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