Microorganism

Adv Colloid Interface Sci, 1999 Jul 1, 81(2), 77 - 165
The cleaning of polymer colloids; Wilkinson MC et al.; The current state of knowledge of the cleaning of polymer colloids is reviewed with regard to a wide range of cleaning and characterisation techniques . The type, level and quantity of impurities involved with different polymer latex formulations varies widely . Even for similar formulations, differences in the nature and number of functional groups reported are often a consequence of sometimes subtle differences in the cleaning procedures employed . Not only may surface functionality be affected but also monomer and oligomer extraction procedures may lead to morphological changes in the particles . No single technique alone is likely to be able to remove all impurities . Care is needed to avoid the introduction of new impurities from the equipment, materials and water used as well as possible contamination from atmospheric carbon dioxide, bacteria and fungi . These factors also need to be considered in the storage of latex particle standards.

Pediatr Dev Pathol, 2000 Mar-Apr, 3(2), 162 - 7
Evaluation of a neuraminidase detection assay for the rapid detection of influenza A and B virus in children; Noyola DE et al.; A prototype version of a new diagnostic assay for influenza A and B (Zstat Flutrade mark) based on detection of viral neuraminidase was evaluated and compared to culture in 196 clinical samples . Children with respiratory illnesses were prospectively evaluated at a pediatrician's office and at a large children's hospital using the neuraminidase assay and viral culture performed on respiratory secretions . Influenza virus was isolated from 51 samples and 83 were positive by the neuraminidase assay . When compared to culture the sensitivity of the assay was 96%, specificity was 77%, positive predictive value was 59%, and negative predictive value was 98% . Testing in the laboratory of pure cultures of bacteria and non-influenza viruses frequently found in the respiratory tract showed 0% cross-reactivity with the neuraminidase assay and 100% specificity for influenza virus in vitro . This new assay provided useful information for the preliminary diagnosis of influenza A and B infections and appears to be suitable for both point-of-care use in the physician's office and rapid diagnosis in a virology laboratory . The high sensitivity makes it particularly useful as a screening test for exclusion of influenza A and B infections . To confirm the diagnosis and exclude a false-positive result, as well as to determine the influenza virus type, a viral culture may be considered.

Proc Natl Acad Sci U S A, 2000 Feb 15, 97(4), 1400 - 5
Paleoproterozoic snowball earth: extreme climatic and geochemical global change and its biological consequences; Kirschvink JL et al.; Geological, geophysical, and geochemical data support a theory that Earth experienced several intervals of intense, global glaciation ("snowball Earth" conditions) during Precambrian time . This snowball model predicts that postglacial, greenhouse-induced warming would lead to the deposition of banded iron formations and cap carbonates . Although global glaciation would have drastically curtailed biological productivity, melting of the oceanic ice would also have induced a cyanobacterial bloom, leading to an oxygen spike in the euphotic zone and to the oxidative precipitation of iron and manganese . A Paleoproterozoic snowball Earth at 2.4 Giga-annum before present (Ga) immediately precedes the Kalahari Manganese Field in southern Africa, suggesting that this rapid and massive change in global climate was responsible for its deposition . As large quantities of O(2) are needed to precipitate this Mn, photosystem II and oxygen radical protection mechanisms must have evolved before 2.4 Ga . This geochemical event may have triggered a compensatory evolutionary branching in the Fe/Mn superoxide dismutase enzyme, providing a Paleoproterozoic calibration point for studies of molecular evolution.

Plant Physiol, 2000 Feb, 122(2), 379 - 88
Cloning and functional characterization of a constitutively expressed nitrate transporter gene, OsNRT1, from rice; Lin CM et al.; Elucidating how rice (Oryza sativa) takes up nitrate at the molecular level could help improve the low recovery rate (<50%) of nitrogen fertilizer in rice paddies . As a first step toward that goal, we have cloned a nitrate transporter gene from rice called OsNRT1 . OsNRT1 is a new member of a growing transporter family called PTR, which consists not only of nitrate transporters from higher plants that are homologs of the Arabidopsis CHL1 (AtNRT1) protein, but also peptide transporters from a wide variety of genera including animals, plants, fungi, and bacteria . However, despite the fact that OsNRT1 shares a higher degree of sequence identity with the two peptide transporters from plants (approximately 50%) than with the nitrate transporters (approximately 40%) of the PTR family, no peptide transport activity was observed when OsNRT1 was expressed in either Xenopus oocytes or yeast . Furthermore, contrasting the dual-affinity nitrate transport activity of CHL1, OsNRT1 displayed only low-affinity nitrate transport activity in Xenopus oocytes, with a K(m) value of approximately 9 mM . Northern-blot and in situ hybridization analysis indicated that OsNRT1 is constitutively expressed in the most external layer of the root, epidermis and root hair . These data strongly indicate that OsNRT1 encodes a constitutive component of a low-affinity nitrate uptake system for rice.

J Biol Chem, 2000 Mar 10, 275(10), 7080 - 6
Macromolecular inhibitors of HIV-1 protease . Characterization of designed heterodimers; Rozzelle JE et al.; Defective variants of human immunodeficiency virus type 1 (HIV-1) protease (HIV PR) have been engineered to inhibit wild-type (wt) HIV PR activity . These variants were designed to promote the formation of heterodimers and to destabilize the formation of inactive variant homodimers of HIV-1 protease through substitutions at Asp-25, Ile-49, and Gly-50 (Babe, L . M., Rose, J., and Craik, C . S . (1995) Proc . Natl . Acad . Sci . U . S . A . 92, 10069-10073; McPhee, F., Good, A . C., Kuntz, I . D., and Craik, C . S . (1996) Proc . Natl . Acad . Sci . U . S . A . 93, 11477-11481) . The mechanism of action of these dominant-negative inhibitors was established using recombinantly expressed defective monomers . The defective monomers were refolded in vitro in the presence of wt HIV PR and showed dose-dependent inhibition of proteolytic activity . This inhibition was shown to result from the formation of inactive heterodimers between defective and wt HIV PR monomers . Heterodimer formation was detected by (i) isolating refolded, inactive heterodimers using histidine-tagged defective monomers and (ii) isolating heterodimers from bacteria coexpressing both wt and defective variants of HIV PR . Single-chain variants of HIV PR, in which the C terminus of the wt HIV PR monomer was covalently tethered to the N terminus of the defective monomer, were also expressed and analyzed . Thermal denaturation of these single-chain heterodimers using differential scanning calorimetry revealed a 1.5-7.2 degrees C greater thermal stability than single-chain wt HIV PR . The thermodynamic trend shown by these three variants mirrors their relative inhibition in provirus transfection assays . These data support the model that the effects seen both in tissue culture and in vitro arise from an increase in stability conferred on these heterodimers by interface mutations and identifies heterodimer formation as their mechanism of inhibition.

Appl Radiat Isot, 2000 Feb, 52(2), 199 - 204
Automatic synthesis of L-{beta-11C}amino acids using an immobilized enzyme column; Sasaki M et al.; We have developed a system for the automatic synthesis of L-{beta-11C}amino acids for i.v . injection by means of enzyme-mediated reactions from 11CO2 via 11CH3I and D,L-{beta-11C}alanine as labeled intermediates . This system, which incorporates an ultrafilter cartridge sterilized by electron beam irradiation and a column packed with immobilized enzymes, was effective for eliminating enzymes and endotoxins that may contaminate the product . Using this system, 1.3 +/- 0.5 GBq of 5-hydroxy-L-{beta-11C}tryptophan with a radiochemical purity of 97.1 +/- 0.6% and a specific activity of 39.6 +/- 8 GBq/mumol a pH value of 4 could be obtained in about 32 min (n = 3, at EOS) . No endotoxin, enzyme, or bacteria was detected in the product . L-{beta-11C}dihydroxyphenylalanine (L-{beta-11C}DOPA) was also synthesized using this system.

J Antibiot (Tokyo), 1999 Dec, 52(12), 1095 - 100
Funalenone, a novel collagenase inhibitor produced by Aspergillus niger; Inokoshi J et al.; Funalenone, a phenalene compound that inhibits type I collagenase (MMP-1), was isolated from mycelium of Aspergillus niger FO-5904 by solvent extaction, ODS column chromatography, Sephadex LH-20 column chromatography and reversed phase HPLC . Funalenone inhibited 50% of type I collagenase activity at a concentration of 170 microM, but inhibited 18.3% and 38.7% against 72 kDa and 92 kDa type IV collagenase, respectively, at a concentration of 400 microM.

J Physiol Pharmacol, 1999 Dec, 50(5), 723 - 33
Epidemiological study on Helicobacter pylori infection and extragastroduodenal disorders in Polish population; Bielanski W; An association between Helicobacter pylori (H . pylori) infection and extragastroduodenal disorders (EGDD) is still not clear . The aim of the study was to investigate the relationship between H . pylori infection and the symptoms of coronary artery disease (CAD), facial dermatological changes (FDC), gastroesophageal reflux diseases (GERD), and periodontal diseases (PD) in Polish population . The study was performed between 1996-1999 year on 7,060 adult inhabitants of municipal area of Krakow (aged 18-76, mean 46.3 year; 55.8% female, 44.2% male): 2,204 subjects with EGDD and 4,856 without symptoms of EGDD . Each patient responded to a detailed questionnaire under supervision of medical staff . The H . pylori status was assessed non-invasively using urea breath test (UBT) with capsulated low-dose 13C-UBT (38 mg) . Exclusion criteria were: recent H . pylori eradication, treatment with PPI, bismuth and/or antibiotics in the last 4 weeks . Four groups of cases with EGDD symptoms were selected . Within each group exclusively only one of studied symptoms was recorded . The study included 328, 138, 688, and 1,050 patients with CAD, FDC, GERD and PD, respectively . For each studied group an age and sex-matched asymptomatic controls were selected (897, 387, 1,083, and 2,489 control patients) . Results: Overall H . pylori infection rate was 69,9% (in 71.4% of 2,204 cases and in 69.31% of 4,856 controls) . In CAD group: 68% of 328 cases were H . pylori (+ve) vs . 70% H . pylori (+ve) of 897 controls . An association was not significant: OR = 0.93 (95% CI, 0.72-1.20) . In 138 of FDC cases, 59% were H . pylori (+ve) vs . 71% H . pylori (+ve) in 387 controls showing the lack of positive association; OR = 0.60 (95% CI, 0.42-0.87) . In GERD, 69% of 688 cases were H . pylori (+ve) vs . 73% of 1,083 H . pylori (+ve) controls and negative association was observed; OR=0.80 (95% CI, 0.65-1.00) . In 1,050 of PD cases 75% were H . pylori (+ve) vs . 68% H . pylori (+ve) of 2,489 controls; positive association was significant; OR = 1.4 (95% CI, 1.16-1.68) . We conclude that in the studied Polish population, no positive association exists between H . pylori positivity and CAD, FDC or GERD possibly due very high overall H . pylori infection rate . The only positive link observed between H . pylori infection and periodontal disease may reflect direct "in situ" H . pylori pathological action of H . pylori in oral cavity . It is not excluded that periodontal diseases may facilitate the H . pylori oro-gastric transmission and colonisation of the bacteria in the digestive tract.

Kansenshogaku Zasshi, 2000 Jan, 74(1), 57 - 63
{Gastrointestinal diseases associated with HIV infection}; Sakamoto M et al.; A clinical studies were carried out on gastrointestinal diseases associated with HIV infection . During the 6 years between January 1993 and December 1998, 71 HIV infected cases visited to Yokohama Municipal Citizen's hospital, and 26 of them developed gastrointestinal complications during the course of their illness . They consisted of 24 males and 2 females, with the mean age of 44.7 years and the medial value of 42.5 years . Of the 26 patients, 21 were Japanese, and the remaining 5 were Southeast Asian . The mean CD4 count was 143/microliter and the medial value was 32/microliter at the time of development of complications . Gastrointestinal complications were esophageal candidiasis in 6 patients, cytomegalovirus (CMV) gastritis and gastric Kaposi's sarcoma in 1 patient each, amebiasis in 8 patients, infectious colitis in 11 patients, and asymptomatic pathogen carriers in 3 patients . Esophageal and gastric complications were common in patients with low count of CD4, and endoscopy was useful for diagnosis . Amebiasis developed even in patients with normal CD4 and was common in males with experience in homosexual contact . It seems that homosexual contact acquire not only HIV infection but also Entamoeba histolytica through sexual contact . Protozoan and acid-fast bacteria were detected at high rate in patients with infectious colitis and asymptomatic pathogen carriers . Besides food-born infections, imported infections were seen in foreign and Japanese patients who had traveled abroad . The gastrointestinal diseases associated with HIV infections for the most part were opportunistic infections or tumors but imported, food-born, and sexually transmitted infections were also observed . It seems necessary to take into consideration of varying background of patients in the treatment of gastrointestinal diseases associated with HIV infections.

Biochemistry, 2000 Mar 7, 39(9), 2420 - 7
Thermodynamics of substrate binding to the chaperone SecB; Panse VG et al.; The thermodynamics of binding of unfolded polypeptides to the chaperone SecB was investigated in vitro by isothermal titration calorimetry and fluorescence spectroscopy . The substrates were reduced and carboxamidomethylated forms of RNase A, BPTI, and alpha-lactalbumin . SecB binds both fully unfolded RNase A and BPTI as well as compact, partially folded disulfide intermediates of alpha-lactalbumin, which have 40-60% of native secondary structure . The heat capacity changes observed on binding the reduced and carboxamidomethylated forms of alpha-lactalbumin, BPTI, and RNase A were found to be -0.10, -0.29, and -0.41 kcal mol(-1) K(-1), respectively, and suggest that between 7 and 29 residues are buried upon substrate binding to SecB . In all cases, binding occurs with a stoichiometry of one polypeptide chain per monomer of SecB . There is no evidence for two separate types of binding sites for positively charged and hydrophobic ligands . Spectroscopic and proteolysis protection studies of the binding of SecB to poly-L-Lys show that binding of highly positively charged peptide ligands to negatively charged SecB leads to charge neutralization and subsequent aggregation of SecB . The data are consistent with a model where SecB binds substrate molecules at an exposed hydrophobic cleft . SecB aggregation in the absence of substrate is prevented by electrostatic repulsion between negatively charged SecB tetramers.

Free Radic Res, 1999 Dec, 31 Suppl, S243 - 9
Catalase-peroxidases in cyanobacteria--similarities and differences to ascorbate peroxidases; Obinger C et al.; Cyanobacteria (blue-green algae) are oxygenic phototrophic bacteria carrying out plant-type photosynthesis . The only hydrogen peroxide scavenging enzymes in at least two unicellular species have been demonstrated to be bifunctional cytosolic catalase-peroxidases (CatPXs) having considerable homology at the active site with plant ascorbate peroxidases (APXs) . In this paper we examined optical and kinetic properties of CatPXs from the cyanobacteria Anacystis nidulans and Synechocystis PCC 6803 and discuss similarities and differences to plant APXs . Both CatPXs and APX showed similar spectra of the ferric enzyme, the redox intermediate Compound I and the cyanide complex, whereas the spectrum of CatPX Compound II had characteristics reminiscent of the spectrum of the native enzyme . Both steady-state and multi-mixing transient-state kinetic studies were performed in order to characterize the kinetic behaviour of CatPXs . Bimolecular rate constants of both formation and reduction of a CatPX Compound I are presented . Because of its intrinsic high catalase activity (which cannot be found in APXs), the rate constants for Compound I formation were measured with peroxoacetic acid and are shown to be 5.9 x 10(4) M(-1) s(-1) for CatPX from A . nidulans and 8.7 x 10(3) M(-1) s(-1) for the Synechocystis enzyme . Compared with o-dianisidine (2.7-6.7 x 10(6) M(-1) s(-1)) and pyrogallol (8.6 x 10(4)-1.6 x 10(5) M(-1) s(-1)), the rate constant for Compound I reduction by ascorbate was extremely low (5.4 x 10(3) M(-1) s(-1) at pH 7.0 and 15 degrees C), in marked contrast to the behaviour of APXs.

Nephrol Dial Transplant, 2000 Mar, 15(3), 379 - 84
Effects of dialyser and dialysate on the acute phase reaction in clinical bicarbonate dialysis; Schouten WE et al.; BACKGROUND: In chronic haemodialysis (HD), morbidity may result from repetitive induction of the acute phase response, caused by a bioincompatible dialysis membrane and/or contaminated dialysate . In the present study, cytokine release (interleukin-6, IL-6) and subsequent production of acute phase proteins (C-reactive protein, CRP and secretory phospholipase A(2), sPLA(2)) were assessed to investigate whether the HD-induced acute phase reaction depends mainly on the type of membrane or on the sterility of the dialysate . METHODS: In 11 patients, IL-6, CRP and sPLA(2) levels were assessed in blood samples drawn before (t(0)), at the end (t(180)) and 24 h after the start of HD (t(1440)) . All patients were dialysed on Cuprammonium (CU) and Polysulphon (PS) dialysers and seven patients underwent an additional HD session on CU plus a dialysate filter (CUf) . RESULTS: IL-6 levels were increased significantly at t(180) compared with t(0) (P<0.02) with both CU and CUf . At t(1440), IL-6 levels had returned to baseline . In contrast, marked fluctuations did not occur during HD with PS . At t(180), IL-6 was significantly greater with CU and CUf devices, than with PS (P<0.02) . Following HD with CU and CUf, a significant increase in CRP was observed at t(1440), compared with postdialysis values (P</=0.05) . In addition, sPLA(2) values were markedly increased at t(1440), compared with t(180), but only significant in the case of CU (P=0.01) . IL-6 levels at t(180) were significantly correlated with CRP (r=0.50, P<0.01) and sPLA(2) (r=0.47, P=0.01) values at t(1440) . During HD with PS membranes, neither CRP nor sPLA(2) values were markedly changed . CONCLUSIONS: In contrast to PS, both CU and CUf resulted in elevated IL-6 plasma levels at the end of HD, compared with t(0), which correlated with increased CRP and sPLA(2) values 24 h later . Therefore, the type of membrane, rather than the bacterial quality of the dialysate, seems to be responsible for the induction of the acute phase response during clinical bicarbonate HD.

J Bacteriol, 2000 Mar, 182(6), 1575 - 9
Identification of the active site of HetR protease and its requirement for heterocyst differentiation in the cyanobacterium Anabaena sp . strain PCC 7120; Dong Y et al.; HetR is a serine-type protease required for heterocyst differentiation in heterocystous cyanobacteria under conditions of nitrogen deprivation . We have identified the active Ser residue of HetR from Anabaena sp . strain PCC 7120 by site-specific mutagenesis . By changing the S152 residue to an Ala residue, the mutant protein cannot be labeled by Dansyl fluoride, a specific serine-type protein inhibitor . The mutant protein showed no autodegradation in vitro . The mutant hetR gene was introduced into Anabaena strain 884a, a hetR mutant . The resultant strain, Anabaena strain S152A, could not form heterocysts under conditions of nitrogen deprivation even though the up-regulation of the mutant hetR gene was induced upon removal of combined nitrogen . The Anabaena strain 216, which carries a mutant hetR gene encoding S179N HetR and could not form heterocysts, also produced HetR protein upon induction . Sequence comparison shows that Ser152 is conserved in all cyanobacterial HetR . Immunoblotting was used to study HetR induction in both the wild-type and mutant strains . The amount of mutant HetR in strain S152A and in strain 216 increased continuously for 24 h after nitrogen step-down, while the amount of HetR in wild-type cells reached a maximum level within 6 h after nitrogen step-down . Our results show the Ser152 is the active site of HetR . The protease activity is required for heterocyst differentiation and might be needed for repression of HetR overproduction under conditions of nitrogen deprivation.

J Bacteriol, 2000 Mar, 182(6), 1507 - 14
A gene cluster involved in metal homeostasis in the cyanobacterium Synechocystis sp . strain PCC 6803; Garcia-Dominguez M et al.; A gene cluster composed of nine open reading frames (ORFs) involved in Ni(2+), Co(2+), and Zn(2+) sensing and tolerance in the cyanobacterium Synechocystis sp . strain PCC 6803 has been identified . The cluster includes an Ni(2+) response operon and a Co(2+) response system, as well as a Zn(2+) response system previously described . Expression of the Ni(2+) response operon (nrs) was induced in the presence of Ni(2+) and Co(2+) . Reduced Ni(2+) tolerance was observed following disruption of two ORFs of the operon (nrsA and nrsD) . We also show that the nrsD gene encodes a putative Ni(2+) permease whose carboxy-terminal region is a metal binding domain . The Co(2+) response system is composed of two divergently transcribed genes, corR and corT, mutants of which showed decreased Co(2+) tolerance . Additionally, corR mutants showed an absence of Co(2+)-dependent induction of corT, indicating that CorR is a transcriptional activator of corT . To our knowledge, CorR is the first Co(2+)-sensing transcription factor described . Our data suggest that this region of the Synechocystis sp . strain PCC 6803 genome is involved in sensing and homeostasis of Ni(2+), Co(2+), and Zn(2+).

J Bacteriol, 2000 Mar, 182(6), 1472 - 80
Three new NifA-regulated genes in the Bradyrhizobium japonicum symbiotic gene region discovered by competitive DNA-RNA hybridization; Nienaber A et al.; The so-called symbiotic region of the Bradyrhizobium japonicum chromosome (C . Kundig, H . Hennecke, and M . Gottfert, J . Bacteriol . 175:613-622, 1993) was screened for the presence of genes controlled by the nitrogen fixation regulatory protein NifA . Southern blots of restriction enzyme-digested cosmids that represent an ordered, overlapping library of the symbiotic region were competitively hybridized with in vitro-labeled RNA from anaerobically grown wild-type cells and an excess of RNA isolated either from anaerobically grown nifA and rpoN mutant cells or from aerobically grown wild-type cells . In addition to the previously characterized nif and fix gene clusters, we identified three new NifA-regulated genes that were named nrgA, nrgB, and nrgC (nrg stands for NifA-regulated gene) . The latter two probably form an operon, nrgBC . The proteins encoded by nrgC and nrgA exhibited amino acid sequence similarity to bacterial hydroxylases and N-acetyltransferases, respectively . The product of nrgB showed no significant similarity to any protein with a database entry . Primer extension experiments and expression studies with translational lacZ fusions revealed the presence of a functional -24/-12-type promoter upstream of nrgA and nrgBC and proved the NifA- and RpoN (sigma(54))-dependent transcription of the respective genes . Null mutations introduced into nrgA and nrgBC resulted in mutant strains that exhibited wild-type-like symbiotic properties, including nitrogen fixation, when tested on soybean, cowpea, or mung bean host plants . Thus, the discovery of nrgA and nrgBC further emphasizes the previously suggested role of NifA as an activator of anaerobically induced genes other than the classical nitrogen fixation genes.

FEBS Lett, 2000 Feb 25, 468(2-3), 231 - 3
A novel covalent modification of nitrogenase in a cyanobacterium; Gallon JR et al.; In extracts of the unicellular cyanobacterium Gloeothece, the Fe-protein of nitrogenase can be separated by SDS-PAGE into two antigenically identifiable components . Unlike the situation in photosynthetic bacteria such as Rhodospirillum rubrum, these two forms do not arise from covalent modification of the protein by ADP-ribosylation . Rather, the Fe-protein of Gloeothece nitrogenase is subjected to modification by palmitoylation.

J Biol Chem, 2000 Mar 3, 275(9), 6047 - 50
Mechanism and cellular applications of a green fluorescent protein-based halide sensor; Jayaraman S et al.; We report the application of a targetable green fluorescent protein-based cellular halide indicator . Fluorescence titrations of the purified recombinant yellow fluorescent protein YFP-H148Q indicated a pK(a) of 7.14 in the absence of Cl(-), which increased to 7.86 at 150 mM Cl(-) . At pH 7.5, YFP-H148Q fluorescence decreased maximally by approximately 2-fold with a K(D) of 100 mM Cl(-) . YFP-H148Q had a fluorescence lifetime of 3.1 ns that was independent of pH and {Cl(-)} . Circular dichroism and absorption spectroscopy revealed distinct Cl(-)-dependent spectral changes indicating Cl(-)/YFP binding . Stopped-flow kinetic analysis showed a biexponential time course of YFP-H148Q fluorescence (time constants <100 ms) in response to changes in pH or {Cl(-)}, establishing a 1:1 YFP-H148Q/Cl(-) binding mechanism . Photobleaching analysis revealed a millisecond triplet state relaxation process that was insensitive to anions and aqueous-phase quenchers . The anion selectivity sequence for YFP-H148Q quenching (ClO(4)(-) approximately I(-) > SCN(-) > NO(3)(-) > Cl(-) > Br(-) > formate > acetate) indicated strong binding of weakly hydrated chaotropic ions . The biophysical data suggest that YFP-H148Q anion sensitivity involves ground state anion binding to a site close to the tri-amino acid chromophore . YFP-H148Q transfected mammalian cells were brightly fluorescent with cytoplasmic/nuclear staining . Ionophore calibrations indicated similar YFP-H148Q pH and anion sensitivities in cells and aqueous solutions . Cyclic AMP-regulated Cl(-) transport through plasma membrane cystic fibrosis transmembrane conductance regulator Cl(-) channels was assayed with excellent sensitivity from the time course of YFP-H148Q fluorescence in response to extracellular Cl(-)/I(-) exchange . The green fluorescent protein-based halide sensor described here should have numerous applications, such as anion channel cloning by screening of mammalian expression libraries and discovery of compounds that correct the cystic fibrosis phenotype by screening of combinatorial libraries.

Acad Emerg Med, 2000 Feb, 7(2), 114 - 9
The effects of epidermal debridement of partial-thickness burns on infection and reepithelialization in swine; Singer AJ et al.; OBJECTIVE: Early postburn debridement of burn blisters is controversial . This study was conducted to compare rates of infection and reepithelialization in debrided vs nondebrided second-degree burns in swine . METHODS: This was a prospective, blinded, controlled, experimental trial using isoflurane-anesthetized swine . Standardized partial-thickness burns were inflicted by applying an aluminum bar preheated to 80 degrees C to the backs and flanks of two young pigs for 20 seconds . In half of the burns the necrotic epidermis was manually debrided . All burns were randomly treated with octylcyanoacrylate spray (OCA) or dry gauze (C) . Full-thickness biopsies were taken at 7, 10, and 14 days for blinded histopathologic evaluation . The primary outcomes were the proportions of infected burns at days 7 and 10 and the proportion of completely reepithelialized burns at day 14 . Burns were considered infected in the presence of intradermal neutrophils containing bacteria (intraobserver agreement, K = 1.00) . A secondary outcome was the proportion of burns with the presence of scar tissue (abnormal collagen under polarized light; intraobserver correlation, K = 0.93) . Chi-square tests were used for group comparisons . This study had 90% power to detect a 40-percentage-point difference in infection rates (alpha = 0.05) . RESULTS: A total of 126 biopsies from 42 burns were available for review . Infection rates were higher in the debrided burns both at day 7 (55% vs 4.5%, p < 0.001) and at day 10 (65% vs 9%, p < 0.001) after injury . The proportion of nondebrided burns that were completely reepithelialized was higher at days 10 (68% vs 0%, p < 0.001) and 14 (100% vs 65%, p = 0.003) . The presence of scar tissue was more common in debrided burns (75% vs 4.5%, p < 0.001) . Burns treated with OCA had fewer infections than controls (4% vs 55%, p < 0.001) . Fewer OCA-treated debrided burns were reepithelialized at 14 days than those that were not debrided (30% vs 100%, p = 0.001) . CONCLUSIONS: Under the current study conditions, early postburn epidermal debridement of second-degree burns resulted in more infections and slower reepithelialization rates in swine . The effects of early postburn epidermal debridement in humans should be explored.

Vet Immunol Immunopathol, 2000 Feb 25, 73(2), 183 - 91
Cloning, sequencing, and analysis of cDNA encoding bovine granulocyte-colony stimulating factor; Heidari M et al.; Neutrophils play a critical role in defending against bacterial infections . Hematopoietic growth factors are a class of regulatory cytokines that are required for stimulation, proliferation, and differentiation of blood cells . Granulocyte colony stimulating factor (G-CSF) is a cytokine that induces proliferation and maturation of precursor myeloid cells in the bone marrow into fully differentiated neutrophils . G-CSF also modulates the functional activity of mature neutrophils . Treatment with G-CSF significantly enhances neutrophil phagocytic activity and killing of bacteria and fungi . We have isolated and sequenced a cDNA clone encoding bovine G-CSF (bG-CSF) from an endothelial cell cDNA library using primers designed from ovine G-CSF . The full length cDNA is 1460 nucleotides with 585 nucleotides comprising the open reading frame . Sequence analysis shows 95% identity with ovine, 89% with porcine, 85% with human, and 76% with murine G-CSF . The deduced G-CSF protein consists of 174 amino acids with 95% identity to ovine, 86% to porcine, 81% to human, and 71% to murine . The signal peptide of G-CSF is 21 amino acids long which is nine amino acids shorter than that of human and murine G-CSF . RT-PCR analysis shows that neither freshly isolated nor ConA stimulated neutrophils express G-CSF mRNA . Mononuclear cells, however, expressed G-CSF mRNA after 48 h incubation with or without ConA stimulation.

Postgrad Med, 2000 Feb, 107(2), 41 - 2, 45-6
Generalized pruritus without primary lesions . Differential diagnosis and approach to treatment; Nowak MA et al.; A 65-year-old man presented with recurrent generalized pruritus and excoriations of many years' duration . He had been treated with antihistamines, topical corticosteroids, and antibiotics for secondary wound infections, but improvement was only temporary . He had also been hospitalized for leg ulcers complicated by cellulitis . Examination revealed multiple oval and linear red papules and nodules measuring 0.5 to 2 cm in diameter . Some of the lesions were eroded and had a central crater and yellowish crust . The patient also had hypopigmented linear scars localized to the posterior scalp, neck, upper back, chest, abdomen, arms, and legs with sparing of the middle and lower back (figures 1 and 2) . An ulcer measuring 1.5 x 2 cm that was surrounded by indurated skin was present on the medial aspect of his right ankle . The ulcer was partially covered by yellow exudate . There was no evidence of cellulitis . Liver enzyme, serum creatinine, and thyrotropin levels, as well as a chest roentgenogram, were normal . Wound cultures for bacteria and fungi were nonsignificant . A punch biopsy from a representative lesion showed an abrupt epidermal defect with sparse superficial lymphocytic infiltrate in the dermis . The patient was admitted to the hospital to isolate him from his home environment . He received a 10-day course of systemic cephalexin, topical clobetasol propionate ointment for the affected skin areas, and oral hydroxyzine for pruritus . Ultraviolet light therapy was instituted once daily and was to continue for 2 months . His lesions had improved moderately by the time he was discharged from the hospital . On follow-up 2 weeks later, his lesions were flat and had resulted in hypopigmented scars . Three months later, however, he had persistent, intense pruritus, and new excoriations had developed on his forearms and back . He improved after receiving treatment with oral doxepin hydrochloride.

Vaccine, 2000 Feb 25, 18(16), 1717 - 20
Human immunosenescence: the prevailing of innate immunity, the failing of clonotypic immunity, and the filling of immunological space; Franceschi C et al.; According to the remodeling theory of aging we proposed several years ago, the current data on human immunosenescence depicts a complex scenario where clonotypical immunity deteriorates, while ancestral innate/natural immunity is largely conserved or even up-regulated with age . Under an evolutionary perspective, antigens are the cause of a persistent life-long antigenic stress, responsible for the accumulation of effector CD8+/CD28- T cells, the decrease of naive T cells (CD95-) and the marked shrinkage of T cell repertoire with age . Concomitantly, NK cytotoxicity, chemotaxis, phagocytosis and complement activities remain unaffected or negligibly affected, in comparison to clonotypical immunity . Thus, immunosenescence is not a random deteriorative phenomenon but appears to inversely recapitulate an evolutionary pattern . On the whole, immunosenescence can be envisaged as the result of the continuous challenge of the unavoidable exposure to a variety of potential antigens (viruses, bacteria, but also food and self molecules among others) . From this perspective antigens are nothing else than a particular type of stressor and immunosenescence appears to be the price paid to immunological memory, i.e . one of the main characteristics of the most evolutionary recent and sophisticated type of immunity . Together with the age-related thymic involution, and the consequent age-related decrease of thymic output of new T cells, this situation leaves the body practically devoid of virgin T cells, and thus likely more prone to a variety of infectious and non infectious diseases.

Blood, 2000 Mar 1, 95(5), 1616 - 25
A soluble form of human Delta-like-1 inhibits differentiation of hematopoietic progenitor cells; Han W et al.; Two Notch ligand families, Delta and Serrate/Jagged, have been identified in vertebrates . Members of the Jagged family have been shown to affect in vitro hematopoiesis . To determine whether members of the Delta family might play a similar role in hematopoiesis, we examined the expression of mouse Delta-like-1 (mDll1) . mDll1 protein was detected in whole marrow and in a marrow stromal cell line MS-5 . At the RNA level, both mDll1 and Notch1 were seen in marrow precursor, differentiated hematopoietic, marrow stromal, and MS-5 cells . We isolated a cDNA encoding the human homologue of mDll1, designated human Delta-like-1 (hDll1) . A soluble form of hDll1, hDll1(NDSL), containing the DSL domain and the N-terminal sequences, was expressed and purified from bacteria as a glutathione S-transferase (GST) fusion protein . We observed that hDll1(NDSL) delayed the acquisition of differentiation markers by murine hematopoietic progenitor cells (Lin-) cultured in vitro with cytokines . In addition, it promoted greater expansion (more than 3 times) of the primitive hematopoietic precursor cell population, measured in high-proliferative potential colony assay and day 12 colony-forming unit spleen (CFU-S) assay, than GST controls . We also observed that the percentage of apoptotic cells decreased and that the number of cells in the S-phase of the cell cycle increased in the cultures of Lin(-) cells with hDll1(NDSL) . The effects of hDll1(NDSL) were blocked by antibody against the mouse counterpart of hDll1(NDSL), mDll1(NDSL) . These observations demonstrate that hDll1 plays a role in mediating cell fate decisions during hematopoiesis . (Blood . 2000;95:1616-1625)

Infect Immun, 2000 Mar, 68(3), 1633 - 48
Comparative genome analysis of the pathogenic spirochetes Borrelia burgdorferi and Treponema pallidum; Subramanian G et al.; A comparative analysis of the predicted protein sequences encoded in the complete genomes of Borrelia burgdorferi and Treponema pallidum provides a number of insights into evolutionary trends and adaptive strategies of the two spirochetes . A measure of orthologous relationships between gene sets, termed the orthology coefficient (OC), was developed . The overall OC value for the gene sets of the two spirochetes is about 0.43, which means that less than one-half of the genes show readily detectable orthologous relationships . This emphasizes significant divergence between the two spirochetes, apparently driven by different biological niches . Different functional categories of proteins as well as different protein families show a broad distribution of OC values, from near 1 (a perfect, one-to-one correspondence) to near 0 . The proteins involved in core biological functions, such as genome replication and expression, typically show high OC values . In contrast, marked variability is seen among proteins that are involved in specific processes, such as nutrient transport, metabolism, gene-specific transcription regulation, signal transduction, and host response . Differences in the gene complements encoded in the two spirochete genomes suggest active adaptive evolution for their distinct niches . Comparative analysis of the spirochete genomes produced evidence of gene exchanges with other bacteria, archaea, and eukaryotic hosts that seem to have occurred at different points in the evolution of the spirochetes . Examples are presented of the use of sequence profile analysis to predict proteins that are likely to play a role in pathogenesis, including secreted proteins that contain specific protein-protein interaction domains, such as von Willebrand A, YWTD, TPR, and PR1, some of which hitherto have been reported only in eukaryotes . We tentatively reconstruct the likely evolutionary process that has led to the divergence of the two spirochete lineages; this reconstruction seems to point to an ancestral state resembling the symbiotic spirochetes found in insect guts.

Infect Immun, 2000 Mar, 68(3), 1297 - 303
Identification of Brucella suis genes affecting intracellular survival in an in vitro human macrophage infection model by signature-tagged transposon mutagenesis; Foulongne V et al.; Bacteria of the genus Brucella are facultative intracellular pathogens which have developed the capacity to survive and multiply in professional and nonprofessional phagocytes . The genetic basis of this aspect of Brucella virulence is still poorly understood . To identify new virulence factors, we have adapted signature-tagged transposon mutagenesis, which has been used essentially in animal models, to an in vitro human macrophage infection model . A library of 1,152 Brucella suis 1330 tagged mini-Tn5 Km2 mutants, in 12 pools, was screened for intracellular survival and multiplication in vitamin D(3)-differentiated THP1 cells . Eighteen mutants were identified, and their attenuation was confirmed in THP1 macrophages and HeLa cells . For each avirulent mutant, a genomic fragment containing the transposon was cloned . The genomic DNA sequence flanking the transposon allowed us to assign functions to all of the inactivated genes . Transposon integration had occurred in 14 different genes, some of which were known virulence genes involved in intracellular survival or biosynthesis of smooth lipopolysaccharide (the virB operon and manB), thus validating the model . Other genes identified encoded factors involved in the regulation of gene expression and enzymes involved in biosynthetic or metabolic pathways . Possible roles in the virulence of Brucella for the different factors identified are discussed.

Curr Microbiol, 2000 Apr, 40(4), 279 - 82
Strains of Xylella fastidiosa rapidly distinguished by arbitrarily primed-PCR; da Costa PI et al.; Genomic DNAs isolated from strains of Xylella fastidiosa that caused citrus variegated chlorosis, coffee leaf scorch, Pierce's Disease of grapevine, and plum leaf scorch were analyzed by arbitrarily primed polymerase chain reaction . Purified DNA was amplified under nonstringent conditions with single primers 21 nucleotides (nt) long . Thirty-nine amplification products were observed that were useful to distinguish among the strains and to derive a similarity matrix and construct a phenogram showing possible relationships among the strains . Strains isolated from diseased coffee and citrus in Brazil were closely related to each other (coefficient of similarity of 0 . 872), but only distantly related to a strain isolated from diseased grapevine in the USA (coefficient of similarity of 0.650) . Strains of Xylella fastidiosa isolated from diseased plums in the USA and Brazil clustered with strains from different hosts isolated from their respective countries of origin . Thus, there may be two quite dissimilar clusters of strains of Xylella fastidiosa, one in North America and the other in South America . Each cluster contains strains that can cause disease in plum . The methods described provide a convenient and rapid method to distinguish between strains of Xylella fastidiosa that cause diseases of coffee and citrus in the same region of Brazil . This has not been possible previously . This will potentially enable the two strains to be distinguished in alternate hosts or in insect vectors.

Proc R Soc Lond B Biol Sci, 2000 Jan 22, 267(1439), 103 - 7
The xylem of rice (Oryza sativa) is colonized by Azorhizobium caulinodans; Gopalaswamy G et al.; Following inoculation with Azorhizobium caulinodans ORS571 (pXLGD4), lateral root development of rice and colonization of lateral root cracks by bacteria were shown to be stimulated by the flavonoid naringenin . Rice seedlings growing aseptically in the presence of naringenin were inoculated with ORS571 (pXLGD4), carrying the lacZ reporter gene . By microscopic analysis of sections of inoculated rice roots, it has been demonstrated that the xylem of rice roots can be colonized by Azorhizobium caulinodans . We discuss whether this colonization of the xylem of rice roots by azorhizobia could provide a suitable niche for endophytic nitrogen fixation.

J Endod, 1999 Oct, 25(10), 676 - 82
Biocompatibility of an adhesive system applied to exposed human dental pulp; Hebling J et al.; Human pulp tissue was directly capped with All Bond 2, or calcium hydroxide and evaluated 7, 30, or 60 days after the procedures . Histological analysis was performed to assess the inflammatory cell response, tissue disorganization, dentin bridging, and the presence of bacteria . At 7 days, with All Bond 2 capping, there was a large area of neutrophilic infiltrate underlying the pulp capping material, and the death of adjacent odontoblasts, was observed . However, with time, the neutrophilic reaction was replaced by fibroblastic proliferation with macrophages and giant cells surrounding globules of resin scattered in the coronal pulp tissue . The persistent inflammatory reaction and hyaline alteration of extracellular matrix inhibited complete pulp repair or dentin bridging . In contrast, at 7 days, the pulp tissue capped with calcium hydroxide exhibited odontoblast-like cells organized underneath coagulation necrosis . Pulp repair evolved into apparent complete dentin bridge formation at 60 days . All Bond 2 did not appear to allow any pulp repair and does not appear to be indicated for direct pulp capping of human teeth.

J Endod, 1999 Jul, 25(7), 486 - 9
Changes in the periodontal membrane due to apical periodontitis; Eberhard J et al.; Teeth with an apical inflammatory lesion were studied by light microscopic morphometrical procedures to estimate the volumetric density of periodontal ligament tissues by point counting . Sixty-four root surfaces were investigated from coronal to apical . The observed tissue changes were similar in groups with and without bacteria, except for an elevated volumetric density of inflammatory cells in the first group . The attachment was lost apically . Principal fibers running to the root surface and extensions in the cementum decreased from coronal to apical, but were replaced by fibers running parallel to cementum and fibers oriented in a network . We suggest that acellular extrinsic fiber cementum was lost, whereas cellular mixed stratified cementum was built . The cellular mixed stratified cementum synthesis was by inclusion of the remaining fibers from the acellular extrinsic fiber cementum . We suspect that the changes were the result of the anti-inflammatory reaction caused by the periapical lesion.

Gen Dent, 1999 Nov-Dec, 47(6), 580 - 8
Not all patients are the same: systemic risk factors for adult periodontitis; Wilson TG; Periodontal diseases are viewed today as multifactoral problems that are initiated and sustained by bacteria but significantly modified by the body's response to bacterial plaque . Local and systemic risk factors are involved in the disease process and both should be included when prognosis and treatment plans are developed . The most significant systemic modifying factors appear to be smoking, diabetes, and a recently discovered genetic marker . Slight changes in genes that produce an important inflammatory mediator, found in one-third of those studied, can lead to major negative outcomes in the way the body responds to bacteria.

Photochem Photobiol, 2000 Feb, 71(2), 135 - 40
Dihydrocercosporin singlet oxygen production and subcellular localization: a possible defense against cercosporin phototoxicity in Cercospora; Daub ME et al.; Fungi in the genus Cercospora produce cercosporin, a potent singlet oxygen (1O2)-generating photosensitizer that plays a critical role in the ability of these fungi to parasitize plants . Although plants, mice, bacteria and many fungi are sensitive to cercosporin, Cercospora species are resistant to its toxicity . The cellular resistance of these fungi to cercosporin has been correlated with fungal cell surface reducing ability and the ability to maintain cercosporin in a chemically reduced state . As a model for reduced cercosporin we employed a reduced, acetylated derivative (hexaacetyl-dihydrocercosporin, HAC) that we tested for 1O2 production in a range of solvents . We found that as a 1O2 photosensitizer, HAC was only moderately effective in organic solvents (phi SO = 0.14-0.18) and very poor in water (phi SO = 0.02-0.04) . By contrast, the 1O2 quantum yield of cercosporin itself was unaffected by solvent (phi SO = 0.84-0.97) . To investigate the localization of reduced cercosporin in fungal cells, we developed a fluorescence assay using laser scanning confocal microscopy . This assay showed a uniform green fluorescence, indicative of reduced cercosporin, in the cytoplasm of hyphal cells treated with cercosporin . We hypothesize that the main protection mechanism against cercosporin phototoxicity in the fungus consists of transformation of cercosporin to a reduced state and localization of this reduced form in the aqueous compartment of the cell, thus decreasing intracellular 1O2 production to levels that can be tolerated by the fungus . In addition, we have, for the first time, directly detected 1O2 phosphorescence from fungal culture, either stained with the photosensitizer rose bengal or actively synthesizing cercosporin, demonstrating 1O2 production in vivo and from cercosporin in culture.

Dis Aquat Organ, 1999 Nov 30, 38(3), 191 - 200
Comparative severity of experimentally induced mycobacteriosis in striped bass Morone saxatilis and hybrid tilapia Oreochromis spp; Wolf JC et al.; Twenty striped bass Morone saxatilis and 20 hybrid tilapia Oreochromis niloticus x O . mossambicus x O . aureus each received a single intramuscular injection of 1.6 x 10(6) colony forming units per gram body weight of Mycobacterium marinum . Striped bass manifested significantly greater clinical and microscopic disease compared to tilapia . Whereas all the striped bass had died or were clinically ill by Day 8 post-infection, there was no apparent disruption of normal behaviour, physical appearance, or growth in any of the sacrificed or surviving tilapia . Histologically, granulomas in striped bass were generally larger and less discrete, with a higher proportion of heavily vacuolated macrophages, and large cores of necrotic cells . Visceral granulomas in tilapia were smaller, with a higher proportion of epithelioid macrophages, more pigment-containing cells, more peripheral lymphocytes, and virtually no central necrosis . Visceral granulomas were 18-fold more numerous in striped bass than in tilapia . Based upon histomorphometric data, mean proportions of acid-fast bacteria within pronephros granulomas were 4-fold greater in striped bass than tilapia, and striped bass granulomas averaged more than twice as large as tilapia granulomas . In the anterior kidney of striped bass, a positive correlation existed between mean mycobacterial proportions and mean necrosis scores . In tilapia, mean mycobacterial proportions correlated negatively with mean granuloma numbers, whereas there was no correlation between these parameters in striped bass . Results suggest that intrinsic functional differences in the immunologic systems of striped bass and hybrid tilapia may contribute to inter-species variation in mycobacteriosis susceptibility.

Blood Purif, 2000, 18(1), 30 - 6
Plasma C-reactive protein in hemodialysis patients: a cross-sectional, longitudinal clinical survey; Panichi V et al.; In hemodialysis patients, C-reactive protein (CRP), an acute-phase reactant, is a sensitive and independent marker of malnutrition, anemia, and amyloidosis . The aim of the present studies was to evaluate CRP and interleukin 6 levels in plasma samples from long-term hemodialysis patients on different extracorporeal modalities associated with or without backfiltration . Two hundred and forty-seven patients were recruited in eight hospital-based centers . All patients had been on their dialytic modality for at least 6 months . At enrollment, 46 hemodialysis patients out of 247 (18.6%) had clinical evidence of pathologies known to be associated with high CRP values . The 201 remaining patients were defined as clinically stable and were on conventional hemodialysis (34%), hemodiafiltration with infusion volumes <10 liters/session (10%), hemodiafiltration with infusion volumes <20 liters/session (32%), and double-chamber hemodiafiltration with infusion volumes <10 liters/session (22%) . Analysis of CRP values in the clinically stable patients showed that an unexpectedly high proportion (47%) of the patients had CRP values higher than 5 mg/l (taken as the upper limit in normal human subjects) . The values of CRP and interleukin 6 were significantly higher in hemodiafiltration with infusion volumes <10 liters/session than in hemodiafiltration with infusion volumes >20 liters/session, in hemodialysis and in double-chamber hemodiafiltration . The same pattern occurred after 6 months of follow-up in 171 out of 201 clinically stable patients . Hemodialytic conditions that expose to the risk of backfiltration such as low exchange volume hemodiafiltration may induce a chronic inflammatory state as reflected by increased plasma values of both CRP and interleukin 6, thus suggesting the need for hemodialytic strategies that reduce (hemodialysis with low-permeability membranes or hemodiafiltration with infusion volumes >20 liters) or eliminate (double-chamber hemodiafiltration) backfiltration of bacteria-derived contaminants .

Biochem Pharmacol, 2000 Mar 15, 59(6), 703 - 12
Comparison of oxidative stress response parameters in newborn mouse liver versus simian virus 40 (SV40)-transformed hepatocyte cell lines; Vasiliou V et al.; Induction of approximately one dozen genes and/or enzyme activities in liver of the untreated newborn c(14CoS)/c(14CoS) mouse-when compared with the c(ch)/c(14CoS) heterozygote or the c(ch)/c(ch) wild-type-is the result of enhanced levels of reactive oxygenated metabolites originating from a block in the tyrosine degradation pathway . Oxidative stress activates genes via the electrophile response element, whereas dioxin activates genes via the receptor-mediated aromatic hydrocarbon response element . Here, we compared several parameters in 14CoS/14CoS versus ch/ch newborn mouse liver with that in simian virus 40 (SV40)-transformed hepatocyte lines that had been derived from newborn liver . We showed in this study that: (a) NADP(H):quinone oxidoreductase and UDP glucuronosyltransferase 1A6 mRNA levels were increased in both the (untreated) 14CoS/14CoS newborn liver and cell line; (b) aldehyde dehydrogenase 3A1 mRNA was increased by both oxidative stress and dioxin in hepatocyte cultures, but was not detectable in liver of the intact mouse; (c) the glutathione S-transferase GSTA1, GSTP1, GSTA3, and GSTM1 mRNA levels were increased by oxidative stress in 14CoS/14CoS newborn liver, but these transcripts were either low or undetectable in the cell lines; (d) GSTA1 mRNA was up-regulated by the absence of cytochrome P450 1A1 (CYP1A1) activity (i.e . the Gsta1 gene is a member of the aromatic hydrocarbon {Ah} battery); and (e) GSTP1 mRNA was not up-regulated by the absence of CYP1A1 activity (i . e . Gstp1 is not a member of the {Ah} battery) . The 14CoS/14CoS and ch/ch hepatocyte established cell lines were transformed with SV40, which expresses large T antigen; this gene product is known to bind to, and interact with, several cell cycle regulatory proteins such as p53 and the retinoblastoma protein-E2F complex . It is therefore likely that differences in the oxidative stress responses between the 14CoS/14CoS newborn liver and the immortalized hepatocyte cell line might be explained by the presence of large T antigen in the established cell line.

Am Nat, 2000 Feb, 155(2), 200 - 218
Effects of Enrichment on Three-Level Food Chains with Omnivory; Diehl S et al.; Although omnivory (the consumption of resources from more than one trophic level) is widespread, this fundamental limitation to the applicability of food chain theory to real communities has received only limited treatment . We investigated effects of enrichment (increasing carrying capacity, K, of the resource) on a system consisting of a resource (R), an intermediate consumer (N), and an omnivore (P) using a general mathematical model and tested the relevance of some of its predictions to a laboratory system of mixed bacteria (=R) and the ciliates Tetrahymena (=N) and Blepharisma (=P) . The model produced six major predictions . First, N may facilitate or inhibit P . Enrichment may revert the net effect of N on P from facilitation to inhibition . Second, along a gradient of K, up to four regions of invasibility and stable coexistence of N and P may exist . At the lowest K, only R is present . At somewhat higher K, N can coexist with R . At intermediate K, either N and P coexist, or either consumer excludes the other depending on initial conditions . At the highest K, N may be excluded through apparent competition and only R and P can coexist . The pattern of persistence of Tetrahymena and Blepharisma along an enrichment gradient conformed fairly well to the scenario allowing coexistence at intermediate K . Third, for stable equilibria of the omnivory system, R always increases and N always decreases with K . The abundances of bacteria and Tetrahymena were suggestive of such a pattern but did not allow a strict test because coexistence occurred at only one level of enrichment . Fourth, an omnivore can invade an R-N system at a lower K than an otherwise identical specialist predator of N . Fifth, an omnivore can always invade a food chain with such a specialist predator . Sixth, over ranges of K where both omnivory systems and otherwise identical three-level food chains are feasible, N is always less abundant in the omnivory system, whereas the relative abundances of R and P in omnivory systems compared to food chains may change with K . It is thus possible that total community biomass at a given K is lower in an omnivory system than in a food chain . Both the model and the experimental results caution that patterns of trophic-level abundances in response to enrichment predicted by food chain theory are not to be expected in systems with significant omnivory.

J Med Virol, 2000 Apr, 60(4), 468 - 74
Evaluation of the antibody specificities of human convalescent-phase sera against the attachment (G) protein of human respiratory syncytial virus: influence of strain variation and carbohydrate side chains; Palomo C et al.; The C-terminal third of the attachment protein (G) of several human respiratory syncytial virus isolates was obtained as either a glycosylated protease-resistant fragment of the purified protein or a nonglycosylated GST fusion protein expressed in bacteria . The reactivity of human convalescent-phase sera with both forms of the protein segment was evaluated in immunoblots . While all serum samples reacted with the mature intact protein of the different isolates, only certain samples reacted with the nonglycosylated C-terminal segment of some viral isolates . The number of human serum samples reacting with the glycosylated C-terminal fragment was even more limited . These results highlight the heterogeneity of the human antibody response against epitopes located in the C-terminal hypervariable region of the G molecule and the influence of carbohydrate side chains for expression of these epitopes . We also have analysed the specificities of human sera by competitive enzyme-linked immunosorbent assay with murine monoclonal antibodies (MAbs) . Most human serum samples inhibited virus binding of MAbs that recognised conserved or group-specific epitopes of the G protein, while only a limited fraction of those samples inhibited binding of MAbs that recognised strain-specific epitopes . These results are discussed in terms of the antibody repertoire induced after human respiratory syncytial virus infection and the relevance of escape mechanisms to preexisting antibodies for the evolution of this virus .

Mycopathologia, 1999, 145(3), 113 - 9
Purification of the endospores and sporangia of Rhinosporidium seeberi on Percoll columns; Atapattu DN et al.; Human rhinosporidial tissue was used as the source of the various developmental stages of Rhinosporidium seeberi--endospores with electron dense bodies, juvenile, and immature sporangia . After homogenisation in phosphate buffered saline (PBS) and removal of tissue fragments by centrifugation, the rhinosporidial bodies were isolated on centrifuged Percoll columns with gradients of densities or on triple-layered columns of varying density . The separated bands, after repeated washing in PBS gave bodies free from human tissue as shown on Leishman and PAS staining and indirect immunofluorescence with rabbit and human patients' anti-rhinosporidial sera . Sonicates of these bodies were tested on agarose gel for precipitation with antisera, and on SDS-PAG electrophoresis and Coomassie Blue staining . Percoll columns were shown to be capable of isolating these stages of R . seeberi, free from human tissue and contaminating bacteria.

J Periodontal Res, 1999 Oct, 34(7), 374 - 8
HLA genetics for diagnosis of susceptibility to early-onset periodontitis; Takashiba S et al.; Human leukocyte antigens (HLA) are essential in the recognition of foreign antigens in humoral immune response, which is genetically predetermined . Susceptibility to certain diseases that involve the immune response has been studied in relation to distinct HLA types . Although some diseases have been found to correlate to specific HLA loci positively, it has been difficult to isolate HLA types that predispose patients to periodontal destruction . Here, we review the current knowledge and recent advances in HLA genetics and its biology, which determine susceptibility to early-onset periodontitis (EOP) . The HLA-DRB1*1501-DQB1*0602 genotype has been found with increasing frequency in EOP patients . This HLA genotype expresses aspartic acid at position 57 and glycine at position 70 on the DQ beta chain, suggesting a capability to bind certain bacterial antigens . The T cell response against the outer membrane protein (Ag53) of Porphyromonas gingivalis was examined via this HLA genotype . Strong T cell response against Ag53 p141-161 was inhibited partially by anti-DR antibody, but not by anti-DQ antibody . Possible host and bacterial peptides capable of binding DRB1*1501 were elucidated when the peptide sequence was compared to gene and protein databases . These results suggest that patients who have the HLA-DRB1*1501-DQB1*0602 genotype may have an accelerated T cell response to certain periodontopathic bacteria such as P . gingivalis in hyperimmune reactions and thus increased susceptibility to EOP.

J Periodontal Res, 1999 Oct, 34(7), 358 - 62
Systemic manifestations of periodontitis in the non-human primate; Ebersole JL et al.; This report describes our findings regarding the potential contribution of periodontitis to atherosclerotic processes using a nonhuman primate model . The goal of the investigations was to target general mechanisms which could describe the association of these disease processes, including: (i) systemic translocation of bacteria/products during periodontitis; (ii) alterations in systemic inflammatory biomarkers during periodontitis; and (iii) the relationship of periodontitis to serum lipids/lipoproteins . Increases in serum endotoxin (e.g . LPS) during ligature-induced periodontitis were observed in these animals . We determined serum levels of various acute phase reactants and chemokines (e.g . CRP, alpha 1-antitrypsin, haptoglobin, fibrinogen, IL-8) . A number of these host factors were significantly increased during gingivitis and/or periodontitis . Finally, we observed specific changes in serum lipid levels (cholesterol, triglycerides, HDL, LDL) and lipoproteins (apoA-I) during periodontitis, which were exacerbated by exposure of the animals to a diet with elevated fat content . Thus, we have described systemic manifestations of periodontitis that include detection of bacterial products, inflammatory biomarkers, and dyslipoproteinemia consistent with an increased atherogenic risk.

J Periodontal Res, 1999 Oct, 34(7), 340 - 5
Association of periodontal infections with atherosclerotic and pulmonary diseases; Scannapieco FA et al.; Chronic infections may influence the severity and/or course of a number of systemic diseases . Periodontal diseases are localized chronic inflammatory conditions of the gingiva and underlying bone and connective tissues induced by bacteria and bacterial products of dental plaque . This paper will discuss the evidence for the role of periodontal disease in the pathogenesis of 2 important systemic diseases, atherosclerosis and pulmonary infections . Both epidemiological and laboratory studies are reviewed to assess the biological basis for the association of periodontal infections and these important diseases . Several potential mechanisms by which periodontal diseases may influence these conditions are also discussed.

J Exp Med, 2000 Feb 21, 191(4), 669 - 82
Fcgamma receptor-mediated phagocytosis in macrophages lacking the Src family tyrosine kinases Hck, Fgr, and Lyn; Fitzer-Attas CJ et al.; Macrophage Fcgamma receptors (FcgammaRs) mediate the uptake and destruction of antibody-coated viruses, bacteria, and parasites . We examined FcgammaR signaling and phagocytic function in bone marrow-derived macrophages from mutant mice lacking the major Src family kinases expressed in these cells, Hck, Fgr, and Lyn . Many FcgammaR-induced functional responses and signaling events were diminished or delayed in these macrophages, including immunoglobulin (Ig)G-coated erythrocyte phagocytosis, respiratory burst, actin cup formation, and activation of Syk, phosphatidylinositol 3-kinase, and extracellular signal-regulated kinases 1 and 2 . Significant reduction of IgG-dependent phagocytosis was not seen in hck(-)(/)-fgr(-)(/)- or lyn(-)(/)- cells, although the single mutant lyn(-)(/)- macrophages did manifest signaling defects . Thus, Src family kinases clearly have roles in two events leading to FcgammaR-mediated phagocytosis, one involving initiation of actin polymerization and the second involving activation of Syk and subsequent internalization . Since FcgammaR-mediated phagocytosis did occur at modest levels in a delayed fashion in triple mutant macrophages, these Src family kinases are not absolutely required for uptake of IgG-opsonized particles.

J Exp Med, 2000 Feb 21, 191(4), 593 - 602
Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells; Asahi M et al.; Attachment of Helicobacter pylori to gastric epithelial cells induces various cellular responses, including the tyrosine phosphorylation of an unknown 145-kD protein and interleukin 8 production . Here we show that this 145-kD protein is the cagA product of H . pylori, an immunodominant, cytotoxin-associated antigen . Epithelial cells infected with various H . pylori clinical isolates resulted in generation of tyrosine-phosphorylated proteins ranging from 130 to 145 kD in size that were also induced in vitro by mixing host cell lysate with bacterial lysate . When epithelial cells were infected with {(35)S}methionine-labeled H . pylori, a radioactive 145-kD protein was detected in the immunoprecipitates with antiphosphotyrosine antibody or anti-CagA (cytotoxin-associated gene A) antibody . Consistently, the 145-kD protein recognized by the anti-CagA and antiphosphotyrosine antibodies was induced in epithelial cells after infection of wild-type H . pylori but not the cagA::Km mutant . Furthermore, the amino acid sequence of the phosphorylated 145-kD protein induced by H . pylori infection was identical to the H . pylori CagA sequence . These results reveal that the tyrosine-phosphorylated 145-kD protein is H . pylori CagA protein, which may be delivered from attached bacteria into the host cytoplasm . The identification of the tyrosine-phosphorylated protein will thus provide further insights into understanding the precise roles of CagA protein in H . pylori pathogenesis.

J Comp Pathol, 2000 Feb-Apr, 122(2-3), 115 - 22
The efficacy of two vaccination schemes against experimental infection with a virulent amyxomatous or a virulent nodular myxoma virus strain; Marlier D et al.; Two types of myxomatosis vaccine are available commercially, namely, vaccine prepared from the Shope fibroma virus (SFV) and that prepared from an attenuated myxoma virus (MV) strain, e.gSG33 . An experiment was designed to compare two vaccination schemes for their ability to protect rabbits against challenge with either a virulent amyxomatous MV strain or a virulent nodular MV strain . Apart from a difference in the cutaneous expression of the disease, the two challenge strains resembled each other in respect of mortality rate, naso-conjunctival shedding of virus, and tissue infection . Vaccination with SFV alone failed to prevent clinical signs, naso-conjunctival shedding or tissue infection . Vaccination with SFV followed by a booster inoculation with SG33 protected rabbits against the development of clinical signs and significantly reduced both viral shedding in naso-conjunctival exudates and viral infection of eyelids, lungs and testes; virus was, however, isolated from testes of some surviving animals.

Pediatr Nephrol, 2000 Feb, 14(2), 152 - 7
Gene expression after intrarenal injection of plasmid DNA in the rat; Kuemmerle NB et al.; Effective gene therapy requires efficient delivery and expression of the necessary genetic information to the target tissue . We demonstrate here that plasmid DNA, injected as naked, uncomplexed DNA into the cortical region of rat kidney, or intravenously, is localized and expressed in the kidney . The plasmid pRSVZ contained the Rous sarcoma virus promoter and a reporter gene, the beta-galactosidase gene, derived from bacteria . The beta-galactosidase gene hydrolyzes the artificial substrate X-gal to produce an intense blue color in cells that have taken up and expressed the plasmid genes . We have used X-gal staining and Western blotting to study plasmid gene expression 1, 4, and 8 days and 6 months after intrarenal injection of 50 microg of plasmid DNA and at 1 and 4 days after intravenous injection . Expression was apparent in the kidneys and several other tissues 24 h after injection and persisted for at least 8 days; expressed proteins could still be detected in the injected kidney 6 months later . These observations were corroborated by use of a plasmid, pEGFP-Puro, harboring the cytomegalovirus promoter in conjunction with a different reporter gene, the green fluorescent protein (GFP) . Histological localization and Western blotting analysis of GFP expression after intrarenal injection of pEGFP-Puro paralleled results obtained with the plasmid pRSVZ . Our findings support the suggestion that intrarenal or intravenous injection of naked plasmid DNA may be an effective means of delivering therapeutic genes to the kidney and several other tissues.

Hua Xi Yi Ke Da Xue Xue Bao, 1997 Jun, 28(2), 117 - 21
{Characterication of reference strains of L . interrogans in China by ISSP-PCR}; Bao L et al.; The gene polymorphism of the DNAs extracted from reference strains of L . interrogans in China was characterized by LSSP-PCR primered by G1 or G2, which were a pair of specific primers for L . interrogans . The fingerprinting produced by LSSP-PCR showed that serovar lai, serovar canicola, serovar pyrogens, serovar autumnalis, serovar australis, serovar linhai, serovar wolffi and serovar haemolytic have similar patterns, but serovar hebdomadis, serovar javanica, serovar ballum, serovar pomona, serovar spaidjin, serovar tarassov and serovar manahao I have different profiles . This result agreed with the classification of genetic species by Yasuda . The utilization of LSSP-PCR banding patterns in the identification of leptospries in blood samples also gained encouraging results . Compared with the routine methods in genetic species classification, LSSP-PCR has the advantages of rapidity, simplicity and low-cost . It appears to be a promising tool in studying such slowly growing bacteria as leptospires . Further exploration of LSSP-PCR in classification and identification of Leptospires is worthwhile.

IUBMB Life, 1999 Dec, 48(6), 625 - 30
Effects of chlorophyll availability on phycobilisomes in Synechocystis sp . PCC 6803; Yu J et al.; Inactivation of the chlL gene in Synechocystis sp . PCC 6803 resulted in negligible chlorophyll content when the mutant was grown in darkness . Upon phycocyanin excitation at 580 nm, the 77K fluorescence spectrum of dark-grown cells showed three peaks at 648 nm, 665 nm, and 685 nm, this last being the largest . This reflects the functional presence of major components of phycobilisomes, including phycocyanin, allophycocyanin, and the terminal emitter, and efficient energy transfer between these components . As expected, no fluorescence emission peaks corresponding to chlorophyll in the photosystems were observed . Intact phycobilisomes could be isolated from the dark-grown chlL-deletion mutant . However, the phycobilisomes had a lower efficiency of energy transfer than did those isolated from the light-grown mutant, probably because of a decreased phycobilisome stability in the absence of chlorophyll . Exposing the dark-grown chlL-deletion mutant to light triggered the biosynthesis of chlorophyll . For the first 6 h in the light, upon phycocyanin excitation at 580 nm, the 77K fluorescence emission spectrum of greening cells was identical to that of dark-grown cells that lacked significant amounts of chlorophyll . With increased chlorophyll synthesis, gradual energy transfer from phycobilisomes to the two photosystems can be demonstrated.

FEBS Lett, 2000 Jan 28, 466(2-3), 359 - 62
Eukaryotic selenocysteine tRNA has the 9/4 secondary structure; Mizutani T et al.; There are two secondary structure models for the eukaryotic selenocysteine (Sec) tRNA(Sec) . One model, the 9/4 structure, was experimentally tested and possesses acceptor and T-stems with 9 and 4 bp, respectively {Sturchler et al., 1993; Hubert et al., 1998} . The other one, the 7/5 secondary structure with a bulge in the T-stem, was derived from theoretical calculation {Ioudovitch and Steinberg, 19991 . In this report, we show more experimental results supporting the 9/4 secondary structure . Several tRNA(Sec) mutants, whose secondary structure can adopt only the 9/4 structure, were active for serylation and selenylation . Some mutants that cannot base-pair between positions 26 and 44 to provide the 6 bp anticodon stem were still active, inconsistent with the model by Steinberg . We also show that the orientation of the V-arm directly or indirectly influences the selenylation activity, and that the rigid 6 bp D-stem is important . Finally, we conclude that all tRNA(Sec) possess the 13 bp domain II made by the stacking of the colinear AA and T-stems, whether they present the 9/4 structure in Eukarya and Archaea or the 8/5 structure in bacteria.

Br J Cancer, 2000 Feb, 82(3), 683 - 90
Interaction of the PA2G4 (EBP1) protein with ErbB-3 and regulation of this binding by heregulin; Yoo JY et al.; The processes by which ErbB-3, an inactive tyrosine kinase, exerts its biological effects are poorly understood . Using the yeast two-hybrid system, we have isolated an ErbB-3 binding protein (Ebp1) that interacts with the juxtamembrane domain of ErbB-3 . This protein is identical to that predicted to be encoded for by the human PA2G4 gene . Ebp1 is the human homologue of a previously identified cell cycle-regulated mouse protein p38-2G4 . Two transcripts of ebp1 mRNA (1.7 and 2.2 kb) were detected in several normal human organs . The interaction of Ebp1 with ErbB-3 was examined in vitro and in vivo . The first 15 amino acids of the juxtamembrane domain of ErbB-3 were essential for Ebp1 binding in vitro . Treatment of AU565 cells with the ErbB-3 ligand heregulin resulted in dissociation of Ebp1 from ErbB-3 . Ebp1 translocated from the cytoplasm into the nucleus following heregulin stimulation . These findings suggest that Ebp1 may be a downstream member of an ErbB-3-regulated signal transduction pathway.

J Mol Med, 1999 Dec, 77(12), 834 - 46
Analysis of the genetic diversity of Helicobacter pylori: the tale of two genomes; Alm RA et al.; Infection with Helicobacter pylori has been linked to numerous severe gastroduodenal diseases including peptic ulcer and gastric cancer . Several techniques have been used to measure the genetic heterogeneity of H . pylori at several different levels and to determine whether there is any correlation with severity of disease . The availability of two completed genome sequences from unrelated strains (J99 and 26,695) has allowed an analysis of the level of diversity from a large-scale yet detailed perspective . Although the two chromosomes are organized differently in a limited number of discrete regions, the genome size and gene order of these two "high-virulence" (cagA+ and vacA+) H . pylori isolates was found to be highly similar . The regions of organizational difference are associated with insertion sequences, DNA restriction/modification genes, repeat sequences, or a combination of the above . A significant level of variation at the nucleotide level is seen across the genome, providing an explanation for why the nucleotide-based typing techniques have such high discriminatory power among independent H . pylori isolates . This nucleotide variation together with the organizational rearrangements appears to have provided an over-estimation of the gene order diversity of H . pylori as assessed by pulse-field gel electrophoresis . Functional assignments are assigned to approximately only 60% of the gene products in each strain, with one-half of the remaining gene products of unknown function having homologues in other bacteria, while the remainder appear to be H . pylori-specific . Between 6% and 7% of the coding capacity of each strain are genes that are absent from the other strain, with almost one-half of these strain-specific genes located in a single hypervariable region called the plasticity zone . The majority of the strain-specific genes in each strain are also H . pylori-specific, with no homologues being identified in the public databases . Significantly, over one-half of the functionally assigned strain-specific genes in both H . pylori J99 and 26695 encode DNA restriction/modification enzymes . Analysis of the level of conservation between orthologues from the two strains indicates that the H . pylori specific genes have a lower level of conservation than those orthologues to which a putative function can be assigned . The plasticity zone represents one of several regions across each genome that is comprised of lower (G+C)% content DNA, some of which has been detected in self-replicating plasmids, suggesting that both horizontal transfer from other species and plasmid integration are responsible for the strain-specific diversity at this locus . These analyses have yielded results with important implications for understanding the genetic diversity of H . pylori and its associated diseases, and imply a need to reassess the respective roles of bacterial and host factors in H . pylori associated diseases.

Ann N Y Acad Sci, 1999, 894, 168 - 80
Agroterrorism . Agricultural infrastructure vulnerability; van Bredow J et al.; The intentional contamination of animal feed to reduce the availability of animal-derived human food or to infect human populations is seldom mentioned, but animal feed could be an easy target for bioterrorists . The period of delay between the contamination of the animal feed and adulteration of the human food product provides an additional degree of uncertainty about the source of the contamination and minimizes the possibility of apprehending the terrorist . The less obvious and more natural the source of biological contamination, the greater the likelihood that the animal feed contamination will be mistaken as a natural phenomenon . However, the problems related to managing natural food contamination and intentional food contamination remain the same . Rapid testing and separation of contaminated feed are important steps, followed by the more specific identification of the contaminant to determine the source of adulteration and/or the possibility of decontamination . At this time identification of the bioagents is dependent on the availability of antibody-specific test systems . The rapid development of specific antibodies for the development of sensitive and specific test kits is the key to identifying contamination and dealing effectively with the disposal or decontamination of the animal feed and, ultimately, preventing the contamination of animal-derived human food products.

Hunan Yi Ke Da Xue Xue Bao, 1998, 23(1), 76 - 8
{Preliminary study on Chlamydia pneumoniae pneumonia}; Su X et al.; In order to know the incidence of Chlamydia pneumoniae (strain TWAR) pneumonia and its clinical features, 93 patients with pneumonia and 93 matched patients with non-respiratory diseases were studied . TWAR antibodies (IgG and IgM) were detected by microimmunofluorescence (MIF) test . The results showed that 19.4% (18 cases) patients with pneumonia were TWAR pneumonia, in which 10 cases accompanied by bacteria infection and 7 cases being simple TWAR pneumonia . There were no significant differences in clinical features between TWAR pneumonia and non-TWAR pneumonia, except dry and moist rales . These data showed that the occurrence percentage of TWAR pneumonia in patients with lung cancer was higher than that in patients with the other respiratory diseases . This study suggests that there are TWAR pneumonia in China.

J Biol Chem, 2000 Feb 25, 275(8), 5687 - 93
Four conserved cytoplasmic sequence motifs are important for transport function of the Leishmania inositol/H(+) symporter; Seyfang A et al.; The protozoan Leishmania donovani has a myo-inositol/proton symporter (MIT) that is a member of a large sugar transporter superfamily . Active transport by MIT is driven by the proton electrochemical gradient across the parasite membrane, and MIT is a prototype for understanding the function of an active transporter in lower eukaryotes . MIT contains two duplicated 6- or 7-amino acid motifs within cytoplasmic loops, which are highly conserved among 50 members of the sugar transporter superfamily and are designated A(1), A(2) ((V)(D/E)(R/K)PhiGR(R/K)), and B(1) (PESPRPhiL), B(2) (VPETKG) . In particular, the three acidic residues within these motifs, Glu(187)(B(1)), Asp(300)(A(2)), and Glu(429)(B(2)) in MIT, are highly conserved with 96, 78, and 96% amino acid identity within the analyzed members of this transporter superfamily ranging from bacteria, archaea, and fungi to plants and the animal kingdom . We have used site-directed mutagenesis in combination with functional expression of transporter mutants in Xenopus oocytes and overexpression in Leishmania transfectants to investigate the significance of these three acidic residues in the B(1), A(2), and B(2) motifs . Alteration to the uncharged amides greatly reduced MIT transport function to 23% (E187Q), 1.4% (D300N), and 3% (E429Q) of wild-type activity, respectively, by affecting V(max) but not substrate affinity . Conservative mutations that retained the charge revealed a less pronounced effect on inositol transport with 39% (E187D), 16% (D300E) and 20% (E429D) remaining transport activity . Immunofluorescence microscopy of oocyte cryosections confirmed that MIT mutants were expressed on the oocyte surface in similar quantity to MIT wild type . The proton uncouplers carbonylcyanide-4-(trifluoromethoxy) phenylhydrazone and dinitrophenol inhibited inositol transport by 50-70% in the wild type as well as in E187Q, D300N, and E429Q, despite their reduced transport activities, suggesting that transport in these mutants is still proton-coupled . Furthermore, temperature-dependent uptake studies showed an increased Arrhenius activation energy for the B(1)-E187Q and the B(2)-E429Q mutants, which supports the idea of an impaired transporter cycle in these mutants . We conclude that the conserved acidic residues Glu(187), Asp(300), and Glu(429) are critical for transport function of MIT.

Proc Natl Acad Sci U S A, 2000 Feb 29, 97(5), 2391 - 6
The cellulose synthase gene of Dictyostelium; Blanton RL et al.; Cellulose is a major component of the extracellular matrices formed during development of the social amoeba, Dictyostelium discoideum . We isolated insertional mutants that failed to accumulate cellulose and had no cellulose synthase activity at any stage of development . Development proceeded normally in the null mutants up to the beginning of stalk formation, at which point the culminating structures collapsed onto themselves, then proceeded to attempt culmination again . No spores or stalk cells were ever made in the mutants, with all cells eventually lysing . The predicted product of the disrupted gene (dcsA) showed significant similarity to the catalytic subunit of cellulose synthases found in bacteria . Enzyme activity and normal development were recovered in strains transformed with a construct expressing the intact dcsA gene . Growing amoebae carrying the construct accumulated the protein product of dcsA, but did not make cellulose until they had developed for at least 10 hr . These studies show directly that the product of dcsA is necessary, but not sufficient, for synthesis of cellulose.

Vet Parasitol, 2000 Feb 29, 88(1-2), 107 - 16
WAAVP/Pfizer award for excellence in veterinary parasitology research . My involvement in, and some thoughts for livestock parasitological research in Australia; Hennessy DR; Being presented with the WAAVP Pfizer award for excellence in parasitological research is the pinnacle of my career . In accepting I acknowledge the support that I have received from workmates, colleagues, friends and family over the years that I have been involved in this field of endeavour . Parasitic disease is the most significant threat to the Australian sheep industry . A lack of understanding of drug action, the absence of epidemiologically-based treatment programs and incorrect or excessive chemical use has resulted in the development of worm, lice and blowfly parasites which are resistant to most existing chemotherapeutic compounds . During the past decade, difficulties in sustainable control of parasitic disease, the decline in demand for wool products and competition from less expensive synthetic fibre has halved the sheep population and profitability of the industry . Notwithstanding this, a 'right-sized', sustainable industry is emerging which will require effective chemotherapy to be the cornerstone of parasite control . Chemical intervention in parasitic disease is therefore here to stay but the paucity of new antiparasitic products in the short term dictates that present therapeutics are all that producers will have for the foreseeable future . This situation will necessitate innovative practices and formulations to provide more cost effective, efficient drug performance and to extend parasiticide life . However, the development of multiple drug resistance and reduction in funds for parasitological research seriously compromises our ability to confront these demands . With the patent life of all but the most recent macrocyclic lactone (ML) compounds lapsing, low cost development of bioequivalent generic formulations and options for innovative strategies to increase performance and market share are eagerly sought . The key to efficient drug use lies in a detailed understanding of the pharmacokinetic principles of drug action and the host animal's physiological responses to identify procedures which maximise drug availability--in essence giving the drug the best chance to work . It is therefore evident that the how, where and why of drug exchange between the bloodstream and the gastrointestinal tract are of such interest . Of particular importance is identification of the kinetic and dynamic behaviour of drug in the gastrointestinal tract (GIT) and the role of biliary secretion and metabolic fate of biliary (and non-biliary) compounds . The extent to which biliary secreted parent drug and/or metabolites are presented to the gut lumen, reabsorbed as free compound or after deconjugation by large bowel bacteria, and participate in the enterohepatic cycle is a major contributor to parasite exposure . Integrating parasiticide disposition with host physiology, particularly relating to such aspects as gastrointestinal function, feed intake and body condition has demonstrated the value of 'whole body' pharmacokinetic studies to identify processes to increase drug efficacy . Novel formulation modifications to provide 'targeted' drug delivery including carrier technology, sustained release devices and site-directed formulations can manipulate pharmacokinetic disposition to direct or extend drug availability to the parasite infection . These research directions should be undertaken as collaborative projects between research organisations and the veterinary pharmaceutical industry . With the identification of methods to improve drug action, emphasis must then be directed towards effective communication for their implementation . It will be vital that parasitologists move out of the laboratory to actively disseminate knowledge to the producer . We are ambassadors for our profession and if we fail to communicate well the perception, and indeed the value, of our work can be at risk.

J Neurosci Res, 2000 Feb 1, 59(3), 365 - 71
Differential effects of TrkC isoforms on sensory axon outgrowth; Ichinose T et al.; Neurotrophins are powerful regulators of neuronal morphology . Several lines of evidence are consistent with the idea that characteristic axonal and dendritic morphologies throughout the nervous system may be determined by local patterns of neurotrophin and neurotrophin receptor expression . Neurotrophin receptor tryosine kinases (Trks) exist in both tyrosine-containing (TK+) and tyrosine-lacking (TK-) isoforms, both of which are expressed in many neuronal populations . However, ratios of TK+ to TK- isoforms may vary at different stages of development and may be differentially distributed to cellular compartments . To test whether these isoforms have different functions related to axon outgrowth, full-length or tyrosine kinase-lacking TrkC receptors were overexpressed in embryonic dorsal root ganglion neurons maintained in explant cultures in neurotrophin-3 (NT-3)-containing media . Neurons were transfected with plasmid DNA encoding enhanced yellow fluorescent protein (EYFP) and TrkC receptor isoforms by particle-mediated gene transfer . Control neurons possessed 3.7 +/- 1.3 primary processes and 113.8 +/- 46 branch points . About 80% of the branches were located along the distal part of the axon . Transfection with the trkC TK+ increased the number of primary processes (6.5 +/- 2.8), whereas transfection with trkC TK- reduced the formation of primary processes (3.0 +/- 1.3) . Surprisingly, the distribution of branch points was shifted to the proximal region of axons in neurons transfected with trkC TK- . These observations are consistent with the idea that differential expression of Trk isoforms during development may sculpt axonal morphology .

Curr Opin Struct Biol, 2000 Feb, 10(1), 124 - 8
DNA helicases: 'inching forward'; Soultanas P et al.; Recently determined crystal structures of PcrA helicase complexed with a DNA substrate have revealed details of the helicase mechanism . PcrA and UvrD helicases have been shown to be functional as monomers, challenging previous suggestions that all helicases are required to be oligomeric . Crystal structures of the hexameric helicases RepA and T7 gene 4 explain the formation of hexameric assemblies from identical monomers with RecA-like folds, but their molecular mechanism remains elusive.

Biochem Biophys Res Commun, 2000 Feb 24, 268(3), 921 - 7
Mouse peroxiredoxin V is a thioredoxin peroxidase that inhibits p53-induced apoptosis; Zhou Y et al.; We have identified human and mouse peroxiredoxin V (Prx-V) by virtue of the sequence homologies to yeast peroxisomal antioxidant enzyme PMP20 . Prx-V represents the fifth of the six currently known subfamilies of mammalian peroxiredoxins . It is a novel organellar enzyme that has orthologs in bacteria . Biochemically, Prx-V is a thioredoxin peroxidase . One important aspect of p53 function in mammalian cells involves induction of apoptosis likely mediated by redox . We show that overexpression of Prx-V prevented the p53-dependent generation of reactive oxygen species . Likewise, Prx-V inhibited p53-induced apoptosis . Thus, Prx-V is critically involved in intracellular redox signaling .

J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 203 - 12
Purification of surface-associated urease from Helicobacter pylori; Rokita E et al.; Helicobacter pylori colonizes the human gastric mucosa and produces large amounts of urease . The enzyme was extracted from the bacteria by distilled water and purified by gel-permeation (Sephacryl S-300), anion-exchange chromatography (Mono Q) and a second gel-permeation (Superdex 200) . Urease enzyme activity was detected with a spectrophotometic assay based on phenol red . The optimal pH for anion-exchange was 6.9 . The recovery of urease was 55-75%, purity 93-98% and the overall protein recovery 0.8-1.4% . The urease in the final extract still had enzymatic activity and showed the typical subunits of Mr 66000 and Mr 30000 when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

J Chromatogr B Biomed Sci Appl, 2000 Jan 14, 737(1-2), 107 - 17
Three-step purification of bacterially expressed human single-chain Fv antibodies for clinical applications; Laroche-Traineau J et al.; We have obtained a cell line which secretes a human monoclonal IgM (B7) reacting with the myosin heavy chain of human heart . We have constructed single-chain fragments (scFv) of B7 . The scFv may be useful for the imaging of myocardial necrosis after myocarditis, cardiac drug toxicosis or graft rejection . The aim of our work was to purify the scFv for immunoscintigraphy . We describe several purification steps including immobilized metal affinity chromatography (IMAC), anti-c-myc monoclonal antibody affinity chromatography, size-exclusion chromatography with Superdex 75 HR 10/30 and ion-exchange chromatography (mini Q TM 30Q).

Int J Pharm, 2000 Feb 15, 195(1-2), 17 - 23
Microcalorimetry does not predict the cellular phagocytosis of latex microspheres; Jones BG et al.; Current literature highlights the potential suitability of microcalorimetry for the investigation of cell-drug interactions . Previous work using bacteria or antigens derived from infectious organisms yielded conclusions that heat production is a quantitative means of measuring phagocytosis . In this study we evaluated the potential of flow-through microcalorimetry as a method of quantifying the phagocytosis of microsphere particulates . The technique avoids the need to incorporate radioactive or fluorescent markers into the particulate formulation, and would be widely applicable in biopharmaceutical research . Using the monocyte cell line Mono Mac 6 a power output of 9.00 microW per million cells was increased significantly on addition of zymosan, lipopolysaccaride (LPS) and phorbol myristate acetate but not following exposure to FITC labelled latex microspheres (LM) . TNFalpha production increased on exposure to zymosan, LPS and LPS-phorbol myristate acetate, though not on exposure to LB . An assay was developed which allowed the quantification of internalised particulates in phagocytic cells using fluorescent activated cell sorting (FACS) . In contrast to the microcalorimetric and TNFalpha data FACS revealed that 20% of the MM6 population phagocytosed a mean of 1.35 LM . Microcalorimetry and measurements of TNFalpha production are assays of cellular activation a phenomenon not necessarily associated with phagocytosis . FACS, however, serves as a specific and quantitative measure of phagocytosis . Microcalorimetry may not be a suitable technique for the quantitative assessment of the phagocytosis of drug delivery particulates.

Gene, 2000 Feb 8, 243(1-2), 105 - 14
Alfalfa (Medicago sativa) carbamoylphosphate synthetase gene structure records the deep lineage of plants; Zhou Z et al.; Given the central role of carbamoylphosphate synthetases in pyrimidine and arginine metabolism in all living organisms, the absence of fundamental information regarding plant CPSase genes is a striking omission {Lawson et al., Mol . Biol . Evol . 13 (1996) 970-977; van den Hoff et al., J . Mol . Evol . 41 (1995) 813-832} . Whereas CPSase gene architecture and aa sequence have proven to be useful characters in establishing ancient and modern genetic affinities, phylogenetic analysis cannot be completed without the inclusion of plant CPSases . We describe the first isolation by molecular cloning of a plant CPSase gene (CPAII) derived from alfalfa (Medicago sativa) . DNA sequence analysis reveals a proteobacterial architecture, namely closely linked carA and carB coding domains separated by a short intergenic region, and transcribed as a polycistronic mRNA . CPAII encodes the amino acid residues that typify a CPSase type II enzyme . In addition, an ancient internal duplication has been retained in the plant carB sequence . Partial nucleotide sequencing of additional clones reveals that the alfalfa genome contains multiple CPSase II gene copies which may be tissue-specific in their expression . It appears that with respect to CPSase genes, CPAII resembles the carAB gene of bacteria, and may have preserved much of this ancient gene structure in the alfalfa genome.

FEBS Lett, 2000 Feb 11, 467(2-3), 245 - 8
Involvement of CDSP 32, a drought-induced thioredoxin, in the response to oxidative stress in potato plants; Broin M et al.; In animal cells, yeast and bacteria, thioredoxins are known to participate in the response to oxidative stress . We recently identified a novel type of plant thioredoxin named CDSP 32 for chloroplastic drought-induced stress protein of 32 kDa . In the present work, we measured comparable increases in the glutathione oxidation ratio and in the level of chlorophyll thermoluminescence, a specific marker for thylakoid lipid peroxidation in Solanum tuberosum plants subjected to drought or oxidative treatments (photooxidative stress, gamma irradiation and methyl viologen spraying) . Further, substantial accumulations of CDSP 32 mRNA and protein were revealed upon oxidative treatments . These data show for the first time in plants the induction of a thioredoxin by oxidative stress . We conclude that CDSP 32 may preserve chloroplastic structures against oxidative injury upon drought.

SAR QSAR Environ Res, 1999 Dec, 10(6), 545 - 56
Using molecular quantum similarity measures as descriptors in quantitative structure-toxicity relationships; Girones X et al.; In this paper molecular quantum similarity measures (MQSM) are used to describe molecular toxicity and to construct Quantitative Structure-Toxicity Relationships (QSTR) models . This study continues the recently described relationships between MQSM and log P values, which permits to use the theoretical MQSM as an alternative to the empirical hydrophobic parameter in QSPR studies . In addition a new type of MQSM is presented in this work: it is based on the expectation value of electron-electron repulsion energy . The molecular properties studied here, as application examples are aquatic toxicity, toxicology on Bacteria and inhibition of a macromolecule employing four different molecular sets.

J Biotechnol, 2000 Jan 28, 77(1), 49 - 64
Relevance and isotopic assessment of hexose-6-phosphate recycling in micro-organisms; Portais JC et al.; Some pathways of hexose-6-phosphate recycling--those involving a breakdown of the hexose skeleton--through carbohydrate metabolism of micro-organisms were analyzed for both metabolic and isotopic effects . Two modes of recycling were proposed based on the degree of alteration of the hexose molecule through the catabolic part of the cycle . Simulated operation of most of these pathways resulted in increased synthesis of hexose-6-phosphate and NADPH, and reduced the NADH and moreover the ATP synthesis within the carbohydrate metabolism . A basic model for the quantitative assessment by means of isotopic studies of the processes of hexose-6-phosphate recycling is presented . The model was initially designed for the study of micro-organisms producing polysaccharides, but it can be extended to other situations.

Crit Rev Food Sci Nutr, 2000 Jan, 40(1), 43 - 81
Fish protein hydrolysates: production, biochemical, and functional properties; Kristinsson HG et al.; Considerable amounts of fish processing byproducts are discarded each year . By developing enzyme technologies for protein recovery and modification, production of a broad spectrum of food ingredients and industrial products may be possible . Hydrolyzed vegetable and milk proteins are widely used food ingredients . There are few hydrolyzed fish protein foods with the exception of East Asian condiments and sauces . This review describes various manufacturing techniques for fish protein hydrolysates using acid, base, endogenous enzymes, and added bacterial or digestive proteases . The chemical and biochemical characteristics of hydrolyzed fish proteins are discussed . In addition, functional properties of fish protein hydrolysates are described, including solubility, water-holding capacity, emulsification, and foam-forming ability . Possible applications of fish protein hydrolysates in food systems are provided, and comparison with other food protein hydrolysates where pertinent.

Br J Plast Surg, 1999 Sep, 52(6), 445 - 7
Reduction of potential contamination of breast implants by the use of 'nipple shields'; Collis N et al.; Forty-three breast implant operations in 25 patients were studied prospectively to determine the effectiveness of covering the nipple-areolar complex with an adhesive film dressing in preventing perioperative expression of bacteria from nipple ducts contaminating the operative field . One swab from the nipple after skin preparation and none of the swabs taken from the outer surface of the film dressing postoperatively yielded any bacterial growth . Fourteen breasts (33%) in 11 patients (44%) yielded bacterial growth from swabs under the film postoperatively . Six of 9 breasts (67%) in 5 patients who had capsulectomies had bacteria isolated from under the film postoperatively . Ten of 14 (71%) control breasts (no shields) in 6 of 7 patients (86%) had positive postoperative swabs . This study confirms the potential risk of bacterial contamination arising from nipple duct flora during intra-operative breast manipulation, and the effectiveness of a perioperative adhesive film placed over the nipple-areolar complex in preventing subclinical bacterial contamination of implanted breast prostheses.

Ann Biol Clin (Paris), 2000 Jan-Feb, 58(1), 29 - 38
{DNA chips}; Delpech M; DNA chips represent a miniaturization of the classical system of reverse dot-blot . They consist of a small size support made of plastic or glass or silicium on which probes are synthesized or immobilized . It is thus possible to fix a few thousand or even a hundred of thousands of probes per cm2 . Practically the chips are hybridized with the nucleic acid to be studied that has been amplified beforehand by PCR and which is generally labelled by a fluorochrome, either during amplification or after hybridization . After washing the hybrids are detected by a system which most of the time consists of a laser and a confocal microscope interfaced with a computer, but many variations exist . It is thus possible to analyse at the same time a considerable number of sequences . The diagnostic applications are only at the prototype stage . Eventually the chips should allow the identification of any point mutation, or the search for bacteria, viruses or parasites in a very short period of time without preliminary cultures . They should also allow many sorts of typing ranging from infectious agents to HLA . They should become a particularly powerful tool in the search for new medicines and in the revealing of their toxicity . A considerable potential market is also the diagnosis of the predisposition to polygenic diseases or to a particular sensitivity to any chemical substance . In fact the chips are only just beginning to be used . One of the foreseeable developments should be the possibility to study nucleic acids without preliminary amplification and we can hope to eventually have at our disposal very cheap, autonomous integrated systems . The technology could eventually extend to fields other than molecular biology such as immunology or biochemistry.

Structure Fold Des, 2000 Jan 15, 8(1), 1 - 12
The high-resolution structure of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase from human heart mitochondria; White SA et al.; BACKGROUND: Transhydrogenase, located in the inner membranes of animal mitochondria and the cytoplasmic membranes of bacteria, couples the transfer of reducing equivalents between NAD(H) and NADP(H) to proton pumping . The protein comprises three subunits termed dI, dII and dIII . The dII component spans the membrane . The dI component, which contains the binding site for NAD(+)/NADH, and the dIII component, which has the binding site for NADP(+)/NADPH, protrude from the membrane . Proton pumping is probably coupled to changes in the binding affinities of dIII for NADP(+) and NADPH . RESULTS: The first X-ray structure of the NADP(H)-binding component, dIII, of human heart transhydrogenase is described here at 2.0 A resolution . It comprises a single domain resembling the classical Rossmann fold, but NADP(+) binds to dIII with a reversed orientation . The first betaalphabetaalphabeta motif of dIII contains a Gly-X-Gly-X-X-Ala/Val 'fingerprint', but it has a different function to that in the classical Rossmann structure . The nicotinamide ring of NADP(+) is located on a ridge where it is exposed to interaction with NADH on the dI subunit . Two distinctive features of the dIII structure are helix D/loop D, which projects from the beta sheet, and loop E, which forms a 'lid' over the bound NADP(+) . CONCLUSIONS: Helix D/loop D interacts with the bound nucleotide and loop E, and probably interacts with the membrane-spanning dII . Changes in ionisation and conformation in helix D/loop D, resulting from proton translocation through dII, are thought to be responsible for the changes in affinity of dIII for NADP(+) and NADPH that drive the reaction.

Biochem Biophys Res Commun, 2000 Jan 27, 267(3), 897 - 905
The lipid component of lipoproteins from Borrelia burgdorferi: structural analysis, antigenicity, and presentation via human dendritic cells; Beermann C et al.; The spirochaetal bacteria Borrelia burgdorferi (Bb) is the tick-borne causative agent of lyme disease . The major membrane immunogens of Bb are outer surface proteins . The lipid component of these lipoproteins is relevant for the immunogenicity of Bb-lipoproteins . To characterize the antigenic properties, the native lipid component of lipoproteins was isolated and the detailed molecular structure was analyzed . The molecular structure of the lipoprotein-lipid component turned out to be S(propane-2',-3'diol)-3-thio-2-aminopropanic acid (S-glyceryl-cysteine) with one ester-linked fatty acid, one acetyl group, and one N-terminal amide-bound fatty acid . Fatty acid analysis of the lipid component indicated a heterogeneous composition comprising C16:0, C18:0, C18:1, C18:2, and C 20:0 . The antigenicity was tested with in vitro bioassays using human blood-derived dendritic cells (DCs) as antigen-presenting cells and autologous Bb-specific T-cells . We found that human DCs present the lipid component of Bb-lipoproteins via MHC class II inducing an antigen-specific T-cell immune response in vitro .

Gut, 2000 Mar, 46(3), 395 - 400
A prospective randomised multicentre trial comparing 10 Fr Teflon Tannenbaum stents with 10 Fr polyethylene Cotton-Leung stents in patients with malignant common duct strictures; England RE et al.; BACKGROUND: Stent blockage is a multifactorial process in which stent design and materials, bacteria, proteins, and bile viscosity play a role . AIMS: To compare the patency of the 10 Fr Teflon Tannenbaum (TT) stent to that of the 10 Fr Cotton-Leung (CL) polyethylene stent with sideholes, in patients with malignant obstructive jaundice . METHODS: Patients were recruited to this prospective multicentre randomised study if they had a newly diagnosed malignant bile duct stricture below the hilum of the liver suitable for stenting with a 10 Fr stent . Data were collected and monitored by a professional monitoring company . Primary patency was the interval between stent placement and first exchange or death without recurrent jaundice . RESULTS: 134 consecutive patients were recruited between November 1994 and June 1997; 65 were randomised to the TT stent and 69 to the CL stent . Median patency and 95% confidence intervals were 181 (59, 303) days for the TT stent and 133 (92, 174) days for the CL stent, with no significant difference between the two stents (p=0.49) . Median survival and 95% confidence intervals were 115 (71, 159) days for the TT stent and 151 (112, 190) days for the CL stent, with no significant difference between the two stents (p=0.765) . CONCLUSION: Neither Teflon as a stent material nor the Tannenbaum design prolong the patency of plastic stents.

J Photochem Photobiol B, 1999 Nov-Dec, 53(1-3), 144 - 52
Analysis of UV-B-induced DNA damage and its repair in heat-shocked skin cells; Schmidt-Rose T et al.; The heat-shock response is a cellular defence mechanism against environmental stresses that is evolutionarily conserved from bacteria to man . Numerous reports demonstrate the beneficial effects of heat-shock protein induction on cell survival under toxic or oxidative stress, e.g., in cardiac and cerebral ischemia or prior to organ transplantation . However, there is little data on the effects of heat treatment on damage caused by UV irradiation . Applying three independent techniques, we have tested the influence of thermal pretreatment of skin cells (1 h, 43 degrees C) on the initial extent of UV-B-induced DNA damage and its subsequent repair . For cultured human epidermal keratinocytes and dermal fibroblasts we can show reduced levels of nucleotide-excision-repair-associated DNA strand incision in the comet assay . Moreover, immunostaining and flow cytometric quantitation of thymidine dimers immediately and one day after irradiation, respectively, reveal that the initial DNA damage is not (keratinocytes) or only moderately (fibroblasts) lower in heat-shocked cells as compared to untreated controls . However, excision repair of dimers is significantly attenuated during the first 24 h in both cell types . Furthermore, using a modified host-cell reactivation assay, we are able to demonstrate that repair of UV-B-damaged plasmid DNA is lower if the transfected cells are previously heat shocked . In summary, heat treatment (1 h, 43 degrees C) inducing heat-shock proteins reduces nucleotide excision repair of UV-B-mediated DNA lesions in fibroblasts and keratinocytes during the following 24 h . This is not necessarily caused by elevated heat-shock protein levels themselves . Possibly the direct thermal damage of repair enzymes is more severe than the potential protective effects of heat-shock proteins.

J Oral Rehabil, 2000 Feb, 27(2), 93 - 102
Pulp reactions to different preparation techniques on teeth exhibiting periodontal disease; Zollner A et al.; To evaluate the histopathological outcome of two preparation techniques (featheredge preparation/shoulder preparation) on teeth exhibiting pulp reactions due to age and periodontal disease, 11 teeth were prepared for full veneer crowns . Laboratory made resin crowns were fixed with a zinc phosphate cement for a period of 90 days . After extraction, adjacent pulpal areas were histopathologically rated according to the BRD criteria comprising the parameters (i) Bacterial invasion, (ii) Regenerative parameters, (iii) Degenerative parameters . Degenerative reactions were more correlated with tooth preparation than with advanced periodontal disease . The severity of endondontal reactions depends more on remaining dentin thickness than on the type of preparation.

Eur J Biochem, 2000 Feb, 267(4), 1050 - 8
Resonance Raman study of multihemic c-type cytochromes from Desulfuromonas acetoxidans; Chottard G et al.; Two multihemic cytochromes c from the sulfur reducing bacteria Desulfuromonas acetoxidans have been studied by optical and resonance Raman spectroscopy: cytochrome c551.5, a trihemic cytochrome and cytochrome c Mr 50 000, a recently isolated high molecular mass cytochrome . The redox and Raman characteristics of cytochrome c551.5 are compared to those of the tetrahemic cytochromes c3 from Desulfovibrio . While the redox behavior, followed by spectroelectrochemistry, is similar to that of cytochrome c3, showing the same conformational change after reduction of the highest potential heme, the Raman data show a contribution from a His- form of the axial ligands and lead to the assignment of a band at 218 cm-1 to the Fe(III)-(His)2 stretching vibration . The Raman data on cytochrome c Mr 50 000 are in favor of an entirely low spin species with two different sets of axial ligands . A partially reduced state is easily accessible by ascorbate addition.

J Biol Chem, 2000 Feb 18, 275(7), 4708 - 12
LexA repressor forms stable dimers in solution . The role of specific dna in tightening protein-protein interactions; Mohana-Borges R et al.; Cooperativity in the interactions among proteins subunits and DNA is crucial for DNA recognition . LexA repressor was originally thought to bind DNA as a monomer, with cooperativity leading to tighter binding of the second monomer . The main support for this model was a high value of the dissociation constant for the LexA dimer (micromolar range) . Here we show that the protein is a dimer at nanomolar concentrations under different conditions . The reversible dissociation of LexA dimer was investigated by the effects of hydrostatic pressure or urea, using fluorescence emission and polarization to monitor the dissociation process . The dissociation constant lies in the picomolar range (lower than 20 pM) . LexA monomers associate with an unusual large volume change (340 ml/mol), indicating the burial of a large surface area upon dimerization . Whereas nonspecific DNA has no stabilizing effect, specific DNA induces tightening of the dimer and a 750-fold decrease in the K(d) . In contrast to the previous model, a tight dimer rather than a monomer is the functional repressor . Accordingly, the LexA dimer only loses its ability to recognize a specific DNA sequence by RecA-induced autoproteolysis . Our work provides insights into the linkage between protein-protein interactions, DNA recognition, and DNA repair.

Am J Respir Crit Care Med, 2000 Feb, 161(2 Pt 2), S20 - 4
Membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) . A widespread protein superfamily; Jakobsson PJ et al.; The members of the MAPEG superfamily have been aligned and found to be distantly related, with a common pattern of hydropathy . Figure 2A shows the multiple sequence alignments of the human members and Figure 2B the corresponding superimposed hydropathy profiles . The alignment in Figure 2A demonstrates a total of six strictly conserved residues . The Arg-51 in LTC4 synthase has been suggested to function as proton donor for the opening of the LTA4 epoxide . This arginine is found in all but the FLAP sequences in accordance with the observation that FLAP has no known enzyme activity . Also the Tyr-93 in LTC4 synthase has been suggested to function as a base for the formation of the thiolate anion of glutathione . This tyrosine is not conserved in MGST1 or MGST1-L1 . Table 1 summarizes some other properties of the individual human proteins . They are all of the same size, ranging from 147 to 161 amino acids . Only FLAP differs in that its isoelectric point is more neutral than that of the other, more basic proteins . The genes encoding these proteins all reside on different chromosomes (when known) (Table 1) . In addition to the human proteins, MAPEG members have been identified in plants, fungi, and bacteria . It is clearly a challenge to elucidate their role in these different phyla in relation to their defined physiological functions in humans.

Eur J Immunol, 2000 Feb, 30(2), 705 - 8
Dendritic cell maturation is induced by mycoplasma infection but not by necrotic cells; Salio M et al.; To identify environmental stimuli that induce dendritic cell (DC) maturation, we exposed human monocyte-derived immature DC to apoptotic or necrotic cells and measured the levels of expression of costimulatory molecules and cytokine production . While most necrotic or apoptotic cells did not have any effect, some induced DC maturation as detected by up-regulation of CD83 and B7.2 and production of IL-12 and IL-6 . The capacity of these cell lines to induce DC maturation was due to their contamination by mycoplasma, since the maturation-inducing effect disappeared when the cells were treated with cyproxin . Furthermore, cell lines deliberately infected with mycoplasma containing supernatant acquired the capacity to induce DC maturation . Our results reveal that DC are able to sense mycoplasma infection and mature as they do in response to most viruses and bacteria . In contrast, apoptotic or necrotic cells fail to induce DC maturation.

J Microbiol Methods, 2000 Feb, 39(3), 179 - 87
Image analysis method to assess adhesion of Helicobacter pylori to gastric epithelium using confocal laser scanning microscopy; Reinhard J et al.; We have used confocal scanning microscopy of FITC-labelled bacteria to assess binding of Helicobacter pylori to stomach sections and to assess the effect of inhibitors on binding to the Lewis antigens . We have quantified the binding using an image manipulation package that is readily available on the web . Our results demonstrate heterogeneity of binding of Helicobacter pylori to tissue sections and that binding can be inhibited using synthetic Lewis B oligosaccharide.

J Am Diet Assoc, 2000 Feb, 100(2), 246 - 53
Position of the American Dietetic Association: food irradiation; Wood OB et al.; Food irradiation has been identified a sa safe technology to reduce the risk of foodborne illness as part of high-quality food production, processing, handling, and preparation . Food irradiation's history of scientific research , evaluation, and testing spans more than 40 countries around the world and it has been endorsed or support by numerous national and international food and organizations and professional groups . Food irradiation utilizes a source of ionizing energy that passes through food to destroy harmful bacteria and other organism . Often referred to as "cold pasteurization," food irradiation offers negligible loss of nutrients or sensory qualities in food as it does not substantially raise the temperature of the food during processing . Food irradiation does not replace proper food production, processing, handling, or preparation, nor can it enhance the quality of or prevent contact with foodborne bacteria after irradiation . In the United States, manufacturers are required to identify irradiated food sold to consumers with an international symbol (Radura) and and terminology describing the process on product labels . In addiction, food irradiation facilities are thoroughly regulated and monitored for worker and environmental safety . Members of The American Dietetic Association (ADA) and other food, nutrition, and health professionals have a responsibility to educate consumers, food processors, manufacturers and retailers about the safety and application of the technology . When consumers are educated about food irradiation, many prefer irradiated products because of their increased safety . It is the position of ADA that food irradiation enhances the safety and quality of the food supply and helps protect consumers from foodborne illness . The ADA encourages the government, food manufactures, food commodity groups, and qualified food and nutrition professionals to work together to educate consumers about this additional food safety tool and make this choice available in the marketplace.

Vet Hum Toxicol, 2000 Feb, 42(1), 1 - 4
Adaptation of pregnant ewes to an exclusive onion diet; Knight AP et al.; A diet consisting entirely of cull onions fed to pregnant ewes produced Heinz body hemolytic anemia in all sheep after 21 d . After 28 d of daily consumption of 20 kg of onions/ewe, the anemia stabilized, and for the remaining 74 d the packed cell volume increased in the majority of sheep, although it did not return to normal . Compared to control ewes fed an alfalfa and grain diet, the onion-fed ewes had comparable body condition scores and fleece weights . There was no significant difference (alpha = 0.05) in pregnancy or lambing rate, number of lambs born/ewe exposed, or number of lambs born/ewe lambing . Greater numbers of sulfate-reducing bacteria (Desulfovibrio spp) and more ruminal hydrogen sulfide were present in onion-fed sheep compared to controls . Although an average 27% reduction in packed cell volume and Heinz body anemia developed in the onion-fed ewes, on the basis of this study it appears that pregnant ewes may be fed a pure onion diet with minimal detrimental effects . This adaptation to a pure onion diet is in part likely due to the apparent ability of the sheep's rumen to quickly develop a population of sulfate-reducing bacteria that decrease the toxicity of onion disulfides.

Eur J Epidemiol, 1999 Nov, 15(10), 907 - 12
Maternal knowledge and environmental factors associated with risk of diarrhea in Israeli Bedouin children; Bilenko N et al.; Diarrhea is still a major cause of morbidity and mortality among children in developing countries . As it is due to multiple causative agents including viruses, bacteria and parasites, biological interventions are not currently available to markedly reduce incidence and severity . We examined maternal knowledge and reported behavior during diarrheal episodes, as well as environmental factors to determine their association with diarrhea . The children and mothers were from a Bedouin township in southern Israel, which has developed preventive and curative health care facilities . The Bedouin population in Israel is in transition from a nomadic to a settled life style . While almost all mothers exhibited good knowledge regarding food storage and prevention of diarrhea episodes in the children, the rate of illness in the children remained relatively high (two episodes per child year of observation) . In a multivariate analysis, cessation of breastfeeding during diarrhea, child sleeping with siblings and lack knowledge about risk factors, were the major risk factors for illness with odds ratios (OR): 4.6, p = 0.02, 5.6, p = 0.03 and 1.7, p = 0.06, respectively . These data indicate that even in this population with free access to preventive medical care, greater efforts should be made to educate mothers regarding risk factor for diarrheal disease identification and the benefits of maintaining breastfeeding during diarrhea episodes.

Arch Surg, 2000 Feb, 135(2), 136 - 40; discussion 141
Internal drainage of giant acute pseudocysts: the role of video-assisted pancreatic necrosectomy; Oria A et al.; BACKGROUND: Internal drainage of giant pancreatic pseudocysts secondary to acute pancreatitis is frequently complicated with postoperative retroperitoneal infection and hemorrhage . Recent data suggest that the risk factor is unrecognized pancreatic necrosis; presumably, pancreatic necrosis becomes infected with bacteria introduced by the cystoenteric anastomosis . HYPOTHESIS: Video-assisted pancreatic necrosectomy, performed at the time of internal drainage, may prevent postoperative retroperitoneal complications in patients with giant acute pseudocysts . DESIGN: A consecutive case-series . SETTING: An urban, university-affiliated, tertiary referral center . PATIENTS: Ten consecutive patients with acute pseudocysts measuring 10 cm or more in major diameter . The mean extent of pancreatic necrosis, as shown by contrast-enhanced computed tomography, was 50% . All patients were operated on electively, at an average time of 7.7 weeks from onset of the attack to surgical treatment . INTERVENTION: Through a midline incision, a 4-cm opening is made at the base of the pseudocyst . Standard laparoscopic instruments are introduced into the pseudocyst and video-assisted pancreatic necrosectomy is performed . The opening is then anastomosed to a Roux-en-Y limb of the jejunum . MAIN OUTCOME MEASURES: Feasibility and safety of video-assisted pancreatic necrosectomy, postoperative morbidity and mortality, hospital stay, and resolution of pseudocysts . RESULTS: Complete necrosectomy was safely performed throughout . There were neither postoperative retroperitoneal complications nor mortality . Mean hospital stay was 8.2 days and all pseudocysts resolved at a mean follow-up of 6.9 months . CONCLUSIONS: Video-assisted pancreatic necrosectomy at the time of internal drainage seems to prevent postoperative retroperitoneal complications in patients with giant acute pseudocysts . Depending on appropriate surgical timing, video-assisted necrosectomy is a feasible and safe procedure.

Histol Histopathol, 2000 Jan, 15(1), 101 - 7
The molecular determinants of the efficiency of green fluorescent protein mutants; Sacchetti A et al.; The Green Fluorescent Protein (GFP) is a spontaneously fluorescent polypeptide of 27 kD from the jellyfish Aequorea victoria that absorbs UV-blue light and emits in the green region of the spectrum . GFP has been successfully expressed both in bacteria and in eukaryotic cells and is widely used to monitor the localization of tagged proteins in living cells . Since wtGFP performs inefficiently in different cellular contexts, efforts have been devoted to the improvement of GFP expression levels and/or fluorescence . We will here review the basic characteristics of wt and mutated GFP, in particular their protein expression vs fluorescent properties . Emphasis will be given to unexpected consequences of mutations of the GFP gene, i.e . on transcription and translation rates and on protein folding in different cell types, and to how these critically reflect on the use of GFP in different cellular environments.

Nature, 2000 Jan 27, 403(6768), 421 - 4
Stimulation by ammonium-based fertilizers of methane oxidation in soil around rice roots; Bodelier PL et al.; Methane is involved in a number of chemical and physical processes in the Earth's atmosphere, including global warming . Atmospheric methane originates mainly from biogenic sources, such as rice paddies and natural wetlands; the former account for at least 30% of the global annual emission of methane to the atmosphere . As an increase of rice production by 60% is the most appropriate way to sustain the estimated increase of the human population during the next three decades, intensified global fertilizer application will be necessary: but it is known that an increase of the commonly used ammonium-based fertilizers can enhance methane emission from rice agriculture . Approximately 10-30% of the methane produced by methanogens in rice paddies is consumed by methane-oxidizing bacteria associated with the roots of rice; these bacteria are generally thought to be inhibited by ammonium-based fertilizers, as was demonstrated for soils and sediments . In contrast, we show here that the activity and growth of such bacteria in the root zone of rice plants are stimulated after fertilization . Using a combination of radioactive fingerprinting and molecular biology techniques, we identify the bacteria responsible for this effect . We expect that our results will make necessary a re-evaluation of the link between fertilizer use and methane emissions, with effects on global warming studies.

Acta Crystallogr D Biol Crystallogr, 2000 Feb, 56 ( Pt 2), 192 - 4
Crystallization and preliminary X-ray diffraction analysis of the catalytic subunit of ADP-glucose pyrophosphorylase from potato tuber; Binderup K et al.; ADP-glucose pyrophosphorylase is the key regulatory enzyme in the biosynthesis of starch in plants and glycogen in bacteria . The enzyme from potato tuber is comprised of a regulatory subunit and a catalytic subunit and is present as a heterotetramer (alpha(2)beta(2)) . The catalytic subunit from potato tuber (50 kDa) was crystallized in four different forms, two of which are suitable for structural studies . A tetragonal crystal form obtained in the presence of the substrate analog Cr-ATP diffracted to 2.2 A and belongs to space group P4(1) (or its enantiomorph), with unit-cell parameters a = b = 110.57, c = 190.14 A . A second crystal form obtained diffracted to 2.8 A and belongs to space group P2, with unit-cell parameters a = 80.06, b = 138.84, c = 92.20 A, beta = 112 . 40 degrees . As this protein displays no significant homology to any currently known protein structure, a search for heavy-atom derivatives has been initiated.

Vet Microbiol, 2000 Jan, 71(1-2), 81 - 7
Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the Apx toxins of Actinobacillus pleuropneumoniae; Nielsen R et al.; The reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae express one or two of three different RTX exotoxins designated Apx I, Apx II and Apx III . The toxins are important virulence factors . In the present study, ELISAs with purified Apx I, Apx II and Apx III, respectively, as antigen were evaluated as candidates for serological diagnosis of Actinobacillus pleuropneumoniae infection in pigs . The pigs were inoculated with biotype 1, serotypes 1-12, and biotype 2, serotype 14, respectively . A strong humoral antibody response was seen to all the three antigens in most pigs irrespective of the serotype used for inoculation . However, titers to the exotoxins secreted by the serotype used for inoculation were generally highest . The results show that toxin proteins of Actinobacillus pleuropneumoniae are antigenically related and that a correlation between serotype and secretion of exotoxin is not revealed serologically in the ELISA test.

Lett Appl Microbiol, 1999 Dec, 29(6), 385 - 8
Change in riverine epilithic extracellular enzyme activity in response to fish farm effluent; Brown SE et al.; Extracellular aminopeptidase and phosphatase activity was measured at the surfaces of submerged stones collected from a river upstream and downstream of a fish farm discharge . Aminopeptidase activity was largely indifferent to the discharge . Phosphatase activity, in contrast, was depressed downstream of the outfall . Thus, there was no evidence of enhanced epilithic extracellular enzyme activity to facilitate biopurification downstream of the fish farm discharge.

Folia Microbiol (Praha), 1999, 44(3), 306 - 10
Production of stress-inducible form of heat-shock protein 70 in mouse peritoneal adherent cells after in vivo infection by Francisella tularensis; Stulik J et al.; Heat-shock proteins (hsp) are ubiquitously produced molecules which participate in the protection of cells from environmental perturbation . Moreover, the members of the heat-shock protein 60 (hsp60) and 70 (hsp70) families play an important role in pathogen-host interactions . We studied in vivo production of the 70-kDa heat-shock proteins in the extract of peritoneal exudate cells (PEC) from mice injected intraperitoneally with an attenuated vaccine strain (LVS) of Francisella tularensis . We found a differential production of a highly stress-inducible member of the hsp70 family, designated hsp72, in three inbred strains of mice exhibiting either resistance or susceptibility to F . tularensis LVS infection . Whereas in tularemia-resistant mice hsp72 was even expressed in PEC without injection of bacteria and its production further increased on day 3 and slowly declined on days 5 and 7 after injection, in susceptible mice hsp72 production was highly inducible and restricted only to day 3 after in vivo infection . Further analysis of hsp72 expression revealed intracellular hsp72 accumulation and its preferential production by peritoneal adherent cells.

Acta Neurochir (Wien), 2000, 142(1), 67 - 74
Candida infection of cerebrospinal fluid shunt devices: report of two cases and review of the literature; Montero A et al.; Use of CSF shunt devices is a common practice in neurosurgery, and infection of the shunt is the most frequent complication . In spite of the fact that bacteria are the most widely implicated pathogens, reports of fungal infections, especially due to Candida sp., have increased in recent years . Their reported frequency ranges between 6% and 17% . Many factors have been implicated in the pathogenesis of Candida meningitis, such as broad spectrum antibiotics used in the treatment of a bacterial meningitis, steroids and indwelling bladder and intravenous catheters . The treatment of Candida meningitis still consists of systemic antifungal agents and removal of the shunt.

Langenbecks Arch Surg, 2000 Jan, 385(1), 39 - 41
Postoperative necrotizing soft-tissue infections of the abdominal wall; Bertram P et al.; OBJECTIVE: The aim of the underlying study was the evaluation of an aggressive surgical regimen for treatment of postoperative necrotizing soft-tissue infection (NSTI) . METHODS: Eight patients with postoperative NSTI of the abdominal wall after emergency (n=6) and elective (n=2) surgery were reviewed over a 9-year period . RESULTS: Initially, three patients presented with general peritonitis . Cultured swabs from the necrotic tissue revealed three to four different types of bacteria in each patient . The mean interval between the primary operation and clinical symptoms of NSTI was 63.0 h . Control of NSTI and intra-abdominal infection was attained by scheduled re-operations on a daily basis accounting for three to six re-interventions in each patient . Temporary closure of the abdominal wall by absorbable polyglactid-acid mesh was used in six cases . Mean hospital stay was 65.3 days (18-110 days) . Two of the eight patients died from cardiac arrest and multiple organ failure, respectively . CONCLUSIONS: Rapid diagnosis and onset of treatment is accomplished by the surgeons' knowledge of this disease and a close follow-up of the patients in an intensive care unit.

Pediatr Surg Int, 2000, 16(1-2), 29 - 34
Reappraisal of the role of the bilioenteric conduit in the pathogenesis of postoperative cholangitis; Chuang JH et al.; The incidence of postoperative cholangitis has changed very little despite progressive improvement in the treatment of biliary atresia . The role of the bilioenteric conduit in its pathogenesis is still uncertain . A retrospective study of 39 patients undergoing either a conventional Kasai operation (group 1, n = 20) or with placement of an antireflux valve (group 2, n = 10) or lengthening (group 3, n = 9) of the jejunal conduit from 40 to 60 cm was performed to compare the incidence of cholangitis . Postoperative cholangitis developed in 18 of the 39 patients (46%) . The incidence was 10/20 (50%) in group 1, 5/10 (50%) in group 2, and 3/9 (33%) in group 3 (P = 0.679) . An animal experiment was conducted concomitantly to compare quantitative bacterial cultures of the bilioenteric anastomosis and the liver before and 1 week after Roux-en-Y hepaticojejunostomy (HPJ) in piglets without (group A, 25 cm) and with (group B, 50 cm) lengthening of the jejunal conduit in a porcine model of extrahepatic biliary obstruction . The growth of bacteria in both the bilioenteric anastomosis and the liver was not affected by lengthening the jejunal conduit from 25 to 50 cm (P = 0.612 and 0 . 057, respectively), despite a geometric increase in bacterial concentrations in both groups after HPJ . It is concluded that neither bacterial growth in the liver nor cholangitis following bile-duct reconstruction was affected by valving or lengthening the bilioenteric conduit.

J Cell Biol, 2000 Feb 7, 148(3), 417 - 26
Organization of the yeast Zip1 protein within the central region of the synaptonemal complex; Dong H et al.; The yeast Zip1 protein is a component of the central region of the synaptonemal complex (SC) . Zip1 is predicted to form an alpha-helical coiled coil, flanked by globular domains at the NH(2) and COOH termini . Immunogold labeling with domain-specific anti-Zip1 antibodies demonstrates that the NH(2)-terminal domain of Zip1 is located in the middle of the central region of the SC, whereas the COOH-terminal domain is embedded in the lateral elements of the complex . Previous studies have shown that overproduction of Zip1 results in the assembly of two types of aggregates, polycomplexes and networks, that are unassociated with chromatin . Our epitope mapping data indicate that the organization of Zip1 within polycomplexes is similar to that of the SC, whereas the organization of Zip1 within networks is fundamentally different . Zip1 protein purified from bacteria assembles into dimers in vitro, and electron microscopic analysis demonstrates that the two monomers within a dimer are arranged in parallel and in register . Together, these results suggest that two Zip1 dimers, lying head-to-head, span the width of the SC.

Biol Chem, 1999 Dec, 380(12), 1413 - 20
Thermodynamic properties and DNA binding of the ParD protein from the broad host-range plasmid RK2/RP4 killing system; Oberer M et al.; ParD is a small, acidic protein from the partitioning system of the plasmid RK2/RP4 . The ParD protein exhibits specific DNA binding activity and, as the antidote component of a toxin-antidote plasmid addiction system, ParD forms a tight complex in solution with its toxin antagonist, the ParE protein . Unopposed ParE acts as a toxin that causes growth retardation and killing of plasmid cured cells . ParD negatively autoregulates its expression by binding to an operator sequence in the parDE promoter region . This DNA binding activity is crucial for the regulation of the relative abundance of toxin and antidote which ultimately determines life or death for the bacterial host and its daughter cells . In light scattering studies and gel filtration chromatography we observed the existence of a stable dimer of ParD in solution . The stoichiometry of ParD-DNA complex formation appeared to be 4:1, the molecular mass of the complex was 72.1 kDa . The alpha-helical content of ParD as determined by CD-spectrometry was 35% . The protein exhibited high thermostability with a T(M) of 64 degrees C and deltaH of 25 kcal/mol as shown by differential scanning calorimetry . Upon complex formation the T(M) increased by 10 degrees C . The thermal unfolding of the ParD protein was highly reversible as observed in repeated DSC scans of the same sample . The recovery of the native fold was proven by CD-spectroscopy.

J Biol Chem, 2000 Feb 11, 275(6), 3879 - 86
The contribution of a conformationally mobile, active site loop to the reaction catalyzed by glutamate semialdehyde aminomutase; Contestabile R et al.; The behavior of glutamate semialdehyde aminomutase, the enzyme that produces 4-aminolevulinate for tetrapyrrole synthesis in plants and bacteria, is markedly affected by the extent to which the central intermediate in the reaction, 4,5-diaminovalerate, is allowed to dissociate . The kinetic properties of the wild-type enzyme are compared with those of a mutant form in which a flexible loop, that reversibly plugs the entrance to the active site, has been deleted by site-directed mutagenesis . The deletion has three effects . The dissociation constant for diaminovalerate is increased approximately 100-fold . The catalytic efficiency of the enzyme, measured as k(cat)/K(m) in the presence of saturating concentrations of diaminovalerate, is lowered 30-fold to 2.1 mM(-1) s(-1) . During the course of the reaction, which begins with the enzyme in its pyridoxamine form, the mutant enzyme undergoes absorbance changes not seen with the wild-type enzyme under the same conditions . These are proposed to be due to abortive complex formation between the pyridoxal form of the enzyme (formed by dissociation of diaminovalerate) and glutamate semialdehyde itself.

Mol Gen Genet, 2000 Jan, 262(6), 1093 - 102
Structure and regulation of the dnaA promoter region in three Streptomyces species; Jakimowicz D et al.; The regulatory region of the Streptomyces dnaA gene comprises a single promoter and two DnaA boxes that are located upstream of the promoter . Comparative analysis of the dnaA promoter region from S . chrysomallus, S . lividans and S . reticuli revealed that the location, spacing and orientation of the DnaA boxes are conserved . In vitro studies demonstrated that efficient binding of the Streptomyces DnaA protein to DNA requires the presence of two DnaA boxes . In vivo analysis of dnaA promoter mutants deleted for one or both DnaA boxes indicated that the dnaA gene is autoregulated . However, the degree of derepression observed is relatively modest.

Xenobiotica, 2000 Jan, 30(1), 103 - 9
Biotransformation of the xanthine oxidase inhibitor BOF-4272 and its metabolites in the liver and by the intestinal flora in rat; Naito S et al.; 1 . BOF-4272, (+/-)-8-(3-methoxy-4-phenylsulfinylphenyl) pyrazolo{1,5-a}-1,3,5-triazine-4(1H)-one), is a new drug intended for the treatment of hyperuricaemia . 2 . This paper describes the detailed biotransformation of BOF-4272 and its metabolites in the liver and by the intestinal flora in rat . 3 . Only the sulphoxide metabolite (M6) was detected in plasma in small amounts after the intravenous administration of M4 (hydroxy-BOF-4272) and in culture medium after the addition of M4 . 4 . Only M3 (the sulphide metabolite of M4) was detected in faeces, and its amount was approximately 50% of the administered dose within 1 day after the intravenous administration of M4 . 5 . These findings suggest that M4, which is excreted in the bile, is metabolized mainly to M3 (the corresponding sulphide of M4) by the intestinal flora in rat, whereas little M4 is metabolized to the sulphoxide (M6) in the rat liver . 6 . M2, which is the demethylated form of BOF-4269, was detected in faeces after the oral administration of BOF-4272 to rat in which the common bile duct was cannulated, suggesting that BOF-4272 is metabolized to BOF-4269 and then to M2 by the intestinal flora . 7 . These findings suggest that in rat the sulphoxide of BOF-4272 and its metabolites are demethylated and reduced by the intestinal flora, with other types of biotransformation occurring in the liver.

J Hosp Infect, 1999 Dec, 43 Suppl, S89 - 92
Where is typing going?
Gemmell CG.
The basic premise in any bacterial typing scheme is that epidemiologically related isolates are derived from the clonal expansion of a single precursor . In simple terms this means that a certain characteristic is more useful than others on the basis that it is conserved within a strain but diverse with a species . Such a definition is appropriate when considering the spread of a species within a ward hospital or even a community . However, as we have developed more and more refined molecular techniques for analysis of the bacterial genome, such a definition has become stressed when considering the evolution that is occurring especially amongst drug-resistant bacteria whose genetic make-up is changing due to the selective pressure induced by drug usage in the hospital or community . Increased sophistication does not necessarily bring improved typing of bacterial strains which might only be variants of the same clonal type . Molecular epidemiology is perhaps too exacting to be practical in certain circumstances and one should be cautious in its interpretation and extrapolation . It is open to debate whether we shall see in the next century unanimity in the choice of pheno- or genotypic tests to be used for typing of bacterial strains.

Microbiology, 2000 Jan, 146 ( Pt 1), 199 - 208
Three pathways for trehalose biosynthesis in mycobacteria; De Smet KA et al.; Trehalose is present as a free disaccharide in the cytoplasm of mycobacteria and as a component of cell-wall glycolipids implicated in tissue damage associated with mycobacterial infection . To obtain an overview of trehalose metabolism, we analysed data from the Mycobacterium tuberculosis genome project and identified ORFs with homology to genes encoding enzymes from three trehalose biosynthesis pathways previously characterized in other bacteria . Functional assays using mycobacterial extracts and recombinant enzymes derived from these ORFs demonstrated that mycobacteria can produce trehalose from glucose 6-phosphate and UDP-glucose (the OtsA-OtsB pathway) from glycogen-like alpha(1-->4)-linked glucose polymers (the TreY-TreZ pathway) and from maltose (the TreS pathway) . Each of the pathways was found to be active in both rapid-growing Mycobacterium smegmatis and slow-growing Mycobacterium bovis BCG . The presence of a disrupted treZ gene in Mycobacterium leprae suggests that this pathway is not functional in this organism . The presence of multiple biosynthetic pathways indicates that trehalose plays an important role in mycobacterial physiology.

J Mol Biol, 2000 Feb 11, 296(1), 269 - 79
Specificity and interactions of the protein OppA: partitioning solvent binding effects using mass spectrometry; Rostom AA et al.; Mass spectrometry (MS) was used to characterise the binding of the 58 kDa protein OppA to 11 peptides with diverse properties . Peptides with two, three and five amino acid residues were added to OppA, and the mass spectra showed that the highest-affinity complexes are formed between OppA and tripeptide ligands . Lower-affinity complexes were observed for OppA and dipeptide ligands, and no complex formation was detected with pentapeptides or a tripeptide in which the N-terminal amino group was acetylated . Tripeptides containing a single d amino acid residue were found not to bind to native OppA . Evidence from the peak width and the, charge in the spectra of the complexes suggests that the bound peptides are encapsulated by the protein in a solvent-filled cavity in the gas phase of the mass spectrometer . Analysis of the proportions of peptide-bound and free proteins under low-energy MS conditions shows a good correlation with solution-phase K(d) measurements where available . Increasing the internal energy of the gas-phase complex led to dissociation of the complex . The ease of dissociation is interpreted in terms of the intrinsic stability of the complex in the absence of the stabilising effects of bulk solvent . The results from this study demonstrate insensitivity to the hydrophobic and ionic properties, of the side-chains of the peptides, in contrast to the investigation of other protein ligand systems by MS . Moreover, these findings are in accord with the physiological role of this protein in allowing into the cell di- and tripeptides containing naturally occurring amino acids, regardless of their sequence, while barring access to potentially harmful peptide mimics .

Genome, 1999 Dec, 42(6), 1255 - 7
Chromosomal location and genetic mapping of the mismatch repair gene homologs MSH2, MSH3, and MSH6 in rye and wheat
Korzun V V, Borner A, Siebert R, Malyshev S, Hilpert M, Kunze R, Puchta H.
The efficiency of homeologous recombination is influenced by mismatch repair genes in bacteria, yeast, and mammals . To elucidate a possible role of these genes in homeologous pairing and cross-compatibility in plants, gene probes of wheat (Triticum aestivum) specific for the mismatch repair gene homologues MSH2, MSH3, and MSH6 were used to map them to their genomic positions in rye (Secale cereale) . Whereas MSH2 was mapped to the short arm of chromosome 1R, MSH3 was mapped to the long arm of chromosome 2R and MSH6 to the long arm of chromosome 5R . Southern blots with nullisomic-tetrasomic (NT) lines of wheat indicated the presence of the sequences on the respective homeologous group of wheat chromosomes . Additionally, an MSH6-specific homologue could also be detected on homoeologous group 3 of wheat . However, in the well-known, highly homoeologous pairing wheat mutant ph1b the MSH6-specific sequence is not within the deleted part of chromosome 5BL, indicating that the pairing phenotype is not due to a loss of one of the mismatch repair genes tested.

Mol Plant Microbe Interact, 2000 Jan, 13(1), 107 - 12
Casuarina glauca prenodule cells display the same differentiation as the corresponding nodule cells; Laplaze L et al.; Recent phylogenetic studies have implied that all plants able to enter root nodule symbioses with nitrogen-fixing bacteria go back to a common ancestor (D.E . Soltis, P.S . Soltis, D.R . Morgan, S.M . Swensen, B.C . Mullin, J.M . Dowd, and P.G . Martin, Proc . Natl . Acad . Sci . USA, 92:2647-2651, 1995) . However, nodules formed by plants from different groups are distinct in nodule organogenesis and structure . In most groups, nodule organogenesis involves the induction of cortical cell divisions . In legumes these divisions lead to the formation of a nodule primordium, while in non-legumes they lead to the formation of a so-called prenodule consisting of infected and uninfected cells . Nodule primordium formation does not involve prenodule cells, and the function of prenodules is not known . Here, we examine the differentiation of actinorhizal prenodule cells in comparison to nodule cells with regard to both symbionts . Our findings indicate that prenodules represent primitive symbiotic organs whose cell types display the same characteristics as their nodule counterparts . The results are discussed in the context of the evolution of root nodule symbioses.

J Antibiot (Tokyo), 1999 Nov, 52(11), 952 - 9
Biomolecular-chemical screening: a novel screening approach for the discovery of biologically active secondary metabolites . II . Application studies with pure metabolites; Maier A et al.; The novel screening strategy called "biomolecular-chemical screening" combines the advantages of the chemical screening approach--the analysis of the chromatographic and chemical behaviour of secondary metabolites on TLC plates--with binding studies of these molecules with bio-macromolecules like DNA . This approach was advantageously used to detect the interaction of pure compounds with DNA . In order to prove the reliability of the biomolecular-chemical screening and to examine DNA-binding properties, 470 pure secondary metabolites were analysed by this method . Besides the confirmation of already known binders with the TLC-based method, for a number of natural products DNA-binding properties were discovered for the first time . In consequence, binding of pure compounds can be measured by 1D TLC in a reliable and easy manner, in which DNA is applied together with the test compound at the starting spot . Analysis is performed via differences in Rf-values in comparison to a reference chromatogram without DNA.

J Clin Pathol, 1999 Sep, 52(9), 693 - 4
Modified Genta triple stain for identifying Helicobacter pylori; el-Zimaity HM et al.; AIM: To evaluate whether lead nitrate could replace uranyl nitrate in the Genta stain for H pylori without sacrificing the advantages of the triple stain (Steiner silver impregnation combined with Alcian blue and haematoxylin/eosin (H&E)) . METHODS: A comparison was made in 16 specimens between the original triple stain and the revised version . One pathologist evaluated all sections . RESULTS: Direct substitution of lead nitrate for uranium nitrate produced well stained organisms without interfering with H&E or Alcian blue staining . No difference was found in the ability to identify bacteria in 11 cases with H pylori density of 1 or 2 (on a scale of 0 to 5) . CONCLUSIONS: The potential chemical and radiological hazards associated with uranium nitrate can be eliminated by using lead nitrate without sacrificing the advantages obtained by using the triple stain.

J Clin Microbiol, 2000 Feb, 38(2), 682 - 7
Sequence variation in the ftsZ gene of Bartonella henselae isolates and clinical samples; Ehrenborg C et al.; In a search for methods for subtyping of Bartonella henselae in clinical samples, we amplified and sequenced a 701-bp region in the 3' end of the ftsZ gene in 15 B . henselae isolates derived from cats and humans in the United States and Europe . The ftsZ sequence variants that were discovered were designated variants Bh ftsZ 1, 2, and 3 and were compared with 16S rRNA genotypes I and II of the same isolates . There was no ftsZ gene variation in the strains of 16S rRNA type I, all of which were Bh ftsZ 1 . The type II strains constituted two groups, with nucleotide sequence variation in the ftsZ gene resulting in amino acid substitutions at three positions, one of which was shared by the two groups . One 16S rRNA type II isolate had an ftsZ gene sequence identical to those of the type I strains . Variants Bh ftsZ 1 and 2 were detected in tissue specimens from seven Swedish patients with diagnoses such as chronic multifocal osteomyelitis, cardiomyopathy, and lymphadenopathy . Patients with similar clinical entities displayed either Bh ftsZ variant . The etiological role of B . henselae in these patients was supported by positive Bartonella antibody titers and/or amplification and sequencing of a part of the B . henselae gltA gene . B . henselae ftsZ gene sequence variation may be useful in providing knowledge about the epidemiology of various B . henselae strains in clinical samples, especially when isolation attempts have failed . This report also describes manifestations of atypical Bartonella infections in Sweden.

Genetics, 2000 Feb, 154(2), 771 - 6
A genetic test of the mechanism of Wolbachia-induced cytoplasmic incompatibility in Drosophila; Presgraves DC; Cytoplasmic bacteria of the genus Wolbachia are best known as the cause of cytoplasmic incompatibility (CI): many uninfected eggs fertilized by Wolbachia-modified sperm from infected males die as embryos . In contrast, eggs of infected females rescue modified sperm and develop normally . Although Wolbachia cause CI in at least five insect orders, the mechanism of CI remains poorly understood . Here I test whether the target of Wolbachia-induced sperm modification is the male pronucleus (e.g., DNA or pronuclear proteins) or some extranuclear factor from the sperm required for embryonic development (e.g., the paternal centrosome) . I distinguish between these hypotheses by crossing gynogenetic Drosophila melanogaster females to infected males . Gynogenetic females produce diploid eggs whose normal development requires no male pronucleus but still depends on extranuclear paternal factors . I show that when gynogenetic females are crossed to infected males, uniparental progeny with maternally derived chromosomes result . This finding shows that Wolbachia impair the male pronucleus but no extranuclear component of the sperm.

Oncogene, 2000 Jan 20, 19(3), 417 - 30
Acidic substitution of the activation loop tyrosines in TrkA supports nerve growth factor-independent cell survival and neuronal differentiation; Gryz EA et al.; TrkA is the receptor tyrosine kinase (RTK) for nerve growth factor (NGF) and stimulates NGF-dependent cell survival and differentiation in primary neurons . TrkA expression in neuronal tumors also supports NGF-dependent differentiation of neuroblastomas and apoptosis of medulloblastomas . Phosphorylation of the activation loop tyrosines in RTK's are essential to activation as well as allosteric changes that facilitate substrate interaction and phosphorylation . Acidic amino acid substitution of the activation loop tyrosines in TrkA, Tyr683Tyr684, was performed to mimic the negative charges normally induced by ligand activation and receptor phosphorylation . A total of eight independent mutants containing single or double substitutions were generated for comparison . Herein, we demonstrate that acidic substitution of the activation loop tyrosines is sufficient to induce allosteric changes required for constitutive TrkA kinase activity as well as phosphorylation of TrkA signaling proteins such as Shc, PLCgamma-1, FRS-2 and erk1/2 . The strongest constitutively active TrkA mutants, GluAsp and AspGlu, support NGF-independent neuritogenesis and cell survival to levels approximately 65 and 80-100%, respectively, of NGF-activated wild type TrkA . Thus, constitutively active TrkA may provide a useful strategy in future therapeutic approaches to limit the development and progression of neuronal tumors.

J Mol Evol, 2000 Jan, 50(1), 45 - 55
Gene expression, amino acid conservation, and hydrophobicity are the main factors shaping codon preferences in Mycobacterium tuberculosis and Mycobacterium leprae; de Miranda AB et al.; Mycobacterium tuberculosis and Mycobacterium leprae are the ethiological agents of tuberculosis and leprosy, respectively . After performing extensive comparisons between genes from these two GC-rich bacterial species, we were able to construct a set of 275 homologous genes . Since these two bacterial species also have a very low growth rate, translational selection could not be so determinant in their codon preferences as it is in other fast-growing bacteria . Indeed, principal-components analysis of codon usage from this set of homologous genes revealed that the codon choices in M . tuberculosis and M . leprae are correlated not only with compositional constraints and translational selection, but also with the degree of amino acid conservation and the hydrophobicity of the encoded proteins . Finally, significant correlations were found between GC3 and synonymous distances as well as between synonymous and nonsynonymous distances.

Appl Environ Microbiol, 2000 Feb, 66(2), 712 - 7
In situ determination of sulfide turnover rates in a meromictic alpine lake; Luthy L et al.; A push-pull method, previously used in groundwater analyses, was successfully adapted for measuring sulfide turnover rates in situ at different depths in the meromictic Lake Cadagno . In the layer of phototrophic bacteria at about 12 m in depth net sulfide consumption was observed during the day, indicating active bacterial photosynthesis . During the night the sulfide turnover rates were positive, indicating a net sulfide production from the reduction of more-oxidized sulfur compounds . Because of lack of light, no photosynthesis takes place in the monimolimnion; thus, only sulfide formation is observed both during the day and the night . Sulfide turnover rates in the oxic mixolimnion were always positive as sulfide is spontaneously oxidized by oxygen and as the rates of sulfide oxidation depend on the oxygen concentrations present . Sulfide oxidation by chemolithotrophic bacteria may occur at the oxicline, but this cannot be distinguished from spontaneous chemical oxidation.

Appl Environ Microbiol, 2000 Feb, 66(2), 664 - 70
A gene encoding a novel multidomain beta-1,4-mannanase from Caldibacillus cellulovorans and action of the recombinant enzyme on kraft pulp; Sunna A et al.; Genomic walking PCR was used to obtained a 4,567-bp nucleotide sequence from Caldibacillus cellulovorans . Analysis of this sequence revealed that there were three open reading frames, designated ORF1, ORF2, and ORF3 . Incomplete ORF1 encoded a putative C-terminal cellulose-binding domain (CBD) homologous to members of CBD family IIIb, while putative ORF3 encoded a protein of unknown function . The putative ManA protein encoded by complete manA ORF2 was an enzyme with a novel multidomain structure and was composed of four domains in the following order: a putative N-terminal domain (D1) of unknown function, an internal CBD (D2), a beta-mannanase catalytic domain (D3), and a C-terminal CBD (D4) . All four domains were linked via proline-threonine-rich peptides . Both of the CBDs exhibited sequence similarity to family IIIb CBDs, while the mannanase catalytic domain exhibited homology to the family 5 glycosyl hydrolases . The purified recombinant enzyme ManAd3 expressed from the cloned catalytic domain (D3) exhibited optimum activity at 85 degrees C and pH 6.0 and was extremely thermostable at 70 degrees C . This enzyme exhibited high specificity with the substituted galactomannan locust bean gum, while more substituted galacto- and glucomannans were poorly hydrolyzed . Preliminary studies to determine the effect of the recombinant ManAd3 and a recombinant thermostable beta-xylanase on oxygen-delignified Pinus radiata kraft pulp revealed that there was an increase in the brightness of the bleached pulp.

Appl Environ Microbiol, 2000 Feb, 66(2), 651 - 8
Genetic variation among endosymbionts of widely distributed vestimentiferan tubeworms; Di Meo CA et al.; Vestimentiferan tubeworms thriving in sulfidic deep-sea hydrothermal vents and cold seeps are constrained by their nutritional reliance on chemoautotrophic endosymbionts . In a recent phylogenetic study using 16S ribosomal DNA, we found that endosymbionts from vent and seep habitats form two distinct clades with little variation within each clade . In the present study, we used two different approaches to assess the genetic variation among biogeographically distinct vestimentiferan symbionts . DNA sequences were obtained for the noncoding, internal transcribed spacer (ITS) regions of the rRNA operons of symbionts associated with six different genera of vestimentiferan tubeworms . ITS sequences from endosymbionts of host genera collected from different habitats and widely distributed vent sites were surprisingly conserved . Because the ITS region was not sufficient for distinguishing endosymbionts from different habitats or locations, we used a DNA fingerprinting technique, repetitive-extragenic-palindrome PCR (REP-PCR), to reveal differences in the distribution of repetitive sequences in the genomes of the bacterial endosymbionts . Most of the endosymbionts displayed unique REP-PCR patterns . A cladogram generated from these fingerprints reflected relationships that may be influenced by a variety of factors, including host genera, geographic location, and bottom type.

Biochemistry, 2000 Feb 8, 39(5), 1100 - 13
Protonation states and pH titration in the photocycle of photoactive yellow protein; Demchuk E et al.; Photoactive yellow protein (PYP) undergoes a light-driven cycle of color and protonation states that is part of a mechanism of bacterial phototaxis . This article concerns functionally important protonation states of PYP and the interactions that stabilize them, and changes in the protonation state during the photocycle . In particular, the chromophore pK(a) is known to be shifted down so that the chromophore is negatively charged in the ground state (dark state) even though it is buried in the protein, while nearby Glu46 has an unusually high pK(a) . The photocycle involves changes of one or both of these protonation states . Calculations of pK(a) values and protonation states using a semi-macroscopic electrostatic model are presented for the wild-type and three mutants, in both the ground state and the bleached (I(2)) intermediate state . Calculations allowing multiple H-bonding arrangements around the chromophore also have been carried out . In addition, ground-state pK(a) values of the chromophore have been measured by UV-visible spectroscopy for the wild-type and the same three mutants . Because of the unusual protonation states and strong electrostatic interactions, PYP represents a severe test of the ability of theoretical models to yield correct calculations of electrostatic interactions in proteins . Good agreement between experiment and theory can be obtained for the ground state provided the protein interior is assumed to have a relatively low dielectric constant, but only partial agreement between theory and experiment is obtained for the bleached state . We also present a reinterpretation of previously published data on the pH-dependence of the recovery of the ground state from the bleached state . The new analysis implies a pK(a) value of 6.37 for Glu46 in the bleached state, which is consistent with other available experimental data, including data that only became available after this analysis . The new analysis suggests that signal transduction is modulated by the titration properties of the bleached state, which are in turn determined by electrostatic interactions . Overall, the results of this study provide a quantitative picture of the interactions responsible for the unusual protonation states of the chromophore and Glu46, and of protonation changes upon bleaching.

Mol Med Today, 2000 Feb, 6(2), 60 - 5
Aquaporins in health and disease; King LS et al.; The molecular basis of membrane water-permeability remained elusive until the recent discovery of the aquaporin water-channel proteins . The fundamental importance of these proteins is suggested by their conservation from bacteria through plants to mammals . Ten mammalian aquaporins have thus far been identified, each with a distinct distribution . In the kidney, lung, eye and brain, multiple water-channel homologs are expressed, providing a network for water transport in those locations . It is increasingly clear that alterations in aquaporin expression or function can be rate-limiting for water transport across certain membranes . Aquaporins are likely to prove central to the pathophysiology of a variety of clinical conditions from diabetes insipidus to various forms of edema and, ultimately, they could be a target for therapy in diseases of altered water homeostasis.

Mol Microbiol, 2000 Jan, 35(2), 260 - 74
The Tat protein export pathway; Berks BC et al.; The Tat (twin-arginine translocation) system is a bacterial protein export pathway with the remarkable ability to transport folded proteins across the cytoplasmic membrane . Preproteins are directed to the Tat pathway by signal peptides that bear a characteristic sequence motif, which includes consecutive arginine residues . Here, we review recent progress on the characterization of the Tat system and critically discuss the structure and operation of this major new bacterial protein export pathway.

Mol Ecol, 2000 Jan, 9(1), 77 - 90
Molecular evidence for multiple infections of a new subgroup of Wolbachia in the European raspberry beetle Byturus tomentosus; Malloch G et al.; Wolbachia, a group of maternally inherited intracellular parasitic bacteria, alter host reproduction, including the induction of thelytokous parthenogenesis, feminization of genetic males, son killing and, most commonly, the induction of cytoplasmic incompatibility (CI), in a diverse array of arthropods . CI can result in infertility and has attracted attention because of its potential in biological control and as an agent in speciation . Although there has been some analysis of overall infection rates in arthropods and within individual insect orders, there has been little exploration of within-species variation . In this study, primers specific for the ftsZ gene of Wolbachia were used to amplify it from different geographical samples of the European raspberry beetle (Byturus tomentosus), confirming the presence of Wolbachia . More than 99% of UK individuals were found to be infected with Wolbachia and 97% of these B . tomentosus beetles harboured multiple infections . Preliminary analysis of B . tomentosus beetles from continental European populations revealed a lower level of infection (24%) than those from the UK . Phylogenetic analysis using the ftsZ DNA sequences places Wolbachia from B . tomentosus into a new clade (Abt) within the A division, with some revisions to the existing Wolbachia phylogeny.

Genes Cells, 2000 Jan, 5(1), 9 - 16
The loose coupling mechanism in molecular machines of living cells; Oosawa F; Living cells have molecular machines for free energy conversion, for example, sliding machines in muscle and other cells, flagellar motors in bacteria, and various ion pumps in cell membranes . They are constructed from protein molecules and work in the nm (nanometer), pN (piconewton) and ms (millisecond) ranges, without inertia . In 1980s, a question was raised of whether the input-output or influx-efflux coupling in these molecular machines is tight or loose, and an idea of loose coupling was proposed . Recently, the long-distance multistep sliding of a single myosin head on an actin filament, coupled with the hydrolysis of one ATP molecule, was observed by Yanagida's group using highly developed techniques of optical microscopy and micromanipulation . This gave direct evidence for the loose coupling between the chemical reaction and the mechanical event in the sliding machine . In this review, I will briefly describe a historical overview of the input-output problem in the molecular machines of living cells.

Photochem Photobiol, 2000 Jan, 71(1), 53 - 9
Photophysical properties of neutral and cationic tetrapyridinoporphyrazines; Marti C et al.; We describe the synthesis and photophysical properties of a series of neutral and cationic 3,4-tetrapyridinoporphyrazines, potential lead photosensitizers for photodynamic inactivation of bacteria . Tetracationic TPyPzs exist essentially as monomers in aqueous systems, but the presence of trialkylated compounds due to incomplete quaternization of the outer nitrogen atoms induces severe aggregation . The absorption, fluorescence, triplet, and singlet oxygen quantum yields for both the neutral and cationic compounds are comparable to those of the related phthalocyanines.

Biotechniques, 2000 Jan, 28(1), 107 - 9
High-throughput method for isolating plasmid DNA with reduced lipopolysaccharide content; Neudecker F et al.; Isolating plasmid DNA from bacteria is a fundamental step in molecular biology . It is often accomplished by an alkaline lysis of bacteria and the subsequent adsorption of nucleic acids to silica oxide in the presence of chaotropic substances . Here we show that the addition of such chaotropic reagents is not required for the efficient DNA isolation with silica oxide . This surprising finding allowed us to purify plasmid DNA with significantly less lipopolysaccharides (LPS), which is otherwise a common bacterial contaminant of silica oxide-isolated DNA and inhibits subsequent applications . In addition, we have implemented a precipitation step that altogether leads to a reduction of the LPS content by a factor of 900 relative to published methods . Our novel protocol facilitates an inexpensive high-throughput analysis of pure plasmids in a 96-well format without the addition of hazardous reagents.

Vaccine, 2000 Jan 18, 18(13), 1186 - 95
Immunogenicity and protective capacity of Mycobacterium bovis BCG after oral or intragastric administration in mice; Lagranderie M et al.; After oral or intragastric administration of BCG to mice, comparable numbers of IFN gamma and TNF gamma producing cells were detected in both local (Peyer's patches) and central (spleen) lymphoid organs . Similar levels of precursors of CD8+ cytotoxic T lymphocytes specific for mycobacterial antigens were also found in the spleen and the mesenteric lymph nodes . These immune responses remained high over the course of 3 months, the duration of observation . Oral administration of BCG led to an enlargement of the cervical lymph nodes, which contained high levels of viable bacteria . In contrast, no adverse effects were observed in mice given the BCG via the intragastric route . These two routes of immunization induced similar levels of protective immunity to those observed in mice immunized via the subcutaneous route against a challenge with a virulent Mycobacterium tuberculosis strain (H37Rv).

Biochemistry (Mosc), 1999 Dec, 64(12), 1342 - 53
UGA: a dual signal for 'stop' and for recoding in protein synthesis; Tate WP et al.; UGA remains an enigma as a signal in protein synthesis . Long recognized as a stop signal that is prone to failure when under competition from near cognate events, there was growing belief that there might be functional significance in the production of small amounts of extended proteins . This view has been reinforced with the discovery that UGA is found at some recoding sites where frameshifting occurs as a regulatory mechanism for controlling the gene expression of specific proteins, and it also serves as the code for selenocysteine (Sec), the 21st amino acid . Why does UGA among the stop signals play this role specifically, and how does it escape being used to stop protein synthesis efficiently at recoding sites involving Sec incorporation or shifts to a new translational frame? These issues concerning the UGA stop signals are discussed in this review.

Curr Opin Urol, 2000 Jan, 10(1), 39 - 44
Influence of urogenital infection on sperm function; Diemer T et al.; Male accessory sex gland infections are considered to be hazards to male fertility . Various pathophysiologic concepts have evolved from experimental and clinical studies that begin to explain the effects of bacteria and immunologic events on spermatozoa . Recent studies have identified and evaluated mediators that are responsible for specific molecular processes in infections that particularly affect the function of spermatozoa.

J Microbiol Immunol Infect, 1999 Dec, 32(4), 223 - 32
Protective effects of partially purified antigens of Actinobacillus pleuropneumoniae on experimentally infected mice; Liu JL et al.; Three different models of protection experiments in mice using partially purified Actinobacillus pleuropneumoniae antigens such as crude culture supernatant extract (CCSE) and partially purified cell extract (PPCE) were attempted . Biochemical analysis showed that these two immunogens had protein concentration of 0.17-0.2 mg/mL and pentose concentration of 0.012-0.014 mg/mL . In the first model intranasal (IN) vaccination with different doses (from 0.01-10 IN-LD50) against IN challenge with the dose of 20 IN-LD50 containing 1.2 x 10(9) colony forming unit (CFU)/50 microL showed that only those with the dose more than 1 IN-LD50 had slight protection in terms of survival index (SI) . In the second model of protection experiment, in which subcutaneous vaccination (s.c.) with the immunogens plus soybean oil against IN challenge with 10 IN-LD50 containing 6 x 10(8) CFU/50 microL, showed that formalin-killed bacteria (bacterin) and CCSE plus PPCE had only a slight protection whereas vaccination with CCSE or PPCE immunogen alone had no protection . In the third model of protection experiment, in which the intramuscular (i.m.) vaccination with the immunogens plus aluminum hydroxide {Al(OH3)} gel against intraperitoneal (i.p.) challenge with the dose of either 2 or 6 i.p.-LD50 containing 1.2-3.6 x 10(8) CFU/0.5 mL of 0.3% mucin saline showed highly effective.

Compend Contin Educ Dent, 1999 May, 20(5), 451 - 6, 458-60, 462; quiz 463
Evaluation of Periostat for patient management; Caton JG; The cause of adult periodontitis involves complex bacteria-host interactions, with modifying influences exerted by genetic and environmental factors . Current thinking indicates that successful, long-term management of adult periodontitis requires a treatment approach that takes into account the various etiologic components of the disease . Recently, a formulation containing a subantimicrobial dose of doxycycline (Periostat) was approved for use as an adjunct to scaling and root planing (SRP) in the treatment of adult periodontitis . As an inhibitor of matrix metalloproteinases that have been implicated in the pathologic degradation of connective tissue collagen in periodontal support structures, Periostat in conjunction with SRP was shown to significantly improve clinical attachment and reduce probing depth compared with placebo in conjunction with SRP . This article reviews the clinical trial results leading to US Food and Drug Administration approved of Periostat and discusses the potential use of this host-modulatory agent as a complementary systemic pharmacotherapy in periodontal management programs.

J Pak Med Assoc, 1999 Nov, 49(11), 273 - 8
Study of the mechanisms of killing of Mycobacterium bovis BCG by apoptosis in J774 murine macrophages; Ahmad A et al.; OBJECTIVE: To determine the relationship between the mechanism of apoptosis and intracellular killing of Mycobacterium bovis BCG . Apoptosis, "programmed cell death"--a physiologically beneficial and distinct form of cell death . SETTING: In vitro study was carried out in murine macrophage cell line J774 that was infected with Mycobacterium bovis BCG in different set of conditions . Percentage of surviving BCG and apoptotic cells was determined . METHODS: IFN-g and/or LPS-activated and non-activated J774 mouse macrophage cells were infected with BCG in a ratio of 1:5 . The morphology of the host cells was studied after 4 hours, 24 hours and 48 hours of infection in cytospins stained with Jenner-Giemsa . Surviving bacteria were counted by incorporation of radiolabelled-uridine after cell lysis . RESULTS: Both in the activated and non-activted J774 cultures some cells undergo apoptosis . In cells activated with IFN-g or LPS without BCG, less than 10% of cells were found to be apoptotic . More apoptosis was seen when LPS-activated cells were infected with BCG . In the cells activated with IFN-g or LPS-activated cells the percentage of apoptotic cells was much higher than in non-activated cells or cells activated with either INF-g or LPS alone . After 24 hours culture, without BCG, about 15% of the cells were found to be apoptotic and with BCG infection this increased to 23% (p < 0.01) . The level further increased after 48 hours of infection . BCG growth inhibition was observed in both non-activated J774 cells and cells activated with LPS, INF-g or both and was sustained to 48 hours of co-culture . CONCLUSION: It is evident that BCG-infected J774 cells undergo apoptosis in the presence of a high concentration of RNI and/or ROI . During this process the cells shrink considerably in volume with the removal of water that may concentrate toxic products in the cell . The increased concentration of toxic species and the disorganisation of the phagocytic vacuoles may account for the enhanced stasis and/or death of the intracellular micro-organisms . We conclude that host cell apoptosis may arrest the growth and account for the death of the intracellular mycobacterial pathogen.

Structure Fold Des, 1999 Dec 15, 7(12), 1473 - 82
Helianthus tuberosus lectin reveals a widespread scaffold for mannose-binding lectins; Bourne Y et al.; BACKGROUND: Heltuba, a tuber lectin from the Jerusalem artichoke Helianthus tuberosus, belongs to the mannose-binding subgroup of the family of jacalin-related plant lectins . Heltuba is highly specific for the disaccharides Man alpha 1-3Man or Man alpha 1-2Man, two carbohydrates that are particularly abundant in the glycoconjugates exposed on the surface of viruses, bacteria and fungi, and on the epithelial cells along the gastrointestinal tract of lower animals . Heltuba is therefore a good candidate as a defense protein against plant pathogens or predators . RESULTS: The 2.0 A resolution structure of Heltuba exhibits a threefold symmetric beta-prism fold made up of three four-stranded beta sheets . The crystal structures of Heltuba in complex with Man alpha 1-3Man and Man alpha 1-2Man, solved at 2.35 A and 2.45 A resolution respectively, reveal the carbohydrate-binding site and the residues required for the specificity towards alpha 1-3 or alpha 1-2 mannose linkages . In addition, the crystal packing reveals a remarkable, donut-shaped, octahedral assembly of subunits with the mannose moieties at the periphery, suggesting possible cross-linking interactions with branched oligomannosides . CONCLUSIONS: The structure of Heltuba, which is the prototype for an extended family of mannose-binding agglutinins, shares the carbohydrate-binding site and beta-prism topology of its galactose-binding counterparts jacalin and Maclura pomifera lectin . However, the beta-prism elements recruited to form the octameric interface of Heltuba, and the strategy used to forge the mannose-binding site, are unique and markedly dissimilar to those described for jacalin . The present structure highlights a hitherto unrecognized adaptability of the beta-prism building block in the evolution of plant proteins.

Nature, 1999 Nov 11, 402(6758), 191 - 5
A plant regulator controlling development of symbiotic root nodules; Schauser L et al.; Symbiotic nitrogen-fixing root nodules on legumes are founded by root cortical cells that de-differentiate and restart cell division to establish nodule primordia . Bacterial microsymbionts invade these primordia through infection threads laid down by the plant and, after endocytosis, membrane-enclosed bacteroids occupy cells in the nitrogen-fixing tissue of functional nodules . The bacteria excrete lipochitin oligosaccharides, triggering a developmental process that is controlled by the plant and can be suppressed . Nodule inception initially relies on cell competence in a narrow infection zone located just behind the growing root tip . Older nodules then regulate the number of nodules on a root system by suppressing the development of nodule primordia . To identify the regulatory components that act early in nodule induction, we characterized a transposon-tagged Lotus japonicus mutant, nin (for nodule inception), arrested at the stage of bacterial recognition . We show that nin is required for the formation of infection threads and the initiation of primordia . NIN protein has regional similarity to transcription factors, and the predicted DNA-binding/dimerization domain identifies and typifies a consensus motif conserved in plant proteins with a function in nitrogen-controlled development.

J Pediatr Surg, 2000 Jan, 35(1), 49 - 55
Effect of repetitive asphyxia on leukocyte-vessel wall interactions in the developing chick intestine; Rouwet EV et al.; BACKGROUND/PURPOSE: Information on leukocyte-vessel wall interactions (LVWI) during development of the immature intestine is scarce . The authors designed an experimental model for studying the microcirculation in the developing intestine of chick fetuses at days 13 (n = 12), 15 (n = 17), and 17 (n = 19) of incubation (0.6, 0.7, and 0.8 of the incubation time, respectively) using intravital microscopy . METHODS: The authors investigated whether episodes of asphyxia increase LVWI and induce tissue damage in the developing intestine . Asphyxia was induced by clamping of the chorioallantoic vein for 6 periods of 5 minutes each, with 5-minute intervals, whereas in sham groups a sham procedure was performed . Video recordings were made before as well as 10, 20, and 30 minutes after the end of the asphyxia or sham protocol . RESULTS: Baseline number of rolling leukocytes per minute significantly increased (P < .001) from 0 at 0.6 incubation to 1.5 and to 4 at 0.7 and 0.8 incubation time, respectively . At 0.6 and 0.7 incubation no adherent leukocytes were observed under baseline conditions, whereas at 0.8 incubation single leukocytes adhered to the venular wall . LVWI variably increased during the course of the experiments . Asphyxia neither enhanced LVWI nor induced histological damage in the intestine . CONCLUSIONS: These findings indicate that (1) leukocyte-vessel wall interactions mature during fetal development, and (2) repetitive episodes of asphyxia induce neither an inflammatory response nor histological tissue injury in the developing intestine from 0.6 to 0.8 incubation . The authors hypothesize that immaturity of leukocyte-vessel wall interactions, as part of the nonspecific host defense to invading bacteria, might play a role in the development of necrotizing enterocolitis in premature neonates.

Laryngoscope, 2000 Jan, 110(1), 107 - 10
Aspiration of nasal secretions into the lungs in patients with acute sinonasal infections; Ozagar A et al.; OBJECTIVES: To compare the amounts of nasal secretions aspirated into the lower airway by patients with acute sinonasal infection with that aspirated by healthy adults during sleep . STUDY DESIGN: Sixteen patients who had received a diagnosis of acute sinonasal infections by accurate history, anterior rhinoscopic examination, and radiological assessment and 13 healthy volunteers, aged 14 to 45 years . METHODS: A 10-mCi dose of technetium 99m-labeled macroaggregated albumin (Tc-99m MAA) with a concentration of 1 mCi/mL was prepared at midnight, just before sleep . Each subject was administered two puffs of this spray . At 8 AM the next morning transmission and emission views of the thorax were taken with a gamma camera . RESULTS: No significant difference between the two groups was observed in the amounts of nasal secretions aspirated into the lungs . CONCLUSIONS: The amount of nasal secretions aspirated does not increase during acute sinonasal infection . However, by irritating the mucosa of the lower respiratory tracts, bacteria, toxins, and inflammatory products existing in purulent secretions may play a major role in the pathophysiology of asthma and sinusitis.

Plant Mol Biol, 1999 Nov, 41(5), 615 - 25
Alternative splicing of two leading exons partitions promoter activity between the coding regions of the maize homeobox gene Zmhox1a and Trap (transposon-associated protein); Comelli P et al.; Elucidation of the exon/intron structure of the maize Zmhox1a homeobox gene revealed two small introns in the homeodomain . Both intron positions are conserved in animal counterparts encoded in the metazoan homeobox gene clusters and thus may indicate a common ancestor . The transcription start of the Zmhox1a gene has been localized far from the protein-coding region . Two distal untranslated leading exons are alternatively spliced to either the Zmhox1a coding exons or an unrelated open reading frame comprising two exons located internally of the large second Zmhox1a intron . Due to significant homology to the C-terminus of the Mutator transposase this alternative gene product was named Trap (transposon-associated protein) . Splice site selection may involve two sequence elements conserved at the splice acceptor sites in front of the Zmhox1a and Trap protein-coding regions . The translation of a mRNA species devoid of exon 3 which encodes the Zmhox1a transcription start codon may give rise to an N-terminal deletion polypeptide, deltaZmhox1a . Ectopic expression experiments in transgenic tobacco indicate a putative function distinct from the full-length Zmhox1a protein.

J Virol, 2000 Feb, 74(4), 1794 - 800
The RNA helicase and nucleotide triphosphatase activities of the bovine viral diarrhea virus NS3 protein are essential for viral replication; Gu B et al.; Helicase/nucleoside triphosphatase (NTPase) motifs have been identified in many RNA virus genomes . Similarly, all the members of the Flaviviridae family contain conserved helicase/NTPase motifs in their homologous NS3 proteins . Although this suggests that this activity plays a critical role in the viral life cycle, the precise role of the helicase/NTPase in virus replication or whether it is essential for virus replication is still unknown . To determine the role of the NS3 helicase/NTPase in the viral life cycle, deletion and point mutations in the helicase/NTPase motifs of the bovine viral diarrhea virus (BVDV) (NADL strain) NS3 protein designed to abolish either helicase activity alone (motif II, DEYH to DEYA) or both NTPase and helicase activity (motif I, GKT to GAT and deletion of motif VI) were generated . The C-terminal domain of NS3 (BVDV amino acids 1854 to 2362) of these mutants and wild type was expressed in bacteria, purified, and assayed for RNA helicase and ATPase activity . These mutations behaved as predicted with respect to RNA helicase and NTPase activities in vitro . When engineered back into an infectious cDNA for BVDV (NADL strain), point mutations in either the GKT or DEYH motif or deletion of motif VI yielded RNA transcripts that no longer produced infectious virus upon transfection of EBTr cells . Further analysis indicated that these mutants did not synthesize minus-strand RNA . These findings represent the first report unequivocably demonstrating that helicase activity is essential for minus-strand synthesis.

Vet Pathol, 2000 Jan, 37(1), 77 - 82
Experimental Brucella abortus induced abortion in a llama: pathologic effects; Gidlewski T et al.; Brucella abortus infection has not been documented in llamas . This report describes the abortion of the only pregnant animal in a group of 12 . The llama was infected by inoculating 1 x 10(8) viable B . abortus organisms into the conjunctival sac . Forty-three days postinfection, the llama aborted a fetus of approximately 8 months gestational age . Brucella organisms were isolated from the placenta and all fetal specimens examined . These organisms were also isolated from the dam's mammary gland and numerous lymph nodes when the llama was necropsied 42 days later . Microscopically, there was a moderate, multifocal, lymphocytic and histiocytic, subacute placentitis with marked loss of trophoblastic epithelial cells . The superficial chorioallantoic stroma contained abundant necrotic and mineralized debris as well as numerous swollen capillaries protruding multifocally from the denuded surface . Immunohistochemistry revealed that these capillaries, as well as sloughed and intact trophoblasts, were expanded by large numbers of Brucella organisms . Brucellar antigen was also detected in occasional macrophages in the fetal kidney and lung . Ultrastructurally, bacteria labeled by an antibody-based colloidal gold procedure were located within degenerate capillaries, within necrotic leukocytes, and extracellularly in the placental stroma.

J Indian Med Assoc, 1999 Aug, 97(8), 299 - 304
HIV/AIDS and ocular manifestations; Chakraborty J; As of December, 1998, worldwide, 33.4 million people have been infected with human immunodeficiency virus (HIV) . A wide variety of ocular disorders is associated with HIV . HIV can affect all structures of the eye . A large number of micro-organisms including virus, bacteria, fungi and protozoa, cause ocular diseases in HIV-infected patients . Out of these, cytomegalovirus (CMV) retinitis is the most common . Ocular tuberculosis (TB), syphilitic retinal diseases and ocular toxoplasmosis are other serious eye problems in HIV patients, especially in the developing countries . HIV can also cause microvascular abnormalities producing cotton-wool spots . Neoplasms and drug-induced ocular disorders may be other problems . Ophthalmologists need to have wide range of information regarding HIV/AIDS for better diagnosis and management of their patients with ocular abnormalities . Further research, data collection, continuing education and the latest information on eye problems of AIDS patients are essential for Indian ophthalmologists.

Comput Chem, 2000 Jan, 24(1), 71 - 94
A global compositional complexity measure for biological sequences: AT-rich and GC-rich genomes encode less complex proteins; Wan H et al.; Different local regions of natural amino acid or nucleotide sequences show remarkable heterogeneity in residue composition, reflecting diversity in evolutionary history and physiochemical constraints . Compositional complexity measures are helpful for describing and understanding this variegation . Motivated by some open problems in comparative genomics and protein folding, we have developed a new 'global' compositional complexity measure, G1, which overcomes a crucial limitation of earlier methods . The 'local' measures used in previous research resemble entropy functions and are inherently dependent on an underlying probability distribution . Local measures cannot rigorously compare complexity across sequences of substantially different size, because real sequences show very irregular heterogeneity and do not have the necessary ergodicity in scaling and asymptotic properties . G1 is a member of a new class of scale-independent, distribution-independent complexity functions . For a sequence S of length L on an N-letter alphabet, G1 is derived from ratios in the integer partition lattice, P inverted question markL,N inverted question mark of L with N parts, where the elements of P inverted question markL,N inverted question mark are the state vectors of S, (n1, n2,..., nN), ranked by an order principle . We present theorems and proofs relating to the metric properties of G1 and its relationship to other state-vector-dependent compositional complexity functions, together with a fully-efficient O(L) algorithm to compute G1 . The distributions of G1 were calculated for the entire sets of translated proteins encoded by extensively sequenced genomes . The results establish the existence of a clear evolutionary principle, common to bacteria, archaea and eukaryotes, that the proteins encoded by more extreme AT-rich and GC-rich genomes have generally lower compositional complexity than those of more typical organisms.

FEMS Immunol Med Microbiol, 2000 Feb, 27(2), 99 - 102
The dprA gene is required for natural transformation of Helicobacter pylori; Smeets LC et al.; Genetic recombination in Helicobacter pylori is believed to be involved in host adaptation of this gastric pathogen and uptake of DNA by natural transformation can result in changes in virulence factors as well as antigenic variation . To elucidate the mechanisms involved in natural transformation we tested two genes with homology to known competence genes (dprA and traG) for their role in this process . Insertion mutants in these genes were constructed in two different H . pylori strains and their competence by natural transformation was compared to the wild-type . Mutation of the traG homolog did not reduce competence . Mutation of the dprA gene, however, severely impaired natural transformation both with plasmid and chromosomal DNA . Our data indicate that dprA and comB3 are essential parts of a common pathway for chromosomal and plasmid transformation.

Mutagenesis, 2000 Jan, 15(1), 91 - 7
Aflatoxin B1-induced mitotic recombination in L5178Y mouse lymphoma cells; Preisler V et al.; Aflatoxin B1 is a human hepatocarcinogen . It is also a known point mutagen in bacteria and mammalian cells . This mutagenic activity may be at least partly responsible for its carcinogenic activity . However, recent studies show that aflatoxin B1 induces mitotic recombination in the yeast Saccharomyces cerevisiae . Because numerous reports have implicated mitotic recombination in mechanisms leading to carcinogenesis and because no one has shown that aflatoxin B1 induces recombination in mammalian cells, we decided to examine the ability of aflatoxin B1 to induce recombination in a mammalian cell line . We used a combination of methods, analysis for loss of heterozygosity and whole chromosome in situ hybridization, to identify mechanisms of chromosome mutation, including mitotic recombination in the mammalian L5178Y mouse lymphoma cell system . Our experiments revealed that mitotic recombination caused approximately 60% or more of the aflatoxin B1-induced mutagenic lesions in this cell system . Thus, mitotic recombination plays an important role in aflatoxin B1-induced mutagenesis in mammalian cells and possibly in chemically induced mutagenesis and carcinogenesis . This work suggests that multiple genetic lesions may be involved in aflatoxin B1-induced pathology.

Int J Biochem Cell Biol, 1999 Dec, 31(12), 1443 - 52
HIV-1 reverse transcriptase is phosphorylated in vitro and in a cellular system; Idriss H et al.; Phosphorylation modulates the activity of many proteins that interact with nucleic acids including DNA and RNA polymerases . The HIV-1 reverse transcriptase (RT) is essential during the replicative cycle of the HIV-1 virus . HIV-1 RT has several potential sites for phosphorylation that could regulate its activities . In this work, the phosphorylation of HIV-1 RT is examined in vitro and in vivo, to evaluate any role for this modification in regulating RT metabolism . Recombinant unphosphorylated HIV-1 RT heterodimer expressed in bacteria can be phosphorylated in vitro by several purified mammalian protein kinases . Seven kinases were tested, and five of these enzymes phosphorylated HIV-1 RT . Using an insect baculovirus expression system, the 66 kDa HIV-1 RT was also phosphorylated in vivo . However, HIV-1 RT immunoprecipitated from H9-lymphoma cells infected with HIV-1 showed negligible phosphorylation . Our results indicate that purified HIV-1 RT can be phosphorylated by several mammalian protein kinases in vitro and during expression in baculovirus infected insect cells.

Infect Immun, 2000 Feb, 68(2), 492 - 501
Host cellular immune response to pneumococcal lung infection in mice; Kadioglu A et al.; Although there is substantial evidence that pneumolysin is an important virulence factor in pneumococcal pneumonia, relatively little is known about how it influences cellular infiltration into the lungs . We investigated how the inability of mutant pneumococci to produce pneumolysin altered the pattern of inflammation and cellular infiltration into the lungs . The effect on bacterial growth in the lungs also was assessed . There were three phases of growth of wild-type bacteria in the lungs: a decline followed by a rapid increase and then stasis or decline . The absence of pneumolysin was associated with a more rapid early decline and then a much slower increase in numbers . The pattern of inflammatory-cell accumulation also had distinct stages, and the timing of these stages was influenced by the presence of pneumolysin . Neutrophils began to accumulate about 12 to 16 h after infection with wild-type pneumococci . This accumulation occurred after the early decline in pneumococcal numbers but coincided with the period of rapid growth . Following infection with pneumococci unable to make pneumolysin, neutrophil influx was slower and less intense . Coincident with the third stage of pneumococcal growth was an accumulation of T and B lymphocytes at the sites of inflammation, but the accumulation was not associated with an increase in the total number of lymphocytes in the lungs . Lymphocyte accumulation in the absence of pneumolysin occurred but was delayed.

Infect Immun, 2000 Feb, 68(2), 960 - 4
Acquired immunity to Chlamydia pneumoniae is dependent on gamma interferon in two mouse strains that initially differ in this respect after primary challenge; Vuola JM et al.; The role of gamma interferon (IFN-gamma) in a Chlamydia pneumoniae mouse model was studied by in vivo neutralization in two inbred mouse strains . During primary C . pneumoniae infection, neutralization of IFN-gamma increased both the numbers of bacteria and the pneumonia score in the lungs of C57BL/6 mice but not BALB/c mice . During reinfection, the bacterial counts in the lungs were increased by IFN-gamma neutralization in both mouse strains . Thus, the effect of IFN-gamma neutralization was dependent on the genetic background in primary infection . However, IFN-gamma appeared to be equally important in both mouse strains during reinfection.

Infect Immun, 2000 Feb, 68(2), 956 - 9
Phagocytosed Bordetella pertussis fails to survive in human neutrophils; Lenz DH et al.; Previous studies have reported that phagocytosed Bordetella pertussis survives in human neutrophils . This issue has been reexamined . Opsonized or unopsonized bacteria expressing green fluorescent protein (GFP) were incubated with adherent human neutrophils . Phagocytosis was quantified by fluorescence microscopy, and the viability of phagocytosed bacteria was determined by colony counts following treatment with polymyxin B to kill extracellular bacteria . Only 1 to 2% of the phagocytosed bacteria remained viable . Opsonization with heat-inactivated immune serum reduced the amount of attachment and phagocytosis of the bacteria but did not alter survival rates . In contrast to previous reports, these data suggest that phagocytosed B . pertussis bacteria are killed by human neutrophils.

Infect Immun, 2000 Feb, 68(2), 877 - 83
A Mycobacterium ulcerans toxin, mycolactone, causes apoptosis in guinea pig ulcers and tissue culture cells; George KM et al.; Mycobacterium ulcerans is the causative agent of Buruli ulcer, a tropical ulcerative skin disease . One of the most intriguing aspects of this disease is the presence of extensive tissue damage in the absence of an acute inflammatory response . We recently purified and characterized a macrolide toxin, mycolactone, from M . ulcerans . Injection of this molecule into guinea pig skin reproduced cell death and lack of acute inflammatory response similar to that seen following the injection of viable bacteria . We also showed that mycolactone causes a cytopathic effect on mouse fibroblast L929 cells that is characterized by cytoskeletal rearrangements and growth arrest within 48 h . However, these results could not account for the extensive cell death which occurs in Buruli ulcer . The results presented here demonstrate that L929 and J774 mouse macrophage cells die via apoptosis after 3 to 5 days of exposure to mycolactone . Treatment of cells with a pan-caspase inhibitor can inhibit mycolactone-induced apoptosis . We demonstrate that injection of mycolactone into guinea pig skin results in cell death via apoptosis and that the extent of apoptosis increases as the lesion progresses . These results may help to explain why tissue damage in Buruli ulcer is not accompanied by an acute inflammatory response.

Infect Immun, 2000 Feb, 68(2), 801 - 8
Alkyl hydroperoxide reductases C and D are major antigens constitutively expressed by Mycobacterium avium subsp . paratuberculosis; Olsen I et al.; Antigens characteristic for Mycobacterium avium subspecies paratuberculosis were identified by crossed immunoelectrophoresis (CIE) and by absorbing out cross-reactive antigens by using a polyclonal and polyvalent Mycobacterium avium subspecies avium antiserum . Two antigens were present in M . avium subsp . paratuberculosis and not detected in Mycobacterium avium subsp . avium . They were identified as antigens 17 and 20 in a CIE reference system for M . avium subsp . paratuberculosis antigens . Purified antigen 20 was identified as alkyl hydroperoxide reductase C (AhpC) while the N-terminal part of purified antigen 17 showed 80% homology with alkyl hydroperoxide reductase D (AhpD) of Mycobacterium tuberculosis . AhpC had a nonreduced mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 45 kDa and is probably a homodimer linked with disulfide bridges in its native form . AhpD had a mobility corresponding to 19 kDa . Monospecific rabbit antiserum against AhpC and AhpD reacted with 9 strains of M . avium subsp . paratuberculosis but not with 20 other mycobacterial strains except for a Mycobacterium gordonae strain, against which a weak cross-reactive band was produced . Goats experimentally infected with M . avium subsp . paratuberculosis had strong gamma interferon (IFN-gamma) responses toward both AhpC and AhpD, and they also had antibodies against AhpC . The ability of AhpC and AhpD to induce IFN-gamma production shows that these proteins potentially could be used in future vaccines or in diagnostic assays . These results further show that AhpC and AhpD are immunologically important proteins which are constitutively and highly expressed in M . avium subsp . paratuberculosis without the bacteria being submitted to oxidative stress and that the specificities of antigens can be a matter of different levels of protein expression in various species as well as distinct structural differences.

Infect Immun, 2000 Feb, 68(2), 767 - 78
Disruption of the genes encoding antigen 85A and antigen 85B of Mycobacterium tuberculosis H37Rv: effect on growth in culture and in macrophages; Armitige LY et al.; The mechanism of pathogenesis of Mycobacterium tuberculosis is thought to be multifactorial . Among the putative virulence factors is the antigen 85 (Ag85) complex . This family of exported fibronectin-binding proteins consists of members Ag85A, Ag85B, and Ag85C and is most prominently represented by 85A and 85B . These proteins have recently been shown to possess mycolyl transferase activity and likely play a role in cell wall synthesis . The purpose of this study was to generate strains of M . tuberculosis deficient in expression of the principal members of this complex in order to determine their role in the pathogenesis of M . tuberculosis . Constructs of fbpA and fbpB disrupted with the kanamycin resistance marker OmegaKm and containing varying amounts of flanking gene and plasmid vector sequences were then introduced as linear fragments into H37Rv by electroporation . Southern blot and PCR analyses revealed disruption of the homologous gene locus in one fbpA::OmegaKm transformant and one fbpB::OmegaKm transformant . The fbpA::OmegaKm mutant, LAa1, resulted from a double-crossover integration event, whereas the fbpB::OmegaKm variant, LAb1, was the product of a single-crossover type event that resulted in insertion of both OmegaKm and plasmid sequences . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis confirmed that expression of the disrupted gene was not detectable in the fbpA and fbpB mutants . Analysis of growth rates demonstrated that the fbpB mutant LAb1 grew at a rate similar to that of the wild-type parent in enriched and nutrient-poor laboratory media as well as in human (THP-1) and mouse (J774.1A) macrophage-like cell lines . The fbpA mutant LAa1 grew similarly to the parent H37Rv in enriched laboratory media but exhibited little or no growth in nutrient-poor media and macrophage-like cell lines . The targeted disruption of two genes encoding mycolyl transferase and fibronectin-binding activities in M . tuberculosis will permit the systematic determination of their roles in the physiology and pathogenesis of this organism.

Infect Immun, 2000 Feb, 68(2), 708 - 15
Identification of an antigen localized to an apparent septum within dividing chlamydiae; Brown WJ et al.; The process of chlamydial cell division has not been thoroughly investigated . The lack of detectable peptidoglycan and the absence of an FtsZ homolog within chlamydiae suggest an unusual mechanism for the division process . Our laboratory has identified an antigen (SEP antigen) localized to a ring-like structure at the apparent septum within dividing chlamydial reticulate bodies (RB) . Antisera directed against SEP show similar patterns of antigen distribution in Chlamydia trachomatis and Chlamydia psittaci RB . In contrast to localization in RB, SEP in elementary bodies appears diffuse and irregular, suggesting that the distribution of the antigen is developmental-stage specific . Treatment of chlamydiae with inhibitors of peptidoglycan synthesis or culture of chlamydiae in medium lacking tryptophan leads to the formation of nondividing, aberrant RB . Staining of aberrant RB with anti-SEP reveals a marked redistribution of the antigen . Within C . trachomatis-infected cells, ampicillin treatment leads to high levels of SEP accumulation at the periphery of aberrant RB, while in C . psittaci, treatment causes SEP to localize to distinct punctate sites within the bacteria . Aberrancy produced via tryptophan depletion results in a different pattern of SEP distribution . In either case, the reversal of aberrant formation results in the production of normal RB and a redistribution of SEP to the apparent plane of bacterial division . Collectively these studies identify a unique chlamydial-genus-common and developmental-stage-specific antigen that may be associated with RB division.

Infect Immun, 2000 Feb, 68(2), 485 - 91
Genital antibody responses in mice after intranasal infection with an attenuated candidate vector strain of Bordetella pertussis; Mielcarek N et al.; Intranasal administration of live attenuated Bordetella pertussis, from which the pertussis toxin gene has been deleted, has previously been shown to give rise to high levels of serum immunoglobulin G (IgG) antibodies against both the protective antigen filamentous hemagglutinin (FHA) and heterologous antigens genetically fused to FHA . Here, we extend these results by demonstrating that anti-FHA IgA and IgG antibodies are also produced in the genital tract of mice, both in the vagina and in the uterus, after a single intranasal administration of B . pertussis . By comparing the immune responses induced after infection with wild-type virulent B . pertussis with that induced by infection with an attenuated pertussis toxin-deficient strain, we conclude that pertussis toxin produced by the virulent bacteria does not modify antibody production to FHA in the genital tract of B . pertussis-infected mice . The intranasal infection with either the attenuated or the virulent B . pertussis strain also led to the development of immunologic memory that could be efficiently boosted with purified FHA administered either intranasally or intravaginally to give rise to a significant increase in the levels of specific IgA and IgG produced locally in the genital tract, as well as of specific antibodies in the serum . These observations suggest that attenuated B . pertussis could be a promising vector for intranasal administration to induce antibody responses against antigens from sexually transmitted pathogens fused to FHA.

Infect Immun, 2000 Feb, 68(2), 470 - 7
Expression of the Helicobacter pylori ureI gene is required for acidic pH activation of cytoplasmic urease; Scott DR et al.; ureI encodes an integral cytoplasmic membrane protein . It is present in the urease gene cluster of Helicobacter pylori and is essential for infection and acid survival, but its role is unknown . To determine the function of UreI protein, we produced H . pylori ureI deletion mutants and measured the pH dependence of urease activity of intact and lysed bacteria and the effect of urea on the membrane potential . We also determined ureI expression, urease activity, and the effect of urea on membrane potential of several gastric and nongastric Helicobacter species . ureI was found to be present in the genome of the gastric Helicobacter species and absent in the nongastric Helicobacter species studied, as determined by PCR . Likewise, Western blot analysis confirmed that UreI was expressed only in the gastric Helicobacter species . When UreI is present, acidic medium pH activation of cytoplasmic urease is found, and urea addition increases membrane potential at acidic pH . The addition of a low concentration of detergent raised urease activity of intact bacteria at neutral pH to that of their homogenates, showing that urease activity was membrane limited . No acidic pH activation or urea induced membrane potential changes were found in the nongastric Helicobacter species . The ureI gene product is probably a pH activated urea transporter or perhaps regulates such a transporter as a function of periplasmic pH.

Proc Natl Acad Sci U S A, 2000 Jan 18, 97(2), 599 - 604
NifS-directed assembly of a transient {2Fe-2S} cluster within the NifU protein; Yuvaniyama P et al.; The NifS and NifU proteins from Azotobacter vinelandii are required for the full activation of nitrogenase . NifS is a homodimeric cysteine desulfurase that supplies the inorganic sulfide necessary for formation of the Fe-S clusters contained within the nitrogenase component proteins . NifU has been suggested to complement NifS either by mobilizing the Fe necessary for nitrogenase Fe-S cluster formation or by providing an intermediate Fe-S cluster assembly site . As isolated, the homodimeric NifU protein contains one {2Fe-2S}(2+, +) cluster per subunit, which is referred to as the permanent cluster . In this report, we show that NifU is able to interact with NifS and that a second, transient {2Fe-2S} cluster can be assembled within NifU in vitro when incubated in the presence of ferric ion, L-cysteine, and catalytic amounts of NifS . Approximately one transient {2Fe-2S} cluster is assembled per homodimer . The transient {2Fe-2S} cluster species is labile and rapidly released on reduction . We propose that transient {2Fe-2S} cluster units are formed on NifU and then released to supply the inorganic iron and sulfur necessary for maturation of the nitrogenase component proteins . The role of the permanent {2Fe-2S} clusters contained within NifU is not yet known, but they could have a redox function involving either the formation or release of transient {2Fe-2S} cluster units assembled on NifU . Because homologs to both NifU and NifS, respectively designated IscU and IscS, are found in non-nitrogen fixing organisms, it is possible that the function of NifU proposed here could represent a general mechanism for the maturation of Fe-S cluster-containing proteins.

J Physiol Pharmacol, 1999 Dec, 50(4), 541 - 50
Endotoxin-endothelium interactions in "low-perfusion state" research; Guc MO; LPS/endotoxin provokes a plethora of pathological events some of which may be considered as examples of "low perfusion state" . These are discussed here . It is well known that hypotension and refractoriness to vasocostrictors are the hallmark of endotoxic shock . Nevertheless, there are some vascular beds, such as mesenteric circulation, that respond with vasoconstriction - not vasodilation to endotoxin . Aminoguanidine, an inhibitor of NOS-2, blocks endotoxin- induced increase of resistance in mesenteric bed and endotoxin-induced translocation of bacteria through the gut wall . It is postulatede that endotoxin has antiarrythmogenic action due to the release of nitric oxide and increase in intracellular cGMP levels . Although we demonstrate that endotoxin increases nitric oxide formation in spleen and liver, its contribution to the injury of these organs by endotoxin is not fully established . In addition, we present our immunochemistry data on nitrotyrosine formation in the liver and spleen of endotoxin-treated animals.

Chirurg, 1999 Dec, 70(12), 1422 - 33
{Supportive measures in palliation: pain therapy and nutrition}; Hohenberger P et al.; Pain relief and nutritional support represent two main efforts of palliative medicine . A considerable proportion of surgical patients might not be treated with adequate analgetic medication . Those patients are often treated too late, too short or with an insufficient amount of drug . Particularly if the treatment goal is palliation problems of drug abuse are of less importance . However, randomized trials aiming at best pain relief have rarely been carried out in oncological patients . Psychological factors (suffering, affective aspects) have to be borne in mind when deciding upon pain treatment . The surgeon often knows best the local problem inducing pain whether it is due to intestinal obstruction, infiltration of bone and joints, arising from the viscera, or resulting from nerve compression . This information is of utmost value to select the most appropriate treatment . Parenteral, local, or regional measures to relief pain can be combined with chemical neurolysis . Receptor-specific drugs may be the analgetics of the future . Regarding nutritional support the patient's acceptance must be respected . Other guidelines concern life expectancy, nutritional status, or intestinal function and influence the decision whether or not nutritional support should be offered . Enteral feeding should always be the treatment of first choice due to economical and logistic reasons but also due to the fact that translocation of bacteria and endotoxin can be minimised with this technique.

J Exp Med, 2000 Jan 17, 191(2), 355 - 64
Tetracycline-controllable selection of CD4(+) T cells: half-life and survival signals in the absence of major histocompatibility complex class II molecules; Witherden D et al.; A system that allows the study, in a gentle fashion, of the role of MHC molecules in naive T cell survival is described . Major histocompatibility complex class II-deficient mice were engineered to express Ealpha chains only in thymic epithelial cells in a tetracycline (tet)-controllable manner . This resulted in tet-responsive display of cell surface E complexes, positive selection of CD4(+)8(-) thymocytes, and generation of a CD4(+) T cell compartment in a class II-barren periphery . Using this system, we have addressed two unresolved issues: the half-life of naive CD4(+) T cells in the absence of class II molecules (3-4 wk) and the early signaling events associated with class II molecule engagement by naive CD4(+) T cells (partial CD3 zeta chain phosphorylation and ZAP-70 association).

J Biol Chem, 2000 Jan 21, 275(3), 1529 - 32
Chloroplast Oxa1p homolog albino3 is required for post-translational integration of the light harvesting chlorophyll-binding protein into thylakoid membranes; Moore M et al.; Multiple sorting pathways operate in chloroplasts to localize proteins to the thylakoid membrane . The signal recognition particle (SRP) pathway in chloroplasts employs the function of a signal recognition particle (cpSRP) to target light harvesting chlorophyll-binding protein (LHCP) to the thylakoid membrane . In assays that reconstitute stroma-dependent LHCP integration in vitro, the stroma is replaceable by the addition of GTP, cpSRP, and an SRP receptor homolog, cpFtsY . Still lacking is an understanding of events that take place at the thylakoid membrane including the identification of membrane proteins that may function at the level of cpFtsY binding or LHCP integration . The identification of Oxa1p in mitochondria, an inner membrane translocase component homologous to predicted proteins in bacteria and to the albino3 (ALB3) protein in thylakoids, led us to investigate the potential role of ALB3 in LHCP integration . Antibody raised against a 50-amino acid region of ALB3 (ALB3-50aa) identified a single 45-kDa thylakoid protein . Treatment of thylakoids with antibody to ALB3-50aa inhibited LHCP integration, whereas the same antibody treatment performed in the presence of antigen reversed the inhibition . In contrast, transport by the thylakoid Sec or Delta pH pathways was unaffected . These data support a model whereby a distinct translocase containing ALB3 is used to integrate LHCP into thylakoid membranes.

Ann Surg, 2000 Jan, 231(1), 88 - 95
Lack of correlation between failure of gut barrier function and septic complications after major upper gastrointestinal surgery; Kanwar S et al.; OBJECTIVE: To determine the influence of abnormal gut barrier function on the risk of septic complications in patients undergoing major resectional surgery for upper gastrointestinal cancer . SUMMARY BACKGROUND DATA: A failure of the gut mucosal barrier to exclude bacteria and endotoxin from the portal and systemic circulation is incriminated in the development of sepsis and multiple organ failure . Although the experimental data is compelling, corroborative evidence from studies in humans is sparse . This study attempted to correlate both preoperative gut barrier dysfunction and the pattern of change after surgery with septic outcome . METHODS: Sixty-eight patients undergoing curative resectional surgery for upper gastrointestinal cancer were monitored for 30-day septic morbidity (intraabdominal abscesses/empyema and pneumonia) . Intestinal permeability, serum IgM and IgG anti-endotoxin antibodies (EndoCAb), and serum C-reactive protein were measured before surgery and on postoperative days 1 and 7 . RESULTS: Increased intestinal permeability before surgery did not predict septic outcome . Major surgery was associated with increased intestinal permeability and evidence of endotoxin exposure . Comparing sepsis and nonsepsis groups, however, there was no significant difference in intestinal permeability, endotoxin exposure, and the acute phase response after surgery . CONCLUSIONS: This study demonstrates that gut barrier dysfunction occurs after surgery, but the magnitude of change does not differentiate patients in whom sepsis develops and those in whom it does not . Preoperative increased intestinal permeability had no predictive value for sepsis . This study failed to support the thesis that gut barrier dysfunction is directly linked to sepsis.

Sci Total Environ, 1999 Dec 15, 243-244, 97 - 105
Assessment of toxicity hazards of dredged lake sediment contaminated by creosote; Hyotylainen T et al.; In order to predict the potential toxicity hazards of sediment remediation by dredging, an experimental laboratory simulation was made by investigating seven ratios of creosote-contaminated sediment (Lake Jamsanvesi, central Finland) and artificial lake water mixtures . Sediment was suspended in water at the ratios of 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 v/v . The elutriates were analysed for the acute toxicity by photoluminescence bacterial and waterflea (Daphnia magna Straus) tests . The concentrations of polycyclic aromatic hydrocarbons (PAHs) are determined by gas chromatography (GC/FID) . The elutriate of ratio 1:2 was most toxic to bacteria (EC50 = 4.5%), whereas the ratio 1:4 was most toxic to waterfleas (EC50 = 21%) . The elutriate of 1:1 contained the highest total PAH-concentration (1.67 mg/l) and total organic carbon (TOC) content (39.4 mg/l) . When compared to the 1:1 ratio, taken as unity, the relative toxic emission yield (RTE) for bacteria was 307 for the ratio 1:128, so the high mixing ratio may cause a considerable ecotoxicological hazard . The highest amounts of PAHs were desorbed from sediment to water layer when the sediment was mixed with water at the ratios 1:1, 1:2 and 1:4 (v/v) . It is assumed that dredging of creosote-contaminated sediment can potentially cause an ecotoxicological risk for a lake system at wide range of suspension ratios . We recommended that basic knowledge for these risks can be produced by simple laboratory simulation.

Quintessence Int, 1999 May, 30(5), 319 - 23
Mechanical effects and volatile sulfur compound-reducing effects of chewing gums: comparison between test and base gums and a control group; Reingewirtz Y et al.; OBJECTIVE: Chewing gum may act as a masking or a therapeutic agent against the different chemical compounds that are responsible for oral malodor . An open-label exploratory study investigated the effect of mastication and aromatic components of chewing gum on reducing oral volatile sulfur compounds . METHOD AND MATERIALS: Twelve dental students (5 males and 7 females) acted as their own controls . Toothbrushing stopped 12 hours before observations . Measurements included organoleptic and volatile sulfur compound scores and the pH of the anterior and posterior zones of the dorsal tongue . Measurements were made at 9 AM and 12 PM on 1 day for 3 successive weeks; week 1, no gum (control); week 2, test gum; week 3, unsweetened gum base . This open-label study was then completed by an observer-blind study, according to the same schedule; the recorded measurement was the plaque index . RESULTS: The pH, volatile sulfur compounds, and organoleptic scores were similar for all groups . The pH was more basic in the posterior part than in the anterior zone of the dorsal tongue, irrespective of time and presence or absence of chewing gum . In addition, the volatile sulfur compound score rose transiently immediately after the test gum, and the organoleptic score fell in the first hour only after the test gum . The two chewing gum groups seemed to have a greater reduction in plaque index than did the control (no gum) group . CONCLUSION: Chewing gum may have a valuable mechanical role in cleaning dental surfaces, and the test gum may temporarily control bad breath . After 3 hours, similar volatile sulfur compound scores were observed for subjects who chewed either test or unsweetened gum base and control subjects.

J Endod, 1999 Aug, 25(8), 555 - 8
Survey for collagenase gene prtC in Porphyromonas gingivalis and Porphyromonas endodontalis isolated from endodontic infections; Odell LJ et al.; Collagenase is a potential virulence factor shown to be expressed by Porphyromonas gingivalis associated with periodontal disease . The purpose of this study was to use the polymerase chain reaction (PCR) to detect the presence of the collagenase gene (prtC) in 21 strains of Porphyromonas species isolated from endodontic infections . Type strains for P . gingivalis (ATCC 33277), P . endodontalis (ATCC 35406), Prevotella intermedia (ATCC 25611), and Prevotella nigrescens (ATCC 33563) were used as controls . When PCR primers specific for the 16S ribosomal RNA gene of P . gingivalis or P . endodontalis were used, 16 of the strains were identified as P . gingivalis, and five strains were identified as P . endodontalis . The presence of the prtC gene for collagenase was detected using PCR . Amplicons were analyzed by agarose gel electrophoresis, with an 815 bp amplicon representing the presence of the collagenase gene . Type strain ATCC 33277 and all 16 clinical isolates of P . gingivalis produced the collagenase gene amplicon . Neither type strain ATCC 35406 nor the five strains from clinical isolates of P . endodontalis produced the collagenase gene amplicon . These results indicate that P . gingivalis from endodontic infections possesses the prtC gene . P . endodontalis does not seem to exhibit prtC . The virulence of P . gingivalis may be related to its production of collagenase.

J Indian Soc Pedod Prev Dent, 1997 Dec, 15(4), 109 - 13
The role of diet, fluoride and saliva in caries prevention; Stephen M; Saliva is rich in calcium and phosphates, facilitating remineralization of early carious lesions . There is evidence that remineralization is associated with an increase in the size of enamel crystals and a consequent increase in resistance to caries . The contribution of sucrose to the implantation, colonization and metabolic activities of cariogenic bacteria has been clearly established, and has led to the search for sucrose substitutes . Recent report from Australia and the United States of America have reconfirmed the safety and efficacy of fluoride in preventing dental caries . The use of fluoride in various forms thus remains the cornerstone of most caries prevention programme.

Am J Gastroenterol, 2000 Jan, 95(1 Suppl), S14 - 5
Diarrheal disease in a developing nation; Ribeiro H Jr; Infants and young children from many developing nations face a serious public health threat from diarrhea . In the northeast region of Brazil, diarrheal disease causes significant morbidity and mortality . Although diarrheal deaths in this region have been declining since the early 1980s, the hospitalization rate remains high . Diarrhea may account for 25% of total healthcare costs in northeast Brazil . Many patients become ill because of poor environmental conditions and because their caregivers lack awareness of basic hygiene . Many also return to the hospital after initial treatment because health workers do not provide appropriate preventive and home care instruction to patients in 99% of cases . Antibiotics have no effect in 85-95% of pediatric diarrheal cases in the region . Improvement of preventive home care management in association with probiotic bacteria, which have been effective against several forms of diarrhea in developing nations, may be useful in managing diarrheal disease in infants.

Korean J Parasitol, 1999 Dec, 37(4), 215 - 28
General properties and phylogenetic utilities of nuclear ribosomal DNA and mitochondrial DNA commonly used in molecular systematics; Hwang UW et al.; To choose one or more appropriate molecular markers or gene regions for resolving a particular systematic question among the organisms at a certain categorical level is still a very difficult process . The primary goal of this review, therefore, is to provide a theoretical information in choosing one or more molecular markers or gene regions by illustrating general properties and phylogenetic utilities of nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) that have been most commonly used for phylogenetic researches . The highly conserved molecular markers and/or gene regions are useful for investigating phylogenetic relationships at higher categorical levels (deep branches of evolutionary history) . On the other hand, the hypervariable molecular markers and/or gene regions are useful for elucidating phylogenetic relationships at lower categorical levels (recently diverged branches) . In summary, different selective forces have led to the evolution of various molecular markers or gene regions with varying degrees of sequence conservation . Thus, appropriate molecular markers or gene regions should be chosen with even greater caution to deduce true phylogenetic relationships over a broad taxonomic spectrum.

Eur J Endocrinol, 2000 Jan, 142(1), 1 - 8
Animal models of Graves' disease; Ludgate M; Graves' disease (GD) is an autoimmune condition in which goitre and hyperthyroidism are induced by thyroid stimulating antibodies (TSAB) which mimic the action of thyrotrophin (TSH) . The target of the autoimmune response is the thyrotrophin receptor (TSHR) and, since its cloning, a number of differing approaches have been adopted in an attempt to develop an animal model of GD . Methods in which synthetic peptides or fragments of the receptor produced in bacteria or insect cells have been injected into animals together with immunological adjuvants have had only limited success in inducing some of the signs and symptoms of GD . Genetic immunisation resulted in thyroiditis in the majority, but TSAB formation in only a minority, of treated inbred mice . Transfer of receptor in vitro primed T cells to syngeneic naive recipients, with priming either using a bacterial fusion protein or genetic immunisation, induced destructive thyroiditis in non-obese diabetic (NOD) mice but lymphocytic thyroiditis in BALBc mice . Furthermore, the orbits of 17/22 of the BALBc animals, but not the NOD animals, with thyroiditis had orbital changes similar to those seen in thyroid eye disease . TSAB and elevated thyroxine levels were induced in AKR/N mice injected with fibroblasts expressing the full length human TSHR and murine major histocompatibility complex (MHC) class II homologous to the recipient mice . No thyroiditis was induced but preliminary results from a different group using the same protocol suggest that receptor autoantibodies and thyroid dysfunction could be transferred using T cells primed in vitro with the receptor and MHC-II expressing cells . The majority of the studies described above have studied inbred mouse strains . In a novel departure, the NMR outbred strain has been treated by genetic immunisation with very promising results, including the induction of increased thyroxine levels in 4/30 female mice, accompanied by TSAB in addition to thyroiditis, and with signs of hyperactivity and orbital pathology . This review discusses the various protocols together with the information regarding the pathogenesis of GD which each has contributed, and concludes with an evaluation of how close we are to mimicking this polygenic, multifactorial disease.

J Bacteriol, 2000 Feb, 182(3), 749 - 57
Discovery of a nonclassical siderophore, legiobactin, produced by strains of Legionella pneumophila; Liles MR et al.; The mechanisms by which Legionella pneumophila, a facultative intracellular parasite and the agent of Legionnaires' disease, acquires iron are largely unexplained . Several earlier studies indicated that L . pneumophila does not elaborate siderophores . However, we now present evidence that supernatants from L . pneumophila cultures can contain a nonproteinaceous, high-affinity iron chelator . More specifically, when aerobically grown in a low-iron, chemically defined medium (CDM), L . pneumophila secretes a substance that is reactive in the chrome azurol S (CAS) assay . Importantly, the siderophore-like activity was only observed when the CDM cultures were inoculated to relatively high density with bacteria that had been grown overnight to log or early stationary phase in CDM or buffered yeast extract . Inocula derived from late-stationary-phase cultures, despite ultimately growing, consistently failed to result in the elaboration of siderophore-like activity . The Legionella CAS reactivity was detected in the culture supernatants of the serogroup 1 strains 130b and Philadelphia-1, as well as those from representatives of other serogroups and other Legionella species . The CAS-reactive substance was resistant to boiling and protease treatment and was associated with the <1-kDa supernatant fraction . As would also be expected for a siderophore, the addition of 0.5 or 2.0 microM iron to the cultures repressed the expression of the CAS-reactive substance . Interestingly, the supernatants were negative in the Arnow, Csaky, and Rioux assays, indicating that the Legionella siderophore was not a classic catecholate or hydroxamate and, hence, might have a novel structure . We have designated the L . pneumophila siderophore legiobactin.

J Bacteriol, 2000 Feb, 182(3), 599 - 606
Environmental regulation of exopolysaccharide production in Sinorhizobium meliloti; Mendrygal KE et al.; Exopolysaccharide production by Sinorhizobium meliloti is required for invasion of root nodules on alfalfa and successful establishment of a nitrogen-fixing symbiosis between the two partners . S . meliloti wild-type strain Rm1021 requires production of either succinoglycan, a polymer of repeating octasaccharide subunits, or EPS II, an exopolysaccharide of repeating dimer subunits . The reason for the production of two functional exopolysaccharides is not clear . Earlier reports suggested that low-phosphate conditions stimulate the production of EPS II in Rm1021 . We found that phosphate concentrations determine which exopolysaccharide is produced by S . meliloti . The low-phosphate conditions normally found in the soil (1 to 10 microM) stimulate EPS II production, while the high-phosphate conditions inside the nodule (20 to 100 mM) block EPS II synthesis and induce the production of succinoglycan . Interestingly, the EPS II produced by S . meliloti in low-phosphate conditions does not allow the invasion of alfalfa nodules . We propose that this invasion phenotype is due to the lack of the active molecular weight fraction of EPS II required for nodule invasion . An analysis of the function of PhoB in this differential exopolysaccharide production is presented.

Scand J Immunol, 2000 Jan, 51(1), 79 - 86
High resolution electroelution of polyacrylamide gels for the purification of single proteins from Mycobacterium tuberculosis culture filtrate; Weldingh K et al.; Culture filtrate from Mycobacterium tuberculosis contains protective molecules which have been used successfully in experimental vaccines against tuberculosis . Despite an increasing number of mycobacterial proteins being characterised, a major effort is still needed to get an overview of the many potentially interesting molecules in culture filtrate . In this study we describe a high throughput method for purification and biological evaluation of protein components in complex protein mixtures . The method presents a new application of the recently developed Mini Whole Gel Eluter and employs this apparatus for the high resolution electroelution of selected molecular mass fractions of protein mixtures previously separated in large polyacrylamide gels . Two novel M . tuberculosis culture filtrate proteins (CspA and TB18.6) were purified by this method, their N-terminal sequences were determined and the open reading frame encoding each of the proteins identified . The immunological recognition of the molecules were evaluated in tuberculosis infected mice and guinea pigs . Both proteins induced DTH responses in guinea pigs and IFN-gamma release from spleen lymphocytes isolated from infected mice.

Eur J Biochem, 2000 Jan, 267(2), 591 - 600
Phosphorylation of the signal transducer PII protein and an additional effector are required for the PII-mediated regulation of nitrate and nitrite uptake in the Cyanobacterium synechococcus sp . PCC 7942; Lee HM et al.; In the cyanobacterium Synechococcus sp . strain PCC 7942, the phosphorylation states of the signal transducer PII protein (GlnB) can change rapidly depending on the nitrogen and carbon supply . A PII-null mutant (MP2) shows no ammonium-dependent inhibition of the nitrate and nitrite uptake, in contrast to the wild-type . New mutants with different types of PII, which may mimic either the phosphorylated (GlnBS49E or GlnBS49D) or unphosphorylated (GlnBS49A) form of the protein, were constructed using site-directed in vitro mutagenesis . Mutant MP2-A (GlnBS49A) grew poorly using nitrate as a nitrogen source and was unable to take up nitrate supplied at 100 microM, even in the absence of externally added ammonium . Mutants MP2-D and MP2-E (GlnBS49D and GlnBS49E, respectively), however, showed nitrate-dependent growth and regulation of nitrate uptake by ammonium, as in the wild-type . Characterization of the mutants also included an analysis of nitrite uptake and of the levels of the nir (nitrate/nitrite assimilation) operon transcripts, the presence of NrtA (nitrate/nitrite transport binding protein), and nitrate and nitrite reductase activities . In vitro, no significant difference was observed in the cooperative binding of ATP and 2-oxoglutarate between the wild-type and the unphosphorylated or phosphorylated-like forms of the mutant PII proteins . The results obtained indicate that both unphosphorylated and phosphorylated-like forms of PII are able to inhibit nitrate uptake in the presence of ammonium, but the unphosphorylated form also has a negative effect in the absence of this nitrogen source . Therefore, an additional effector, possibly 2-oxoglutarate, is required for the PII protein to relieve inhibition of nitrate uptake in the absence of ammonium.

Eur J Biochem, 2000 Jan, 267(2), 361 - 9
A transglycosylating 1,3(4)-beta-glucanase from rhodothermus marinus NMR analysis of enzyme reactions; Petersen BO et al.; The enzymatic hydrolysis of polysaccharides by the 1, 3(4)-beta-glucanase (LamR) from Rhodothermus marinus has been explored . The enzyme cleaves the 1,3-beta-linkages of 3-O-substituted glucose units in 1,3-beta-glucans such as laminarin and curdlan, and also the 1,4-beta-linkages of 3-O-substituted beta-glucose in beta-glucans such as lichenin and 1,3-1, 4-beta-glucan from the cell walls of barley endosperm . The polysaccharide substrates (laminarin, curdlan and barley beta-glucan) were characterised using NMR spectroscopy . The reaction of LamR with its substrates was followed by recording one-dimensional and two-dimensional 1H-NMR and 13C-NMR spectra at suitable time intervals after addition of the enzyme . It is shown that hydrolysis occurs with retention of the anomeric configuration and that LamR performs transglycosylation to generate both 1, 3-beta-glycosidic and 1,4-beta glycosidic linkages . The transglycosylation results in, e.g . formation of the trisaccharide 4-O-glucosyl-laminaribiose from exclusively 1,3-beta-oligoglucosides . When barley 1,3-1,4-beta-glucan was incubated with LamR the beta-1, 4-linkages of 3-O-substituted beta-glycosyl residues were rapidly hydrolysed . Simultaneously de novo formation of 1,3-beta-glycosidic linkages was observed which, however, were cleaved during prolonged incubations . It is shown that a laminaribiosyl unit is the minimum requirement for formation of an enzyme-substrate complex and subsequent hydrolysis/transglycosylation.

Aliment Pharmacol Ther, 2000 Jan, 14(1), 7 - 14
Review article: nitroimidazole resistance in Helicobacter pylori; Van Der Wouden EJ et al.; The efficacy of a nitroimidazole-containing regimen for the treatment of Helicobacter pylori infection is decreased by nitroimidazole resistance . Nitroimidazoles are meta- bolized by H . pylori by several nitro-reductases of which an oxygen-insensitive NADPH nitroreductase encoded by the rdxA gene is the most important . Null mutations in this gene are associated with resistance . Susceptibility testing to nitroimidazoles may give variable results . This is not only related to the slow growth under specific conditions, but also to variability in the activity of the other nitroreductases and the ability to deactivate toxic metabolites of an NI and to repair DNA damage . Moreover, co-infections with resistant and susceptible bacteria are frequently found . The presence of nitroimidazole resistance is related to the previous use of this drug . The prevalence of resistance is rising and nowadays 10-50% of the isolates are resistant . Resistance reduces the efficacy of a treatment regimen to a variable degree . This is related to efficacy of the other components of the regimen and the treatment duration . Whether a nitroimidazole is still effective in resistant strains remains unresolved . When nitroimidazole resistance is present, a nitro-imidazole-containing regimen should be avoided or a regimen with other highly effective components should be used.

J Periodontol, 1999 Dec, 70(12), 1472 - 8
Periodontal pathogens stimulate CC-chemokine production by mononuclear and bone-derived cells; Jiang Y et al.; BACKGROUND: Chemokines are chemotactic cytokines that stimulate recruitment of leukocytes . Monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and RANTES (regulated on activation, normal T cell expressed, and secreted) are 3 well-characterized CC-chemokines that regulate mononuclear cell recruitment . The recruitment of inflammatory cells is of particular importance in the oral cavity because of the likelihood that cells will be challenged with bacteria either during acute infection or following surgical procedures . Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans are putative periodontal pathogens that may be harbored in subgingival and supragingival plaque . The capacity of the host to respond to these bacteria by the elaboration of chemoattractants may represent an important defense mechanism . METHODS: In the present study, we examined CC-chemokine production by human mononuclear cells and bone-derived cells in response to P . gingivalis, A . actinomycetemcomitans and lipopolysaccharides (LPS) stimulation in vitro . The chemokines produced were measured by ELISA . RESULTS: The results demonstrate that P . gingivalis and A . actinomycetemcomitans induce high levels of MIP-1alpha in mononuclear cells . P . gingivalis and A . actinomycetemcomitans stimulated high levels of MCP-1 in bone-derived cells and induced moderate levels of RANTES production in both mononuclear and osteoblastic cells . In mononuclear cells, LPS induced high levels of MIP-1alpha and RANTES and moderate levels of MCP-1; in osteoblasts LPS only induced MCP-1 . CONCLUSIONS: The capacity of bacteria to induce a given chemokine depends upon the cell type stimulated . That different cell types would exhibit differences in the CC-chemokines produced under the same stimulus provides a mechanism to explain tissue-specific recruitment of leukocytes.

J Periodontol, 1999 Dec, 70(12), 1464 - 71
Regulation of gingival fibroblast interleukin-6 secretion by cyclosporine A; Morton RS et al.; BACKGROUND: Cyclosporine A (CsA) is a widely used immunosuppressant, with clinical applications ranging from organ transplants to chronic inflammatory diseases . One of the side effects associated with CsA treatment is the development of gingival overgrowth . Exuberant growth of connective tissue within the periodontium can result from hyperactivity of resident fibroblasts . Fibroblasts are capable of secreting interleukin-6 (IL-6), which has been shown to enhance proliferation as well as collagen and glycosaminoglycan synthesis by these cells . We tested the hypothesis that one of the pathogenetic mechanisms underlying CsA-induced fibrosis is an enhanced IL-6 secretion by gingival fibroblasts (GF) in response to this drug . METHODS: The ability of CsA to upregulate GF IL-6 secretion alone or in combination with bacterial challenge or other inflammatory cytokines was tested in an in vitro system . Fibroblast cultures were established from systemically healthy gingival tissue donors and were challenged with CsA in the absence or presence of bacteria, IL-1beta, or tumor necrosis factor (TNF) alpha as co-stimulants . Nifedipine and phenytoin were also tested to further support findings with CsA . After 72 hours of incubation, culture supernatants were collected and analyzed for IL-6 content by ELISA . RESULTS: We have shown that GF respond to CsA with an increase in IL-6 secretion . The magnitude of this response varies among cultures derived from different tissue donors . We have also demonstrated that GF IL-6 responses to bacterial challenge or TNFalpha are downregulated by CsA . However, CsA synergizes with IL-1beta to further upregulate IL-6 secretion, and this effect is shared by phenytoin and nifedipine . CONCLUSIONS: We conclude that one of the pathogenetic mechanisms underlying drug-induced gingival overgrowth may be enhanced secretion of IL-6 by GF in response to these medications . This is the first report on direct and indirect effects of gingival overgrowth-related medications on GF IL-6 metabolism . This work will lay the foundation for future studies directed towards the development of prevention or treatment modalities for gingival overgrowth based on blocking the fibrogenic activities of IL-6 at the cellular level.

J Periodontol, 1999 Dec, 70(12), 1443 - 8
Chlorhexidine-induced changes to human gingival fibroblast collagen and non-collagen protein production; Mariotti AJ et al.; BACKGROUND: Chlorhexidine (CHX) has been used extensively as an adjunctive therapy in the treatment of periodontal disease . It is well known that chlorhexidine is toxic to bacteria, but recent evidence has suggested that chlorhexidine may also have deleterious effects on gingival fibroblast proliferation as well as collagen and non-collagen protein production in cell culture . The purpose of this study was to examine the effects of chlorhexidine on gingival fibroblast proliferation as well as collagen and non-collagen protein production in cell culture . METHODS: Human gingival fibroblasts were incubated in MEM containing chlorhexidine concentrations ranging from 1 microM to 1300 microM at 37 degrees C for 1, 5, or 15 minutes . Control cells received an equal volume of MEM without chlorhexidine for similar times at 37 degrees C . Following incubation, the media were removed, cells washed twice with MEM supplemented with 10% FBS, and fibroblasts in treatment and control groups were allowed to recover in the same media for 24 hours . RESULTS: In all strains, cellular proliferation was dependent on the concentration of solubilized chlorhexidine in cell culture but independent of the duration of chlorhexidine exposure . The average inhibitory concentration necessary to reduce cellular proliferation by 50% (ID50) was 222.1 microM . In regard to collagen and non-collagen protein production, fibroblasts exposed to chlorhexidine concentrations (1 microM) well below the ID50 had a 65% reduction in collagen production and a 57% reduction in noncollagen protein production . CONCLUSIONS: These results suggest that chlorhexidine will induce a dose dependent reduction in cellular proliferation and that concentrations of chlorhexidine that have little effect on cellular proliferation can significantly reduce both collagen and noncollagen protein production of human gingival fibroblasts in vitro . Hence, the introduction of commercially available concentrations (0.12%) or diluted commercial concentrations (as low as 0.00009%) of chlorhexidine to surgical sites for short periods of time prior to wound closure can conceivably have serious toxic effects on gingival fibroblasts and may negatively affect wound healing.

Biophys Chem, 1999 Dec 13, 82(2-3), 165 - 71
Proportion of membrane proteins in proteomes of 15 single-cell organisms analyzed by the SOSUI prediction system; Mitaku S et al.; A software system, SOSUI, was previously developed for discriminating between soluble and membrane proteins and predicting transmembrane regions (Hirokawa et al., Bioinformatics, 14 (1998) 378-379) . The performance of the system was 99% for the discrimination between two types of proteins and 96% for the prediction of transmembrane helices . When all of the amino acid sequences from 15 single-cell organisms were analyzed by SOSUI, the proportion of predicted polytopic membrane proteins showed an almost constant value of 15-20%, irrespective of the total genome size . However, single-cell organisms appeared to be categorized in terms of the preference of the number of transmembrane segments: species with small genomes were characterized by a significant peak at a helix number of approximately six or seven; species with large genomes showed a peak at 10 or 11 helices; and species with intermediate genome sizes showed a monotonous decrease of the population of membrane proteins against the number of transmembrane helices.

Mutat Res, 1999 Dec 6, 430(2), 211 - 9
Combined effects of space flight factors and radiation on humans; Todd P et al.; The probability that a dose of ionizing radiation kills a cell is about 10,000 times the probability that the cell will be transformed to malignancy . On the other hand, the number of cells killed required to significantly impact health is about 10,000 times the number that must be transformed to cause a late malignancy . If these two risks, cell killing and malignant transformation, are about equal, then the risk that occurs during a mission is more significant than the risk that occurs after a mission . The latent period for acute irradiation effects (cell killing) is about 2-4 weeks; the latent period for malignancy is 10-20 years . If these statements are approximately true, then the impact of cell killing on health in the low-gravity environment of space flight should be examined to establish an estimate of risk . The objective of this study is to synthesize data and conclusions from three areas of space biology and environmental health to arrive at rational risk assessment for radiations received by spacecraft crews: (1) the increased physiological demands of the space flight environment; (2) the effects of the space flight environment on physiological systems; and (3) the effects of radiation on physiological systems . One physiological system has been chosen: the immune response and its components, consisting of myeloid and lymphoid proliferative cell compartments . Best-case and worst-case scenarios are considered . In the worst case, a doubling of immune-function demand, accompanied by a halving of immune capacity, would reduce the endangering dose to a crew member to around 1 Gy.

Exp Parasitol, 2000 Jan, 94(1), 33 - 41
BCG expressing LCR1 of Leishmania chagasi induces protective immunity in susceptible mice; Streit JA et al.; Cellular immune responses are required for protective immunity against Leishmania chagasi . Immunization strategies using live intracellular bacteria (e.g., bacille-Calmette Guerin strain of Mycobacterium bovis) expressing recombinant antigens can induce cellular immune responses to these antigens . Previous studies demonstrated that the L . chagasi antigen LCR1 stimulates IFN-gamma production from T cells of infected BALB/c mice, and immunization with recombinant LCR1 partially protects against L . chagasi infection . To determine whether live bacteria could enhance the immunization potential of LCR1, we engineered BCG expressing LCR1 (BCG-LCR1) . Subcutaneous immunization with BCG-LCR1, but not with BCG containing plasmid only (BCG-pMV261), elicited better protective immunity against L . chagasi infection than LCR1 protein alone . BCG-LCR1 administered intraperitoneally did not protect . Splenocytes from mice immunized s.c . with either BCG-LCR1 or BCG-pMV261 and then infected with L . chagasi promastigotes had increased antigen-induced IFN-gamma and reduced IL-10 production compared to splenocytes of control mice . We propose that BCG-LCR1 promotes a Th1-type protective immune response, and it may be a useful component of a Leishmania vaccine .

Yakugaku Zasshi, 1999 Dec, 119(12), 921 - 8
{Quantitative analysis of factors to influence the environment of the clean room and clean bench during preparation of intravenous hyperalimentation (IVH) admixtures}; Hotoda S et al.; We quantitatively studied factors influencing the environment cleanliness for intravenous hyperalimentation (IVH) admixing . The environment cleanliness was evaluated by measuring the counts of particles (> 0.5 micron) and bacteria floating in 1 ft3 of the air inside the clean room (23.6 m3) and in the clean bench built in the department of pharmacy, The University of Tokyo Hospital in 1998 . The number of particles at the center of the clean room during IVH admixing by 4 pharmacists was higher than that at the medicine passing area (150 +/- 50/ft3 vs . 260 +/- 60/ft3; mean +/- S.D., n = 12) . The cleanliness inside the clean room was improved as the measurement point became higher from the floor (600 +/- 180/ft3, 150 +/- 50/ft3, and 35 +/- 15/ft3 at 50, 100, and 150 cm height, respectively) and the number of persons working inside the room decreased . The changes in the counts of floating bacteria were similar to that of floating particles under the same conditions . In addition the effect of disinfection on the counts of bacteria was clearly observed . When the cleanliness of the room became lower by turning off the air conditioning, the particle counts inside the clean bench became lower along with the distance from the front glass becoming deeper (i.e., 1400 +/- 550/ft3, 140 +/- 70/ft3, and 40 +/- 30/ft3 at 0, 5, and 15 cm, respectively) . From these lines of evidence, the following items were suggested in order to maintain the environment cleanliness for IVH admixing . First, the number of persons residing in the clean room should be kept to be minimum . Second, the clean bench should be set up in the center of the clean room . Finally IVH admixing operation should be performed at more than 15 cm depth inside the front glass surface of the clean bench . Moreover, the effect of mopping-up of the clean room with 0.1% benzethonium chloride clearly demonstrated the importance of disinfection on a routine basis.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2000 Jan, 89(1), 99 - 103
Apical terminus location of root canal treatment procedures; Wu MK et al.; The apical termination of root canal treatment is considered an important factor in treatment success . The exact impact of termination is somewhat uncertain; most publications on outcomes are based on retrospective findings . After vital pulpectomy, the best success rate has been reported when the procedures terminated 2 to 3 mm short of the radiographic apex . With pulpal necrosis, bacteria and their byproducts, as well as infected dentinal debris may remain in the most apical portion of the canal; these irritants may jeopardize apical healing . In these cases, better success was achieved when the procedures terminated at or within 2 mm of the radiographic apex (0 to 2 mm) . When the therapeutic procedures were shorter than 2 mm from or past the radiographic apex, the success rate for infected canals was approximately 20% lower than that when the procedures terminated at 0 to 2 mm . Clinical determination of apical canal anatomy is difficult . An apical constriction is often absent . Based on biologic and clinical principles, instrumentation and obturation should not extend beyond the apical foramen.

Mol Cell Biochem, 1999 Nov, 201(1-2), 159 - 67
Characterization of phospholipase A1, A2, C activity in Ureaplasma urealyticum membranes; DeSilva NS et al.; The presence of endogenous phospholipase A (PL-A) activity of U . urealyticum hydrolyzing the acyl ester bond and phospholipase C (PL-C) activity hydrolyzing the phosphodiester bond is primarily localized in the membranes of ureaplasmas . Characterization of the membrane PL-A and PL-C activity in exponential growing cells of serovars 3, 4, and 8 was investigated . The pH optimum was about 8.5-9 for phospholipase A1 (PL-A1) in the three serovars . A more acidic pH optimum of 6 was observed for phospholipase A2 (PL-A2) enzymes in serovars 3 and 4 . However, a very significant stimulation of PL-A2 activity in serovar 8 occurred around pH 7 . The specific activity of PL-A2 was always 50-100 fold higher than PL-A1 activity in the pH range studied . Ca2+ ions only slightly stimulated PL-A1 activity in all 3 serovars . PL-A1 activity was stimulated about 6-fold from 0.5-0.8 mM Ca2+ ion concentrations for serovar 3 and 12-fold for serovar 8 . Only lower concentrations (0.2-0.4 mM) of calcium stimulated PL-A2 activity in serovar 4 . EDTA inhibition corresponded to Ca2+ stimulation for PL-A1 activity for serovars 3 and 8 . A general stimulation of PL-A1 activity by diethyl ether was evident but the degree of stimulation varied with the serovar . Sodium deoxycholate enhanced PL-A activity of serovars 4 and 3, but partially inhibited that of serovar 8 . PL-A activity in the three serovars were not significantly affected by p-hydroxymercuribenzoate, a marker of -SH groups in the enzyme . All 3 serovars were inactivated by heat . A broad pH optimum for PL-C activity was evident around 7-8 . Diethyl ether enhanced PL-C activity of serovar 8 . Sodium deoxycholate and heat were inhibitory to PL-C activity . The results demonstrate that the major characteristics of ureaplasma membrane bound PL-A and PL-C are basically similar to those of other mollicutes and bacteria . However, the major differences in the specific characteristics of specially PL-A1 and PL-A2 suggest that the ureaplasma phospholipases are unique enzymes different from the phospholipases of bacteria . Both the PL-A and PL-C enzymes function over the broad range at which ureaplasma can grow, pH 5-9 essential for survival . The ureaplasma PL-As are also markedly different from one serovar to another . This variation in specific activity could contribute significantly to differences in virulence among serovars in specific host milieus . There is significant variation from acidic pH of the vagina and alveolar surface of the lung to a more neutral pH of the endometrium and placenta . There are marked differences in calcium concentrations under specific circumstances in various host tissues . Thus the differences in specific activity among the phospholipases of the serovars of U . urealyticum may be of physiological importance in interactions with host tissues and pathogenesis of disease.

J Invest Surg, 1999 Nov-Dec, 12(6), 319 - 25
Vitamin A deficiency impairs colonic healing but not adhesion formation in germ-free and conventional rats; Okada M et al.; Vitamin A is well recognized as a factor of major importance in epithelial and connective tissue repair mechanisms . Recently it was shown that vitamin A deficiency caused overgrowth and translocation of intestinal bacteria in rats . The aim of this study was to investigate the healing of colonic anastomoses and formation of postsurgical adhesions in vitamin A-deficient germ-free and conventional rats . Fourteen germ-free and 10 conventional rats were allocated to four groups: germ-free rats not given vitamin A, germ-free rats given vitamin A, conventional rats not given vitamin A, and conventional rats given vitamin A . All rats underwent surgery for colonic anastomosis . Seven days afterward, they were euthanized, and the bursting pressure of colonic anastomosis and formation of peritoneal adhesions were evaluated . The bursting pressures in groups not given vitamin A were lower than in groups given vitamin A . The adhesion scores in germ-free groups were lower than in conventional groups . These findings demonstrated that vitamin A had an important role in healing of colonic anastomoses whether in the presence or absence of intestinal flora, and that intestinal bacteria had a greater effect than vitamin A on formation of postsurgical adhesions . This may suggest that the mechanism of healing of colonic anastomoses differs from that of postsurgical adhesion formation.

J Bacteriol, 2000 Jan, 182(2), 543 - 5
A cyanobacterial gene, sqdX, required for biosynthesis of the sulfolipid sulfoquinovosyldiacylglycerol; Guler S et al.; The sulfolipid sulfoquinovosyldiacylglycerol is present in the photosynthetic membranes of plants and many photosynthetic bacteria . A novel gene, sqdX, essential for sulfolipid biosynthesis in the cyanobacterium Synechococcus sp . strain PCC7942 is proposed to encode the cyanobacterial sulfolipid synthase catalyzing the last reaction of the pathway.

J Bacteriol, 2000 Jan, 182(2), 522 - 5
DNA-Binding proteins possibly involved in regulation of the post-logarithmic-phase expression of lipoprotein P35 in Borrelia burgdorferi; Indest KJ et al.; Previously, we have shown that the transcription of p35, a lipoprotein gene of Borrelia burgdorferi, is upregulated or initiated during the post-logarithmic bacterial growth phase in vitro . To identify potential regulatory factors, we examined the formation of DNA-protein complexes by electromobility shift assay, using a 157-bp DNA fragment that spans the p35 promoter region and cell-free extracts of spirochetes harvested from both logarithmic and stationary growth phases . The binding properties of the extracts with the promoter region of the flaB gene, a constitutively expressed, growth-phase-independent gene, were also compared . The results from these experiments demonstrate that B . burgdorferi stationary-phase cell-free extracts have a growth-phase-dependent DNA binding protein that interacts specifically with the p35 promoter region . We show, in addition, that a segment from the 157-bp p35 promoter region which contains both a T-rich stretch and an inverted repeat is able to compete off the stationary-phase-specific complex when the segment is present in molar excess.

J Bacteriol, 2000 Jan, 182(2), 504 - 7
Proteolysis of the McpA chemoreceptor does not require the Caulobacter major chemotaxis operon; Tsai JW et al.; The degradation of the McpA chemoreceptor in Caulobacter crescentus accompanies the swarmer cell to the stalked-cell differentiation event . To further analyze the requirements for its degradation, we have constructed a series of strains that have deletions in the mcpA gene and in the mcpA chemotaxis operon . Internal deletions of the mcpA gene demonstrate that the highly conserved domain (signalling unit) and the methylation domains are not required for cell cycle-regulated proteolysis . The deletion of the chemotaxis operon, which is absolutely required for chemotaxis and McpA chemoreceptor methylation, has no effect on McpA proteolysis.

Arch Microbiol, 2000 Nov, 174(5), 322 - 33
Maintenance and control of redox poise in Rhodobacter capsulatus strains deficient in the Calvin-Benson-Bassham pathway; Tichi MA et al.; Carbon dioxide serves as the preferred electron acceptor during photoheterotrophic growth of nonsulfur purple photosynthetic bacteria such as Rhodobacter capsulatus and Rhodobacter sphaeroides . This CO2, produced as a result of the oxidation of preferred organic carbon sources, is reduced through reactions of the Calvin-Benson-Bassham reductive pentose phosphate pathway . This pathway is thus crucial to maintain a balanced intracellular oxidation-reduction potential (or redox poise) under photoheterotrophic growth conditions . In the absence of a functional Calvin-Benson-Bassham pathway, either an exogenous electron acceptor, such as dimethylsulfoxide, must be supplied or the organism must somehow develop alternative electron acceptor pathways to preserve the intracellular redox state of the cell . Spontaneous variants of Rba . capsulatus strains deficient in the Calvin-Benson-Bassham pathway that have become photoheterotrophically competent (in the absence of an exogenous electron acceptor) were isolated . These strains (SBP-PHC and RCNd1, RCNd3, and RCNd4) were shown to obviate normal ammonia control and derepress synthesis of the dinitrogenase enzyme complex for the dissipation of excess reducing equivalents and generation of H2 gas via proton reduction . In contrast to previous studies with other organisms, the dinitrogenase reductase polypeptides were maintained in an active and unmodified form in strain SBP-PHC and the respective RCNd strains . Unlike the situation in Rba . sphaeroides, the Rba . capsulatus strains did not regain full ammonia control when complemented with plasmids that reconstituted a functional Calvin-Benson-Bassham pathway . Moreover, dinitrogenase derepression in Rba . capsulatas was responsive to the addition of the auxiliary electron acceptor dimethylsulfoxide . These results indicated a hierarchical control over the removal of reducing equivalents during photoheterotrophic growth that differs from strains of Rba . sphaeroides and Rhodospirillum rubrum deficient in the Calvin-Benson-Bassham pathway.

Acta Vet Scand, 2000, 41(3), 283 - 97
The anatomy of the oesophagus, stomach and intestine in common wolffish (Anarhichas lupus L.): a basis for diagnostic work and research; Hellberg H et al.; The alimentary canal of laboratory-reared common wolffish (Anarhichas lupus L.) was studied using light and electron microscopy . In the oesophagus, a simple columnar microvillous epithelium with transport characteristics was observed in addition to the main striated squamous epithelium . An osmoregulatory function is proposed for the simple columnar epithelium, which was supported by wide, thin-walled vessels . In the stomach, a separate type of neck cells was observed leading into the acinar gastric glands, which morphologically consist of one cell type: chief cells . The intestine was divided into a proximal and distal segment by an intestinal valve . Pyloric caeca were not present . We propose that shallow, crypt-like structures in the intestinal mucosa are the sites of epithelial-cell proliferation in juveniles and adults . The length of microvilli decreased from approximately 4 microns in the cranial part of the proximal intestine, to 1.5 microns in the distal intestine . In the distal intestine, rod-shaped bacteria were observed between microvilli . An extensive system of thin-walled vessels was observed in the submucosa of juvenile and adult wolffish stomach and intestine . Eosinophilic granular cells were numerous in the perivascular connective tissue in the gastric and intestinal submucosa of adults and juveniles, but were not observed in larvae.

Nucleic Acids Res, 2001 Jan 1, 29(1), 171 - 2
tmRDB (tmRNA database); Knudsen B et al.; The tmRNA database (tmRDB) is maintained at the University of Texas Health Science Center at Tyler, Texas, and accessible on the World Wide Web at the URL +html . Mirror sites are located at Auburn University, Auburn, Alabama and the Institute of Biological Sciences, Aarhus, Denmark . The tmRDB provides information and citation links about tmRNA, a molecule that combines functions of tRNA and mRNA in trans-translation . tmRNA is likely to be present in all bacteria and has been found in algae chloroplasts, the cyanelle of Cyanophora paradoxa and the mitochondrion of the flagellate Reclinomonas americana . This release adds 26 new sequences and corresponding predicted tmRNA-encoded tag peptides for a total of 86 tmRNAs, ordered alphabetically and phylogenetically . Secondary structures and three-dimensional models in PDB format for representative molecules are being made available . tmRNA alignments prove individual base pairs and are generated manually assisted by computational tools . The alignments with their corresponding structural annotation can be obtained in various formats, including a new column format designed to improve and simplify computational usability of the data.

Nucleic Acids Res, 2001 Jan 1, 29(1), 44 - 8
Proteome Analysis Database: online application of InterPro and CluSTr for the functional classification of proteins in whole genomes; Apweiler R et al.; The SWISS-PROT group at EBI has developed the Proteome Analysis Database utilising existing resources and providing comparative analysis of the predicted protein coding sequences of the complete genomes of bacteria, archaea and eukaryotes . The two main projects used, InterPro and CluSTr, give a new perspective on families, domains and sites and cover 31-67% (InterPro statistics) of the proteins from each of the complete genomes . CluSTr covers the three complete eukaryotic genomes and the incomplete human genome data . The Proteome Analysis Database is accompanied by a program that has been designed to carry out InterPro proteome comparisons for any one proteome against any other one or more of the proteomes in the database.

Nucleic Acids Res, 2001 Jan 1, 29(1), 22 - 8
The COG database: new developments in phylogenetic classification of proteins from complete genomes; Tatusov RL et al.; The database of Clusters of Orthologous Groups of proteins (COGs), which represents an attempt on a phylogenetic classification of the proteins encoded in complete genomes, currently consists of 2791 COGs including 45 350 proteins from 30 genomes of bacteria, archaea and the yeast Saccharomyces cerevisiae . In addition, a supplement to the COGs is available, in which proteins encoded in the genomes of two multicellular eukaryotes, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, and shared with bacteria and/or archaea were included . The new features added to the COG database include information pages with structural and functional details on each COG and literature references, improvements of the COGNITOR program that is used to fit new proteins into the COGs, and classification of genomes and COGs constructed by using principal component analysis.

Proc Nutr Soc, 2000 Nov, 59(4), 587 - 94
Micronutrients and immune function in cattle; Spears JW; Complex inter-relationships exist between certain micronutrients, immune function and disease resistance in cattle . Several micronutrients have been shown to influence immune responses . The relationship between deficiencies of some micronutrients and disease resistance is less clear . A number of studies have indicated that Cr supplementation may improve cell-mediated and humoral immune response as well as resistance to respiratory infections in stressed cattle . With respiratory-disease challenge models Cr generally does not affect disease resistance . Deficiencies of Cu, Se, vitamin E and Co in cattle reduce the ability of isolated neutrophils to kill yeast and/or bacteria . Cu deficiency reduces antibody production, but cell-mediated immunity is generally not altered . However, Cu deficiency appears to reduce production of interferon and tumour necrosis factor by mononuclear cells . Numerous studies have linked low vitamin E and/or Se status to increased susceptibility of dairy cows to intramammary infections . In contrast to findings in laboratory animals, marginal Zn deficiency does not appear to impair antibody production or lymphocyte responsiveness to mitogen stimulation in ruminants . Co deficiency has been associated with reduced resistance to parasitic infections . It is well documented that vitamin A-deficient animals are more susceptible to various types of infections . beta-Carotene, possibly via its antioxidant properties, may affect immune function and disease resistance independent of its role as a precursor of vitamin A.

Proc Nutr Soc, 2000 Nov, 59(4), 565 - 72
Role of breast-feeding in managing malnutrition and infectious disease; Filteau SM; Breast-feeding policy tends to be an emotive issue . International agencies recommend exclusive breast-feeding for 4-6 months followed by continued partial breast-feeding into the second year of life in order to promote infant and child health and minimize the damage caused by the malnutrition-infection cycle . To what extent are these recommendations supported by the experimental evidence? Are they a simplification for emotional reasons or public health purposes? Breast-feeding is believed to benefit infants because breast milk contains the ideal mix of nutrients for infants, because it contains factors which promote development of the infant's gut and immune system and which prevent pathogen invasion, and because exclusive breast-feeding prevents intake of pathogens in food or water . However, some apparently contradictory evidence exists . First, in environments which are not highly contaminated breast-fed infants tend to growth falter relative to those fed formula . Second, in such environments partial breast-feeding is not associated with significantly increased gut damage relative to exclusive breast-feeding, suggesting that active promotion of gut development by breast-feeding is more important than simple avoidance of pathogens from other foods . Third, many immune factors in breast milk are probably present primarily to protect the mother, not the infant . Finally, breast milk itself may contain bacteria or viruses . This problem has come to the fore with the human immunodeficiency virus epidemic, since it is clear breast-feeding is an important mode of mother-to-child transmission . The present review will examine these challenges to the basis of the international infant feeding recommendations and will suggest that the science does actually support the policy.

Proc Natl Acad Sci U S A, 2000 Dec 5, 97(25), 13720 - 5
Targeted modification and transportation of cellular proteins; Colas P et al.; Peptide aptamers are proteins selected from combinatorial libraries that display conformationally constrained variable regions . Peptide aptamers can disrupt specific protein interactions and thus represent a useful method for manipulating protein function in vivo . Here, we describe aptamer derivatives that extend the range of functional manipulations . We isolated an aptamer with increased affinity for its Cdk2 target by mutagenizing an existing aptamer and identifying tighter binding mutants with calibrated two-hybrid reporter genes . We used this and other anti-Cdk2 aptamers as recognition domains in chimeric proteins that contained other functional moieties . Aptamers fused to the catalytic domain of a ubiquitin ligase specifically decorated LexA-Cdk2 with ubiquitin moieties in vivo . Aptamers against Cdk2 and another protein, Ste5, that carried a nuclear localization sequence transported their targets into the nucleus . These experiments indicate that fusion proteins containing aptameric recognition moieties will be useful for specific modification of protein function in vivo.

Pediatrics, 2000 Dec, 106(6), 1505 - 10
Technical report: irradiation of food . Committee on Environmental Health; Shea KM; Recent well-publicized outbreaks of foodborne illness have heightened general interest in food safety . Food irradiation is a technology that has been approved for use in selected foods in the United States since 1963 . Widespread use of irradiation remains controversial, however, because of public concern regarding the safety of the technology and the wholesomeness of irradiated foods . In this report, we describe the technology, review safety and wholesomeness issues, and give a historical perspective of the public controversy regarding food irradiation.

J Cell Biochem, 1999, Suppl 32-33, 84 - 94
A functional-phylogenetic system for the classification of transport proteins; Saier MH Jr; Twenty completely sequenced genomes of bacteria, archaea, and eukaryotes have been surveyed for the presence of genes encoding homologues of known solute transport proteins . These analyses and others have demonstrated the presence of nearly 250 families of sequence-related transporters . All such proteins have been classified according to the system we call the transporter classification system of the transport commission (TC) . This short summary article describes the main features of this system; the families are presented in tabular form . Detailed information concerning these families and their constituent transporters is available on our web sites . J . Cell . Biochem . Suppls . 32/33:84-94, 1999 .

Ned Tijdschr Geneeskd, 1999 Dec 11, 143(50), 2511 - 4
{Intestinal infections due to inhibition of gastric acid secretion in reflux disease}; Otten MH; Gastric acid stimulates the absorption of nutrients and is the most important non-immunological defence system against the constant bacterial invasion of our digestive tract . Patients with achlorhydria and resected stomachs have excessive growth of bacteria in the digestive tract and a much higher incidence of gastrointestinal infections . Modern treatment of reflux oesophagitis with acid secretion inhibitors creates a similar low acid state . Suppression of gastric acid secretion causes a dose dependent increased risk of a wide variety of intestinal infections especially for people over 65, immune compromised persons, sick patients with a reduced resistance and travellers to tropical areas . The potentially dangerous infections can be reduced by adequate counselling about the importance of gastric acid, the precaution of improved hygiene, on demand use of especially proton pump inhibitors and prescription of antacids and less potent acid inhibitors.

Microbiology, 1999 Dec, 145 ( Pt 12), 3487 - 95
The PE-PGRS glycine-rich proteins of Mycobacterium tuberculosis: a new family of fibronectin-binding proteins?
Espitia C, Laclette JP, Mondragon-Palomino M, Amador A, Campuzano J, Martens A, Singh M, Cicero R, Zhang Y, Moreno C.
A clone was isolated by screening of a cosmid library of Mycobacterium tuberculosis with an oligonucleotide designed from the N-terminal sequence of a previously reported proline-rich protein . Characterization of the 4481 bp insert showed the presence of polymorphic CG-repetitive sequences (PGRSs) with an ORF of 2.7 kb, encoding a 81.3 kDa protein (PE-PGRS81) . Southern blot analysis and BLAST-p searches revealed several homologous sequences in the genome of M . tuberculosis . The deduced amino acid sequence was highly similar to a stretch of about 98 residues in the N-terminus present in several members of the PE-PGRS family available in the GenBank database, including 100% identity with the partial amino acid sequence of the potential protein encoded by orf3' as well as with the Rv0278c sequence . A neighbour-joining analysis of the 99 PE-PGRS sequences available in the database indicated that PE-PGRS81 is included in a group where its closest relatives are the sequences orf3', Rv0278c, Rv0279c, Rv1759c, Rv3652 and Rv0747 . Probing with the complete coding regions of PE-PGRS81 and Rv1759c in Southern blot assays, on samples of genomic DNA from M . tuberculosis H37Rv, Mycobacterium bovis BCG and M . tuberculosis clinical isolates, showed a complex hybridization pattern for all strains . This shows the existence of intrastrain PGRS variability as reported for other PGRS members . In contrast, probing with the short conserved N-terminal region of Rv1759c reduced the hybridization to a single band . This marker allowed identification of M . tuberculosis clinical strains that lack Rv1759c . A recombinant C-terminal fragment of Rv1759c showed fibronectin-binding properties and was recognized by sera from patients infected with M . tuberculosis, suggesting that at least this member of the PE-PGRS is expressed in tuberculosis infection.

IARC Sci Publ, 1999, (150), 17 - 27
Chemistry and biology of DNA damage by malondialdehyde; Marnett LJ; Malondialdehyde is a naturally occurring product of lipid peroxidation and prostaglandin biosynthesis which is mutagenic and carcinogenic . It reacts with DNA to form adducts to deoxyguanosine and deoxyadenosine . The major adduct to DNA is a pyrimidopurinone called pyrimido{1,2-a}purin-10(3H)-one (M1G) . Studies of site-specific mutagenesis indicate that M1G is mutagenic in bacteria and is repaired by the nucleotide excision repair pathway . M1G has been detected in liver, leukocytes, pancreas and breast from healthy human beings at levels ranging from 1 to 120 per 10(8) nucleotides . Several assays for M1G have been described which are based on mass spectrometry, 32P-postlabelling or immunochemical techniques . M1G appears to be a major endogenous DNA adduct in human beings that may contribute significantly to cancer linked to lifestyle and dietary factors . Recent advances in the chemistry and biology of M1G are reviewed.

J Biol Chem, 2000 Jan 14, 275(2), 1377 - 83
Pantothenate kinase regulation of the intracellular concentration of coenzyme A; Rock CO et al.; Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway in bacteria and is thought to play a similar role in mammalian cells . We examined this hypothesis by identifying and characterizing two murine cDNAs that encoded PanK . The two cDNAs were predicted to arise from alternate splicing of the same gene to yield different mRNAs that encode two isoforms (mPanK1alpha and mPanK1beta) with distinct amino termini . The predicted protein sequence of mPanK1 was not related to bacterial PanK but exhibited significant similarity to Aspergillus nidulans PanK . mPanK1alpha was most highly expressed in heart and kidney, whereas mPanK1beta mRNA was detected primarily in liver and kidney . Pantothenate was the most abundant pathway component (42.8%) in normal cells providing clear evidence that pantothenate phosphorylation was a rate-controlling step in CoA biosynthesis . Enhanced mPanK1beta expression eliminated the intracellular pantothenate pool and triggered a 13-fold increase in intracellular CoA content . mPanK1beta activity in vitro was stimulated by CoA and strongly inhibited by acetyl-CoA illustrating that differential modulation of mPanK1beta activity by pathway end products also contributed to the management of CoA levels . These data support the concept that the expression and/or activity of PanK is a determining factor in the physiological regulation of the intracellular CoA concentration.

J Biol Chem, 2000 Jan 14, 275(2), 729 - 34
Multimeric self-assembly equilibria involving the histone-like protein H-NS . A thermodynamic study; Ceschini S et al.; The thermodynamic parameters affecting protein-protein multimeric self-assembly equilibria of the histone-like protein H-NS were quantified by "large zone" gel-permeation chromatography . The abundance of the different association states (monomer, dimer, and tetramer) were found to be strictly dependent on the monomeric concentration and affected by physical (temperature) and chemical (cations) parameters . On the basis of the results obtained in this study and the available structural information concerning this protein, a mechanism is proposed to explain the association behavior also in relation to the functional properties of the protein.

Immunity, 1999 Dec, 11(6), 743 - 52
Separate pathways for antigen presentation by CD1 molecules; Sugita M et al.; The ability to sample relevant intracellular compartments is necessary for effective antigen presentation . To detect peptide antigens, MHC class I and II molecules differentially sample cytosolic and endosomal compartments . CD1 constitutes another lineage of lipid antigen-presenting molecules . We show that CD1b traffics deeply into late endosomal compartments, while CD1a is excluded from these compartments and instead traffics independently in the recycling pathway of the early endocytic system . Further, CD1b but not CD1a antigen presentation is dependent upon vesicular acidification . Since lipids and various bacteria are known to traffic differentially, either penetrating deeply into the endocytic system or following the route of recycling endosomes, these findings elucidate efficient monitoring of distinct components of the endocytic compartment by CD1 lipid antigen-presenting molecules.

Biochemistry, 2000 Jan 11, 39(1), 205 - 12
Demonstration of unidirectional single-stranded DNA translocation by PcrA helicase: measurement of step size and translocation speed; Dillingham MS et al.; Using a fluorescent sensor for inorganic phosphate, the kinetics of ATP hydrolysis by PcrA helicase were measured in the presence of saturating concentrations of oligonucleotides of various lengths . There is a rapid phase of inorganic phosphate release that is equivalent to several turnovers of the ATPase, followed by slower steady-state ATP hydrolysis . The magnitude of the rapid phase is governed by the length of single-stranded DNA, while the slow phase is independent of its length . A kinetic model is presented in which the rapid phase is associated with translocation along single-stranded DNA, after the PcrA binds randomly along the DNA . There is a linear relationship between the length of single-stranded DNA and both the duration and amplitude of the rapid phase . These data suggest that the translocation activity occurs at 50 bases/s in unidirectional single-base steps, each requiring the hydrolysis of 1 ATP molecule.

J Clin Anesth, 1999 Nov, 11(7), 536 - 9
High-quality filtration allows reuse of anesthesia breathing circuits resulting in cost savings and reduced medical waste; Daggan R et al.; STUDY OBJECTIVES: To determine if the new Filta-Therm filter prevents contamination and allows the reuse of breathing circuit with considerable cost and environmental savings . DESIGN: Prospective study . PATIENTS: 52 ASA physical status I, II, III, and IV patients, aged 18 to 75 years . INTERVENTIONS: Each morning a new breathing circuit was assembled . The Filta-Therm filter (Intersurgical, Inc., Liverpool, NY) elbow, and mask, but not the circuit, were changed between patients . The filter was placed between the Y-piece and the elbow of the breathing circuit . Prior to anesthesia, samples were obtained at the Y-piece, and the inspiratory and expiratory ports of breathing circuit . Following anesthesia, samples were obtained at the Murphy eye of endotrachael tube, and at the Y-piece . The samples were incubated, and the results examined at 24 and 48 hours . MEASUREMENTS AND MAIN RESULTS: Prior to anesthesia, cultures of the Y-piece and the inspired and expired ports samples showed no growth . Following anesthesia, all 52 samples obtained at the endotracheal tube were contaminated with various organisms, while all 52 Y-piece samples showed negative growth . CONCLUSIONS: The single use of Filta-Therm filter prevents bacterial contamination and allows reuse of breathing circuit at least twice, resulting in significant cost savings ($50,778 per year) . Further studies are needed to establish the safety of reusing breathing circuits when appropriate bacterial filters are used.

J Immunol, 2000 Jan 15, 164(2), 966 - 72
Lipopolysaccharide activates distinct signaling pathways in intestinal epithelial cell lines expressing Toll-like receptors; Cario E et al.; LPS elicits several immediate proinflammatoy responses in peripheral blood leukocytes via a recently described pathway including CD14, Toll-like receptors (TLR), serine-threonine kinases, and NF-kappaB transcription factor . However, the functional responses of intestinal epithelial cells (IEC) to stimulation with LPS are unknown . Expression of mRNA and protein for CD14 and TLRs were assessed by RT-PCR, immunoblotting, and immunohistochemistry in mouse and human IEC lines . LPS-induced activation of signaling pathways (p42/p44 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK), p38, p65, NF-kappaB) were assessed by immunoblotting and gel shifts . CD14 mRNA and protein expression were not detectable in IEC . However, human TLR2, TLR3, and TLR4 mRNA were present in IEC . TLR4 protein was expressed in all cell lines; however, TLR2 protein was absent in HT29 cells . Immunofluorescent staining of T84 cells demonstrated the cell-surface presence of the TLRs . LPS-stimulation of IEC resulted in activation (>1.5-fold) of the three members of the MAPK family . In contrast, LPS did not significantly induce activation of JNK and p38 in CMT93 cells, p38 in T84 cells and MAPK and JNK in HT29 cells . Downstream, LPS activated NF-kappaB in IEC in a time-, dose-, and serum-dependent manner . IEC express TLRs that appear to mediate LPS stimulation of specific intracellular signal transduction pathways in IEC . Thus, IEC may play a frontline role in monitoring lumenal bacteria.

J Virol, 2000 Jan, 74(2), 669 - 75
Profilin is required for optimal actin-dependent transcription of respiratory syncytial virus genome RNA; Burke E et al.; Transcription of human respiratory syncytial virus (RSV) genome RNA exhibited an obligatory need for the host cytoskeletal protein actin . Optimal transcription, however, required the participation of another cellular protein that was characterized as profilin by a number of criteria . The amino acid sequence of the protein, purified on the basis of its transcription-optimizing activity in vitro, exactly matched that of profilin . RSV transcription was inhibited 60 to 80% by antiprofilin antibody or poly-L-proline, molecules that specifically bind profilin . Native profilin, purified from extracts of lung epithelial cells by affinity binding to a poly-L-proline matrix, stimulated the actin-saturated RSV transcription by 2.5- to 3-fold . Recombinant profilin, expressed in bacteria, stimulated viral transcription as effectively as the native protein and was also inhibited by poly-L-proline . Profilin alone, in the absence of actin, did not activate viral transcription . It is estimated that at optimal levels of transcription, every molecule of viral genomic RNA associates with approximately the following number of protein molecules: 30 molecules of L, 120 molecules of phosphoprotein P, and 60 molecules each of actin and profilin . Together, these results demonstrated for the first time a cardinal role for profilin, an actin-modulatory protein, in the transcription of a paramyxovirus RNA genome.

J Virol, 2000 Jan, 74(2), 593 - 9
The VP5 domain of VP4 can mediate attachment of rotaviruses to cells; Zarate S et al.; Some animal rotaviruses require the presence of sialic acid (SA) on the cell surface to infect the cell . We have isolated variants of rhesus rotavirus (RRV) whose infectivity no longer depends on SA . Both the SA-dependent and -independent interactions of these viruses with the cell are mediated by the virus spike protein VP4, which is cleaved by trypsin into two domains, VP5 and VP8 . In this work we have compared the binding characteristics of wild-type RRV and its variant nar3 to MA104 cells . In a direct nonradioactive binding assay, both viruses bound to the cells in a saturable and specific manner . When neutralizing monoclonal antibodies directed to both the VP8 and VP5 domains of VP4 were used to block virus binding, antibodies to VP8 blocked the cell attachment of wild-type RRV but not that of the variant nar3 . Conversely, an antibody to VP5 inhibited the binding of nar3 but not that of RRV . These results suggest that while RRV binds to the cell through VP8, the variant does so through the VP5 domain of VP4 . This observation was further sustained by the fact that recombinant VP8 and VP5 proteins, produced in bacteria as fusion products with glutathione S-transferase, were found to bind to MA104 cells in a specific and saturable manner and, when preincubated with the cell, were capable of inhibiting the binding of wild-type and variant viruses, respectively . In addition, the VP5 and VP8 recombinant proteins inhibited the infectivity of nar3 and RRV, respectively, confirming the results obtained in the binding assays . Interestingly, when the infectivity assay was performed on neuraminidase-treated cells, the VP5 fusion protein was also found to inhibit the infectivity of RRV, suggesting that RRV could bind to the cell through two sequential steps mediated by the interaction of VP8 and VP5 with SA-containing and SA-independent cell surface receptors, respectively.

Biotechnol Bioeng, 2000 Feb 20, 67(4), 417 - 23
Effects of bioaugmentation strategies in UASB reactors with a methanogenic consortium for removal of phenolic compounds; Hajji KT et al.; The removal of phenol, ortho- (o-) and para- (p-)cresol was studied with two series of UASB reactors using unacclimatized granular sludges bioaugmented with a consortium enriched against these substances . The parameters studied were the amount of inoculum added to the sludges and the method of immobilization of the inoculum . Two methods were used, adsorption to the biomass or encapsulation within calcium alginate beads . In the bioaugmentation by adsorption experiment, and with a 10% inoculum, complete phenol removal was obtained after 36 d, while 178 d were required in the control reactor . For p-cresol, 95% removal was obtained in the bioaugmented reactor on day 48 while 60 d were required to achieve 90% removal in the control reactor . For o-cresol, the removals were only marginally better with the bioaugmented reactors . Tests performed with the reactors biomass under non-limiting substrate concentrations showed that the specific activities of the bioaugmented biomasses were larger than the original biomass for phenol, and p-cresol even after 276 of operations, showing that the inoculum bacteria successfully colonized the sludge granules . Immobilization of the inoculum by encapsulation in calcium alginate beads, was performed with 10% of the inoculum . Results showed that the best activities were obtained when the consortium was encapsulated alone and the beads added to the sludges . This reactor presented excellent activity and the highest removal of the various phenolic compounds a few days after start-up . After 90 d, a high-phenolic compounds removal was still observed, demonstrating the effectiveness of the encapsulation technique for the start-up and maintenance of high-removal activities .

FEBS Lett, 1999 Dec 3, 462(3), 442 - 6
Proteolytic cleavage of protein kinase Cmu upon induction of apoptosis in U937 cells; Haussermann S et al.; Treatment of U937 cells with various apoptosis-inducing agents, such as TNFalpha and beta-D-arabinofuranosylcytosine (ara-C) alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), bryostatin 1 or cycloheximide, causes proteolytic cleavage of protein kinase Cmu (PKCmu) between the regulatory and catalytic domain, generating a 62 kDa catalytic fragment of the kinase . The formation of this fragment is effectively suppressed by the caspase-3 inhibitor Z-DEVD-FMK . In accordance with these in vivo data, treatment of recombinant PKCmu with caspase-3 in vitro results also in the generation of a 62 kDa fragment (p62) . Treatment of several aspartic acid to alanine mutants of PKCmu with caspase-3 resulted in an unexpected finding . PKCmu is not cleaved at one of the typical cleavage sites containing the motif DXXD but at the atypical site CQND378/S379 . The respective fragment (amino acids 379-912) was expressed in bacteria as a GST fusion protein (GST-p62) and partially purified . In contrast to the intact kinase, the fragment does not respond to the activating cofactors TPA and phosphatidylserine and is thus unable to phosphorylate substrates effectively.

Int J Clin Pract, 1998 Nov-Dec, 52(8), 529 - 32
Contamination of the surgeon's bare and gloved fingertips in cardiac operations; Goldsmith I et al.; The surgeon's bare fingertips and the external surface of gloved fingertips were examined for contamination by bacteria during the course of 27 cardiac surgical operations . Following the surgical scrub, positive colony counts were obtained in 20 (74%) of bare fingertip impressions (median colony count 4 {inter-quartile range, IQR, 0-9)}, while at the conclusion of surgery positive counts were obtained in 15 (55.6%) fingertip impressions (median count 1 {IQR 0-6}; range 0-247; paired Wilcoxon test p = NS) . Furthermore, positive colony counts at the start of the operation were obtained in none of the gloved fingertip impressions and at conclusion of surgery in 17 (62.9%) of the gloved fingertip impressions (median count 2 {IQR 0-6} p = 0.0002) . There was no significant relationship between the total colony count on gloved fingertip impressions and the length of surgery (Pearson's r = 0.17, p = NS) . Contrary to expectations there was no significant increase in the colony count on the bare fingertips at the conclusion of surgery . Although there was an increase in the bacterial count on the surgeon's gloved fingertips, this increase did not correlate with the length of surgery.

Insect Mol Biol, 1999 Nov, 8(4), 501 - 18
Diversity of odourant binding proteins revealed by an expressed sequence tag project on male Manduca sexta moth antennae; Robertson HM et al.; A small expressed sequence tag (EST) project generating 506 ESTs from 375 cDNAs was undertaken on the antennae of male Manduca sexta moths in an effort to discover olfactory receptor proteins . We encountered several clones that encode apparent transmembrane proteins; however, none is a clear candidate for an olfactory receptor . Instead we found a greater diversity of odourant binding proteins (OBPs) than previously known in moth antennae, raising the number known for M . sexta from three to seven . Together with evidence of seventeen members of the family from the Drosophila melanogaster genome project, our results suggest that insects may have many tens of OBPs expressed in subsets of the chemosensory sensilla on their antennae . These results support a model for insect olfaction in which OBPs selectively transport and present odourants to transmembrane olfactory receptors . We also found five members of a family of shorter proteins, named sensory appendage proteins (SAPs), that might also be involved in odourant transport . This small EST project also revealed several candidate odourant degrading enzymes including three P450 cytochromes, a glutathione S-transferase and a uridine diphosphate (UDP) glucosyltransferase . Several first insect homologues of proteins known from vertebrates, the nematode Caenorhabditis elegans, yeast and bacteria were encountered, and most have now also been detected by the large D . melanogaster EST project . Only thriteen entirely novel proteins were encountered, some of which are likely to be cuticle proteins.

BJU Int, 2000 Jan, 85(1), 130 - 8
Establishment and characterization of seven human renal cell carcinoma cell lines; Shin KH et al.; OBJECTIVE: To establish human renal cell carcinoma (RCC) cell lines, and to investigate the cell phenotypes and molecular characteristics of human RCC cell lines and their corresponding tumour tissues . MATERIALS AND METHODS: Seven human RCC cell lines from pathologically proven RCCs were established . The histopathology of the primary tumours, in vitro growth characteristics and status of tumour suppressor genes, mismatch repair genes and microsatellite instability (MSI) were examined in cell lines and their corresponding tumour tissues . Five of the cell lines were derived from clear cells (SNU-228, -267, -328, -349, and -1272), one from granular cells (SNU-482), and one from mixed clear and granular cell types (SNU-333) . The mutational status was compared for von Hippel-Lindau (VHL), p53, TGF-beta type II receptor (TGF-betaRII), hMSH2, and hMLH1 genes in the cell lines and their corresponding tumour tissues . The MSI status of the cell lines was determined by screening for adenine repeat sequences, e.g . BAT-25, BAT-26, and BAT-40 . RESULTS: All lines showed different doubling times and were confirmed by DNA fingerprinting analysis to be unique . Contamination by mycoplasma or bacteria was excluded . In two cell lines (SNU-349 and -1272) and their tumour tissues, mutations in the VHL gene were found . The SNU-267 line had a frameshift mutation in the p53 gene . A missense mutation of the TGF-betaRII gene was detected in the SNU-1272 line and the corresponding tissue . Analysis of the repeat sequences showed one cell line (SNU-349) to have MSI and the other six to have microsatellite stability . As MSI is a hallmark of the inactivation of mismatch repair genes, the presence of hMSH2 and hMLH1 mutations was investigated in all seven cell lines . An inactivating homozygous single base-pair deletion of the hMLH1 gene was found only in the SNU-349 cell line and corresponding tissue . Moreover, a frameshift mutation within an 8-bp polyadenine repeat present in the hMSH3 coding region was found only in the MSI cell line and tumour tissue . CONCLUSION: These newly established RCC cell lines should provide a useful in vitro model for studies related to human RCC . The SNU-349 cell line should be especially useful for studies of MSI and mismatch repair-defective RCCs.

Am J Respir Crit Care Med, 2000 Jan, 161(1), 104 - 9
Efficiency and safety of mechanical ventilation with a heat and moisture exchanger changed only once a week; Ricard JD et al.; The cost of mechanical ventilation (MV) is high . Efforts to reduce this cost, as long as they are not detrimental for the patients, are needed . MV with heat and moisture exchangers (HME) changed every 48 h is safe, efficient, and cost-effective . Preliminary reports suggest that the life span of these filters may be prolonged . We determined prospectively whether a hygroscopic and hydrophobic HME (Hygrobac-Dar; Mallinckrodt) provided safe and efficient heating and humidification of the inspired gases when changed only once a week . Patients who were considered to require mechanical ventilation for more than 48 h were included in the study . HMEs were initially set for 7 d . Efficient airway heating and humidification were assessed by clinical parameters (number of tracheal suctionings and instillations required, peak airway pressures) and hygrometric measurements performed by psychrometry . Resistance was measured from Day 0 to Day 7 . Bacterial colonization of circuits and HMEs was studied . A total of 377 days of mechanical ventilation with 60 HMEs was studied . Clinical parameters and hygrometric measurements did not change between Day 0 and Day 7 . Mean absolute humidity was 30.3 +/- 1.3 mg H(2)O/L on Day 0 and 30.8 +/- 1.5 mg H(2)O/L on Day 7 (p = 0.7) . Endotracheal tube occlusion never occurred . Three HMEs were replaced prematurely because of insufficient absolute humidity . This rare event occurred only in patients with COPD and after the third day of use . In addition, the absolute humidity delivered by the HMEs was significantly lower in patients with COPD than in the rest of the population . Resistance did not change from Day 0 to Day 7 (2.4 +/- 0.3 versus 2.7 +/- 0.3 cm H(2)O/L/s; p = 0.4) . Bacterial samples of both circuits and ventilator sides of HMEs were sterile in most cases . We conclude that mechanical ventilation can be safely conducted in non-COPD patients using an HME changed only once a week, leading to substantial cost savings (about $110,000 per year if these findings were applied to the university-affiliated hospitals in Paris).

Vaccine, 2000 Feb 14, 18(15), 1473 - 84
Phase I clinical trials of aroA aroD and aroA aroD htrA attenuated S . typhi vaccines; effect of formulation on safety and immunogenicity; Dilts DA et al.; PBCC211, an aroA aroD derivative of S . typhi strain CDC10-80, was tested in phase I trials as a single dose typhoid fever vaccine . Three different vaccine preparations, reconstituted lyophilized bacteria, freshly grown bacteria or lyophilized bacteria reconstituted from sachets, were orally administered to a total of 86 adult volunteers . An aroA aroD htrA strain, PBCC222, was also tested in 38 volunteers . Formulation impacted on the determination of a safe and immunogenic dose; reconstituted lyophilized cultures required higher doses than the broth cultures to stimulate seroconversion . In general, doses which seroconverted the majority of group members produced undesirable symptoms regardless of attenuation or formulation . The inability to separate the presence of symptoms from achieving significant immunogenicity in these aroA aroD or aroA aroD htrA strains precludes their use as single dose typhoid vaccines in the formulations tested . Multiple doses of these strains at a lower, safe level may be effective as vectors for foreign antigens.

Clin Diagn Lab Immunol, 2000 Jan, 7(1), 6 - 8
Serum cytokine responses during acute human granulocytic ehrlichiosis; Dumler JS et al.; Human granulocytic ehrlichiosis (HGE) is caused by obligate intracellular bacteria in the Ehrlichia phagocytophila group . The disease ranges from subclinical to fatal . We speculated that cell-mediated immunity would be important for recovery from and potentially in the clinical manifestations of HGE; thus, serum tumor necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), gamma interferon (IFN-gamma), IL-10, and IL-4 concentrations were studied . IFN-gamma (1,035 +/- 235 pg/ml {mean +/- standard error of the mean}) and IL-10 (118 +/- 46 pg/ml) concentrations were elevated in acute-phase sera versus convalescent sera and normal subjects (P </= 0.013 and P </= 0.018, respectively) . TNF-alpha, IL-1beta, and IL-4 levels were not elevated . Cytokine levels in severely and mildly affected patients were not different . HGE leads to induction of IFN-gamma-dominated cell-mediated immunity associated with clinical manifestations, recovery from infection, or both.

Cell, 1999 Dec 23, 99(7), 735 - 45
An enzymatic activity in the yeast Sir2 protein that is essential for gene silencing; Tanny JC et al.; Despite its conservation in organisms from bacteria to human and its general requirement for transcriptional silencing in yeast, the function of the Sir2 protein is unknown . Here we show that Sir2 can transfer labeled phosphate from nicotinamide adenine dinucleotide to itself and histones in vitro . A modified form of Sir2, which results from its automodification activity, is specifically recognized by anti-mono-ADP-ribose antibodies, suggesting that Sir2 is an ADP-ribosyltransferase . Mutation of a phylogenetically invariant histidine residue in Sir2 abolishes both its enzymatic activity in vitro and its silencing functions in vivo . However, the mutant protein is associated with chromatin and other silencing factors in a manner similar to wild-type Sir2 . These findings suggest that Sir2 contains an ADP-ribosyltransferase activity that is essential for its silencing function.

Mol Cell, 1999 Nov, 4(5), 673 - 82
Dynamic movement of the ParA-like Soj protein of B . subtilis and its dual role in nucleoid organization and developmental regulation; Marston AL et al.; The Spo0J and Soj proteins of B . subtilis belong to a widespread family of bacterial proteins required for accurate segregation of plasmids and chromosomes . Spo0J binds to several sites around the oriC region of the chromosome, which are organized into compact foci that may play a centromere-like role in active chromosome segregation . We now show that Soj has a role in organization or compaction of Spo0J-oriC complexes and possibly other regions of the nucleoid . This activity is accompanied by a dynamic localization pattern in which Soj protein undergoes assembly and disassembly into large nucleoid-associated patches on a timescale of minutes . The dynamic behavior of Soj, like its previously described transcriptional repression activity, is controlled by Spo0J . These interactions may constitute a checkpoint coupling developmental transcription to cell cycle progression.

Proc Natl Acad Sci U S A, 2000 Jan 4, 97(1), 151 - 6
Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins; Schwille P et al.; Fast excitation-driven fluctuations in the fluorescence emission of yellow-shifted green fluorescent protein mutants T203Y and T203F, with S65G/S72A, are discovered in the 10(-6)-10(-3)-s time range, by using fluorescence correlation spectroscopy at 10(-8) M . This intensity-dependent flickering is conspicuous at high pH, with rate constants independent of pH and viscosity with a minor temperature effect . The mean flicker rate increases linearly with excitation intensity for at least three decades, but the mean dark fraction of the molecules undergoing these dynamics is independent of illumination intensity over approximately 6 x 10(2) to 5 x 10(6) W/cm(2) . These results suggest that optical excitation establishes an equilibration between two molecular states of different spectroscopic properties that are coupled only via the excited state as a gateway . This reversible excitation-driven transition has a quantum efficiency of approximately 10(-3) . Dynamics of external protonation, reversibly quenching the fluorescence, are also observed at low pH in the 10- to 100-microseconds time range . The independence of these two bright-dark flicker processes implies the existence of at least two separate dark states of these green fluorescent protein mutants . Time-resolved fluorescence measurements reveal a single exponential decay of the excited state population with 3.8-ns lifetime, after 500-nm excitation, that is pH independent . Our fluorescence correlation spectroscopy results are discussed in terms of recent theoretical studies that invoke isomerization of the chromophore as a nonradiative channel of the excited state relaxation.

Appl Environ Microbiol, 2000 Jan, 66(1), 453 - 4
Multiregional evaluation of the SimPlate heterotrophic plate count method compared to the standard plate count agar pour plate method in water; Jackson RW et al.; A new SimPlate heterotrophic plate count (HPC) method (IDEXX Laboratories, Westbrook, Maine) was compared with the pour plate method at 35 degrees C for 48 h . Six laboratories tested a total of 632 water samples . The SimPlate HPC method was found to be equivalent to the pour plate method by regression analysis (r = 0 . 95; y = 0.99X + 0.06).

J Biol Chem, 2000 Jan 7, 275(1), 619 - 23
Cox11p is required for stable formation of the Cu(B) and magnesium centers of cytochrome c oxidase; Hiser L et al.; Assembly of the core subunits of the aa(3)-type cytochrome c oxidase in mitochondria and aerobic bacteria such as Rhodobacter sphaeroides requires the association of three subunits and the formation of five to seven metal centers . Several assembly proteins are required for the late stages of oxidase assembly in eukaryotes; some of these are also present in Rb . sphaeroides . To investigate the role of one of these proteins, Cox11p, the mitochondrial-like oxidase of Rb . sphaeroides was overexpressed and purified from cells that lacked cox11, the gene for Cox11p . The oxidase that assembled in the absence of Cox11p lacked Cu(B) at the active site and contained greatly reduced amounts of metal at the magnesium/manganese-binding site between subunits I and II . This inactive oxidase, however, did contain hemes a and a(3), Cu(A), and all three subunits . These results indicate that Cox11p is required at a late, perhaps final, step in the assembly of cytochrome oxidase, most likely the insertion of Cu(B) . Oxidase which assembled in a strain with a low copy number of cox11 appeared nearly wild type, suggesting that Cox11p is required in substoichiometric amounts for its role in oxidase assembly.

J Biotechnol, 1999 Oct 8, 75(2-3), 81 - 97
Adaptive responses of Ralstonia eutropha to feast and famine conditions analysed by flow cytometry; Muller S et al.; Results obtained by flow cytometry allow conclusions to be drawn about how the physiological states of Ralstsonia eutropha JMP134 are connected with survival strategies under distinct growth conditions . During both feast and famine conditions the cells were found to proceed through sharply separated phases of life . Two sources of carbon and energy, one poor (0.02% phenol) and one rich (0.2% pyruvate and 0.1% yeast extract) were chosen to study the cellular responses . Despite the major differences in carbon source, when growth stages of the bacteria on the two substrates were characterised in batch growth, only minor differences were found in the time course of the membrane potential related fluorescence intensity (MPRFI) . This also applied to the rRNA content and the size-correlated forward scatter (FSC) signal of the cells, both of which increased to high levels during the (early) exponential growth phase . On the rich medium, DNA synthesis initially occurred in an uncoupled manner, then a high rate of PHB formation followed when nutrients began to be limiting . Under famine conditions, the cellular responses were much more complex . PHB was synthesised, then DNA synthesis occurred in a 'eukaryotic' mode, to be succeeded by renewed PHB synthesis . To obtain defined cell physiological states, the chemostat technique was used in addition to batch experiments . The results obtained clearly indicated that key events in cell physiology, including initiation of DNA replication and overflow metabolism, occurred in a hierarchically ordered manner and were tightly correlated with changes in the environmental conditions of the bacterial cells.

J Ethnopharmacol, 1999 Oct, 67(1), 37 - 44
Biological activities of crude plant extracts from Vitex trifolia L . (Verbenaceae); Hernandez MM et al.; Biological assays of Vitex trifolia L . organic extracts have shown relevant activities . Hexanic and dichloromethanic (DCM) extracts, when prepared from stems and foliage, have proved to be very toxic against several cancer cell lines in culture (SQC-1 UISO, OVCAR-5, HCT-15 COLADCAR, and KB) . Also, an important antifeeding activity against the insect pest Spodoptera frugiperda (Lepidoptera: Noctuidae) was recorded . The hexanic extract from leaves completely inhibited the growth of the fungal plant pathogen Fusarium sp . within the first 2 days of the experiment, but dropped significantly at day 6 (15% inhibition) . The potential of V . trifolia for several uses is discussed.

J Gastroenterol, 1999, 34 Suppl 11, 37 - 42
Production of secretory component and pathogenesis of gastric cancer in Helicobacter pylori-infected stomach; Handa Y et al.; The purpose of this study was to examine the production of secretory component (SC) and immunoglobulin A (IgA) in the gastric mucosa with Helicobacter pylori infection and to investigate the influence of immunological reactions on various phases of infection (gastritis, intestinal metaplasia, gastric cancer) . Production of SC and IgA was assessed by immunohistochemical staining in (1) endoscopic biopsy samples of H . pylori-eradicated cases (n = 25), and (2) surgically resected stomach tissues of H . pylori-positive gastric cancer cases, intestinal type (IGC, n = 25) and diffuse type (DGC, n = 25) . Before eradication therapy, all samples showed positive staining of SC and IgA in epithelial cells, and IgA was also positive in plasma cells in the mucosal layer . H . pylori bacteria were positively stained for SC and IgA . After treatment, the degree of SC and IgA staining in epithelial cells was reduced with successful eradication; but with intestinal metaplasia, SC staining was positive regardless of the results of treatment . In nonmetaplastic mucosa, SC-positive cells were increased in the glandular neck zone to the surface mucosal layer; and the intensity of SC staining was increased in proportion to the degree of mucosal inflammation and IgA-positive cell aggregation . In intestinal metaplasia, SC was positive irrespective of the degree of inflammation . Most cancer foci also showed positive staining of SC, irrespective of histological type . Production of SC and IgA was thought to be a specific reaction against H . pylori infection, occurring from the early to the late stages and not limited to intestinal metaplasia . It was suggested that immunological reactions against H . pylori infection might generally be involved with the pathogenesis of intestinal metaplasia and both histological types of gastric cancer (IGC and DGC).

J Gastroenterol, 1999, 34 Suppl 11, 18 - 23
Chemotaxis and motility of Helicobacter pylori in a viscous environment; Yoshiyama H et al.; The chemotactic activity of Helicobacter pylori is important for its colonization . H . pylori exhibited chemotactic responses to urea and potassium bicarbonate, which can be supplied from human gastric epithelium . The chemotactic activities of H . pylori in a fluid environment were higher on the urease-positive strain than on the isogenic urease-negative strain . In a viscous solution containing 3% polyvinylpyrrolidone, the urease-positive strain showed stimulated chemotactic activity, whereas the urease-negative mutant did not show such stimulation . These results were in accordance with the fact that the mutant strain did not show swarming, which is a form of bacterial active motility in the viscous environment in soft agar regardless of having flagella . Incubation of the wild-type strain with urease inhibitors partially inhibited the chemotactic activities in the viscous solution . Inhibition of the chemotactic activity by urease inhibitors paralleled the inhibition of urease activity . The chemotactic activity of H . pylori has been shown to utilize proton motive force for motility . These results highlighted the importance of cytoplasmic urease for chemotactic motility of H . pylori possibly by an increase in the proton motive force under a condition that mimics the gastric mucus layer, in which the bacteria reside . These results indicated a possible application of drugs having urease-inhibiting potential for eradicating H . pylori . The significance of swarming in the expression of bacterial virulence was also discussed.

Lancet . 2000 Jan 1;355(9197):44.
Potential risk of cross-infection during peripheral-venous access by contamination of tourniquets; Golder M et al.; We found that a high proportion of reusable tourniquets are contaminated with blood and bacterial pathogens . Their use contravenes hospital cross-infection control protocols and we therefore recommend the use of disposable tourniquets.

Nat Genet, 2000 Jan, 24(1), 27 - 35
MLH3: a DNA mismatch repair gene associated with mammalian microsatellite instability; Lipkin SM et al.; DNA mismatch repair is important because of its role in maintaining genomic integrity and its association with hereditary non-polyposis colon cancer (HNPCC) . To identify new human mismatch repair proteins, we probed nuclear extracts with the conserved carboxy-terminal MLH1 interaction domain . Here we describe the cloning and complete genomic sequence of MLH3, which encodes a new DNA mismatch repair protein that interacts with MLH1 . MLH3 is more similar to mismatch repair proteins from yeast, plants, worms and bacteria than to any known mammalian protein, suggesting that its conserved sequence may confer unique functions in mice and humans . Cells in culture stably expressing a dominant-negative MLH3 protein exhibit microsatellite instability . Mlh3 is highly expressed in gastrointestinal epithelium and physically maps to the mouse complex trait locus colon cancer susceptibility I (Ccs1) . Although we were unable to identify a mutation in the protein-coding region of Mlh3 in the susceptible mouse strain, colon tumours from congenic Ccs1 mice exhibit microsatellite instability . Functional redundancy among Mlh3, Pms1 and Pms2 may explain why neither Pms1 nor Pms2 mutant mice develop colon cancer, and why PMS1 and PMS2 mutations are only rarely found in HNPCC families.

Vet Immunol Immunopathol, 1999 Dec 15, 72(1-2), 183 - 8
Cytokine therapy: a natural alternative for disease control; Lowenthal JW et al.; Disease control in food production animals is normally mediated through the use of vaccines, chemicals and antibiotics . However, the extensive use of antibiotics and chemicals in livestock has resulted in environmental and human health concerns, particularly with regard to the emergence of drug-resistant bacteria in the food chain . In fact, the World Health Organisation (WHO) has now urged meat producers to use environmentally-friendly alternative methods to control disease . Cytokines, as natural mediators of the immune response, offer exciting alternatives to conventional therapeutics . The utilisation of cytokines is becoming more feasible with the recent cloning of a number of cytokine genes . Since the chicken's immune system is similar to that of mammals, they offer an attractive model system with which to study the effectiveness of cytokine therapy in the control of disease in intensive livestock . In this report we will review our recent studies on the therapeutic potential of chicken interferon gamma (ChIFN-gamma) as a vaccine adjuvant and a growth promoter.

Enferm Infecc Microbiol Clin, 1999 Nov, 17(9), 434 - 8
{Seroepidemiology of Bartonella henselae infection in HIV-infected patients}; Blanco JR et al.; BACKGROUND: Bartonella henselae infections are closely related to numerous clinical infections of growing interest in Spain . Since immunosuppressed patients are a potential risk group for infection by this bacteria, the aim of the present was to study the seroepidemiology of B . henselae infection in a risk group (patients with HIV infection) and in a control group (donors) . PATIENTS AND METHODS: In October, 1997, antibodies versus B . henselae were determined at different dilutions (cut off > or = 1:64) by immunofluorescence in 52 patients with HIV infection and 85 donors . An epidemiologic study included age, sex, smoking, alcohol intake, INVDA, HIV infection, AIDS stage, cutaneous anergy, CD4 lymphocyte count, antiretroviral treatment and chemoprophylaxis versus P . carinii . RESULTS: Nine of the patients with HIV infection (17.3%) and five donors (5.88%) presented titers > or = 1:64 with no significant differences (p = 0.06) (adjusted OR: 1.7; CI 95%: 0.34-8.54) . Moreover, multiple logistic regression analysis did not show any risk or protection factor associated with B . henselae infection in patients with HIV infection . CONCLUSIONS: A high level of seroprevalence of antibodies versus B . henselae was observed in patients with HIV infection . No risk or protection factors associated with B . henselae infection in patients with HIV infection were found.

J Bacteriol, 2000 Jan, 182(1), 164 - 70
Straight and curved conformations of FtsZ are regulated by GTP hydrolysis; Lu C et al.; FtsZ assembles in vitro into protofilaments that can adopt two conformations-the straight conformation, which can assemble further into two-dimensional protofilament sheets, and the curved conformation, which forms minirings about 23 nm in diameter . Here, we describe the structure of FtsZ tubes, which are a variation of the curved conformation . In the tube the curved protofilament forms a shallow helix with a diameter of 23 nm and a pitch of 18 or 24 degrees . We suggest that this shallow helix is the relaxed structure of the curved protofilament in solution . We provide evidence that GTP favors the straight conformation while GDP favors the curved conformation . In particular, exclusively straight protofilaments and protofilament sheets are assembled in GMPCPP, a nonhydrolyzable GTP analog, or in GTP following chelation of Mg, which blocks GTP hydrolysis . Assembly in GDP produces exclusively tubes . The transition from straight protofilaments to the curved conformation may provide a mechanism whereby the energy of GTP hydrolysis is used to generate force for the constriction of the FtsZ ring in cell division.

J Bacteriol, 2000 Jan, 182(1), 14 - 22
Role of HrcA and CIRCE in the heat shock regulatory network of Bradyrhizobium japonicum; Minder AC et al.; A large number of bacteria regulate chaperone gene expression by the CIRCE-HrcA system in which a DNA element called CIRCE serves as binding site for the repressor protein HrcA under non-heat-shock conditions . We have cloned the two consecutive genes hrcA and grpE of Bradyrhizobium japonicum by using a complementation approach that screened for GrpE function . In vivo and in vitro transcript mapping demonstrated that both genes are transcribed separately from RpoH (sigma(32))-dependent promoters . To investigate the supposed negative regulatory function of HrcA, we compared the expression of putative target genes in the wild type with that in an hrcA mutant . Transcription of the CIRCE-associated chaperonin operons groESL(4) and groESL(5), as well as the beta-galactosidase activity derived from corresponding groE-lacZ fusions, was strongly elevated in the hrcA mutant even at physiological temperatures . Expression of other heat shock regulons (RpoH or ROSE dependent) was not affected . To study the activity of HrcA in vitro, we purified a histidine-tagged version of the protein under nondenaturing conditions . Specific binding to the CIRCE element was obtained with a soluble fraction of HrcA in gel retardation experiments.

FEMS Microbiol Lett, 2000 Jan 1, 182(1), 45 - 9
Mycobacterium avium resists exposure to the acidic conditions of the stomach; Bodmer T et al.; Organisms of the Mycobacterium avium complex are common pathogens in immunosuppressed patients such as individuals with AIDS . There is evidence that in AIDS patients, the main route for M . avium infection is the gastrointestinal tract . The stomach is a formidable barrier to pathogens and the ability to resist exposure to pH lower than 3 has been shown to be a virulence determinant of enteric pathogens . Incubation of three clinical isolates of M . avium under acidic pH revealed resistance of M . avium grown both to the exponential and stationary phase at pH 2.2 for 2 h . Inhibition of protein synthesis had no effect on the acid tolerance . When the duration of the incubation at pH 2.2 was extended to 24 h, bacteria grown to the stationary phase had a significantly greater tolerance to acid than exponential phase bacteria . M . avium incubated with acid in the presence of water was significantly more resistant to pH 2.2 than M . avium in the presence of buffer . Pre-adaptation in water prior to exposure to acidic conditions was also associated with increased resistance to pH 2.2 . Isoosmolarity of Hank's balanced salt solution appears to be responsible for the impaired resistance to acid between 2 and 24 h of incubation . These findings indicate that M . avium is naturally tolerant to pH<3 and that pre-adaptation under conditions similar to the conditions where M . avium is found in the environment results in increased acid resistance.

Mar Biotechnol (NY), 1999 Nov, 1(6), 509 - 532
Cultivation of Marine Sponges; Osinga R et al.; There is increasing interest in biotechnological production of marine sponge biomass owing to the discovery of many commercially important secondary metabolites in this group of animals . In this article, different approaches to producing sponge biomass are reviewed, and several factors that possibly influence culture success are evaluated . In situ sponge aquacultures, based on old methods for producing commercial bath sponges, are still the easiest and least expensive way to obtain sponge biomass in bulk . However, success of cultivation with this method strongly depends on the unpredictable and often suboptimal natural environment . Hence, a better-defined production system would be desirable . Some progress has been made with culturing sponges in semicontrolled systems, but these still use unfiltered natural seawater . Cultivation of sponges under completely controlled conditions has remained a problem . When designing an in vitro cultivation method, it is important to determine both qualitatively and quantitatively the nutritional demands of the species that is to be cultured . An adequate supply of food seems to be the key to successful sponge culture . Recently, some progress has been made with sponge cell cultures . The advantage of cell cultures is that they are completely controlled and can easily be manipulated for optimal production of the target metabolites . However, this technique is still in its infancy: a continuous cell line has yet to be established . Axenic cultures of sponge aggregates (primmorphs) may provide an alternative to cell culture . Some sponge metabolites are, in fact, produced by endosymbiotic bacteria or algae that live in the sponge tissue . Only a few of these endosymbionts have been cultivated so far . The biotechnology for the production of sponge metabolites needs further development . Research efforts should be continued to enable commercial exploitation of this valuable natural resource in the near future.

Microbes Infect, 1999 Dec, 1(15), 1313 - 25
TGF-beta in infections and infectious diseases; Reed SG; Since it was first described as having the ability to inhibit macrophage activation, transforming growth factor-beta (TGF-beta) has been analyzed for its role in regulating immune responses to a variety of pathogens, including viruses, bacteria, yeast, and protozoa . Most of the studies have involved organisms that infect macrophages, and this discussion will attempt to highlight these findings . Perhaps the most work has been performed with protozoan pathogens, including Trypanosoma cruzi and a variety of Leishmania species, so the discussion will begin with these organisms . Other studies have focused on mycobacteria and viruses, including human immunodeficiency virus, so these areas will also be emphasized in the discussion . For the most part, investigators have reported that TGF-beta has, as expected, a negative influence on host responses and a beneficial effect on the survival and growth of intracellular pathogens . However, other studies have found that TGF-beta may have a positive or beneficial effect in some models of infection . This review will attempt to highlight studies and conclusions on the roles of TGF-beta in infection.

Clin Orthop, 1999 Dec, (369), 103 - 9
Operating room environment; Ritter MA; Sepsis after total joint replacement is related directly to environmental contamination . Therefore, to control the source of environmental contamination, and ultimately sepsis, it must be realized that the operating room personnel are the major source of the bacteria as evidence by the rise in the colony forming units per square foot per hour from 13 units in an operating room without people to greater than 400 units during actual surgery . The use of inclusive gowns, such as hooded body exhaust, is most helpful . However, all operating room personnel including anesthesia personnel, circulating nurses, visitors, and the operating room team must wear inclusive gowns . Face masks and head covers offer no environmental protection . Some type of an environmental control, such as laminar airflow or ultraviolet light, is the most helpful with greater than 90% reduction of airborne bacteria at the wound and 60% reduction of airborne bacteria in the operating room . Therefore, to reduce environmental bacteria contamination the number of personnel in the operating room and the length of time for the actual surgery should be reduced, because wound contamination occurs first by direct fall out from the environment and second by contaminated equipment and gloved hands that initially were contaminated by the environment.

Proc Natl Acad Sci U S A, 1999 Dec 21, 96(26), 15368 - 73
Empirical laws of survival and evolution: their universality and implications; Azbel' MY; Presented analysis of human and fly life tables proves that with the specified accuracy their entire survival and mortality curves are uniquely determined by a single point (e.g., by the birth mortality q(0)), according to the law, which is universal for species as remote as humans and flies . Mortality at any age decreases with the birth mortality q(0) . According to life tables, in the narrow vicinity of a certain q(0) value (which is the same for all animals of a given species, independent of their living conditions), the curves change very rapidly and nearly simultaneously for an entire population of different ages . The change is the largest in old age . Because probability to survive to the mean reproductive age quantifies biological fitness and evolution, its universal rapid change with q(0) (which changes with living conditions) manifests a new kind of an evolutionary spurt of an entire population . Agreement between theoretical and life table data is explicitly seen in the figures . Analysis of the data on basic metabolism reduces it to the maximal mean lifespan (for animals from invertebrates to mammals), or to the maximal mean fission time (for bacteria), and universally scales them with the total number of body atoms only . Phenomenological origin of this unification and universality of metabolism, survival, and evolution is suggested . Their implications and challenges are discussed.

Proc Natl Acad Sci U S A, 1999 Dec 21, 96(26), 14706 - 11
Structural details of an interaction between cardiolipin and an integral membrane protein; McAuley KE et al.; Anionic lipids play a variety of key roles in biomembrane function, including providing the immediate environment for the integral membrane proteins that catalyze photosynthetic and respiratory energy transduction . Little is known about the molecular basis of these lipid-protein interactions . In this study, x-ray crystallography has been used to examine the structural details of an interaction between cardiolipin and the photoreaction center, a key light-driven electron transfer protein complex found in the cytoplasmic membrane of photosynthetic bacteria . X-ray diffraction data collected over the resolution range 30.0-2.1 A show that binding of the lipid to the protein involves a combination of ionic interactions between the protein and the lipid headgroup and van der Waals interactions between the lipid tails and the electroneutral intramembrane surface of the protein . In the headgroup region, ionic interactions involve polar groups of a number of residues, the protein backbone, and bound water molecules . The lipid tails sit along largely hydrophobic grooves in the irregular surface of the protein . In addition to providing new information on the immediate lipid environment of a key integral membrane protein, this study provides the first, to our knowledge, high-resolution x-ray crystal structure for cardiolipin . The possible significance of this interaction between an integral membrane protein and cardiolipin is considered.

Gastroenterology, 2000 Jan, 118(1), 90 - 100
Glutamine metabolism stimulates intestinal cell MAPKs by a cAMP-inhibitable, Raf-independent mechanism; Rhoads JM et al.; BACKGROUND & AIMS: Infectious diarrhea caused by viruses plus enterotoxigenic bacteria is often more severe than diarrhea induced by either pathogen alone . We postulated that the increased cell adenosine 3',5'-cyclic monophosphate (cAMP) concentration observed during infection by enterotoxigenic organisms retards the intestinal repair process by blocking activation of mitogen-activated protein kinases (MAPKs) in proliferating intestinal cells . METHODS: We evaluated the effects of glutamine on MAPK activity, thymidine incorporation, and cell number in glutamine-starved and -sufficient rat intestinal crypt cells (IEC-6) . RESULTS: In glutamine-starved cells, 10 mmol/L glutamine in the absence of serum stimulated {(3)H}thymidine incorporation 8-fold . This effect was inhibited by 60% with 8-(4-chlorophenylthio) (8-CPT)-cAMP (100 micromol/L) + isobutyl methylxanthine (100 micromol/L) . In cells not starved of glutamine, glutamine stimulated thymidine incorporation by 3-fold, and 8-CPT-cAMP completely blocked the mitogenic effect . Inhibition of proliferation by cAMP persisted for at least 68 hours after cAMP removal . In vitro kinase assays showed that glutamine signaling requires an intact ERK (extracellular signal-related kinase) pathway in unstarved cells . In starved cells, at least one other pathway (JNK) was activated by glutamine, and the mitogenic inhibition by 8-CPT-cAMP was incomplete . Other intestinal fuels (glucose and acetate) were not mitogenic . CONCLUSIONS: Increased levels of intracellular cAMP inhibit ERKs but only partially reduce glutamine-stimulated proliferation in enterocytes adapted to low glutamine.

J Biomol NMR, 1999 Jun, 14(2), 123 - 32
Completeness of NOEs in protein structure: a statistical analysis of NMR; Doreleijers JF et al.; The completeness of experimentally observed NOE restraints of a set of 97 NMR protein structures deposited in the PDB has been assessed . Completeness is defined as the ratio of the number of experimentally observed NOEs and the number of 'expected NOEs' . A practical definition of 'expected NOEs' based on inter-proton distances in the structures up to a given cut-off distance is proposed . The average completeness for the set of 97 structures is 68, 48, and 26% up to 3, 4, and 5 A cut-off distances, respectively . For recent state-of-the-art structures these numbers are approximately 90, 75, and 45% . Almost 20% of the observed NOEs are between atoms that are further than 5 A apart in the final structures . The completeness is independent of the relative surface accessibility and does not depend strongly on residue type, secondary structure or local precision, although the number of observed NOEs in these classes varies considerably . The completeness of NOE restraints is a useful quality criterion in the course of structure refinement . The completeness per residue is more informative than the number of NOEs per residue, which makes it a useful tool to assess the quality of the NMR data set in relation to the resulting structures.

Biofactors, 1999, 10(2-3), 99 - 104
Biological functions of carotenoids--diversity and evolution; Vershinin A; Carotenoids first emerged in archaebacteria as lipids reinforcing cell membranes . To serve this function their long molecules have extremely rigid backbone due to the linear chain of usually 10 to 11 conjugated C=C bonds in transconfiguration--the length corresponding the thickness of hydrophobic zone of membrane which they penetrate as "molecular rivets" . Carotenoids retain their membrane-reinforcing function in some fungi and animals . The general structure of carotenoid molecule, originally having evolved for mechanical functions in membranes, possess a number of other properties that were later used for independent functions . The most striking fact is that these properties proved to fit some new functions to perfection . The polyene chain of 9-11 double bonds absorbs light precisely in the gap of chlorophyll absorption--function as accessory light-harvesting pigments in all plants; Unique arrangement of electronic levels owing to the by polyene chain structure makes carotenoids the only natural compounds capable of excitation energy transfer both (i) from carotenoid excited state to chlorophyll in the light-harvesting complex and (ii) from triplet chlorophyll or singlet oxygen to carotenoid in photosynthetic reaction centers--protection of RC from photodamage . The linear system of conjugated C=C bonds provides high reducing potential of carotenoid molecules making them potent antioxidants in lipid formations . Still, there is a lack of evidence of the chemical antioxidant function of carotenoids, especially in higher organisms; most data demonstrate an antioxidant ability rather than a function . Carotenoids have many other independent biological functions, including: specific coloration patterns in plants and animals, screening from excessive light and spectral filtering, defense of egg proteins from proteases in some invertebrates; the direct carotenoid derivative--retinal--acts as visual pigment in all animals and as chromophore in bacteriorhodopsin photosynthesis, retinoic acid in animals and abscisic acid in plants serve as hormones . All these functions utilize various properties (mechanical, electronic, stereospecific) of a single structure evolved in bacteria for a single membrane-reinforcing function, thus demonstrating an example of pure evolutionary preadaptation . One of the practical conclusions that can be reached by reviewing uniquely diverse properties and functions of carotenoids is that, when considering possible mechanisms of their effects in organisms (e.g., anticarcinogenic action), all their functional traits should be taken into account.

Microbiol Immunol, 1999, 43(11), 1009 - 15
Enhancement of the growth of Helicobacter pylori in Brucella broth by hydrogen peroxide; Murano A et al.; We found that a sub-lethal concentration of hydrogen peroxide (HPOx) enhanced the growth of Helicobacter pylori in Brucella broth supplemented with 10% fetal bovine serum (BB/FBS) . The enhancement was evident at 0.1 mM HPOx and reached a maximun at 3.5 mM . The growth stimulation was dependent on the basal media used; when brain heart infusion broth (BHIB) was used instead of BB, the growth was not altered regardless of the presence or absence of HPOx . Furthermore, the growth in BHIB/FBS was comparable to that in BB/FBS plus 3.5 mM HPOx . This suggested that the enhancement of growth by HPOx resulted from the derepression of the inhibitory factor existing in BB by HPOx . The inhibitory substance seemed to be bisulfite salt since the bacteria grew to a similar extent in bisulfite-less Brucella broth (BLBB0)/FBS compared to the bacterial growth in BHIB/FBS and BB/FBS plus HPOx . These results indicate that the detoxification of bisulfite in BB can be easily achieved by simply adding HPOx to the medium, which causes the oxidation of bisulfite to bisulfate, a less-toxic compound to the bacterial growth . Since we also found that the morphology and cellular protein profile of BB/FBS-cultured bacteria were apparently different from those cultured in BLBB/FBS, we propose that the use of BB for primary isolation and cultivation of H . pylori should be limited on certain occasions, or if necessary, BB can be used after detoxification of the bisulfite by the addition of a low concentration of HPOx.

Curr Biol, 1999 Dec 2, 9(23), R883 - 6
Lessons from the Aeropyrum pernix genome; Faguy DM et al.; Aeropyrum pernix is the first crenarchaeote and first aerobic member of the Archaea for which the complete genome sequence has been determined . The sequence confirms the distinct nature of crenarchaeotes and provides new insight into the relationships between the three domains: Bacteria, Archaea and Eukaryotes.

Med Hypotheses, 1999 Oct, 53(4), 345 - 6
Red blood cells, hemoglobin and the immune system; Bishlawy IM; The paramount function of the red blood cells (RBCs) is generally reckoned to be oxygen carriage . The possibility that they now participate in the defensive mechanism of the human body is ignored and neglected, despite the fact that the RBCs are both mechanical and biochemical barriers against infections, bacteria, and blood parasites . Immune reactions are regulated to ensure harmony between the red and white blood cell populations . This hypothesis will lead to better comprehension of blood cell functions in various physiological and pathological conditions.

Curr Opin Microbiol, 1999 Dec, 2(6), 630 - 5
Swarming motility; Fraser GM et al.; Swarming involves differentiation of vegetative cells into hyperflagellated swarm cells that undergo rapid and coordinated population migration across solid surfaces . Cell density, surface contact, and physiological signals all provide critical stimuli, and close cell alignment and the production of secreted migration factors facilitate mass translocation . Flagella biogenesis is central to swarming, and the flhDC flagellar master operon is the focal point of a regulatory network governing differentiation and migration.

Curr Opin Microbiol, 1999 Dec, 2(6), 624 - 9
Motility in Myxococcus xanthus and its role in developmental aggregation; Ward MJ et al.; The Frz signal transduction system of Myxococcus xanthus was originally thought to be a simple variation of the well-characterized Che system of the enteric bacteria . Recently, however, many additional Frz proteins, along with alternative signal transduction systems, have been discovered . Together these signal transduction pathways coordinate cell-cell behavior, permitting the complex interactions required for developmental aggregation and fruiting body formation.

Curr Opin Genet Dev, 1999 Dec, 9(6), 649 - 56
Shaping the genome--restriction-modification systems as mobile genetic elements; Kobayashi I et al.; A restriction enzyme gene is often linked to a modification methylase gene the role of which is to protect a recognition site on DNA from breakage by the former . Loss of some restriction-modification gene complexes leads to cell death through restriction breakage in the genome . Their behavior as genomic parasites/symbionts may explain the distribution of restriction sites and clarify certain aspects of bacterial recombination repair and mutagenesis . A comparison of bacterial genomes supports the hypothesis that restriction-modification gene complexes are mobile elements involved in various genome rearrangements and evolution.

Curr Biol, 1999 Dec 2, 9(23), 1373 - 81
Developmental and cell biological functions of the Drosophila DEAD-box protein abstrakt; Irion U et al.; BACKGROUND: DEAD-box proteins are a large family of proteins found in bacteria, plants and animals, but only few have been analysed functionally . They are involved in the regulation of various aspects of RNA processing and metabolism, including splicing, transport and translation . The study of their function in multicellular organisms has been restricted to a few special cases, such as the Vasa protein in the fruit fly Drosophila . RESULTS: We show that abstrakt, a gene originally identified genetically by its effect on axon outgrowth and fasciculation of the Bolwig nerve, encodes a new Drosophila DEAD-box protein of which the closest homologue is a human gene of unknown function . Using temperature-sensitive alleles to assay its function, we found that abstrakt is essential for survival at all stages throughout the life cycle of the fly . Mutants show specific defects in many developmental processes, including cell-shape changes, localisation of RNA and apoptosis . CONCLUSIONS: Abstrakt is not globally required for RNA splicing, transport, subcellular localisation or translation . Nevertheless, there is a widespread requirement for Abstrakt during post-transcriptional gene expression . Abstrakt must affect processing of specific subsets of RNAs, suggesting that differential post-translational control during development is more common than previously suspected.

J Colloid Interface Sci, 1999 Dec 15, 220(2), 229 - 234
Ice/Water Interface: Zeta Potential, Point of Zero Charge, and Hydrophobicity; Drzymala J et al.; The ice/water interface is a common and important part of many biological, environmental, and technological systems . In contrast to its importance, the system has not been extensively studied and is not well understood . Therefore, in this paper the properties of the H(2)O ice/water and D(2)O ice/water interfaces were investigated . Although the zeta potential vs pH data points were significantly scattered, it was determined that the isoelectric point (iep) of D(2)O ice particles in water at 3.5 degrees C containing 10(-3) M NaCl occurs at about pH 3.0 . The negative values of the zeta potential, calculated from the electrophoretic mobility, seem to decrease with decreasing content of NaCl, while the iep shifts to a higher pH . The point of zero charge (pzc) of D(2)O ice and H(2)O ice, determined by changes in pH of 10(-4) M NaCl aqueous solution at 0.5 degrees C after the ice particle addition, was found to be very different from the iep and equal to pH 7.0 +/- 0.5 . The shift of the iep with NaCl concentration and the difference in the positions of the iep and pzc on the pH scale point to complex specific adsorption of ions at the interface . Interestingly, similar values of iep and pzc were found for very different systems, such as hydrophilic ice and highly hydrophobic hexadecane droplets in water . A comparison of the zeta potential vs pH curves for hydrophilic ice and hydrophobic materials that do not possess dissociative functional groups at the interface (diamond, air bubbles, bacteria, and hexadecane) indicated that all of them have an iep near pH 3.5 . These results indicate that the zeta potential and surface charge data alone cannot be used to delineate the electrochemical properties of a given water/moiety interface because similar electrical properties do not necessary mean a similar structure of the interfacial region . A good example is the aliphatic hydrocarbon/water interface in comparison to the ice/water interface . Although the experiments were carried out with care, both the zeta potential, measured with a precise ZetaPlus meter, and DeltapH values (a measure of surface charge) vs pH were significantly scattered, and the origin of dissemination of the data points was not established . Differently charged ice particles and not fully equilibrium conditions at the ice/water interface may have been responsible for the dissemination of the data .

Jpn J Physiol, 1999 Dec, 49(6), 467 - 78
Central control mechanisms of circulation in the medulla oblongata by nitric oxide; Maeda M et al.; Nitric oxide (NO) is involved in numerous physiological functions . Besides its role as an endothelium-dependent relaxing factor (EDRF), NO inhibits platelet aggregation, contributes to cytotoxicity against bacteria, is active in synaptic transmission within the brain, etc . NO synthase (NOS) is distributed in brain regions related to the regulation of cardiovascular functions . NO has been inferred not only to act directly on vascular vessels, but also to regulate circulation within the brain . In this review paper, we mainly consider the functions of NO in the cardiovascular center of the medulla oblongata . That is, we describe the anatomical distribution of NOS in the brain, effects of intravenous and intracerebroventricular administration of NOS inhibitors on the circulation, effects of microinjection of NO donors and NOS inhibitors into the nucleus tractus solitarius (NTS) and ventrolateral medulla (VLM), the results of electrophysiological studies on these areas, and finally, the data obtained by new molecular biological techniques.

Microbes Infect, 1999 Apr, 1(5), 395 - 404
Magic bullets need accurate guns--syphilis eradication, elimination, and control; Garnett GP et al.; When cases of early syphilis are treated promptly, the spread of the bacteria within a population is interrupted . However, if complacency is induced by successful control, then upsurges in syphilis incidence can occur . The methods and aims of syphilis control in industrialised countries are reviewed in the light of the potential for regional elimination and global eradication programmes . While the medical means to eliminate syphilis are at hand, acceptable means for finding and treating cases that transmit infection need to be developed, particularly in the marginalized communities with limited access to care.

Microbes Infect, 1999 Apr, 1(5), 385 - 94
Molecular basis of defence against oxidative stress in Entamoeba histolytica and Giardia lamblia; Tekwani BL et al.; Gastrointestinal protozoa, viz . Entamoeba histolytica and Giardia, are able to survive in a microaerobic environment . The role of parasitic factors, particularly cysteine, cysteine-rich proteins, superoxide dismutase, and certain alternative mechanisms, has been described in the defence against oxidative stress . The role of the host-derived factors, particularly the phagocytosis of bacteria or host erythrocytes (intact or their enzymatic/non enzymatic components), in the detoxification of reactive oxygen metabolites in E . histolytica may provide novel approaches for the chemotherapy of invasive amoebiasis.

Oncogene, 1999 Nov 22, 18(49), 6853 - 66
Activators and target genes of Rel/NF-kappaB transcription factors; Pahl HL; The vertebrate transcription factor NF-kappaB is induced by over 150 different stimuli . Active NF-kappaB, in turn, participates in the control of transcription of over 150 target genes . Because a large variety of bacteria and viruses activate NF-kappaB and because the transcription factor regulates the expression of inflammatory cytokines, chemokines, immunoreceptors, and cell adhesion molecules, NF-kappaB has often been termed a 'central mediator of the human immune response' . This article contains a complete listing of all NF-kappaB inducers and target genes described to date . The collected data argue that NF-kappaB functions more generally as a central regulator of stress responses . In addition, NF-kappaB activation blocks apoptosis in several cell types . Coupling stress responsiveness and anti-apoptotic pathways through the use of a common transcription factor may result in increased cell survival following stress insults.

Eur J Biochem, 2000 Jan, 267(1), 104 - 9
Structure and interaction of VacA of Helicobacter pylori with a lipid membrane; Pagliaccia C et al.; In its mature form, the VacA toxin of Helicobacter pylori is a 95-kDa protein which is released from the bacteria as a low-activity complex . This complex can be activated by low-pH treatment that parallels the activity of the toxin on target cells . VacA has been previously shown to insert itself into lipid membranes and to induce anion-selective channels in planar lipid bilayers . Binding of VacA to lipid vesicles and its ability to induce calcein release from these vesicles were systematically compared as a function of pH . These two phenomena show a different pH-dependence, suggesting that the association with the lipid membrane may be a two-step mechanism . The secondary and tertiary structure of VacA as a function of pH and the presence of lipid vesicles were investigated by Fourier-transform infrared spectroscopy . The secondary structure of VacA is identical whatever the pH and the presence of a lipid membrane, but the tertiary structure in the presence of a lipid membrane is dependent on pH, as evidenced by H/D exchange.

Chemotherapy, 2000 Jan-Feb, 46(1), 15 - 22
Detection of rokitamycin-induced morphostructural alterations in Helicobacter pylori by atomic force microscopy; Braga PC et al.; The subinhibitory and suprainhibitory concentrations of many antibiotics are capable of interfering with the morphology of bacteria, thus disrupting the integrity of their structures and reducing bacterial virulence . Studies of this type have so far been performed using optical or scanning electron microscopes, but a new type of lens-free microscope, the atomic force microscope (AFM), has recently been introduced which is capable of investigating fine three-dimensional surface topography . The present study made use of this new technique to investigate the morphological alterations in Helicobacter pylori as a bacterial specimen exposed or not to different concentrations of rokitamycin . The produced images clearly show that AFM is a very useful tool for obtaining fine-quality three-dimensional images of bacterial morphology . The breakthrough in applying this new type of microscopy is also due to the fact that the images can be digitally collected at very high resolution in the z-axis, without the need to adopt the critical point-drying method or vacuum conditions, and without the need to coat the surface of the sample with a metal layer, a result not otherwise attainable with other microscopy techniques .

J Exp Biol, 2000 Jan, 203(Pt 1), 51 - 9
Crucial role of the membrane potential for ATP synthesis by F(1)F(o) ATP synthases; Dimroth P et al.; ATP, the universal carrier of cell energy, is manufactured from ADP and phosphate by the enzyme ATP synthase using the free energy of an electrochemical gradient of protons (or Na(+)) . The proton-motive force consists of two components, the transmembrane proton concentration gradient (delta pH) and the membrane potential . The two components were considered to be not only thermodynamically but also kinetically equivalent, since the chloroplast ATP synthase appeared to operate on delta pH only . Recent experiments demonstrate, however, that the chloroplast ATP synthase, like those of mitochondria and bacteria, requires a membrane potential for ATP synthesis . Hence, the membrane potential and proton gradient are not equivalent under normal operating conditions far from equilibrium . These conclusions are corroborated by the finding that only the membrane potential induces a rotary torque that drives the counter-rotation of the a and c subunits in the F(o) motor of Propionigenium modestum ATP synthase.

Arkh Patol, 1999 Sep-Oct, 61(5), 81 - 4
{Tuberculosis: socio-medical aspects}; Berestova AV; Tuberculosis is an ubiquitous infection . The epidemiological situation is now critical . 2.5 million people die every year in the world due to different types of tuberculosis . In Russia, tuberculosis morbidity and mortality are also on the increase . Clinical manifestations of the disease became grave while the treatment less efficient . The key element in tuberculosis pathogenesis is interaction between bacteria, T-lymphocytes and macrophages with cytokines participation this resulting in elimination of the bacteria and formation of hypersensitivity . One of the main control mechanisms may be macrophage apoptosis in the center of the granulomas following with caseous necrosis . Methods of molecular biology open new perspectives in diagnosis, understanding of pathogenesis and tuberculosis treatment.

Equine Vet J, 1999 Nov, 31(6), 478 - 82
Encephalopathy with idiopathic hyperammonaemia and Alzheimer type II astrocytes in equidae; Hasel KM et al.; In 3 mature female horses of varying breeds, episodes of colic and depression for 14 days preceded an encephalopathic disorder with maniacal behaviour, anxiety, profuse sweating and, in one case, terminal opisthotonus . Blood ammonia levels were elevated approximately 10-fold . At necropsy, there were gastrointestinal serosal and mesenteric haemorrhages . Histologically, all 3 cases revealed diffuse Alzheimer type II astrocytes in the cerebral grey matter . Alzheimer type II astrocytes were glial fibrillary acidic protein (GFAP) negative or only weakly positive, weakly S-100 positive, and vimentin negative . In the absence of primary hepatic and/or renal lesions, an increase in intestinal ammonia absorption due to ileus or increased ammonia production by colonic bacteria is hypothesised.

J Am Acad Dermatol, 2000 Jan, 42(1 Pt 1), 132 - 3
Pustular tinea pedis; Hirschmann JV et al.; Pustules are uncommon in tinea pedis and may suggest a bacterial infection . We describe a patient with large pustules on his feet that contained hyphae on Gram's stain of the pus and on a potassium hydroxide preparation of the pustule roof . Cultures were negative for bacteria, but grew Trichophyton rubrum.

FEBS Lett, 1999 Dec 17, 463(3), 216 - 20
Protein PII regulates both inorganic carbon and nitrate uptake and is modified by a redox signal in synechocystis PCC 6803; Hisbergues M et al.; In Synechocystis PCC 6803 as in other cyanobacteria, involvement of protein PII in the co-regulation of inorganic carbon and nitrogen metabolism was established based on post-translational modifications of the protein resulting from changes in the carbon/nitrogen regimes . Uptake of bicarbonate and nitrate in response to changes of the carbon and/or nitrogen regimes is altered in a PII-null mutant, indicating that both processes are under control of PII . Modulation of electron flow by addition of methyl viologen with or without duroquinol, or in a NAD(P)H dehydrogenase-deficient mutant, affects the phosphorylation level of PII . The redox state of the cells would thus act as a trigger for PII phosphorylation.

Biochemistry, 1999 Dec 21, 38(51), 16794 - 801
Crystal structure of eosinophil cationic protein at 2.4 A resolution; Boix E et al.; Eosinophil cationic protein (ECP) is located in the matrix of the eosinophil's large specific granule and has marked toxicity for a variety of helminth parasites, hemoflagellates, bacteria, single-stranded RNA virus, and mammalian cells and tissues . It belongs to the bovine pancreatic ribonuclease A (RNase A) family and exhibits ribonucleolytic activity which is about 100-fold lower than that of a related eosinophil ribonuclease, the eosinophil-derived neurotoxin (EDN) . The crystal structure of human ECP, determined at 2.4 A, is similar to that of RNase A and EDN . It reveals that residues Gln-14, His-15, Lys-38, Thr-42, and His-128 at the active site are conserved as in all other RNase A homologues . Nevertheless, evidence for considerable divergence of ECP is also implicit in the structure . Amino acid residues Arg-7, Trp-10, Asn-39, His-64, and His-82 appear to play a key part in the substrate specificity and low catalytic activity of ECP . The structure also shows how the cationic residues are distributed on the surface of the ECP molecule that may have implications for an understanding of the cytotoxicity of this enzyme.

J Food Prot, 1999 Dec, 62(12), 1404 - 10
A new medium for determining the total plate count in food; Smith CF et al.; SimPlate for Total Plate Count-Color Indicator (TPC-CI, IDEXX Laboratories, Inc., Westbrook, Me.) is a new medium that incorporates the redox dye resazurin to detect and quantify bacteria in food . Enumeration is achieved by the most probable number method using a SimPlate device . Viable bacteria are detected in each well of the SimPlate device by the biochemical reduction of resazurin, which is blue, to the pink resorufin or the clear dihydroresorufin indicators . Results after 24 h of incubation for TPC-CI are highly correlated with standard plate count agar after 48 h of incubation . Correlation coefficients from studies conducted at five laboratories ranged from 0.94 to 0.98 in side-by-side comparisons against standard plate count agar . Four additional test sites, using alternative methods for determining the aerobic plate count in food, reported similar results in comparison studies (r = 0.91 to 0.97) . The slopes from linear regression analysis at all sites ranged from 0.91 to 0.98, with y intercepts ranging from 0.11 to 0.84 . Samples used for the validation of TPC-CI included raw food products (i.e., liver and grains), which may contain natural enzymes that interfere with enzyme-based detection methods . No interference was seen from the foods tested . These results suggest that TPC-CI is a suitable alternative to existing plate count methods and has reduced incubation time.

Int J Biochem Cell Biol, 1999 Nov, 31(11), 1255 - 60
Human membrane cofactor protein (MCP, CD46): multiple isoforms and functions; Seya T et al.; Human membrane cofactor protein (MCP, CD46) is a 45-70 kDa protein with genetic and tissue-specific heterogeneity, and is expressed on all nucleated cells . MCP consists from N-terminus of 4 short consensus repeats (SCRs), 1-3 serine/threonine-rich (ST) domains, a transmembrane domain (TM) and a cytoplasmic tail (CYT) . More than 8 isoforms are generated secondary to alternative splicing due to combinations of various exons encoding the ST, TM and CYT domains . It serves as a cofactor of serine protease factor I for inactivation of complement C3b and C4b . Its primary role is to protect host cells from homologous complement attack by inactivating C3b/C4b deposited on the membrane . It also acts as receptors for measles virus (MV), some kinds of bacteria and for a putative ligand on oocytes . MV infection causes temporal host immune suppression, which may appear secondary to signaling events through MCP on macrophages and dendritic cells . These functional properties of human MCP may facilitate xenotransplantation and may be useful in the generation of animal models of measles by creating human MCP-expressing animals.

Voen Med Zh, 1999 Oct, 320(10), 65 - 7, 96
{The effect of thymogen on the state of immunity in destructive pulmonary tuberculosis}; Medus AI et al.; Publication Types:

Clinical Trial

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