The first group served as a control group . The cavities were prepared in the classical manner by using a diamond, beveled and subsequently conditioned by the etching method . In group 2, the cavities were prepared and conditioned exclusively with the Er,Cr:YSGG laser . In group 3, laser preparation was supplemented by conditioning of the cavity with phosphoric acid . RESULTS: No significant difference could be found between the classical preparation technique in combination with etching and the laser preparation method with supplementary etching (group 3) . The degree of dye penetration was highest in the group undergoing laser-prepared restoration without additional etching (group 2) (Wilcoxon test, P < 0.017) . CONCLUSIONS: Although it was found in previous studies that there is no significant difference between bond strength of acid etched enamel and Er,Cr:YSGG laser etched enamel, the dye penetration rate differs . On the basis of the results of our study, the additional use of etching after Er,Cr:YSGG laser preparation is recommended as it is used in the classical cavity preparation technique.

Trends Biochem Sci, 2001 May, 26(5), 275 - 7
Type II CAAX prenyl endopeptidases belong to a novel superfamily of putative membrane-bound metalloproteases; Pei J et al.; In this article, a novel, large and diverse superfamily of putative membrane-bound proteins that includes the type II CAAX prenyl endopeptidases is described . The majority of the members of this superfamily are hypothetical proteins from bacteria and plants . Analysis of the conserved motifs, combined with available experimental data, suggests that these proteins are putative metal-dependent proteases that are potentially involved in protein and/or peptide modification and secretion.

J Exp Med, 2001 May 7, 193(9), 995 - 1004
Testicular damage by microcirculatory disruption and colonization of an immune-privileged site during Borrelia crocidurae infection; Shamaei-Tousi A et al.; The agent of African relapsing fever, Borrelia crocidurae, causes reversible multiple organ damage . We hypothesize that this damage is caused when the spirochete forms aggregate with erythrocytes in vivo, creating rosettes that plug the microcirculatory system . To test this hypothesis, we compared testicular microcirculation over an extended time period in two groups of rats: one experimentally inoculated with B . crocidurae, the other with the nonerythrocyte rosette-forming Borrelia hermsii . In the B . crocidurae group, erythrocyte rosettes formed during spiro-chetemia blocked precapillary blood vessels and reduced the normal pattern of microcirculatory blood flow . After spirochetemia, erythrocyte rosettes disappeared and flow was normalized . Decreased blood flow and focal vascular damage with increased permeability and interstitial bleeding adjacent to the erythrocyte microemboli induced cell death in seminiferous tubules . Interestingly, we found that B . crocidurae could penetrate the tubules and remain in the testis long after the end of spirochetemia, suggesting that the testis can serve as a reservoir for this bacteria in subsequent relapses . The group infected with B . hermsii displayed normal testicular blood flow and vasomotion at all selected time points, and suffered no testicular damage . These results confirmed our hypothesis that the erythrocyte rosettes produce vascular obstruction and are the main cause of histopathology seen in model animal and human infections.

J Biol Chem, 2001 Jul 13, 276(28), 25654 - 60 Epub 2001 May 07.
Replication protein A in Pyrococcus furiosus is involved in homologous DNA recombination; Komori K et al.; Single-stranded DNA-binding protein in Bacteria and replication protein A (RPA) in Eukarya play crucial roles in DNA replication, repair, and recombination processes . We identified an RPA complex from the hyperthermophilic archaeon, Pyrococcus furiosus . Unlike the single-peptide RPAs from the methanogenic archaea, Methanococcus jannaschii and Methanothermobacter thermoautotrophicus, P . furiosus RPA (PfuRPA) exists as a stable hetero-oligomeric complex consisting of three subunits, RPA41, RPA14, and RPA32 . The amino acid sequence of RPA41 has some similarity to those of the eukaryotic RPA70 subunit and the M . jannaschii RPA . On the other hand, RPA14 and RPA32 do not share homology with any known open reading frames from Bacteria and Eukarya . However, six of eight archaea, whose total genome sequences have been published, have the open reading frame homologous to RPA32 . The PfuRPA complex, but not each subunit alone, specifically bound to a single-stranded DNA and clearly enhanced the efficiency of an in vitro strand-exchange reaction by the P . furiosus RadA protein . Moreover, immunoprecipitation analyses showed that PfuRPA interacts with the recombination proteins, RadA and Hjc, as well as replication proteins, DNA polymerases, primase, proliferating cell nuclear antigen, and replication factor C in P . furiosus cells . These results indicate that PfuRPA plays important roles in the homologous DNA recombination in P . furiosus.

Mutat Res, 2001 Feb 25, 485(1), 61 - 7
The two-step model for translesion synthesis: then and now; Bridges B; The formation of base substitution mutations following exposure of bacteria to ultraviolet light and many other mutagens occurs during translesion synthesis opposite a photoproduct or other lesion in the template strand of DNA . This process requires the UmuD(2)' UmuC complex, only formed to a significant extent in SOS-induced cells . The "two-step" model proposed that there were two steps, insertion of a wrong base (misincorporation) and use of the misincorporated base as a primer for further chain extension (bypass) . The original evidence suggested that UmuD(2)' UmuC was needed only for the second step and that in its absence other polymerases such as DNA polymerase III could make misincorporations . Now we know that the UmuD(2)' UmuC complex is DNA polymerase V and that it can carry out both steps in vitro and probably does both in vivo in wild-type cells . Even so, DNA polymerase III clearly has an important accessory role in vitro and a possibly essential role in vivo, the precise nature of which is not clear . DNA polymerases II and IV are also up-regulated in SOS-induced cells and their involvement in the broader picture of translesion synthesis is only now beginning to emerge . It is suggested that we need to think of the chromosomal replication factory as a structure through which the DNA passes and within which as many as five DNA polymerases may need to act . Protein-protein interactions may result in a cassette system in which the most appropriate polymerase can be engaged with the DNA at any given time . The original two-step model was very specific, and thus an oversimplification . As a general concept, however, it reflects reality and has been demonstrated in experiments with eukaryotic DNA polymerases in vitro.

J Biol Chem, 2001 Jul 6, 276(27), 24931 - 6 Epub 2001 May 04.
Elk-1, C/EBPalpha, and Pit-1 confer an insulin-responsive phenotype on prolactin promoter expression in Chinese hamster ovary cells and define the factors required for insulin-increased transcription; Jacob KK et al.; The transcription factor(s) that mediate insulin-increased gene transcription are not well defined . These studies use phenotypic conversion of Rat2 and Chinese hamster ovary (CHO) cells with transcription factors to identify components required for regulation of prolactin promoter activity and its control by insulin . The pituitary-derived GH4 cells contain all of the transcription factors required for insulin-increased prolactin-chloramphenicol acetyltransferase (CAT) expression while HeLa cells require only Pit-1, a pituitary-specific factor . However, Rat2 and CHO cells require additional factors . We had determined previously that the transcription factor that mediates insulin-increased prolactin gene expression was likely an Ets-related protein . Elk-1 and Sap-1 were the only Ets-related transcription factors tested as chimeras with LexA DNA-binding domain that were able to mediate insulin-increased expression of a LexA-CAT reporter plasmid . Elk-1 and Sap-1 are expressed in GH4 and HeLa cells but Rat2 and CHO cells express Sap-1, but not Elk-1 . Expression of Elk-1 made Rat2 cells (but not CHO cells) insulin responsive . C/EBPalpha also binds to the prolactin promoter at a sequence overlapping the binding site for Elk-1 . Expression of both C/EBPalpha and Pit-1 in CHO cells is required for high basal transcription of prolactin-CAT . Expression of Elk-1 converts CHO cells into a phenotype in which prolactin gene expression is increased by insulin treatment . Finally, antisense mediated reduction of Elk-1 in GH4 cells decreased insulin-increased prolactin gene expression and confirmed the requirement for Elk-1 for insulin-increased prolactin gene expression . Thus, both C/EBPalpha and Pit-1 were required for high basal transcription while insulin sensitivity required Elk-1.

Exp Cell Res, 2001 May 15, 266(1), 135 - 41
RasG regulates discoidin gene expression during Dictyostelium growth; Secko DM et al.; Activated rasG, rasG(G12T), was expressed in Dictyostelium cells under the control of the folate-repressible discoidin promoter (pVEII-rasG(G12T)) and found to have a unique pattern of expression when cells were transferred to folate-deficient media: an initial increase of RasG(G12T) resulting from the removal of folate, followed by a rapid decline while cells were still in the early exponential phase of growth . Discoidin levels were considerably lower and declined more rapidly in the pVEII-rasG(G12T) transformant than they did in the wild type, suggesting that RasG(G12T) represses discoidin expression . This was independently confirmed by placing the rasG(G12T) gene under the control of the ribonucleotide reductase (rnrB) promoter . Exposure of cells to 10 mM methyl methanesulfonate (MMS) rapidly generated RasG(G12T) and this was accompanied by an equally rapid decrease in discoidin mRNA levels . rasG null cells also contained decreased levels of discoidin under all conditions tested, indicating that RasG is essential for optimum discoidin expression . However, rasG null cells showed normal regulation of discoidin expression in response to PSF, CMF, folate, bacteria, and axenic media, indicating that RasG is not necessary for any of these responses . These results reveal a role for RasG in regulating discoidin gene expression and add a further level of complexity to the regulation of the discoidin promoter .

J Periodontol, 2001 Apr, 72(4), 461 - 9
Cyclooxygenase-2 is upregulated in inflamed gingival tissues; Morton RS et al.; BACKGROUND: Increased release of prostaglandins (PG) within periodontal tissues is considered to play a pathogenetic role during periodontal disease progression . The rate-limiting step in the formation of PG from arachidonic acid is catalyzed by cyclooxygenase (COX) . Currently there are 2 known isoforms of the enzyme . COX-1 is constitutively expressed in various tissues whereas COX-2 is an inducible enzyme believed to be responsible for PG synthesis at sites of inflammation . The purpose of this study was to compare COX-2 expression in inflamed and healthy human gingiva and further explore some of the pathogenetic mechanisms which may lead to elevated COX-2 expression in vivo . METHODS: Thirty-two gingival biopsies were obtained during routine oral surgical procedures and were processed histologically using hematoxylin and eosin to determine the degree of inflammation . Of these biopsies, 7 with low and 7 with high histological levels of inflammation were further processed immunohistochemically in order to assess the levels of COX-2 expression in situ . To explore some potential mechanisms of COX-2 upregulation, gingival connective tissue primary cell cultures were established and challenged with periodontal bacteria or proinflammatory cytokines in vitro . The levels of COX-2 expression were analyzed by Western blot of cell lysates . COX-2 activity was assessed by quantifying prostaglandin E2 (PGE2) levels in culture supernatants by competitive EIA . RESULTS: We have shown by immunohistochemistry that COX-2 expression was significantly higher (P < 0.01) in tissues with higher levels of inflammatory infiltrates . Expression of COX-2 was detected in gingival epithelium, endothelial cells as well as cells with fibroblast morphology . In vitro studies indicated that gingival fibroblasts (GF) did not express COX-2 constitutively . However, when these cells were challenged with interleukin (IL)-1 beta or bacterial cells (A . actinomycetemcomitans JP2 or B . forsythus ATCC 43037), COX-2 expression as well as COX-2 activity were upregulated . COX-2 expression was upregulated as early as 2 hours post IL-1 beta challenge and was accompanied by a sustained PGE2 release in the culture supernatants . Cyclosporin A (CsA) did not inhibit COX-2 expression induced by bacterial challenge . In contrast, NS-398, a selective inhibitor of COX-2 activity, almost completely abolished PGE2 synthesis by these cells in response to bacterial or cytokine challenge . CONCLUSIONS: We conclude that COX-2 expression is significantly upregulated in inflamed periodontal tissues . Both inflammatory cytokines such as IL-1 beta and bacterial constituents may be responsible for the enhanced COX-2 expression and PGE2 synthesis in vivo.

J Periodontol, 2001 Apr, 72(4), 425 - 37
Heterogeneity of host immunological risk factors in patients with aggressive periodontitis; Takahashi K et al.; BACKGROUND: The pathogenesis of early-onset periodontitis (EOP) can be explained by various host risk factors . Previous studies have focused on a single (among many possible) immunological risk factor and the association among the factors has not been assessed . We comprehensively investigated the associations among multiple host immunological risk factors in EOP patients to further elucidate their role in the pathogenesis of EOP . METHODS: Sixty-eight EOP patients (50 generalized EOP, 18 localized EOP), 51 EOP-suspected patients (S-EOP), 43 adult periodontitis (AP) patients, and 36 periodontally healthy subjects (HS) participated in this cross-sectional study . We examined peripheral neutrophil functions, phenotypic and functional characterization of peripheral lymphocytes (lymphocyte subsets, T-cell proliferative activity), cytokine productivity (interleukin {IL}-1, IL-2, tumor necrosis factor {TNF}-alpha, interferon {IFN}-gamma, IL-4 and IL-6), serum immunoglobulin G (IgG) antibody titers against 12 periodontal bacteria, and HLA class II genotypes . RESULTS: G-EOP, S-EOP, and AP patient groups showed significantly lower percentages of pan T cells and CD8-positive cells (P < 0.02) compared with the HS group . L-EOP patients showed depressed IL-4 and TNF-alpha productivity compared with the HS group (P < 0.02) . The EOP group showed significantly elevated antibody levels against Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, and Fusobacterium nucleatum compared with the HS group (P < 0.05) . The frequency with DQB1*0503 was significantly higher in the EOP patient group than the HS group (P = 0.045) due to the higher frequency in L-EOP patients than the HS group (P = 0.035) . There were wide interindividual variations in each of the tests among patient and HS groups; however, EOP patients showed wider intradiagnostic group variations in certain host defensive cell functions than the other groups . There were some EOP patients who showed extremely low or high values in some tests; the EOP patients could be further divided into subgroups according to their host defensive and immunological profiles . However, there was heterogeneity in some of the other host immunological tests even in the subgroups . CONCLUSIONS: The association of host immunological risk factors in EOP patients is widely varied and more complex than previously thought . These results indicate the difficulty of explaining the pathogenesis of EOP based on a single host risk factor and also emphasize the importance of critical assessment of not only EOP patient groups, but also individual patients.

J Biol Chem, 2001 Jul 13, 276(28), 26492 - 8 Epub 2001 May 03.
Identification of active site residues in glucosylceramide synthase . A nucleotide-binding catalytic motif conserved with processive beta-glycosyltransferases; Marks DL et al.; Glucosylceramide synthase (GCS) transfers glucose from UDP-Glc to ceramide, catalyzing the first glycosylation step in the formation of higher order glycosphingolipids . The amino acid sequence of GCS was reported to be dissimilar from other proteins, with no identifiable functional domains . We previously identified His-193 of rat GCS as an important residue in UDP-Glc and GCS inhibitor binding; however, little else is known about the GCS active site . Here, we identify key residues of the GCS active site by performing biochemical and site-directed mutagenesis studies of rat GCS expressed in bacteria . First, we found that Cys-207 was the primary residue involved in GCS N-ethylmaleimide sensitivity . Next, we showed by multiple alignment that the region of GCS flanking His-193 and Cys-207 (amino acids 89-278) contains a D1,D2,D3,(Q/R)XXRW motif found in the putative active site of processive beta-glycosyltransferases (e.g . cellulose, chitin, and hyaluronan synthases) . Site-directed mutagenesis studies demonstrated that most of the highly conserved residues were essential for GCS activity . We also note that GCS and processive beta-glycosyltransferases are topologically similar, possessing cytosolic active sites, with putative transmembrane domains immediately N-terminal to the conserved domain . These results provide the first extensive information on the GCS active site and show that GCS and processive beta-glycosyltransferases possess a conserved substrate-binding/catalytic domain.

Annu Rev Plant Physiol Plant Mol Biol, 2001 Jun, 52, 469 - 497
TONOPLAST TRANSPORTERS: Organization and Function; Maeshima M; Regulation of the contents and volume of vacuoles in plant cells depends on the coordinated activities of transporters and channels located in the tonoplast (vacuolar membrane) . The three major components of the tonoplast are two proton pumps, the vacuolar H+-ATPase (V-ATPase) and H+-pyrophosphatase (V-PPase), and aquaporins . The tertiary structure of the V-ATPase complex and properties of its subunits have been characterized by biochemical and genetic techniques . These studies and a comparison with the F-type ATPase have enabled estimation of the dynamics of V-ATPase activity during catalysis . V-PPase, a simple proton pump, has been identified and cloned from various plant species and other organisms, such as algae and phototrophic bacteria, and functional motifs of the enzyme have been determined . Aquaporin, serving as the water channel, is the most abundant protein in the tonoplast in most plants . A common molecular architecture of aquaporins in mammals and plants has been determined by two-dimensional crystallographic analysis . Furthermore, recent molecular biological studies have revealed several other types of tonoplast transporters, such as the Ca2+-ATPase, Ca2+/H+ antiporter and Na+/H+ antiporter . Many other transporters and channels in the tonoplast remain to be identified; their activities have already been detected . This review presents an overview of the field and discusses recent findings on the tonoplast protein components that have been identified and their physiological consequences.

Biomol Eng, 2001 Jun, 17(6), 183 - 92
PNA oligomers as tools for specific modulation of gene expression; Pooga M et al.; Small synthetic molecules that can specifically inhibit translation and/or transcription have shown great promise as potential antisense/antigene drugs . Peptide nucleic acid (PNA), an oligonucleotide mimic, has a non-charged achiral polyamide backbone to which the nucleobases are attached . PNA oligomers are extremely stable in biological fluids and they specifically hybridise to DNA or RNA in a complementary manner, forming very strong heteroduplexes . Some of the mRNAs have yet undetermined and possibly long half-lives, successful down regulation of gene expression by antisense oligonucleotides (ON) requires that the antisense agent is long lived . PNA fulfils this requirement better than phosphodiester or phosphorothioate ONs . PNA can inhibit transcription and translation of respective genes by tight binding to DNA or mRNA . First in vitro experiments to specifically down regulate protein expression by PNA have been followed by successful antisense and antigene application of PNA oligomers in vivo . This review discusses the principles of the in vitro and in vivo use of PNA oligonucleotides.

Comp Biochem Physiol B Biochem Mol Biol, 2001 May, 129(1), 109 - 20
Oxyconformity in the intertidal worm Sipunculus nudus: the mitochondrial background and energetic consequences; Buchner T et al.; The energetic consequences of strict oxyconformity in the intertidal worm S . nudus were studied by characterizing the Po2 dependence of respiration in mitochondria isolated from the body wall tissue . Mitochondrial respiration rose in a Po2 range between 2.8 and 31.3 kPa from a mean of 56.5 to 223.9 nmol O mg protein(-1) h(-1) . Respiration was sensitive to both salicylhydroxamic acid (SHAM) and KCN . Po2 dependence remained unchanged with saturating and non-saturating substrate levels (malate, glutamate and ADP) . A concomitant decrease of the ATP/O ratio revealed a lower ATP yield of aerobic metabolism at elevated Po2 . Obviously, oxyconforming respiration implies progressive uncoupling of mitochondria . The decrease in ATP/O ratios at higher Po2 was completely reversible . Addition of 90.9 micromol H2O2 l(-1) did not inhibit ATP synthesis . Both observations suggest that oxidative injury did not contribute to oxyconformity . The contribution of the rates of mitochondrial ROS production and proton leakiness to mitochondrial oxygen consumption and uncoupling was investigated by using oligomycin as a specific inhibitor of the ATP synthase . The maximum contribution of oligomycin independent respiration to state 3 respiration remained below 6% and showed a minor, insignificant increase at elevated Po2, at a slope significantly lower than the increment of state 3 respiration . Therefore, Po2 dependent mitochondrial proton leakage or ROS production cannot explain oxyconformity . In conclusion Po2 dependent state 3 respiration likely relates to the progressive contribution of an alternative oxidase (cytochrome o), which is characterized by a low affinity to oxygen and an ATP/O ratio similar to the branched respiratory system of bacteria . The molecular nature of the alternative oxidase in lower invertebrates is still obscure.

Scand J Gastroenterol, 2001 Apr, 36(4), 367 - 71
Fructose malabsorption is associated with decreased plasma tryptophan; Ledochowski M et al.; BACKGROUND: Fructose malabsorption is characterized by the inability to absorb fructose efficiently . As a consequence fructose reaches the colon where it is broken down by bacteria to short fatty acids, CO2, H2, CH4 and lactic acid . Bloating, cramps, osmotic diarrhea and other symptoms of irritable bowel syndrome are the consequence and can be seen in about 50% of fructose malabsorbers . Recently it was found that fructose malabsorption was associated with early signs of depressive disorders . Therefore, it was investigated whether fructose malabsorption is associated with abnormal tryptophan metabolism . METHODS: Fifty adults (16 men, 34 women) with gastrointestinal discomfort were analyzed by measuring breath hydrogen concentrations after an oral dose of 50 g fructose after an overnight fast . They were classified as normals or fructose malabsorbers according to their breath H2 concentrations . All patients filled out a Beck depression inventory questionnaire . Blood samples were taken for plasma tryptophan and kynurenine measurements . RESULTS: Fructose malabsorption (breath deltaH2 production >20 ppm) was detected in 35 of 50 individuals (70%) . Subjects with fructose malabsorption showed significantly lower plasma tryptophan concentrations and significantly higher scores in the Beck depression inventory compared to those with normal fructose absorption . CONCLUSIONS: Fructose malabsorption is associated with lower tryptophan levels that may play a role in the development of depressive disorders . High intestinal fructose concentration seems to interfere with L-tryptophan metabolism, and it may reduce availability of tryptophan for the biosynthesis of serotonin (5-hydroxytryptamine) . Fructose malabsorption should be considered in patients with symptoms of depression and disturbances of tryptophan metabolism.

FEMS Immunol Med Microbiol, 2001 Apr, 30(3), 173 - 9
Flow cytometric analysis of the localization of Helicobacter pylori antigens during different growth phases; Blom K et al.; Previous studies on the localization of several different Helicobacter pylori antigens have been contradictory . We have therefore examined by using both one- and two-color flow cytometry (FCM), immunofluorescence (IF), and immunoelectron microscopy (IEM), the possible surface localization of some H . pylori antigens that may be important virulence factors . All four methods detected the lipopolysaccharide and the N-acetyl-neuroaminyllactose-binding hemagglutinin protein (HpaA) as surface-exposed, while the urease enzyme was not detected at all and the neutrophil activating protein only in low concentration on the surface of the H . pylori bacteria during culture of H . pylori in liquid broth for 11 days . The FCM analysis was found to be quite sensitive and specific and also extremely fast compared with IF and IEM, and therefore the preferred method for detection of surface-localized antigens of H . pylori.

Biochim Biophys Acta, 2001 May 2, 1512(1), 1 - 14
Amino acid distributions in integral membrane protein structures; Ulmschneider MB et al.; Advances in structure determination of membrane proteins enable analysis of the propensities of amino acids in extramembrane versus transmembrane locations to be performed on the basis of structure rather than of sequence and predicted topology . Using 29 available structures of integral membrane proteins with resolutions better than 4 A the distributions of amino acids in the transmembrane domains were calculated . The results were compared to analysis based on just the sequences of the same transmembrane alpha-helices and significant differences were found . The distribution of residues between transmembrane alpha-helices and beta-strands was also compared . Large hydrophobic (Phe, Leu, Ile, Val) residues showed a clear preference for the protein surfaces facing the lipids for beta-barrels, but in alpha-helical proteins no such preference was seen, with these residues equally distributed between the interior and the surface of the protein . A notable exception to this was alanine, which showed a slight preference for the interior of alpha-helical membrane proteins . Aromatic residues were found to follow saddle-like distributions preferring to be located in the lipid/water interfaces . The resultant 'aromatic belts' were spaced more closely for beta-barrel than for alpha-helical membrane proteins . Charged residues could be shown to generally avoid surfaces facing the bilayer although they were found to occur frequently in the transmembrane region of beta-barrels . Indeed detailed comparison between alpha-helical and beta-barrel proteins showed many qualitative differences in residue distributions . This suggests that there may be subtle differences in the factors stabilising beta-barrels in bacterial outer membranes and alpha-helix bundles in all other membranes.

J Autoimmun, 2001 May, 16(3), 347 - 53
Protective role of infections and vaccinations on autoimmune diseases; Bach JF; Infectious agents may induce autoimmune disease through several mechanisms, notably antigen mimicry and inflammation of the target organ; conversely, infections may protect from autoimmune diseases . This paradoxical effect has been demonstrated for a number of bacteria, viruses and parasites on a variety of spontaneous or experimentally induced animal models of autoimmune diseases (e.g . experimental allergic encephalomyelitis, lupus mice, non-obese diabetic mice) . The mechanisms of the protection are still ill-defined, and probably vary according to models . Stimulation of immunoregulatory CD4 T cells has been shown to play a central role in several major models . The role of superantigens is also important, like that of Toll-like receptors . Antigen competition is another major mechanism, itself open to several interpretations . Epidemiological data support a protective role of infections on human allergic and autoimmune diseases . These diseases are much more common in countries with high socio-economic development (typically Northern countries in Europe) . The reason for this cannot be fully explained by genetic differences because migrating populations develop these diseases with the same incidence of the adoptive country rather than that of the country of origin . It is interesting that the frequency of these diseases has been increasing in developed countries over the last 20 years but not in undeveloped ones .

Res Commun Mol Pathol Pharmacol, 2000, 107(1-2), 21 - 32
Detection of restricted specificity of endonucleases by non-poisson analysis of their enzymatic digestion products; Lambert PJ et al.; Endonucleases, enzymes of the hydrolase class that cleave DNA or RNA within the substrate molecule, play numerous critical roles in molecular biologic, pharmacologic, and pathologic events as well as constitute invaluable tools used in genetic analysis, gene cloning and DNA sequencing . These enzymes have two critical parameters, activity and site specificity . The former is easily measured in commonly used assays, but the latter must be analyzed by more complex, indirect assays that are often not even carried out . We now show how both parameters can be measured directly and simultaneously in the same assay, which is not significantly more labor intensive than the commonly used activity assay . The effectiveness and accuracy of this assay was tested on a series of five bacterial restriction enzymes which recognized, respectively, 1, 2, 3, 4, and a large number (26) of reactive sites on a small, uniform DNA substrate molecule . We found that the distributions of the reaction products of all five enzymes precisely followed that predicted by the Binominal and Poisson distributions for the number of sites recognized by each enzyme, and that these distributions could be used to determine this number experimentally . In this way a measure of site specificity can be made for each enzyme as well as its activity . This new assay should be used routinely in a number of laboratories that now employ conventional assays for endonuclease activity.

Int Microbiol, 2000 Dec, 3(4), 213 - 23
Canaleparolina darwiniensis, gen . nov., sp . nov., and other pillotinaceous spirochetes from insects; Wier A et al.; We describe two new pillotinaceous spirochetes (Canaleparolina darwiniensis, Diplocalyx cryptotermitidis) and identify for the first time Hollandina pterotermitidis from both the subterranean termite Cryptotermes cavifrons and the wood-eating cockroach Cryptocercus punctulatus based on morphometric analysis of transmission electron micrographic thin sections . C . darwiniensis, gen . nov., sp . nov., limited to near Darwin, Australia, invariably is present on the surface of the treponeme-studded trichomonad Mixotricha paradoxa, a consistent inhabitant of the hindgut of healthy termite Mastotermes darwiniensis . The spirochete both attached to the surface of protists and free-swimming in the paunch (hindgut) lumen of the insect has 16 periplasmic flagella (16:32:16) and imbricated wall structures that resemble flattened crenulations of Pillotina . The flagella surround half the protoplasmic cylinder . C . darwiniensis is the largest (0.5 microm diameter x 25 microm length) of the three epibiotic bacteria (two spirochetes, one rod) that comprise the complex cortex of its host Mixotricha paradoxa . Several criteria distinguish Diplocalyx cryptotermitidis sp . nov . isolated from Cryptotermes cavifrons intestine: smaller diameter, fewer flagella, absence of inner and outer coats of the outer membrane, wider angle subtended by its flagella and, most notably, cytoplasmic tubule-associated centers, which are periodic electron dense spheres within the protoplasmic cylinder from which emanate cytoplasmic tubules up to 24 nm in diameter . This is also the first report of abundant populations of Hollandina in Cryptotermes cavifrons (those populations belong to the species H . pterotermitidis) . Morphometric analysis of the first thin sections of any spirochetes (published nearly 40 years ago by A.V . Grimstone) permits us to identify the large (0.9 microm diameter) free-swimming intestinal symbiont of Cryptocercus punctulatus also as Hollandina pterotermitidis.

Environ Health Perspect, 2001 Mar, 109(3), 209 - 12
Sawmill chemicals and carcinogenesis; Huff J; Workers in wood industries are exposed to variable medleys of chemicals, both natural and synthetic . Additional exposures include fungi, bacteria, bark and wood dusts, solvents, paints, and various other wood coatings . These individual and conglomerate exposures have been associated with diverse occupational illnesses and hazards, including cancers . In this commentary, I summarize both experimental and epidemiologic carcinogenesis results for several chemicals used in the wood industry, as well as for wood dust . Working in the wood industries entails excess risks of cancers, among other diseases and workplace injuries . A key to preventing occupationally and environmentally associated cancers, as in the wood industries, is avoiding exposures to chemicals and wood dusts and, in particular, chemicals known to cause cancer in animals or/and humans.

J Dent Res, 2001 Feb, 80(2), 470 - 5
Evidence for reactive nitrogen species formation in the gingivomucosal tissue; Lohinai Z et al.; An increase in nitric oxide production has been demonstrated in periodontitis . Here we investigated the potential role of nitric-oxide-derived nitrating species (such as peroxynitrite) in a rat model of ligature-induced periodontitis . Formation of 3-nitrotyrosine, the stable product formed from tyrosine reacting with nitric-oxide-derived nitrating species, was detected in the gingivomucosal tissue . 3-Nitrotyrosine immunohistochemical analysis revealed a significant elevation in the number of immunopositive leukocytes, and higher immunoreactivity of the gingival ligaments and epithelium in the ligated than in the contralateral (control) side . On both sides, several 3-nitrotyrosine-positive bands and, on the ligated side, a unique 52-kDa 3-nitrotyrosine-positive band were detected by Western blot . However, in the sterile gingivomucosal tissue of rat pups, no 3-nitrotyrosine or inducible nitric oxide synthase immunoreactivity was found . Analysis of these data suggests that resident bacteria of the gingivomucosal tissue induce an increase in reactive nitrogen species, which is greatly enhanced by plaque formation in periodontitis.

Dtsch Med Wochenschr, 2001 Mar 30, 126(13), 360 - 3
{Abdominal tuberculosis: a rare differential diagnosis of pancreatic carcinoma}; Enders M et al.; HISTORY AND ADMISSION FINDINGS: A 79-year-old local resident, presenting with abdominal pain, sweating and weight loss and suspected of having cancer of the pancreas was referred for diagnosis and treatment . Physical examination was negative except for pain on pressure over the right upper abdomen and the epigastrium . INVESTIGATIONS: Erythrocyte sedimentation rate was increased; as were the transaminases and cholestasis parameters . Ultrasonography and computed tomography of the abdomen revealed an echo-poor mass with cystic areas in the region of the head of the pancreas, as well as extra- and intrahepatic dilatation of the biliary tract . Endoscopic retrograde cholangiopancreatography failed to demonstrate a ductal pancreatic carcinoma . Biopsies of a macroscopically peculiar-looking duodenal ulcer demonstrated a noncaseous epithelioid granuloma . A fine-needle biopsy was performed for further diagnosis . DIAGNOSIS, TREATMENT AND COURSE: Histological examination of the needle biopsy revealed a caseous granuloma and acid-fast bacteria . The tuberculin test (GTI) was strongly positive (14-15 mm), indicating tuberculosis of the pancreas and duodenum . Multiple tuberculostatics rapidly improved the patient's symptoms, and the further course was without complications . CONCLUSION: Tuberculosis should be included in the differential diagnosis of consumptive disease with an atypical presentation, especially because treatment could well be curative.

J Interferon Cytokine Res, 2001 Mar, 21(3), 137 - 46
Regulation by IFN-beta of inducible nitric oxide synthase and interleukin-12/p40 in murine macrophages cultured in the presence of Chlamydia pneumoniae antigens; Yao SY et al.; Chlamydia pneumoniae has been demonstrated in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) . Interferon-beta (IFN-beta) has favorable effects on the clinical course of MS . We investigated whether the beneficial effects of IFN-beta in MS may involve its role in regulating nitric oxide (NO) and interleukin-12 (IL-12) in macrophages, as these immune modulators form part of the innate immune response to intracellular pathogens, such as C . pneumoniae . Murine macrophages in cultures exposed to elementary body antigens or recombinant major outer membrane protein (rMOMP) of C . pneumoniae demonstrate a significant increase in NO as well as production of IL-12/p40 in culture supernatants compared with basal levels . Addition of murine IFN-beta increased NO activity in murine macrophages cultured with chlamydial antigens . Addition of neutralizing anti-IFN-beta antibody prevented the NO increase . In contrast to its effect on inducible NO synthase (iNOS), IFN-beta reduced induction of IL-12/p40 following culture with either elementary body antigens or rMOMP . Inhibition was reversed with anti-IFN-beta antibody . If C . pneumoniae infection is responsible for the inflammatory response in the pathogenesis of MS, the beneficial effects of IFN-beta in MS may be due to its enhancing intracellular NO activity while inhibiting secretion of the proinflammatory cytokine, IL-12.

Appl Microbiol Biotechnol, 2001 Mar, 55(2), 210 - 3
Screening of micro-organisms for decolorization of melanins produced by bluestain fungi; Ratto M et al.; A total of 17 fungi and four bacteria were screened for their ability to decolorize melanin, using isolated extracellular melanin of the bluestain fungus Aureobasidium pullulans as substrate . On agar media, decolorization was observed by four fungal strains: Bjerkandera adusta VTT-D-99746, Galactomyces geotrichum VTT-D-84228, Trametes hirsuta VTT-D-95443 and Trametes versicolor VTT-D-99747 . The four fungi were more efficient on nitrogen-limited medium than on complete medium . The melanin-decolorizing activity of G . geotrichum appeared to be located on the mycelium and could be liberated into the medium enzymatically.

Biosci Biotechnol Biochem, 2001 Mar, 65(3), 555 - 62
Mechanism of growth inhibition by tungsten in Acidithiobacillus ferrooxidans; Sugio T et al.; Cell growth of three hundred iron-oxidizing bacteria isolated from natural environments was inhibited strongly by 0.05 mM, and completely by 0.2 mM of sodium tungstate (Na2WO4), respectively . Since no great difference in the level of tungsten inhibition was observed among the 300 strains tested, the mechanism of inhibition by Na2WO4 was studied with Acidithiobacillus ferrooxidans strain AP19-3 . When resting cells of AP19-3 were incubated in 0.1 M beta-alanine-SO4(2-) buffer (pH 3.0) with 0.1 mM Na2WO4 for 1 h, the amount of tungsten bound to the cells was 12 microg/mg protein . The optimum pH for tungsten binding to the resting cells was 2 to approximately 3 . Approximately 2 times more tungsten bound to the cells at pH 3.0 than at pH 6.0 . The tungsten binding was specifically inhibited by sodium molybdenum . However, copper, nickel, cadmium, zinc, manganese, cobalt, and vanadate did not disturb tungsten binding to the resting cells . The iron-oxidizing activity of AP19-3 was inhibited 24, 62, and 77% by 1, 5, and 10 mM of Na2WO4, respectively . Among the components of iron oxidation enzyme system, iron:cytochrome c oxidoreductase activity was not inhibited by 10 mM of Na2WO4 . In contrast, the activity of cytochrome c oxidase purified highly from the strain was inhibited 50 and 72%, respectively, by 0.05 and 0.1 mM of Na2WO4 . The amounts of tungsten bound to plasma membrane, cytosol fraction, and a purified cytochrome c oxidase were 8, 0.5, and 191 microg/mg protein, respectively . From the results, the growth inhibition by Na2WO4 observed in A . ferrooxidans is explained as follows: tungsten binds to cytochrome c oxidase in plasma membranes and inhibits cytochrome c oxidase activity, and as a results, the generation of energy needed for cell growth from the oxidation of Fe2+ is stopped.

Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 55 - 8
{Human angiogenin: expression, purification, biological assay}; Yang H et al.; Angiogenin cDNA was obtained by RT-PCR, and cloned into the fusion expression vector pRSETB . The recombinant Angiogenin protein was fused with His6 at its N-terminal and expressed as inclusion body . The expression level was about 10% of the total bacteria protein . After dissolved in 8 mol/L urea, the recombinant protein was purified by Ni2(+)-NTA chelating resin, according to the high affinity of His6 with Ni2+ . The biological assay indicated that purified rhANG could induced the new blood vessel formation of CAM and degraded tRNA in vitro.

BMC Microbiol . 2001;1(1):4 . Epub 2001 Apr 24.
Envelope structure of Synechococcus sp . WH8113, a nonflagellated swimming cyanobacterium; Samuel AD et al.; BACKGROUND: Many bacteria swim by rotating helical flagellar filaments . Waterbury et al . discovered an exception, strains of the cyanobacterium Synechococcus that swim without flagella or visible changes in shape . Other species of cyanobacteria glide on surfaces . The hypothesis that Synechococcus might swim using traveling surface waves prompted this investigation . RESULTS: Using quick-freeze electron microscopy, we have identified a crystalline surface layer that encloses the outer membrane of the motile strain Synechococcus sp . WH8113, the components of which are arranged in a rhomboid lattice . Spicules emerge in profusion from the layer and extend up to 150 nm into the surrounding fluid . These spicules also send extensions inwards to the inner cell membrane where motility is powered by an ion-motive force . CONCLUSION: The envelope structure of Synechococcus sp . WH8113 provides new constraints on its motile mechanism . The spicules are well positioned to transduce energy at the cell membrane into mechanical work at the cell surface . One model is that an unidentified motor embedded in the cell membrane utilizes the spicules as oars to generate a traveling wave external to the surface layer in the manner of ciliated eukaryotes.

Biochemistry, 2001 Feb 20, 40(7), 2176 - 85
Triplet formation on a monomeric chlorophyll in the photosystem II reaction center as studied by time-resolved infrared spectroscopy; Noguchi T et al.; The process of formation of the triplet state of chlorophyll in the photosystem II (PS II) reaction center complex was studied by means of time-resolved infrared (IR) spectroscopy . Using a dispersive-type IR spectrometer with a time resolution of approximately 55 ns, transient spectra in the C=O stretching region (1760--1600 cm(-1)) were measured at 77 K . The data were analyzed by singular-value decomposition and subsequent least-squares fitting . Two distinct spectral components having different kinetic behaviors were resolved . One had spectral features characterized by negative peaks at 1740 and 1680 cm(-1) and an overall positive background and was assigned to the P(680)(+)Phe(-)/P(680)Phe radical pair by static FTIR measurements of the P(680)(+)/P(680) and Phe(-)/Phe differences . The other had prominent negative and positive peaks at 1668 and 1628 cm(-1), respectively, which were previously assigned to the keto C==O change upon triplet formation of the monomeric chlorophyll denoted as Chl(T) {Noguchi, T., Tomo, T., and Inoue, Y . (1998) Biochemistry 37, 13614-13625} . The former component of P(680)(+)Phe(-)/P(680)Phe exhibited a multiphasic decay with time constants of 77 ns (75%), 640 ns (18%), 8.3 micros (4%), and 0.3 ms (3%), while the latter component of (3)Chl(T)/Chl(T) was formed with a single-exponential rise with a time constant of 57 ns and had a lifetime of 1.5 ms . From the observations that only the two spectral components were resolved without any other triplet intermediates and the time constant of (3)Chl(T) formation roughly agreed with or seemed even faster than that of the major phase of the P(680)(+)Phe(-) decay, two alternative mechanisms of triplet formation are proposed . (i) (3)Chl(T) is directly formed from P(680)(+)Phe(-) by charge recombination at Chl(T), and (ii) (3)P(680) is formed, and then the triplet is transferred to Chl(T) with a time constant of much less than 50 ns . The location of Chl(T) in the D1 subunit as the monomer chlorophyll corresponding to the accessory bacteriochlorophyll in the L subunit of purple bacteria is favored to explain the former mechanism as well as the triplet properties reported in the literature . The physiological role of the triplet formation on Chl(T) is also discussed.

Nat Biotechnol, 2001 May, 19(5), 434 - 9
A strategy for disease gene identification through nonsense-mediated mRNA decay inhibition; Noensie EN et al.; Premature termination codons (PTCs) have been shown to initiate degradation of mutant transcripts through the nonsense-mediated messenger RNA (mRNA) decay (NMD) pathway . We report a strategy, termed gene identification by NMD inhibition (GINI), to identify genes harboring nonsense codons that underlie human diseases . In this strategy, the NMD pathway is pharmacologically inhibited in cultured patient cells, resulting in stabilization of nonsense transcripts . To distinguish stabilized nonsense transcripts from background transcripts upregulated by drug treatment, drug-induced expression changes are measured in control and disease cell lines with complementary DNA (cDNA) microarrays . Transcripts are ranked by a nonsense enrichment index (NEI), which relates expression changes for a given transcript in NMD-inhibited control and patient cell lines . The most promising candidates can be selected using information such as map location or biological function; however, an important advantage of the GINI strategy is that a priori information is not essential for disease gene identification . GINI was tested on colon cancer and Sandhoff disease cell lines, which contained previously characterized nonsense mutations in the MutL homolog 1 (MLH1) and hexosaminidase B (HEXB) genes, respectively . A list of genes was produced in which the MLH1 and HEXB genes were among the top 1% of candidates, thus validating the strategy.

Mol Endocrinol, 2001 May, 15(5), 695 - 703
Binding of agonist but not antagonist leads to fluorescence resonance energy transfer between intrinsically fluorescent gonadotropin-releasing hormone receptors; Horvat RD et al.; We have used spot fluorescence photobleaching recovery methods to measure the lateral diffusion of GnRH receptor (GnRHR) fused at its C terminus to green fluorescent protein (GFP) after binding of either GnRH agonists or antagonist . Before ligand binding, GnRHR-GFP exhibited fast rates of lateral diffusion (D = 18 +/- 2.8 x 10(-10)cm2 x sec(-1)) and high values for fractional fluorescence recovery (%R) after photobleaching (73 +/- 1%) . Increasing concentrations of agonists, GnRH or D-Ala6-GnRH, caused a dose-dependent slowing of receptor lateral diffusion as well as a decreased fraction of mobile receptors . Increasing concentrations of the GnRH antagonist Antide slowed the rate of receptor diffusion but had no effect on the fraction of mobile receptors, which remained high . To determine whether the decrease in %R caused by GnRH agonists was due, in part, to increased receptor self-association, we measured the fluorescence resonance energy transfer efficiency between GnRHR-GFP and yellow fluorescent protein-GNRHR: There was no energy transfer between GnRHR on untreated cells . Treatment of cells with GnRH agonists led to a concentration-dependent increase in the energy transfer between GnRH receptors to a maximum value of 16 +/- 1% . There was no significant energy transfer between GnRH receptors on cells treated with Antide, even at a concentration of 100 nM . These data provide direct evidence that, before binding of ligand, GnRHR exists as an isolated receptor and that binding of GnRH agonists, but not antagonist, leads to formation of large complexes that exhibit slow diffusion and contain receptors that are self-associated.

Helicobacter, 2001 Mar, 6(1), 15 - 23
New approaches for validation of lethal phenotypes and genetic reversion in Helicobacter pylori; McDaniel TK et al.; BACKGROUND: Because of limited genetic tools for use in Helicobacter pylori, tests routinely applied in other bacteria for demonstrating a gene's role in viability and other phenotypes have not been applied to this organism . In a mutational study of putative response regulator genes, we aimed to develop such tools for H . pylori . MATERIALS AND METHODS: We attempted to mutate five response regulator genes by allelic exchange insertional mutagenesis . For genes that yielded no viable mutants, a second copy of the gene was inserted into the chromosome via a suicide vector, and it was seen if providing the second copy would permit the gene's disruption . For genes that yielded mutants with selectable phenotypes, a strategy was developed for reversion whereby an intact copy of the gene is introduced to the organism by transformation with PCR products . Following this procedure, revertants were selected by phenotypic tests then tested for genetic reversion . RESULTS: After failure to attain transformants upon attempted mutation of genes HP0166 and HP1365, we inserted a second copy of each gene within the H . pylori chromosome . In each case the second copy relieved the block of transformation . Mutation of genes HP0703 and HP1021 gave non-motile and small-colony phenotypes, respectively . Following transformation with PCR products containing intact copies of the genes, both phenotype and genotype had reverted following phenotypic selections . CONCLUSIONS: The methods used in this study provide new approaches for confirming suspected genotype/phenotype associations and should be widely applicable in the study of H . pylori.

Helicobacter, 2001 Mar, 6(1), 1 - 14
Helicobacter pylori infection in the cat: evaluation of gastric colonization, inflammation and function; Simpson KW et al.; BACKGROUND: Further elucidation of the consequences of Helicobacter pylori infection on gastric mucosal inflammation and gastric secretory function would be facilitated by an animal model that is susceptible to infection with H . pylori, is broadly similar in gastric physiology and pathology to people, and is amenable to repeated non-invasive evaluation . The goal of this study was to examine the interrelationship of bacterial colonization, mucosal inflammation and gastric secretory function in cats with naturally acquired H . pylori infection . MATERIALS AND METHODS: Twenty clinically healthy cats with naturally acquired H . pylori infection (cagA-, picB) and 19 Helicobacter-free cats were evaluated . Gastric colonization was determined by tissue urease activity, light microscopy, culture and PCR . The mucosal inflammatory response was evaluated by light microscopy, and by RT-PCR of the pro-inflammatory cytokines IL-1alpha, IL-1beta, IL-8 and TNF-alpha in gastric mucosa . Gastric secretory function was assessed by measuring pentagastrin-stimulated acid secretion, fasting plasma gastrin, and antral mucosal gastrin and somatostatin immunoreactivity . RESULTS: H . pylori colonized the pylorus, fundus and cardia in similar density . Bacteria were observed free in the lumen of gastric glands and were also tightly adherent to epithelial cells where they were associated with microvillus effacement . Mononuclear inflammation, lymphoid follicle hyperplasia, atrophy and fibrosis were observed primarily in H . pylori-infected cats, with the pylorus most severely affected . Neutrophilic and eosinophilic infiltrates, epithelial dysplasia, and up-regulation of mucosal IL-1beta and IL-8 were observed solely in infected cats . Fasting plasma gastrin concentrations and pentagastrin-stimulated acid output were similar in both infected and uninfected cats . There was no relationship of bacterial colonization density or gastric inflammation to plasma gastrin concentrations or gastric acid output . CONCLUSIONS: The pattern of colonization and the mucosal inflammatory response in cats with naturally acquired H . pylori are broadly similar to those in infected people, particularly children, and non-human primates . The upregulation of IL-8 in infected cats was independent of cagA and picB . Our findings argue against a direct acid-suppressing effect of H . pylori on the gastric secretory-axis in chronically infected cats.

J Mol Biol, 2001 May 4, 308(3), 449 - 55
Complexation precedes phosphorylation for two-component regulatory system FixL/FixJ of Sinorhizobium meliloti; Tuckerman JR et al.; The FixL/FixJ two-component regulatory system of Sinorhizobium meliloti controls the expression of nitrogen fixation genes in response to O2 . When phosphorylated, the transcription factor FixJ binds to the nifA and fixK promoters in S . meliloti and induces expression of the corresponding genes, both of which encode key transcription activators . Phosphorylation of FixJ has been proposed to occur via the following cascade . The sensor kinase FixL reacts with ATP independently of FixJ, transferring a phosphoryl group to one of its own histidine residues . Dissociation of O2 from a heme-binding PAS domain in FixL greatly accelerates the rate of this autophosphorylation . The phosphoryl group is rapidly transferred from phospho-FixL to an aspartate residue on FixJ . The resulting phospho-FixJ is short-lived, due to a FixL-catalyzed hydrolysis of the aspartyl phosphate . Here, we show that phosphorylation of FixLJ, i.e . the complex of FixL with FixJ, is at least tenfold faster than the phosphorylation of FixL without FixJ . We further show that a phospho-FixJ phosphatase, thought to reside in FixL, is absent from this complex . These results indicate that FixLJ reacts with ATP as a unit and much more efficiently than FixL alone, and that autophosphorylation and phosphoryl transfer do not occur independently, in sequence, but rather in a closely coupled processive reaction . These findings highlight the possible influence of synergistic interactions of the regulatory components in two-component-system signal transduction .

IUBMB Life, 2000 Dec, 50(6), 347 - 54
Macromolecular mimicry in translation initiation: a model for the initiation factor IF2 on the ribosome; Moreno JM et al.; Protein biosynthesis in bacteria is controlled by a number of translation factors . Recent data based on comparison of sequence and structure data of translation factors have established a novel hypothesis for their interaction with the ribosome: initiation, elongation, and termination factors may use a common or partly overlapping binding site on the ribosome in a process of macromolecular mimicry of an A-site-bound tRNA . This paper reviews structural knowledge and tRNA macromolecular mimicry involvement of translation initiation factor IF2 . Furthermore, a model is proposed for the factor and its interaction with the ribosome during the formation of the translation initiation complex.

J Anat, 2001 Apr, 198(Pt 4), 497 - 500
Neutrophil migration in tonsils; Ebenfelt A et al.; Recent studies have indicated the existence of an active cellular defence in the secretion on the tonsillar surface . This defence seems to consist partly of physiologically active neutrophils and is present in health and during disease . The present study was undertaken to examine the migration of these neutrophils to the secretion on the mucosal surface . Tonsils from 6 patients with acute tonsillitis and 5 patients with snoring problems were removed and sectioned . Sections were stained immunohistochemically against CD15 to visualise neutrophils . Other sections were stained with acridine orange to detect bacteria . Clusters of neutrophils were frequently seen in tonsils both from patients with acute tonsillitis and from snorers . They were observed to be accumulated within the tonsillar epithelial layer . Streaks of neutrophils could be observed running not only from vessels mainly near or within the epithelium to the epithelial surface, but also from vessels far from the epithelium through the extrafollicular areas to the epithelial surface . Bacteria were not present in the epithelium . We consider that the findings indicate an active physiological migration of neutrophils to the tonsillar surface.

J Periodontol, 2001 Mar, 72(3), 361 - 7
Periodontal treatment with an Er: YAG laser compared to scaling and root planing . A controlled clinical study; Schwarz F et al.; BACKGROUND: The aim of the present study was to compare the effectiveness of an Er:YAG laser to that of scaling and root planing for non-surgical periodontal treatment . METHODS: Twenty patients with moderate to advanced periodontal destruction were treated under local anesthesia and the quadrants were randomly allocated in a split-mouth design to either Er:YAG laser using an energy level of 160 mJ/pulse and 10 Hz or scaling and root planing (SRP) using hand instruments . Clinical assessments of plaque index (PI), gingival index (GI), bleeding on probing (BOP), probing depth (PD), gingival recession (GR), and clinical attachment level (CAL) were made prior to and at 3 and 6 months after treatment . Subgingival plaque samples were taken at each appointment and analyzed using darkfield microscopy for the presence of cocci, non-motile rods, motile rods, and spirochetes . Differences in clinical parameters and prevalence of bacterial species were analyzed using the paired t-test . RESULTS: The PI remained nearly unchanged while a significant reduction of the GI occurred in both groups after 6 months (P < or =0.001, P< or =0.001, respectively) . The mean value of BOP decreased in the laser group from 56% at baseline to 13% after 6 months (P < or =0.001) and in the SRP group from 52% at baseline to 23% after 6 months (P < or =0.001) . The mean value of the PD decreased in the laser group from 4.9+/-0.7 mm at baseline to 2.9+/-0.6 mm after 6 months (P< or =0.001) and in the SRP group from 5.0+/-0.6 mm at baseline to 3.4+/-0.7 mm after 6 months (P < or =0.001) . The mean value of the CAL decreased in the laser group from 6.3+/-1.1 mm at baseline to 4.4+/-1.0 mm after 6 months (P < or =0.001) and in the SRP group from 6.5+/-1.0 mm at baseline to 5.5+/-1.0 after 6 months (P < or =0.001) . The reduction of the BOP score and the CAL improvement was significantly higher in the laser group than in the SRP group (P < or =0.05, P < or =0.001, respectively) . Both groups showed a significant increase of cocci and non-motile rods and a decrease in the amount of motile rods and spirochetes . CONCLUSIONS: An Er:YAG laser may represent a suitable alternative for non-surgical periodontal treatment.

Zhonghua Jie He He Hu Xi Za Zhi, 1998 Jul, 21(7), 392 - 4
{Polymerase chain reaction technique in monitoring treatment of bacillary pulmonary tuberculosis}; An S et al.; OBJECTIVE: To explore the relationship between sputum Mycobacterium tuberculosis as well as its DNA negative conversion and relapse in bacillary pulmonary tuberculosis during chemotherapy and 2 years after completion of treatment, and to evaluate the value of polymerase chain reaction (PCR) technique in monitoring the efficacy of treatment . METHOD: Eighty-seven patients with bacillary pulmonary tuberculosis were monthly examined by PCR technique, smear and culture methods, and were followed up for 2 years after treatment . RESULT: The duration of sputum negative conversion by PCR technique was 1-3 months later than that by smear and culture methods . The more the sputum bacteria, the longer the duration of PCR positive results . Positive PCR results maintained in 10 out of 87 patients for more than 1 year, among them 3 (30%) relapsed respectively at 8, 12 and 16 months after treatment . One PCR negative conversion case relapsed at 18 months after treatment . These 4 cases who regained sputum positive results and showed deterioration in chest X-ray films were admitted to hospital again . CONCLUSION: PCR technique is more practical than smear and culture methods in monitoring efficacy of the treatment of bacillary pulmonary tuberculosis, and is useful for evaluating cases with possible relapse.

Cleve Clin J Med, 2001 Apr, 68(4), 325 - 9, 333-4, 336
Handwashing compliance: what works?
Serkey JM, Hall GS.
Health care personnel--particularly physicians--do a poor job of complying with national handwashing guidelines, yet handwashing is the cornerstone of infection control . New products designed to increase compliance are available, such as automated handwashing machines, but their clinical benefits have not been fully studied . The best solution for now may be to continue awareness campaigns and education programs, ensure access to sinks and appropriate antiseptic products, and promote the use of alcohol disinfectants when handwashing is not possible . KEY POINTS: Antiseptic products are now preferred over handwashing with plain soap, which does not reliably prevent transmission of bacteria . Because 100% compliance may not be realistic, interventions that improve compliance, such as the use of alcohol sanitizing products when handwashing is not possible, may be the best solution . A number of barriers deter compliance, including lack of access to handwashing stations and lack of time . Gloves are not a substitute for handwashing because they are not fully protective.

Dis Aquat Organ, 2001 Mar 9, 44(2), 121 - 6
Characterization of attenuated Renibacterium salmoninarum strains and their use as live vaccines; Daly JG et al.; Two nutritionally mutant strains of Renibacterium salmoninarum (Rs) were isolated that grew on tryticase soy agar (Rs TSA1) or brain heart infusion agar (Rs BHI1) . These 2 strains could be continuously cultured on these media, whereas typical R . salmoninarum would only grow on KDM-2 agar . We determined no other phenotypic difference that could be used to distinguish them from wild-type R . salmoninarum . Both strains were found to be avirulent when 5 x 10(6) bacteria were intraperitoneally (i.p.) injected into Atlantic salmon . Rs TSA1, Rs BHI1, and Rs MT-239 (a R . salmoninarum strain previously shown to be attenuated) were tested as live vaccines in 2 separate trials . The best protection was seen with Rs TSA1 . Vaccinated Atlantic salmon had relative percent survival (RPS) of 50 at 74 d post-challenge in Trial 1 and 76 at 60 d post-challenge in Trial 2 . In both trials, 100% of the control salmon died from bacterial kidney disease (BKD) (within 40 d for Trial 1 and 50 d for Trial 2) after i.p . challenge with 5 x 10(6) live cells of the virulent isolate Rs Margaree.

J Mass Dent Soc, 2000 Autumn, 49(3), 26 - 30
Is periodontitis genetic? The answer may be Yes!
Kornman KS, Knobelman C, Wang HY.
Specific bacteria cause periodontitis by activating immuno-inflammatory responses in the tissues . There are certain risk factors that significantly affect the disease process by altering the immuno-inflammatory response and increasing the likelihood of severe periodontitis . These risk factors are smoking, diabetes, IL-1 genotype, and perhaps others . Today about 20 percent of the population smokes at a level that should make a difference relative to periodontitis . About 30 to 33 percent of the Caucasian population is IL-1 genotype positive . There are compelling reasons to look at these risk factors in your practice to help formulate a complete treatment plan for your patients.

Trends Immunol, 2001 May, 22(5), 227 - 9
The human genome: an immuno-centric view of evolutionary strategies; Liu Y et al.; A hallmark of modern biology is the realization of the fundamental unity of biological processes in all life forms . Consequently, the complete genome sequencing of various bacteria, yeast (Saccharomyces cerevisiae), fly (Drosophila melanogaster) and worm (Caenorhabditis elegans) over the past five years has already had an impact on all of biology . "Model organisms" have contributed a great deal to immunology; for example, the Toll receptors of the fly provided the impetus for the investigation of Toll-like receptors, which proved to be fundamental elements in the mammalian innate immune system . The recent release of a draft sequence of the human genome provides the first panoramic view of the 30000-35000 human genes in the human genetic blueprint and provides a plethora of new details, the significance of which will take some time to appreciate . The over-riding concepts that emerge from these studies relate primarily to general evolutionary processes that are equally as relevant to immunology as they are to other disciplines of biology.

Eur J Biochem, 2001 May, 268(9), 2540 - 6
Activation of pro-astacin . Immunological and model peptide studies on the processing of immature astacin, a zinc-endopeptidase from the crayfish Astacus astacus; Mohrlen F et al.; To contribute knowledge of the processing and activation of invertebrate proteolytic enzymes, we studied the metalloprotease astacin, a digestive enzyme from the freshwater crayfish Astacus astacus (decapod crustacean) . It is the prototype of the protein family of astacins, members of which occur in organisms from bacteria to man and are involved in a variety of physiological reactions . According to its genomic structure, astacin is produced as a zymogen {Geier, G., Jacob, E., Stocker, W . & Zwilling, R . (1997) Arch . Biochem . Biophys . 337, 300-307} . To localize and follow the processing of pro-astacin in different parts of the digestive tract, we synthesized two peptides covering the pro part of pro-astacin and raised antibodies against them . In addition, antiserum against the whole active astacin was produced . Using immunohistochemical investigation, we detected pro-astacin in the F cells of the hepatopancreas and all the way into the tubular lumen and the collecting ducts of this gland . Immunoblot assays revealed only active astacin, and never pro-astacin, present in the cardiac stomach . We conclude from these studies that astacin is secreted into the lumen of the hepatopancreatic tubules in its pro form and is activated on its way to the stomach . To investigate which of the two endopeptidases found in the digestive tract of crayfish, astacin or trypsin, is responsible for cleaving the propeptide from pro-astacin, we synthesized different peptides that mimick the activation site . MS analysis of the cleavage products of astacin and trypsin showed that astacin is capable of catalyzing its own activation . Any contribution of trypsin would require the successive action of an aminopeptidase . Substituting glycine for arginine at position -1 of the activation site does not prevent astacin activity . As most members of the astacin protein family have basic amino-acid residues in this position, in these cases also astacin-specific cleavage would be possible.

Wei Sheng Yan Jiu, 2001 Mar, 30(2), 74 - 6
{Disinfection for cistern water}; Ling B et al.; Rainwater is often collected into cisterns (pits or tanks) for household using as drinking water source in the rural areas of the northwest and the southeast coast in China, where no enough fresh water resource is available . However, the total number of bacteria and coliforms in the cisterns water was higher than the standard of that in drinking water . In order to ensure the safety for drinking, the effectiveness, conditions of treatment and cost for such disinfection methods compared with solar radiation, ultraviolet (UV), chloridation, micro-filteration and KDF were studied in 10 households in Cixi of Zhejiang Province and Weiyuan of Gansu Provinces, respectively . The micro-filteration is more compatible for bacteria removal in the tanks, while chloridation more for disinfection in the underground pits.

J Mol Microbiol Biotechnol, 2001 Apr, 3(2), 287 - 93
SigB, SigC, and SigE from Myxococcus xanthus homologous to sigma32 are not required for heat shock response but for multicellular differentiation; Ueki T et al.; Myxococcus xanthus has been known to have multiple sigma factors which are considered to play important roles in regulation of gene expression in development . A new gene encoding a putative sigma factor, sigE, was cloned by using a degenerate oligonucleotide corresponding to the conserved region 2.2 of M . xanthus SigA . In the 2.0-kb nucleotide sequence, an open reading frame consisting of 280 amino acid residues was identified . The amino acid sequence of SigE shows high similarity to heat shock sigma factors in bacteria . However, the sigE gene is not induced by heat shock and deletion of sigE does not affect production of heat shock proteins . SigE is expressed during both vegetative growth and fruiting body development . In the deletion mutant of the sigE gene fruiting body formation is initiated earlier and fewer spores are produced than in the parent strain . Interestingly, the deltasigE mutant shows defects in fruiting body formation at 37 degrees C . In addition to SigE, SigB and SigC show high sequence similarity to heat shock sigma factors . However, even if all three sigma factor genes are disrupted, heat shock proteins are still normally induced . A deltasigBdeltasigCdeltasigE triple deletion strain forms fruiting bodies earlier, but sporulats later than the parent strain . Spores from the triple deletion mutant are aberrant and their viability is less than 0.001% compared with that of the parent strain, suggesting that these sigma factors may have redundant functions in multicellular differentiation of M . xanthus.

Int J Syst Evol Microbiol, 2001 Mar, 51(Pt 2), 611 - 21
Methylosarcina fibrata gen . nov., sp . nov . and Methylosarcina quisquiliarum sp.nov., novel type 1 methanotrophs; Wise MG et al.; Two novel species of obligate methane-oxidizing bacteria, isolated from landfill soil, were characterized . Both strains were unusual in that some members of the population grew in irregularly shaped, refractile cell packets that resembled sarcina-like clusters . Electron microscopy revealed that the cell packets were covered with a slime layer and the cells contained many large granular inclusion bodies . The individual cells of each strain were sometimes motile and had differing morphologies . Isolate AML-C10T was always coccoidal in shape, and the cells were covered with extracellular fibrils . Isolate AML-D4T was pleomorphic, changing from rod to coccal form, sometimes exhibiting an unusual fusiform morphology . AML-D4T lacked the extensive fibrillar matrix observed with AML-C10T . Both strains utilized only methane and methanol as carbon sources . In stationary phase, the cells of each strain swelled in size and formed cysts . Aside from morphological differences, strains could also be distinguished from each other by cellular protein patterns, as well as by temperature and pH tolerances . 16S rDNA phylogenetic analysis showed that these are type I methanotrophs (family: Methylococcaceae) most closely related to the Methylobacter/Methylomicrobium clade, although they form a monophyletic grouping supported by moderately high bootstrap values . By 16S rDNA database searches, the most similar species to both isolates were Methylobacter spp . However, partial particulate methane monooxygenase sequence analysis suggested that these bacteria might be more closely related to Methylomicrobium than Methylobacter . Furthermore, cellular fatty acid profiles of the strains more closely resemble those of Methylomicrobium, although the absence of significant levels of 16:1omega5c argues for the uniqueness of these two strains . On the basis of the results described here, it is proposed that a new genus should be created, Methylosarcina gen . nov., harbouring two species, Methylosarcina fibrata sp . nov . (type species) and Methylosarcina quisquiliarum sp . nov . The type strains are AML-C10T (= ATCC 700909T = DSM 13736T) and AML-D4T (= ATCC 700908T = DSM 13737T), respectively.

Int J Syst Evol Microbiol, 2001 Mar, 51(Pt 2), 401 - 7
Schineria larvae gen . nov., sp . nov., isolated from the 1st and 2nd larval stages of Wohlfahrtia magnifica (Diptera: Sarcophagidae); Toth E et al.; Four bacterial strains were isolated from the fly larvae of an obligate parasitic fly, Wohlfahrtia magnifica (Diptera: Sarcophagidae) . These isolates were characterized by a polyphasic approach and represent a new lineage of gamma-Proteobacteria as their closest relative is Xylella fastidiosa (87.1% 16S rDNA similarity) . The four strains are identical at the 16S rDNA level, the level of similarity between them, based on DNA-DNA hybridization, is high (97.8-102.5%) and they are similar in their physiological and biochemical characteristics, although they differ in their utilization of different sole carbon sources . All produce chitinase . They are obligately aerobic: no growth is detected under anaerobic conditions, even in the presence of NO3- as terminal electron acceptor . Their predominant respiratory quinone is Q-8 . The G+C content of their DNA is 42 mol% . Their cell membrane contains phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine and two unknown polar lipids . Their main fatty acids are C18:1, C16:0 and C14:0 . To accommodate these bacteria, a new genus, Schineria gen . nov., with the type species Schineria larvae sp . nov., is proposed.

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