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