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| J Biol Chem, 2003 Aug 1, 278(31), 28882 - 91 Epub 2003 May 19. DEN1 is a dual function protease capable of processing the C terminus of Nedd8 and deconjugating hyper-neddylated CUL1; Wu K et al.; Nedd8 activates ubiquitination by increasing the efficiency of polyubiquitin chain assembly through its covalent conjugation to cullin molecules . Here we report the isolation, cloning, and characterization of a novel human Nedd8-specific protease called DEN1 . Human DEN1 is encoded by AAH31411.1, a previously uncharacterized protein of 212 amino acids that shares homology with the Ulp1 cysteinyl SUMO deconjugating enzyme family . Recombinant human DEN1, purified from bacteria, selectively binds to Nedd8 and hydrolyzes C-terminal derivatives of Nedd8 . Interestingly, DEN1 deconjugates cullin 1 (CUL1)-Nedd8 in a concentration-dependent manner . At a low concentration, DEN1 processes hyper-neddylated CUL1 to yield a mononeddylated form, which presumably contains the Lys-720CUL1-Nedd8 linkage . At elevated concentrations, DEN1 is able to complete the removal of Nedd8 from CUL1 . These activities distinguish DEN1 from the COP9 signalosome, which is capable of efficiently cleaving the Lys-720CUL1-Nedd8 conjugate, but lacks Nedd8 C-terminal hydrolytic activity and poorly processes hyperneddylated CUL1 . These results suggest a unique role for DEN1 in regulating the modification of cullins by Nedd8. J Biol Chem, 2003 Aug 1, 278(31), 28711 - 8 Epub 2003 May 20. The amino-terminal GAF domain of Azotobacter vinelandii NifA binds 2-oxoglutarate to resist inhibition by NifL under nitrogen-limiting conditions; Little R et al.; The expression of genes required for the synthesis of molybdenum nitrogenase in Azotobacter vinelandii is controlled by the NifL-NifA transcriptional regulatory complex in response to nitrogen, carbon, and redox status . Activation of nif gene expression by the transcriptional activator NifA is inhibited by direct protein-protein interaction with NifL under conditions unfavorable for nitrogen fixation . We have recently shown that the NifL-NifA system responds directly to physiological concentrations of 2-oxoglutarate, resulting in relief of NifA activity from inhibition by NifL under conditions when fixed nitrogen is limiting . The inhibitory activity of NifL is restored under conditions of excess combined nitrogen through the binding of the signal transduction protein Av GlnK to the carboxyl-terminal domain of NifL . The amino-terminal domain of NifA comprises a GAF domain implicated in the regulatory response to NifL . A truncated form of NifA lacking this domain is not responsive to 2-oxoglutarate in the presence of NifL, suggesting that the GAF domain is required for the response to this ligand . Using isothermal titration calorimetry, we demonstrate stoichiometric binding of 2-oxoglutarate to both the isolated GAF domain and the full-length A . vinelandii NifA protein with a dissociation constant of approximately 60 microm . Limited proteolysis experiments indicate that the binding of 2-oxoglutarate increases the susceptibility of the GAF domain to trypsin digestion and also prevents NifL from protecting these cleavage sites . However, protection by NifL is restored when the non-modified (non-uridylylated) form of Av GlnK is also present . Our results suggest that the binding of 2-oxoglutarate to the GAF domain of NifA may induce a conformational change that prevents inhibition by NifL under conditions when fixed nitrogen is limiting. Microbes Infect, 2003 May, 5(6), 535 - 44 Endophytes as sources of bioactive products; Strobel GA; An increase in the number of people in the world having health problems caused by various cancers, drug-resistant bacteria, parasitic protozoans, and fungi is a cause for alarm . An intensive search for newer and more effective agents to deal with these disease problems is now under way and endophytes are a novel source of potentially useful medicinal compounds. Trends Plant Sci, 2003 May, 8(5), 238 - 44 Quorum quenching and proactive host defense; Zhang LH; Both plants and humans have inducible defense mechanisms . This passive defense strategy leaves the host unprotected for a period of time until resistance is activated . Moreover, many bacterial pathogens have evolved cell-cell communication (quorum-sensing) mechanisms to mount population-density-dependent attacks to overwhelm the host's defense responses . Several chemicals and enzymes have been investigated for years for their potential to target the key components of bacterial quorum-sensing systems . These quorum-quenching reagents, which block bacterial cell-cell communications, can disintegrate a bacterial population-density-dependent attack . It has now been shown that a quorum-quenching mechanism can be engineered in plants and might be used as a strategy in controlling bacterial pathogens and to build up a proactive defense barrier. Aquat Toxicol, 2003 May 29, 63(4), 373 - 89 Effects of the pyrethroid insecticide cypermethrin on a freshwater community studied under field conditions . II . Direct and indirect effects on the species composition; Wendt-Rasch L et al.; The effects of cypermethrin, a commonly used pyrethroid insecticide, were studied in small in situ enclosures situated in an eutrophic lake over an 11-day period . The experimental design used a regression principle that included three untreated controls and a gradient of six unreplicated cypermethrin concentrations, ranging from 0.01 to 6 microg/l . This paper is the second in a series of two and describes the effects on the species composition of the crustacean, rotifer, periphyton and phytoplankton communities . Multivariate ordination technique (redundancy analysis (RDA) combined with Monte Carlo permutation tests) showed that exposure to cypermethrin caused significant changes in the species composition of the communities . Changes in the structure of the communities were observed following exposure to a nominal concentration of 0.13 microg cypermethrin per litre above . The direct acute effect of exposure to cypermethrin was a rapid decrease of many species of crustacean zooplankton . The alterations in crustacean species composition were probably due to variations in susceptibility to the direct toxic effects of cypermethrin . No effects concentration (NEC) for individual zooplankton species were calculated using inverse regression and revealed that copepod nauplii were the most sensitive (NEC=0.01 microg/l) of the crustacean groups examined . The observed alterations of the species composition of the autotrophic communities as well as of the rotifers were most likely caused indirectly by cypermethrin, mediated through the direct negative effects of the insecticide on the crustacean grazers . The results of this experiment provide further knowledge about the direct and indirect effects of pesticide stress on the ecosystem level . They also show that there is a variation in sensitivity between different species of zooplankton under natural conditions and thus exemplify the necessity of multispecies approaches in the risk assessment of pesticides. Aquat Toxicol, 2003 May 29, 63(4), 357 - 71 Effects of the pyrethroid insecticide, cypermethrin, on a freshwater community studied under field conditions . I . Direct and indirect effects on abundance measures of organisms at different trophic levels; Friberg-Jensen U et al.; The effects of the pyrethroid insecticide cypermethrin on a natural freshwater community were studied in small in situ enclosures over an 11-day period . The experiment was conducted in a eutrophic lake using a regression design that included three untreated controls and a gradient of six unreplicated cypermethrin concentrations, ranging from 0.01 to 6.1 microg/l . This paper is the first in a series of two, and describes the fate of cypermethrin and its effects on the abundance of crustaceans, rotifers, protozoans (cilliates and heterotrophic nanoflagellates (HNF)) and bacteria and the biomass of periphytic and planktonic algae . The concentration of cypermethrin decreased quickly during the experiment, with a half-life of 48 h for the total and 25 h for the dissolved fractions of cypermethrin, respectively . Cypermethrin proved to be acutely toxic to crustaceans in enclosures receiving nominal cypermethrin concentrations of >/=0.13 microg/l . No Effect Concentration (NEC) and median Effect Concentration (EC(50)) for the total crustacean community and cladoceran and copepod subgroups ranged between 0.02-0.07 and 0.04-0.17 microg/l, respectively, with copepods being less sensitive than cladocerans . The abundance of rotifers, protozoans and bacteria and the chlorophyll-a concentration of planktonic and periphytic algae was significantly related to the concentration of cypermethrin . All groups proliferated within 2-7 days after the cypermethrin application in those enclosures where the abundance of crustaceans was seriously affected by cypermethrin (i.e . >/=0.13 microg/l) . We hypothesise that the proliferation of rotifers, protozoans, bacteria and algae was due to a reduced grazer control from crustaceans and thereby mediated indirectly by cypermethrin . The results of this experiment provide knowledge on how an entire microplankton community may respond to pyrethroids in nature, and the indirect effects observed on the community clearly demonstrates the necessity of multispecies field experiments in ecotoxicological risk assessment. FEMS Microbiol Lett, 2003 May 16, 222(1), 33 - 7 Sulerythrin, the smallest member of the rubrerythrin family, from a strictly aerobic and thermoacidophilic archaeon, Sulfolobus tokodaii strain 7; Wakagi T; A protein corresponding to the N-terminal domain of rubrerythrin was isolated from a strictly aerobic archaeon, Sulfolobus tokodaii strain 7 . The molecular mass was found to be 15.8 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 16278 Da by time-of-flight mass spectrometry and 34.5 kDa by gel filtration chromatography, suggesting that the protein is dimeric . Two mol iron and 1-2 mol zinc mol(-1) protein were detected . On addition of the azide ion, the absorption spectrum was greatly affected . The far UV circular dichroism spectrum suggested that the protein was mostly composed of alpha-helices . The N-terminal sequence completely matched the open reading frame, st2370, recently found on genome analysis of the organism . The protein was homologous to rubrerythrin but lacked a C-terminal rubredoxin domain . It was found in the genus Sulfolobus and therefore named sulerythrin; it is the smallest and first aerobic member of the rubrerythrin family. Dtsch Tierarztl Wochenschr, 2003 Apr, 110(4), 158 - 60 {Acute herpesvirus-gastritis in a cat}; Breuer W et al.; Gastritis in cats is caused, among other things, by infectious agents, like bacteria, metazoic parasites or viruses . Herpesvirus-gastritis has not as yet been documented in cats . Therefore in this paper such a case will be described . In this case the mucous membrane of the stomach shows multifocal acute necroses with evidence of intranuclear inclusion bodies in epithelial cells of the gastric glands . By means of electron microscopy the causative virus can be specified as herpesvirus. J Plant Physiol, 2003 Apr, 160(4), 377 - 86 Nitrogen fixation in transposon mutants from Bradyrhizobium japonicum USDA 110 impaired in nitrate reductase; Camacho M et al.; Tn5 transposon mutagenesis was carried out in Bradyrhizobium japonicum strain USDA 110 to produce defective mutants . From over one thousand clones expressing low levels of nitrate reductase activity as free-living bacteria, approximately five percent had significantly different ratios of nodulation, N2 fixation or nitrate reductase activity compared to the wild strain when determined in bacteroids from soybean nodules . Tn5 insertions were checked previously and mutants were arranged into four different groups . Only one of these groups, designated AN, was less effective at N2 fixation than the wild strain, suggesting a mutation in a domain shared by nitrogenase and NR . The remaining groups of insertions successfully nodulated and were as effective at N2 fixation as the wild strain, but showed diminished ability to reduce nitrate both in nodules and in the isolated bacteroids when assayed in vitro with NADH or methyl viologen as electron donors . PCR amplification demonstrated that Tn5 insertions took place in different genes on each mutant group and the type of mutant (CC) expressing almost no nitrate reductase activity under all treatments seemed to possess transposable elements in two genes . Induction of nitrate reductase activity by nitrate was observed only in those clones expressing a low constitutive activity (AN and AE) . Nitrate reductase activity in bacteroids along nodule growth decreased in all groups including the ineffective AN group, whose nodulation was highly inhibited by nitrate at 5 mmol/L N . Host-cultivar interaction seemed to influence the regulation of nitrate reductase activity in bacteroids . Total or partial repression of nitrate reductase activity in bacteroids unaffected by N2 fixation (CC, AJ and AE groups) improved nodule resistance to nitrate and N yields of shoots over those of the wild strain . These observations may suggest that some of the energy supplied to bacteroids was wasted by its constitutive NRA. Vet Ther, 2003 Spring, 4(1), 12 - 23 Evaluation of an ear cleanser for the treatment of infectious otitis externa in dogs; Cole LK et al.; Thirty-one ears (16 dogs) with otitis externa originating from bacterial or yeast infections were enrolled in a study to evaluate the in vivo efficacy of an ear cleanser containing 2.5% lactic acid and 0.1% salicylic acid for the treatment of infectious otitis externa . The affected ears were treated with the ear cleanser twice daily for 2 weeks and evaluated after 1 and 2 weeks of treatment . The ear cleanser was effective in resolution of infection in 67.7% of the ears, and clinical signs of infectious otitis externa were significantly reduced within 2 weeks. J Vasc Surg, 2003 May, 37(5), 1082 - 90 Neutrophil survival on biomaterials is determined by surface topography; Chang S et al.; PURPOSE: Cardiovascular device-centered infections are a major cause of hospital morbidity, mortality, and expense . Caused by opportunistic bacteria, this phenomenon is thought to arise because of a defect in neutrophil bacterial killing . We have shown that neutrophils that adhere to polystyrene remain viable, whereas neutrophils that adhere to the vascular biomaterials expanded polytetrafluoroethylene (ePTFE) and Dacron undergo a rapid nonapoptotic death . This study was designed to test the hypothesis that surface topography is a determinant of the nonapoptotic death response of neutrophils to biomaterials . METHODS: We took advantage of the ease with which a polystyrene surface can be manipulated to examine the effect of surface topography on neutrophil viability . Neutrophils were exposed to smooth or roughened polystyrene surfaces both in vivo and in vitro . Changes in cell membrane permeability and production of reactive oxygen species by individual cells were monitored with fluorescent dyes . RESULTS: Host cells and isolated human neutrophils died rapidly after adhesion to roughened polystyrene . Neutrophils adherent to roughened surfaces produced more reactive oxygen intermediates than those adherent to smooth surfaces and were first to die . The cell death response precipitated by expanded polytetrafluoroethylene, Dacron, or the roughened surfaces was significantly reduced with treatment of the neutrophils with catalase, diphenylene iodonium, or the src kinase inhibitor PP2 before adhesion . CONCLUSIONS: Neutrophil adhesion to roughened materials triggers rapid production of reactive oxygen species and precipitates a nonapoptotic cell death . Understanding the material properties that trigger these responses is essential to development of the next generation of implantable biomaterials. J Helminthol, 2003 Jun, 77(2), 147 - 53 Causality or coincidence: may the slow disappearance of helminths be responsible for the imbalances in immune control mechanisms? Palmas C, Gabriele F, Conchedda M, Bortoletti G, Ecca AR. Intestinal infection continues to be a problem worldwide and helminths, which currently infect billions of individuals, are primary culprits . The major burden of disease falls on the populations of developing countries, given that over the last four to five decades helminth infections are disappearing in industrialized societies . In developing countries, a major source of immunomodulatory signals in post-natal life are parasites, particularly helminths, which, unlike most bacteria and viruses, selectively stimulate Th2 function . Helminths and their eggs are probably the most potent stimulators of mucosal Th2 responses . Responses elicited by worms can modulate immune reactions to other parasites, bacterial, viral infections and several unrelated diseases . Bacterial and protozoal infections may also protect against atopy and asthma, through the induction of the Th1 regulatory responses . Today, people in developed countries often live in ultra-hygienic environments, avoiding exposure to viruses, bacteria, ectoparasites and endoparasites, particularly helminths . Perhaps failure to acquire worms and experience mucosal Th2 conditioning predisposes to unrelated diseases . In contrast to this hypothesis it has also been suggested that Th2 responses can make the host more susceptible to other important diseases and to contribute to the spread of them. Indoor Air, 2003 Jun, 13(2), 136 - 47 The 3-year follow-up study in a block of flats - experiences in the use of the Finnish indoor climate classification; Tuomainen M et al.; Indoor climate of two new blocks of flats was investigated . The case building was built for people with respiratory diseases by following the instructions of the Finnish Classification of Indoor Climate, Construction and Finishing Materials, while the control building was built using conventional building technology . The main indoor air parameters (temperature, relative humidity and levels of CO, CO2, ammonia, total volatile organic compounds, total suspended particles, fungal spores, bacteria and cat, dog and house dust mite allergens) were measured in six apartments of both the buildings on five occasions during the 3-year occupancy . In addition, a questionnaire to evaluate symptoms of the occupants and their satisfaction with their home environment was conducted in connection with indoor air quality (IAQ) measurements . The levels of indoor air pollutants in the case building were, in general, lower than those in the control building . In addition, the asthmatic occupants informed that their symptoms had decreased during the occupancy in the case building . This case study showed that high IAQ is possible to reach by careful design, proper materials and equipment and on high-quality construction with reasonable additional costs . In addition, the study indicated that good IAQ can also be maintained during the occupancy, if sufficient information on factors affecting IAQ and guidance on proper use and care of equipment are available for occupants. Environ Microbiol, 2003 Jun, 5(6), 523 - 33 Phylogeny and distribution of nitrate-storing Beggiatoa spp . in coastal marine sediments; Mussmann M et al.; Filamentous sulphide-oxidizing Beggiatoa spp . often occur in large numbers in the coastal seabed without forming visible mats on the sediment surface . We studied the diversity, population structure and the nitrate-storing capability of such bacteria in the Danish Limfjorden and the German Wadden Sea . Their distribution was compared to the vertical gradients of O2, NO3- and H2S as measured by microsensors . The main Beggiatoa spp . populations occurred in a 0.5-3 cm thick intermediate zone, below the depth of oxygen and nitrate penetration but above the zone of free sulphide . The Beggiatoa spp . filaments were found to store nitrate, presumably in liquid vacuoles up to a concentration of 370 mM NO3-, similar to the related large marine sulphur bacteria, Thioploca and Thiomargarita . The observations indicate that marine Beggiatoa spp . can live anaerobically and conserve energy by coupling sulphide oxidation with the reduction of nitrate to dinitrogen and/or ammonia . Calculations of the diffusive nitrate flux and the potential sulphide oxidation by Beggiatoa spp . show that the bacteria may play a critical role for the sulphur cycling and the nitrogen balance in these coastal environments . 16S rDNA sequence analysis shows a large diversity of these uncultured, nitrate-storing Beggiatoa spp . Smaller (9-17 micro m wide) and larger (33-40 micro m wide) Beggiatoa spp . represent novel phylogenetic clusters distinct from previously sequenced, large marine Beggiatoa spp . and Thioploca spp . Fluorescence in situ hybridization (FISH) of the natural Beggiatoa spp . populations showed that filament width is a conservative character of each phylogenetic species but a given filament width may represent multiple phylogenetic species in a mixed population. Biochemistry, 2003 May 27, 42(20), 6201 - 8 EPR and ENDOR evidence for a 1-His, hydroxo-bridged mixed-valent diiron site in Desulfovibrio vulgaris rubrerythrin; Smoukov SK et al.; Key features differentiating the coordination environment of the two irons in the mixed-valent (Fe(2+),Fe(3+)) diiron site of Desulfovibrio vulgaris rubrerythrin (Rbr(mv)) were determined by continuous wave (CW) and pulsed ENDOR spectroscopy at 35GHz . (14)N ENDOR evidence indicates that a nitrogen is bound only to the Fe(2+) ion of the mixed-valent site . Assuming that this nitrogen is from His131Ndelta, the same one that furnishes an iron ligand in the crystal structure of the diferric site, the ENDOR data allow us to specify the Fe(2+) and Fe(3+) positions within the molecular reference frame . In addition, the (1,2)H ENDOR on Rbr(mv) indicates the presence of a solvent-derived aqua/hydroxo ligand bound either terminally or in a bridging mode to Fe(3+) in the mixed-valent site . The relatively large g anisotropy of Rbr(mv) and weak antiferromagnetic coupling, J approximately -8 cm(-)(1) (in the 2JS(1)*S(2) formalism), between the irons is more consistent with a bridging than terminal hydroxo ligand . gamma-Irradiation was used to cryoreduce Rbr at 77 K, thereby producing a mixed-valent diiron site {(Rbr(ox))(mv)} that retains the structure of the diferric site . The EPR spectrum of (Rbr(ox))(mv) was nearly identical to that of the as-isolated or chemically reduced samples . This near identity implies that the structure of the mixed-valent Rbr diiron site is essentially identical to that of the diferric site, except for protonation of the oxo bridge, which apparently occurred via a proton jump from hydrogen-bonded solvent at 77 K . The EPR spectrum of (Rbr(ox))(mv) thus supports the (14)N ENDOR-assigned His131 ligation to Fe(2+) and assignment of the solvent-derived ligand observed in the (1,2)H ENDOR to a hydroxo bridge between the irons of the mixed-valent diiron site. Antonie Van Leeuwenhoek, 2003, 83(1), 21 - 6 Streptomyces yatensis sp . nov., a novel bioactive streptomycete isolated from a New-Caledonian ultramafic soil; Saintpierre D et al.; The taxonomic position of an actinomycete isolated from an ultramafic soil in New Caledonia was examined using a polyphasic approach . The organism, which was designated SFOCin 76, was found to have chemical and morphological properties typical of streptomycetes and formed a distinct phyletic line in the Streptomyces violaceusniger clade of the 16S rDNA tree . It also showed a unique pattern of phenotypic properties that distinguished it from representatives of all of the validly described species classified in this clade . It is, therefore, proposed that strain SFOCin 76 be classified in the genus Streptomyces as Streptomyces yatensis sp . nov. Int J Med Microbiol, 2003 Apr, 293(1), 77 - 86 Intestinal flora and mucosal immune responses; Heller F et al.; The normal intestinal flora and the mucosal immune system exist in close spatial proximity . A normal structure and function of both very complex systems is required for health and develops in a constant and interactive process . An abnormal host response to the normal intestinal flora leads to chronic intestinal inflammation . Probiotic bacteria may modulate the intestinal flora and the mucosal immune response and are an effective therapy for remission maintenance of ulcerative colitis and pouchitis. Am J Vet Res, 2003 May, 64(5), 538 - 43 Neutrophil function and plasma opsonic capacity in colostrum-fed and colostrum-deprived neonatal kittens; Hanel RM et al.; OBJECTIVE: To determine whether passive transfer of IgG in neonatal kittens affects plasma opsonic capacity and neutrophil phagocytic and oxidative burst responses to bacteria in vitro . ANIMALS: 22 kittens from 6 specific pathogen-free queens . PROCEDURE: Kittens were randomized at birth into the following treatment groups: colostrum-fed, colostrum-deprived, or colostrum-deprived supplemented with feline or equine IgG . Blood samples were collected at intervals from birth to 56 days of age . Plasma IgG concentrations were determined by radial immunodiffusion assay . Neutrophil function was assessed by a flow cytometry assay providing simultaneous measurement of bacteria-induced phagocytosis and oxidative burst . The opsonic capacity of kitten plasma was determined in an opsonophagocytosis assay with bacteria incubated in untreated or heat-inactivated plasma . RESULTS: Among treatment groups, there were no significant differences in neutrophil phagocytic and oxidative burst responses to bacteria or opsonic capacity of plasma . In all samples of plasma, inactivation of complement and other heat-labile opsonins significantly reduced the opsonic capacity . Plasma IgG concentrations in kittens did not correlate with neutrophil function or plasma opsonic capacity before or after inactivation of complement . CONCLUSIONS AND CLINICAL RELEVANCE: The plasma opsonic capacity and neutrophil phagocytic and oxidative burst responses in vitro of kittens receiving passive transfer of IgG via colostrum intake or IgG supplementation and those deprived of colostrum were similar . The alternate complement pathway or other heat-labile opsonins may be more important than IgG in bacterial opsonization and phagocytosis. East Afr Med J, 2003 Jan, 80(1), 30 - 5 Pneumocystis carinii pneumonia in HIV/AIDS patients at an urban district hospital in Kenya; Chakaya JM et al.; BACKGROUND: Pneumocystis carinii pneumonia has generally been regarded to be an uncommon opportunistic infection in HIV infected individuals in sub-Saharan Africa . The reason for this has not been clear but postulates included a lack of suitable pathogenic types in the African environment, diagnostic difficulties and the more commonly held belief that African HIV infected individuals were dying early from common non-opportunistic pathogens before severe degrees of immunosuppression occured . Recently a trend has emerged at the Mbagathi district hospital whereby an increasing number of HIV infected patients are empirically treated for Pneumocystis carinii pneumonia (PCP) based on clinical and radiological features . OBJECTIVE: To determine the prevalence of PCP and clinical outcomes of HIV infected patients presenting at the Mbagathi District Hospital, Nairobi with the presumptive diagnosis of PCP . SETTING: Mbagathi District Hospital, a 169-bed public hospital in Nairobi, Kenya . METHODS: Patients presenting with a sub-acute onset of cough and dyspnoea were eligible for the study if they were found to have bilateral pulmonary shadows and had negative sputum smears for AFBS . Consenting patients who had no contraindication to fiberoptic bronchoscopy had a clinical evaluation which was followed with a fiberoptic bronchoscopy procedure where bronchoalveolar lavage fluid (BALF) was obtained . BALF was examined for cysts of P . carinii using toluidine blue stain and immunofluorescent antibody test (IFAT) . BALF was also processed for fungi, bacteria and mycobacteria using routine procedures . Standard treatment with high dose cotrimoxazole was offered to all patients who were then followed up until discharge from hospital or death whichever came first . RESULTS: Between June 1999 and August 2000 a total of 63 patients were referred for bronchoscopy . Of these four declined to undergo the fiberoptic bronchoscopy procedure, four died before the procedure could be done, one was judged too sick to undergo the procedure and three had been on cotrimoxazole for longer than five days . Thus 51 patients underwent bronchoscopy . Pneumocystis carinii stain was positive in 19 (37.2%) while death occured in 16 (31.4%) of the 51 patients . There were more deaths in those without PCP but this difference was not statistically significant (odds ratio 0.68 (95% CI 0.35-1.32; P=0.2) . CONCLUSION: PCP was found to be common in HIV infected patients presenting with clinical and radiological features of the disease . The mortality rate for patients with a presumptive diagnosis of PCP is high . This study suggests that cotrimoxazole preventive therapy may be a useful intervention in symptomatic HIV infected patients in Kenya for the prevention of PCP and may avert deaths from this disease. Gac Med Mex, 2003 Mar-Apr, 139(2), 118 - 22 {Mycoses frequency in three communities in the North mountain of the State of Puebla}; Mendez-Tovar LJ et al.; In order to know mycosis frequency in the North of the State of Puebla, Mexico, in habitants from the communities of Ayotoxco, Mazatepec and Zacatipan were studied . Previous medical study biological samples were submitted to direct examination, smear and culture . Histoplasmin and sporotrichin skin test were applied to 57 individual from Zacatipan . From 110 patients 146 mycological studies were performed . Eighty six cases (59%) of mycosis were detected: 43 finger or toenails onychomycosis, 25 tinea pedis, seven tinea capitis, four cases of tinea manum and, finally, five cases of seborrhoeic dermatitis and two of pitiriasis versicolor . We isolated: 18 streins of dermatophytes, mainly Trichophyton rubrum and T . mentagrophytes (11 and 5 strains respectively); 12 cultures of non-dermatophytes filamentous fungi; six cases of mycelia sterile; six yeast strains, most of them Candida spp but none C . albicans . From 57 patients to whom skin tests were applied, five of them (8.8%) were positive to both antigens; ten positive (17.6%) only to histoplasmin and eight (14%) to sporotrichin . This study showed that rural population from Puebla present a high frequency of superficial mycosis (61% of mycological studies) . Considering the percentage of positive skin test we suppose that there are many not diagnosed sporotrichosis and histoplasmosis cases. C R Biol, 2003 Feb, 326(2), 141 - 7 Does the size of small objects influence chemical reactivity in living systems? Sanfeld A, Steinchen A. Previous theoretical works showed that chemical reactions in micro- and nano-droplets, bubbles and solid particles were strongly affected by their confinement . In particular, the smallness of the systems leads to high internal pressure compared to the external pressure, which then significantly modifies the values of chemical equilibrium and kinetic constants . In addition, surface tension or surface stress, reactional dilatation and surface charge play also a major role on the chemical reactivity . As living systems are also made of very complex dispersed subsystems, i.e . organelles, it seemed obvious to illustrate our theory by some biological actual examples encountered in pulmonary alveolae, in vacuolae and in medical applications, such as dissolution of gallstones. Microb Ecol, 2003 Jul, 46(1), 22 - 32 Epub 2003 May 21. Distribution of exopolymeric substances in the littoral sediments of an oligotrophic lake; Hirst CN et al.; Bacteria and algae release exopolymeric substances (EPS) that perform a wide range of important functions in aquatic and terrestrial systems . In this study we measured EPS in sediments at nine littoral sites around a shallow oligotrophic basin, and tested whether the concentration and composition of EPS was related to sediment characteristics . The concentrations of both loosely bound (colloidal) and tightly bound (capsular) EPS carbohydrates ranged up to ~800 micro g glucose equiv . cm(-2) and were well within the range of concentrations reported from marine intertidal flats, where EPS play an important role in stabilizing sediments, affecting nutrient exchanges between sediments and the water column, feeding benthic invertebrates, and sequestering and increasing the transfer of contaminants to food webs . Proteins were an important component of the EPS in these littoral sediments, with protein:carbohydrate ratios of approximately 0.4 . In summer, the concentrations of most EPS fractions were positively related ( P < 0.05) to the porewater and organic matter content of the sediments . Capsular EPS concentrations were lower in the fall, with a simultaneous increase in colloidal proteins but not in colloidal carbohydrates . This suggests that the carbohydrates in this colloidal EPS may be more labile than the proteins . Our results suggest that exopolymeric substances could be an important, but neglected, component of littoral sediments in lakes. FEBS Lett, 2003 May 22, 543(1-3), 42 - 6 Transcriptional effects of the signal transduction protein P(II) (glnB gene product) on NtcA-dependent genes in Synechococcus sp . PCC 7942; Paz-Yepes J et al.; P(II) proteins signal the cellular nitrogen status in numerous bacteria, and in cyanobacteria P(II) is subjected to serine phosphorylation when the cells experience a high C to N balance . In the unicellular cyanobacterium Synechococcus sp . PCC 7942, the P(II) protein (glnB gene product) is known to mediate the ammonium-dependent inhibition of nitrate and nitrite uptake . The analysis of gene expression through RNA/DNA hybridization indicated that a P(II)-null mutant was also impaired in the induction of NtcA-dependent, nitrogen assimilation genes amt1 (ammonium permease), glnA (glutamine synthetase) and nir (nitrite reductase), as well as of the N-control gene ntcA, mainly under nitrogen deprivation . This gene expression phenotype of the glnB mutant could be complemented by wild-type P(II) protein or by modified P(II) proteins that cannot be phosphorylated and mimic either the phosphorylated (GlnB(S49D) and GlnB(S49E)) or unphosphorylated (GlnB(S49A)) form of P(II) . However, strains carrying the GlnB(S49D) and GlnB(S49E) mutant proteins exhibited higher levels of expression of nitrogen-regulated genes than the strains carrying the wild-type P(II) or the GlnB(S49A) protein. Plant J, 2003 May, 34(4), 427 - 40 Ku80- and DNA ligase IV-deficient plants are sensitive to ionizing radiation and defective in T-DNA integration; Friesner J et al.; Double-strand break (DSB) repair pathways catalyze the rejoining of broken chromosomes and the integration of transforming DNAs . These processes have been well characterized in bacteria, fungi, and animals . Plants are generally thought primarily to utilize a non-homologous end joining (NHEJ) pathway to repair DSBs and integrate transgenes, as transforming DNAs with large tracts of homology to the chromosome are integrated at random . In order to test the hypothesis that NHEJ is an important pathway for the repair of DSBs in plants, we isolated T-DNA insertion mutations in the Arabidopsis homologs of the Ku80 and DNA ligase IV genes, required for the initiation and completion, respectively, of NHEJ . Both mutants were hypersensitive to the cytostatic effects of gamma radiation, suggesting that NHEJ is indeed a critical pathway for the repair of DSBs . T-DNA insertion rates were also decreased in the mutants, indicating that Ku80 and DNA ligase IV play an important role in either the mechanism or the regulation of T-DNA integration in Arabidopsis. J Periodontal Res, 2003 Jun, 38(3), 276 - 81 Serum antibody reactivity against recombinant PrtC of Porphyromonas gingivalis following periodontal therapy; Beikler T et al.; OBJECTIVES: In 34 patients with chronic periodontitis, the presence of IgA, IgG, and IgG subclass serum antibodies against recombinant PrtC (rPrtC) of Porphyromonas gingivalis was assessed by immunoblot analysis 24 months after therapy . METHODS: rPrtC was produced from P . gingivalis ATTC 33277 using the plasmid pGEX-2T . In addition, intraoral colonization with P . gingivalis was detected by PCR in subgingival plaque and swab samples from buccal mucosae, tonsils and tongue at baseline, 10 d, and 3, 6, 9, 12, 18, and 24 months . RESULTS: All patients were found to harbor P . gingivalis in the oral cavity at least once during the observation period . The identified antibody responses against the rPrtC of P . gingivalis were IgA (97%, i.e . 33/34 patients) and IgG (100%, i.e . 34/34), with an IgG subclass distribution of IgG2 (65%, i.e . 22/34 patients) > IgG3 (47%, i.e . 16/34) > IgG1 (38%, i.e . 13/34) > IgG4 (29%, i.e . 10/34) . Anti-rPrtC IgA and IgG antibody reactivity was found in all but one patients (anti-rPrtC IgA negative), who tested negative for P . gingivalis at all of the assessed intraoral sites for at least 6 months before sera collection . There was no association between IgG subclass reactivity against the rPrtC of P . gingivalis and progression of periodontal attachment loss . CONCLUSION: The results indicated that anti-rPrtC IgA and IgG antibodies may serve as an indicator for past or present intraoral colonization with P . gingivalis. J Periodontal Res, 2003 Jun, 38(3), 262 - 8 Generation of gingival T cell lines/clones specific with Porphyromonas gingivalis pulsed dendritic cells from periodontitis patients; Aroonrerk N et al.; OBJECTIVES AND BACKGROUND: It is well documented that in periodontitis lesions, most infiltrated gingival T cells are antigen-specific memory T cells . These cells play an important role as regulators and effector cells in the pathogenesis of periodontitis . In this study, we used dendritic cells (DCs) as antigen-presenting cells to generate human gingival T cell lines and clones specific for Porphyromonas gingivalis from periodontitis patients . METHODS: Autologous DCs were derived from the patients' adherent monocytes using granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4 . Lymphocytes were isolated from gingival biopsies using collagenase enzyme digestion and the number was increased by subsequent culturing in IL-2-containing medium . T cells were then negatively sorted using flow cytometry, cocultured with P . gingivalis-pulsed DCs and subsequently expanded in the culture medium containing IL-2 . T cells were kept viable and active by periodic exposure to antigen-pulsed DCs . The specificity of the T cell lines was tested against four plaque bacteria: P . gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia and Actinomyces viscosus . The established T cell lines were then cloned . Three P . gingivalis-specific T cell lines and 12 gingival T cell clones were generated . They all showed good specificity against P . gingivalis but not to other plaque bacteria . RESULTS: All T cell clones were positive for CD4 and the majority of them produced interferon gamma, but a minimal or negligible amount of IL-5 . CONCLUSIONS: The data obtained clearly showed that monocyte-derived DCs could be used as powerful antigen-presenting cells to generate antigen-specific T cells from periodontitis tissues. Mol Ecol, 2003 Apr, 12(4), 1049 - 60 Wolbachia infection complexity among insects in the tropical rice-field community; Kittayapong P et al.; Wolbachia are a group of intracellular bacteria that cause reproductive alterations in their arthropod hosts . Widely discordant host and Wolbachia phylogenies indicate that horizontal transmission of these bacteria among species sometimes occurs . A likely means of horizontal transfer is through the feeding relations of organisms within communities . Feeding interactions among insects within the rice-field insect community have been well documented in the past . Here, we present the results of a polymerase chain reaction-based survey and phylogenetic analysis of Wolbachia strains in the rice-field insect community of Thailand . Our field survey indicated that 49 of 209 (23.4%) rice-field insect species were infected with Wolbachia . Of the 49 infected species, 27 were members of two feeding complexes: (i) a group of 13 hoppers preyed on by 2 mirid species and parasitized by a fly species, and (ii) 2 lepidopteran pests parasitized by 9 wasp species . Wolbachia strains found in three hoppers, Recilia dorsalis, Nephotettix malayanus and Nisia nervosa, the two mirid predators, Cyrtorhinus lividipennis and Tytthus chinensis, and the fly parasitoid, Tomosvaryella subvirescens, were all in the same Wolbachia clade . In the second complex, the two lepidopteran pests, Cnaphalocrocis medinalis and Scirpophaga incertulas, were both infected with Wolbachia from the same clade, as was the parasitoid Tropobracon schoenobii . However, none of the other infected parasitoid species in this feeding complex was infected by Wolbachia from this clade . Mean (+/- SD) genetic distance of Wolbachia wsp sequences among interacting species pairs of the hopper feeding complex (0.118 +/- 0.091 nucleotide sequence differences), but not for the other two complexes, was significantly smaller than that between noninteracting species pairs (0.162 +/- 0.079 nucleotide sequence differences) . Our results suggest that some feeding complexes, such as the hopper complex described here, could be an important means by which Wolbachia spreads among species within arthropod communities. Mol Microbiol, 2003 May, 48(4), 901 - 11 Sequence-specific recognition but position-dependent cleavage of two distinct telomeres by the Borrelia burgdorferi telomere resolvase, ResT; Tourand Y et al.; An unusual feature of bacteria in the genus Borrelia (causative agents of Lyme disease and relapsing fever) is a segmented genome consisting of multiple linear DNA molecules with covalently closed hairpin ends, known as telomeres . The hairpin telomeres are generated by a DNA breakage and reunion process (telomere resolution) promoted by ResT, an enzyme using an active site related to that of tyrosine recombinases and type IB topoisomerases . In this study, we define the minimal sequence requirements for a functional telomere and identify specific basepairs that appear to be important for telomere resolution . In addition, we show that the two naturally occurring and distinct telomere spacings found in B . burgdorferi can both be efficiently processed by ResT . This flexibility for substrate utilization by ResT supports the argument for a single telomere resolvase in Borrelia . Furthermore, although telomere recognition requires sequence specificity in part of the substrate, DNA cleavage is instead position dependent and occurs at a fixed distance from the axis of symmetry and the conserved sequence of box 3 in the different replicated telomere substrates . This positional dependence for DNA cleavage has not been observed previously for a tyrosine recombinase. Helicobacter, 2003 Jun, 8(3), 186 - 91 Is the recurrence of Helicobacter pylori infection after eradication therapy resultant from recrudescence or reinfection, in Japan; Okimoto T et al.; BACKGROUND: Reinfection of Helicobacter pylori after eradication is rare in developed countries but most often occurs within 1 year . In the present study, we attempted to differentiate between reinfection and recrudescence of H . pylori strains between 6 months and 6 years after successful eradication in Japan, a country with a high prevalence of H . pylori infection . MATERIALS AND METHODS: After successful eradication of H . pylori, 274 patients were followed up by endoscopy and urea breath test . In recurrent patients, H . pylori strains isolated initially and after recurrence were compared using PCR-based restriction fragment length polymorphism (RFLP) analysis . RESULTS: Recurrence of H . pylori occurred in 15 of 274 patients (5.5%) at 6 months after eradication and the annual recurrence rate was 2.0% per patient year (between 1 and 6 years) . PCR-based RFLP analysis of H . pylori strains isolated initially and after recurrence showed that 62.5% (at 6 months) and 100% (after 1 years) of bacteria were of different strains . CONCLUSION: Reinfection of H . pylori was not as rare at 6 months after eradication as reported previously, and up to 6 years after eradication, the annual reinfection rate is 2.0% per patient year in Japan. Scand J Infect Dis, 2003, 35(3), 189 - 96 Disseminated Nocardia transvalensis infection with osteomyelitis and multiple brain abscesses; Montoya JP et al.; A case of Nocardia transvalensis osteomyelitis and cerebral abscesses is described . To the authors' knowledge, the patient described here represents the first reported case of primary N . transvalensis leg abscess with contiguous osteomyelitis of the femur and secondary brain abscesses . The literature on nocardial osteomyelitis and cerebral nocardiosis is reviewed, and treatment of both conditions is discussed . The interactions between the immune system and nocardial organisms are described, as well as the host defense defects in diabetic patients as they relate to pathogenicity of the bacteria . Finally, the patient's paradoxical therapeutic response of resolving brain abscesses with concomitant unresolving osteomyelitis is discussed, as well as its implications for managing similar cases in the future. Int J Neurosci, 2003 Feb, 113(2), 259 - 77 Hypothalamic mechanisms of immunity; Baciu I et al.; The present article considers a synthetical analysis of the results reported by our laboratory in the last twenty years in the field of neuroimmunomodulation . The studies we discuss here continue a previous research activity, a synthesis of which has also been published in this journal (Baciu, 1988) . In that paper, we reported data concerning the role of the hypothalamic tubero-mammillary area in triggering of the phagocytic and of the secondary immune specific response . Here, we present an analysis of experimental facts gathered after 1988, and also of some prior to that date, which were not included in the above-mentioned review . They regard localizations, attained with stereotactical methods, of hypothalamic areas involved in maintenance of basal phagocytosis and of its circadian rhythm, of the phagocytic and of the primary and secondary specific response . We attempted to re-analyze these data in an integrative view, and accomplish a coherent image of the hypothalamic mechanisms of the nonspecific and specific immune response . The conclusion we draw is that the nervous system may exert its modulatory action upon the immune response in several ways: i) subsequent to a direct hypothalamic stimulation (electrical or through bacteria or bacterial products) or to a cortico-hypothalamic stimulation; ii) depending on the nature, intensity, duration, and frequency of the appropriate stimulus, it may either enhance the immune response, via neural and humoral pathways, or depress it; iii) via the hypothalamus-pituitary-adrenal axis . Nervous triggering and enhancement of the immune response are essential, their occurrence in the initial stages ensuring its favorable course . The finding that repeated electroconvulsant shocks, employed for hypothalamus stimulation in dogs of different breed, age, weight, and individual history, are followed by extremely variable changes of the phagocytic activity raises the question on the individuality of the immune response. Rev Inst Med Trop Sao Paulo, 2003 Jan-Feb, 45(1), 53 - 4 Epub 2003 Apr 03. P system antigenic determiners expression in Ascaris lumbricoides; Ponce de Leon P et al.; The P System antigens have been detected in numerous parasites, bacteria and viruses, nevertheless the clinical significance is still unknown . The aim was to study the presence of P1 antigenic determiners in A . lumbricoides extracts by means of the use of 6 different monoclonal antibodies of well-known concentrations and Ig class . We worked with 14 A . lumbricoides extracts . Inhibition Agglutination Test was made in a bromelin enzymatic medium and 4 degrees C temperature . Titre, Score and Sensitivity Parameter were determined for each monoclonal antibody against red cells suspension used as revealing system . Ten extracts inhibited the agglutination of all anti P1 monoclonal antibodies . The 4 remaining extracts only inhibited the agglutination of some of them . It is demonstrated that the extracts have P1 activity . This activity is independent of titre, Score, Sensitivity Parameter, concentration and Ig class and it depends on the epitope at which the monoclonal antibody is directed. Mikrobiologiia, 2003 Mar-Apr, 72(2), 250 - 8 {The distribution of bacterioplankton and mesozooplankton biomass in the White and Barents Seas coastal water}; Chikin SM et al.; The total population density and the biomass of bacterioplankton, mesozooplankton, and phosphate-accumulating bacteria (PAB) were estimated during the 2000-2001 summer-autumn seasons in the coastal waters of the White and Barents Seas, which are subjects to the action of tidal and sea currents, the inflow of riverine waters, and anthropogenic impact . In the shallow estuarine waters with salinities of 6.5-32@1000 near the Chernaya, Pesha, and Pechora River mouths, the population of PAB fluctuated from 0.1 to 9.1 million cells/ml (0-36% of the total bacterial population) . In pelagic seawaters, which are low in phosphates (12-50 micrograms/l) and are characterized by an increased iron/phosphorus ratio (2.0-3.6), bacterioplankton amounted to 0.1-1.6 million cells/ml and was mainly represented by small organisms with a volume of 0.08-0.15 micron 3, commonly lacking intracellular polyphosphates . In the pelagic zone of the Barents Sea, the biomass of mesozooplankton (Bz) was comparable with that of bacterioplankton (Bb = 39-175 mg/m3), the Bb/Bz ratio being 1.4-4.6 . Off the Varandeiskii, Pechora, and Kolguyev oil terminals, Bb increased to 155-300 mg/m3 and the Bb/Bz ratio rose to 1.4 to 50.3 (with an average value of 20.9), presumably due to the severe anthropogenic impact on these waters . In this case, the dense population of bacterioplankton (0.9-7.6 million cells/ml) was mainly represented by large cells (0.12-0.76 micron 3 in volume), most of which (3-43% of the total bacterioplankton population) contained polyphosphates . The chemical composition of these waters was characterized by an elevated content of the total phosphorus (65-128 micrograms/l) and by a low iron/phosphorus ratio (0.9-1.2). Mikrobiologiia, 2003 Mar-Apr, 72(2), 149 - 60 {The biology of aerobic methylobacteria capable of degrading halomethanes}; Trotsenko IuA et al.; Recent data on the biology of aerobic methylotrophic bacteria capable of utilizing toxic halogenated methane derivatives as sources of carbon and energy are reviewed, with particular emphasis on the taxonomic, physiological, and biochemical diversity of mono- and dihalomethane-degrading methylobacteria and the enzymatic and genetic aspects of their primary metabolism . The initial steps of chloromethane dehalogenation to formate and HCl through a methylated corrinoid and methyletrahydrofolate are catalyzed by inducible cobalamin methyl transferase, made up of two proteins (CmuA and CmuB) encoded by the cmuA and cmuB genes . At the same time, the primary dehalogenation of dichloromethane to formaldehyde and HCl is catalyzed by cytosolic glutathione transferase with S-chloromethylglutathione as an intermediate . The latter enzyme is encoded by the structural dcmA gene and is under the negative control of the regulatory dcmR gene . In spite of considerable progress in the study of halomethane dehalogenation, some aspects concerning the structural and functional organization of this process and its regulation remain unknown, including the mechanisms of halomethane transport, the release of toxic dehalogenation products (S-chloromethylglutathione, CH2O, and HCl) from cells, and the maintenance of intracellular pH . Of particular interest is quantitative evaluation of the ecophysiological role of aerobic methylobacteria in the mineralization of halomethanes and protection of the biosphere from these toxic pollutants. Oecologia, 2003 Aug, 136(3), 431 - 8 Epub 2003 May 15. Nitrogen fixation and nitrogen limitation of primary production along a natural marsh chronosequence; Tyler AC et al.; Nitrogen (N) limitation of primary production is common in temperate salt marshes, even though conservative N recycling can fulfill a large proportion of plant N demand . In nutrient poor young marshes, N limitation may be more severe and new N sources, such as N fixation, more important for plant growth . We measured N fixation and the response of salt marsh primary producers (Spartina alterniflora and benthic microalgae) to N fertilization in one mature (>150 year) and two young (7 and 15 year) naturally developing marshes at the Virginia Coast Reserve LTER site . S . alterniflora aboveground biomass in the mature marsh (1,700+/-273 g m(-2)) was 1.8 and 2.8 times higher than in the 15 year and 7 year old marshes, respectively . Fertilization significantly increased S . alterniflora biomass in the two young marshes (160-175%) and areal aboveground tissue N in the youngest marsh (260%) . Microalgal chlorophyll a (Chl a) in the mature marsh was nearly 2-fold lower than in the 7-year-old marsh, and there was no evidence that this was due to light limitation . However, Chl a in fertilized plots was 30% higher than control plots at the youngest site . Daily N fixation decreased with increasing marsh age in summer, when rates were highest at all sites . Autotrophic N fixation (difference between rates in the light and dark) was most important in the summer, but we saw no indication of a shift in dominance between autotrophic and heterotrophic N fixers during marsh development . Estimated annual N fixation was 2- to 3-fold higher in the young marsh (18.3+/-1.5 g N m(-2) year(-1)), than in the intermediate-aged (9.0+/-0.7) or mature marsh (6.1+/-0.5) . In the young marshes, N fixation was sufficient to provide a substantial proportion of aboveground S . alterniflora N demand . Our results suggest that both benthic microalgae and S . alterniflora in young salt marshes are N limited, and that this limitation decreases as the marsh matures . The high rates of N fixation by autotrophic and heterotrophic bacteria in the sediment could provide an important source of N for primary producers during marsh development. Appl Microbiol Biotechnol, 2003 Nov, 63(1), 75 - 80 Epub 2003 May 15. Degradation of an endocrine disrupting chemical, DEHP {di-(2-ethylhexyl)-phthalate}, by Fusarium oxysporum f . sp . pisi cutinase; Kim YH et al.; The efficiency of two lypolytic enzymes (fungal cutinase, yeast esterase) in the degradation of di-(2-ethylhexyl)-phthalate (DEHP) was investigated . The DEHP-degradation rate of fungal cutinase was surprisingly high, i.e . almost 70% of the initial DEHP (500 mg/l) was decomposed within 2.5 h and nearly 50% of the degraded DEHP disappeared within the initial 15 min . With the yeast esterase, despite the same concentration, more than 85% of the DEHP remained even after 3 days of treatment . During the enzymatic degradation of DEHP, several DEHP-derived compounds were detected and time-course changes in composition were also monitored . During degradation with fungal cutinase, most DEHP was converted into 1,3-isobenzofurandione (IBF) by diester hydrolysis . In the degradation by yeast esterase, two organic chemicals were produced from DEHP: IBF and an unidentified compound (X) . The final chemical composition after 3 days was significantly dependent on the enzyme used . Fungal cutinase produced IBF as a major degradation compound . However, in the DEHP degradation by yeast esterase, compound X was produced in abundance in addition to IBF . The toxic effects of the final degradation products were investigated, using various recombinant bioluminescent bacteria and, as a result, the degradation products from yeast esterase were shown to contain a toxic hazard, causing oxidative stress and damage to protein synthesis. Proc Natl Acad Sci U S A, 2003 May 27, 100(11), 6325 - 30 Epub 2003 May 15. Spontaneous mitotic homologous recombination at an enhanced yellow fluorescent protein (EYFP) cDNA direct repeat in transgenic mice; Hendricks CA et al.; A transgenic mouse has been created that provides a powerful tool for revealing genetic and environmental factors that modulate mitotic homologous recombination . The fluorescent yellow direct-repeat (FYDR) mice described here carry two different copies of expression cassettes for truncated coding sequences of the enhanced yellow fluorescent protein (EYFP), arranged in tandem . Homologous recombination between these repeated elements can restore full-length EYFP coding sequence to yield a fluorescent phenotype, and the resulting fluorescent recombinant cells are rapidly quantifiable by flow cytometry . Analysis of genomic DNA from recombined FYDR cells shows that this mouse model detects gene conversions, and based on the arrangement of the integrated recombination substrate, unequal sister-chromatid exchanges and repair of collapsed replication forks are also expected to reconstitute EYFP coding sequence . The rate of spontaneous recombination in primary fibroblasts derived from adult ear tissue is 1.3 +/- 0.1 per 106 cell divisions . Interestingly, the rate is approximately 10-fold greater in fibroblasts derived from embryonic tissue . We observe an approximately 15-fold increase in the frequency of recombinant cells in cultures of ear fibroblasts when exposed to mitomycin C, which is consistent with the ability of interstrand crosslinks to induce homologous recombination . In addition to studies of recombination in cultured primary cells, the frequency of recombinant cells present in skin was also measured by direct analysis of disaggregated cells . Thus, the FYDR mouse model can be used for studies of mitotic homologous recombination both in vitro and in vivo. News Physiol Sci, 2003 Jun, 18, 104 - 8 Immune adherence revisited: novel players in an old game; Hess C et al.; Erythrocytes bind immune complexes (ICs) composed of antibodies binding their respective antigen (e.g., bacteria, parasites, viruses, or autoantigen) plus complement proteins via complement receptors {immune adherence (IA)} . In vivo studies have shown that erythrocytes act as an inert shuttle, targeting ICs to fixed macrophages in liver and spleen . Here we outline established and emerging implications of IA in health and disease. Exp Cell Res, 2003 Jun 10, 286(2), 276 - 87 Nucleocytoplasmic shuttling of antigen in mammalian cells conferred by a soluble versus insoluble single-chain antibody fragment equipped with import/export signals; Sibler AP et al.; The ectopic expression of antibody fragments within mammalian cells is a challenging approach for interfering with or even blocking the biological function of the intracellular target . For this purpose, single-chain Fv (scFv) fragments are generally preferred . Here, by transfecting several mammalian cell lines, we compared the intracellular behavior of two scFvs (13R4 and 1F4) that strongly differ in their requirement of disulphide bonding for the formation of active molecules in bacteria . The scFv 13R4, which is correctly folded in the bacterial cytoplasm, was solubly expressed in all cell lines tested and was distributed in their cytoplasm and nucleus, as well . In addition, by appending to the 13R4 molecules the SV40 T-antigen nuclear localisation signal (NLS) tag, cytoplasmic-coexpressed antigen was efficiently retargeted to the nucleus . Compared to the scFv 13R4, the scFv 1F4, which needs to be secreted in bacteria for activity, accumulated, even with the NLS tag, as insoluble aggregates within the cytoplasm of the transfected cells, thereby severely disturbing fundamental functions of cell physiology . Furthermore, by replacing the NLS tag with a leucine-rich nuclear export signal (NES), the scFv 13R4 was exclusively located in the cytoplasm, whereas the similarly modified scFv 1F4 still promoted cell death . Coexpression of NES-tagged 13R4 fragments with nuclear antigen promoted its efficient retargeting to the cytoplasm . This dominant effect of the NES tag was also observed after exchange of the nuclear signals between the scFv 13R4 and its antigen . Taken together, the results indicate that scFvs that are active in the cytoplasm of bacteria may behave similarly in mammalian cells and that the requirement of their conserved disulphide bridges for activity is a limiting factor for mediating the nuclear import/export of target in a mammalian cell context . The described shuttling effect of antigen conferred by a soluble scFv may represent the basis of a reliable in vivo assay of effective protein- protein interactions. Biotechnol Appl Biochem, 2003 Oct, 38(Pt 2), 123 - 30 Maize (Zea mays)-derived bovine trypsin: characterization of the first large-scale, commercial protein product from transgenic plants; Woodard SL et al.; Bovine trypsin (EC 3.4.21.4) is an enzyme that is widely used for commercial purposes to digest or process other proteins, including some therapeutic proteins . The biopharmaceutical industry is trying to eliminate animal-derived proteins from manufacturing processes due to the possible contamination of these products by human pathogens . Recombinant trypsin has been produced in a number of systems, including cell culture, bacteria and yeast . To date, these expression systems have not produced trypsin on a scale sufficient to fulfill the need of biopharmaceutical manufacturers where kilogram quantities are often required . The present paper describes commercial-level production of trypsin in transgenic maize (Zea mays) and its physical and functional characterization . This protease, the first enzyme to be produced on a large-scale using transgenic plant technology, is functionally equivalent to native bovine pancreatic trypsin . The availability of this reagent should allow for the replacement of animal-derived trypsin in the processing of pharmaceutical proteins. Eur J Ophthalmol, 2003 Apr, 13 Suppl 3, S5 - 10 Introduction to apoptosis in ophthalmology; Carella G; Apoptosis represents a mode of cellular death genetically programmed to maintain homeostasis of tissues . In specific pathologic circumstances, the death program may be activated by various environmental factors such as exposure to toxic substances or bacteria or deprivation of nutrients . From this point of view, apoptosis is considered the final event in several pathologies . In ophthalmology, experimental evidence has confirmed that apoptosis is a type of cellular death involved in various pathologic processes including glaucoma, retinitis pigmentosa, ischemic retinopathy, corneal reparative processes, cataract, and retinoblastoma . The aim of this article is to review the most recent results published in this field and to describe some of the molecular mechanisms responsible for the activation of the apoptotic program in some important ocular disorders . The understanding of such mechanisms could outline new therapeutic strategies for the prevention of cellular death in ophthalmology. Parasitol Res, 2003 Aug, 90(5), 405 - 8 Epub 2003 May 14. No evidence of Wolbachia endosymbiosis with Loa loa and Mansonella perstans; Grobusch MP et al.; Endosymbiotic Wolbachia bacteria from different filarial species, including major pathogens of humans such as Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus, seem to play an important role in the development, viability and fertility of these worms . Wolbachia trigger inflammatory host responses as well as adverse reactions against standard treatment regimens and are therefore under investigation as novel treatment targets . We investigated whether Wolbachia are also endosymbiotic in Loa loa and Mansonella perstans . In both male and female adult L . loa, we found no evidence of bacteria by light or transmission electron microscopy . Furthermore, Wolbachia-specific PCR was negative in both L . loa and M . perstans microfilariae . The absence of Wolbachia in both filarial species therefore discourages the use of antibiotics as an adjunct or alternative approach to current treatment concepts for both loiasis and mansonelliasis perstans. Adv Biochem Eng Biotechnol, 2003, 82, 135 - 82 Monitoring and control of anaerobic reactors; Pind PF et al.; The current status in monitoring and control of anaerobic reactors is reviewed . The influence of reactor design and waste composition on the possible monitoring and control schemes is examined . After defining the overall control structure, and possible control objectives, the possible process measurements are reviewed in detail . In the sequel, possible manipulated variables, such as the hydraulic retention time, the organic loading rate, the sludge retention time, temperature, pH and alkalinity are evaluated with respect to the two main reactor types: high-rate and low-rate . Finally, the different control approaches that have been used are comprehensively described . These include simple and adaptive controllers, as well as more recent developments such as fuzzy controllers, knowledge-based controllers and controllers based on neural networks. Adv Biochem Eng Biotechnol, 2003, 81, 57 - 93 Kinetics and modeling of anaerobic digestion process; Gavala HN et al.; Anaerobic digestion modeling started in the early 1970s when the need for design and efficient operation of anaerobic systems became evident . At that time not only was the knowledge about the complex process of anaerobic digestion inadequate but also there were computational limitations . Thus, the first models were very simple and consisted of a limited number of equations . During the past thirty years much research has been conducted on the peculiarities of the process and on the factors that influence it on the one hand while an enormous progress took place in computer science on the other . The combination of both parameters resulted in the development of more and more concise and complex models . In this chapter the most important models found in the literature are described starting from the simplest and oldest to the more recent and complex ones. Syst Appl Microbiol, 2003 Mar, 26(1), 54 - 64 Sinorhizobium americanus sp . nov., a new Sinorhizobium species nodulating native Acacia spp . in Mexico; Toledo I et al.; The sinorhizobia isolated from root nodules of Acacia species native of Mexico constitute a diverse group of bacteria on the basis of their metabolic enzyme electromorphs but share restriction patterns of the PCR products of 16S rRNA genes and a common 500 kb symbiotic plasmid . They are distinguished from other Sinorhizobium species by their levels of DNA-DNA hybridization and the sequence of 16S rRNA and nifH genes . nolR gene hybridization patterns were found useful to identify sinorhizobia and characterize species . A new species, Sinorhizobium americanus, is described and the type strain is CFNEI 156 from Acacia acatlensis. Clin Exp Rheumatol, 2003 Mar-Apr, 21(2), 185 - 92 Dendritic cells co-localize with activated CD4+ T cells in giant cell arteritis; Wagner AD et al.; OBJECTIVE: Giant cell arteritis (GCA) is a vasculitis predominantly affecting medium- and large-sized arteries . Recent data show the co-localization of dendritic cells and Chlamydia pneumoniae in vascular biopsies from GCA patients . Here we define the topographical relation of dendritic cells and these activated T-cells to determine the antigen presenting cell in GCA, and to examine several auxiliary biochemical and genetic aspects relating to the role of bacteria such as C . pneumoniae in eliciting GCA . METHODS: 18 paraffin-embedded temporal artery biopsy specimens from 14 patients with GCA that were PCR-positive for C . pneumoniae were examined by two-color immunohistochemistry for the topographical relationship between dendritic cells and activated T-cells . In addition the presence of GTP-binding proteins . Tumor necrosis factor alpha (TNF alpha), and Toll-like receptor 4 (TLR4) was investigated . 15 temporal artery specimens from 10 patients without GCA served as controls . RESULTS: In all GCA specimens, dendritic cells co-localized in the immediate vicinity of activated CD4+ Talin-expressing T cells, and these were predominantly found in granulomatous infiltrates . Confocal microscopy confirmed the cell-cell contact of dendritic cells with activated T cells . Results further showed that RhoA and Rac1 were predominantly present in the region of granulomatous infiltrates . TNF alpha production and expression was found in dendritic cells and macrophages, predominantly in granulomatous infiltrates and in endothelial cells of the vasa vasorum dispersed in the adventitial and medial layers of the temporal artery . No control specimens showed TNF alpha expression . More than 95% of dendritic cells were positive for TLR4; macrophages and endothelial cells localized in the adventitia showed TLR4 production . CONCLUSIONS: The immediate co-localization of dendritic cells and activated T cells indicate a high probability that the former represent the antigen presenting cells in GCA . In addition, because of the presence of Rho A and Rac1 in the granulomatous infiltrates, we speculate that they provide the right environment for cell-cell contact and adhesion, and that they may promote the internalization of bacteria . TNF alpha is expressed at high levels in the granulomatous infiltrates of temporal artery specimens from patients with GCA . Since TLR4 is produced in the same cell types, and predominantly in the adventitial layer of the temporal artery, we suggest that these receptors are coupled to signal transduction pathways that control TNF alpha expression. Mol Biotechnol, 2003 Jun, 24(2), 203 - 20 Multilocus enzyme electrophoresis: a practical guide; Stanley TG et al.; Multilocus enzyme electrophoresis (MEE) uses the relative electrophoretic mobilities of intracellular enzymes to characterize and differentiate organisms by generating an electromorph type (ET) . This article presents the chemical conditions that may be useful, a guide to the successful practice of the electrophoretic technique, and analysis of the results. J Biol Chem, 2003 Jul 25, 278(30), 28274 - 83 Epub 2003 May 13. Oligomerization of dopamine transporters visualized in living cells by fluorescence resonance energy transfer microscopy; Sorkina T et al.; To examine the oligomeric state and trafficking of the dopamine transporter (DAT) in different compartments of living cells, human DAT was fused to yellow (YFP) or cyan fluorescent protein (CFP) . YFP-DAT and CFP-DAT were transiently and stably expressed in porcine aortic endothelial (PAE) cells, human embryonic kidney (HEK) 293 cells, and an immortalized dopaminergic cell line 1RB3AN27 . Fluorescence microscopic imaging of cells co-expressing YFP-DAT and CFP-DAT revealed fluorescence resonance energy transfer (FRET) between CFP and YFP, which is consistent with an intermolecular interaction of DAT fusion proteins . FRET signals were detected between CFP- and YFP-DAT located at the plasma membrane and in intracellular membrane compartments . Phorbol esters or amphetamine induced the endocytosis of YFP/CFP-DAT to early and recycling endosomes, identified by Rab5, Rab11, Hrs and EEA.1 proteins . Interestingly, however, DAT was mainly excluded from Rab5- and Hrs-containing microdomains within the endosomes . The strongest FRET signals were measured in endosomes, indicative of efficient oligomerization of internalized DAT . The intermolecular DAT interactions were confirmed by co-immunoprecipitation . A DAT mutant that was retained in the endoplasmic reticulum (ER) after biosynthesis was used to show that DAT is oligomeric in the ER . Moreover, co-expression of an ER-retained DAT mutant and wild-type DAT resulted in the retention of wild-type DAT in the ER . These data suggest that DAT oligomers are formed in the ER and then are constitutively maintained both at the cell surface and during trafficking between the plasma membrane and endosomes. J Biol Chem, 2003 Aug 22, 278(34), 32397 - 404 Epub 2003 May 12. Crystal structure of the tRNA processing enzyme RNase PH from Aquifex aeolicus; Ishii R et al.; RNase PH is one of the exoribonucleases that catalyze the 3' end processing of tRNA in bacteria . RNase PH removes nucleotides following the CCA sequence of tRNA precursors by phosphorolysis and generates mature tRNAs with amino acid acceptor activity . In this study, we determined the crystal structure of Aquifex aeolicus RNase PH bound with a phosphate, a co-substrate, in the active site at 2.3-A resolution . RNase PH has the typical alpha/beta fold, which forms a hexameric ring structure as a trimer of dimers . This ring structure resembles that of the polynucleotide phosphorylase core domain homotrimer, another phosphorolytic exoribonuclease . Four amino acid residues, Arg-86, Gly-124, Thr-125, and Arg-126, of RNase PH are involved in the phosphate-binding site . Mutational analyses of these residues showed their importance in the phosphorolysis reaction . A docking model with the tRNA acceptor stem suggests how RNase PH accommodates substrate RNAs. Int J Neurosci, 2003 May, 113(5), 621 - 30 Membrane Na+-K+ ATPase inhibition mediated quantal model for brain evolution; Kurup RK et al.; The hypothalamus secretes an endogenous membrane Na+-K+ ATPase inhibitor, digoxin . A digoxin-mediated model of quantal perception is proposed . In the quantal state, self replication of self-organized macromolecules is possible . This leads to the origin of molecular organisms like prions . Macromolecules group together to form organelle, which in evolutionary terms are independent bacteria . The organelle/bacteria symbiotically cluster together to form the cell . The human organism, including the brain, can be visualized as an organized cluster or colony of unicellular, symbiotically grouped flagellated bacteria . Synaptic connections form in the bacterial cluster leading to the evolution of the primitive neuronal networks, and later the human brain . The role of quantal perception and the observer function of consciousness in the origin of matter is important . Symbiotically clustered intergalactic magnetotactic bacterial networks are important in the evolution of the universe. Int J Environ Health Res, 2003 Jun, 13(2), 169 - 79 Effect of aerobic and anaerobic digestion on the viability of Cryptosporidium parvum oocysts and Ascaris suum eggs; Kato S et al.; The viability of Cryptosporidium parvum oocysts and Ascaris suum eggs inoculated into aerobic and anaerobic digesters was measured . The digesters were maintained at 37 degrees C, 47 degrees C, and 55 degrees C, with 10-day detention times . Eggs and oocysts were added to each digester in a single spike or in chambers placed in the digesters for varying periods . Oocysts were inactivated very rapidly in all systems as determined by a dye permeability assay, > 99% inactivated after 10 days at 37 degrees C, 4 days at 47 degrees C, and 2 days at 55 degrees C . Eggs were more rapidly inactivated in anaerobic digesters than in aerobic digesters . At 55 degrees C, eggs in both anaerobic and aerobic digesters were > 99% inactivated within 1 h . At 47 degrees C, anaerobic digestion inactivated around 95% eggs in 2 days, but around 25% of the eggs were still viable after 10 days in aerobic digesters . At 37 degrees C, anaerobic digestion inactivated more than 75% of the eggs after 10 days, but in the aerobic digester at 37 degrees C, 10 days of treatment had no effect on viability . The oocysts and eggs added in chambers appeared to behave similarly to these pathogens added directly to the biosolids within the digesters. Mol Plant Microbe Interact, 2003 Apr, 16(4), 342 - 51 New NodW- or NifA-regulated Bradyrhizobium japonicum genes; Caldelari Baumberger I et al.; A cluster of genes coding for putative plant cell-wall degrading enzymes (i.e., genes for two endoglucanases {gunA and gunA2}, one pectinmethylesterase {pme}, and one polygalacturonase {pgl}) was identified by sequence similarities in the symbiotic region of the Bradyrhizobium japonicum chromosome . In addition, a systematic screen of the region revealed several genes potentially transcribed by the sigma(54)-RNA polymerase and activated by the transcriptional regulator NifA (i.e., genes for proteins with similarity to outer membrane proteins {id117 and id525} and a citrate carrier {id331 or citA} and one open reading frame without similarity to known proteins {id747}) . Expression studies using transcriptional lacZ fusions showed that gunA2 and pgl were strongly induced by the isoflavone genistein in a NodW-dependent manner, suggesting a role of the gene products in early events of the nodulation process; by contrast, gunA and pme expression was very weak in the conditions tested . The gunA2 gene product was purified and was shown to have cellulase activity . beta-Galactosidase activity expressed from transcriptional lacZ fusions to id117, id525, and id747 in the wild type and in nifA and rpoN mutant backgrounds confirmed that their transcription was dependent on NifA and sigma(54) . Despite the presence of a -24/-12-type promoter and a NifA binding site upstream of citA, no regulation could be demonstrated in this case . Null mutations introduced in gunA, gunA2, pgl, pme, citA, id117, id525, and id747 did not impair the symbiosis with the host plants. Am J Dent, 2003 Feb, 16(1), 47 - 52 Resin adhesion to carious dentin; Yoshiyama M et al.; PURPOSE: To investigate the microtensile bond strength (microTBS) of a self-etching priming adhesive system to normal, caries-affected and caries-infected dentin, and to observe the ultrastructure of the resin-dentin interface by transmission electron microscopy (TEM) . MATERIALS AND METHODS: Twelve extracted human molar teeth with deep occlusal caries were stained with caries detector solution and ground flat occlusally . The red-stained soft dentin was classified as caries-infected . The surrounding discolored dentin was classified as caries-affected dentin . The surrounding normal dentin served as a control . The entire flat surface was bonded with Clearfil Liner Bond 2V (CV) and covered with resin composite to form a composite crown 5 mm high . One day later the specimens were serially sectioned vertically into multiple slabs 0.8 mm thick . Under microscopic observation, the specimens were divided into normal or caries-infected or caries-affected dentin . These regions were isolated by cutting away the remaining dentin to form hour-glass shapes with the smallest surface area at the test site . After measuring the areas, the specimens were fixed to a microtensile tester and pulled under tension to failure . Additional slabs that were not used for bond strength tests were processed for TEM . Bond strength data were analyzed by Kruskal-Wallis one-way ANOVA and Student-Newman-Keuls multiple comparisons . RESULTS: The microTBS of CV to normal, caries-affected and caries-infected dentin were 45 +/- 10 MPa, 30 +/- 10 MPa, 10 +/- 5 MPa, respectively . TEM images showed that CV formed thin hybrid layers that were less than 1 microm thick in normal dentin, but that were between 6-8 microm thick in caries-affected dentin . Bacteria were only sparsely observed in the dentin tubules of bonded caries-affected dentin . However, in caries-infected dentin, an unusual interface was seen in which carious bacteria within disorganized non-banded collagen fibrils could be seen embedded by the adhesive . The hybrid layer in caries-infected dentin was found to be 30-60 microm thick. Am J Dent, 2003 Feb, 16(1), 28 - 32 Resin-dentin interfaces of endodontically-treated restored teeth; Mannocci F et al.; PURPOSE: To compare by confocal and scanning electron microscopy the post-resin-dentin interfaces of endodontically-treated teeth which had been restored with dentin-bonding systems and composite and needed to be extracted after 6 months to 6 years of clinical service . MATERIALS AND METHODS: Sixteen single rooted teeth showing proximal lesions penetrating the pulp chamber were included in the investigation and divided into two groups . Group 1 teeth were endodontically-treated, restored with carbon fiber posts and resin-based composite . Group 2 teeth were treated in the same way and covered with porcelain fused-to-metal crowns . RESULTS: Two thirds of the observed interfaces showed resin penetration of the root dentin in the middle and coronal third of the post space preparation, while only one third of the interface showed resin infiltration in the apical third of the post space preparation . Debonding of the adhesive from the resin infiltrated dentin area and debonding of the composite cement from the adhesive were the most frequently observed failure modes and were observed in one third of the observed interfaces . There was no difference between the post-resin-dentin interfaces of teeth covered and teeth not covered with porcelain-fused to metal crowns . Teeth extracted after longer periods of clinical service showed more abundant areas of debonding of the adhesive from the resin-dentin interdiffusion zone . The presence of bacteria in resin infiltrated dentin tubules was observed in two specimens. Parasitol Res, 2003 May, 90(1), 52 - 6 Epub 2003 Jan 30. Endocytobiont KC5/2 induces transformation into sol-like cytoplasm of its host Acanthamoeba sp . as substrate for its own development; Michel R et al.; New investigations of a novel, recently described, non-cultivable endocytobiont of Acanthamoeba sp . reveal at least three hitherto unobserved developmental stages which shed some light on the nature of this peculiar organism . The development of the endocytobiont is closely connected with conspicuous changes in the host amoeba, inducing the transformation from gel to sol-like cytoplasm which bulges like a balloon inside the host cell . Young and transitory developmental stages were found within the homogenous, sol-like cytoplasm . The infectious stages, with their voluminous cell wall and a conspicuous ostiole, could be observed within all parts of the cytoplasm with the exception of the nucleus . It is a remarkable adaptation for this parasite to be able to induce this gel-sol transformation in order to facilitate its own development . The fate of the heavily infected host amoebae is death by rupture or lysis after being overcrowded with parasites . As no structures could be observed within the endoparasites that were comparable to other bacteria, the real nature and taxonomic position of these peculiar organisms remain obscure. J Gastroenterol, 2003, 38(4), 390 - 4 A decreased number of c- kit-expressing cells in a patient with afferent loop syndrome; Kiyohara T et al.; Following gastrectomy, stasis in the afferent jejunal loop accompanied by an overgrowth of bacteria leads to a number of clinical symptoms, including the so-called afferent loop syndrome . The disturbances in intestinal motility may be related to stagnation of the intestinal contents in the afferent loop . The pacemaker cells for the basic contractile activity of the intestine are thought to be the interstitial cells of Cajal (ICCs) . We and others have reported that ICCs express the c- kit receptor, and that a decreased number of c- kit-expressing ICCs is generally thought to result in disturbed intestinal motility . We report here a patient with postgastrectomy afferent loop syndrome with a decreased number of c- kit-expressing cells in the external muscle layer of the dilated intestine, suggesting damage to the ICCs. J Cell Biol, 2003 May 12, 161(3), 461 - 2 Amyloid as a natural product; Kelly JW et al.; Amyloid fibrils, such as those found in Alzheimer's and the gelsolin amyloid diseases, result from the misassembly of peptides produced by either normal or aberrant intracellular proteolytic processing . A paper in this issue by Marks and colleagues (Berson et al., 2003) demonstrates that intra-melanosome fibrils are formed through normal biological proteolytic processing of an integral membrane protein . The resulting peptide fragment assembles into fibrils promoting the formation of melanin pigment granules . These results, along with the observation that amyloid fibril formation by bacteria is highly orchestrated, suggest that fibril formation is an evolutionary conserved biological pathway used to generate natural product nanostructures. EMBO J, 2003 May 15, 22(10), 2387 - 99 Conventional kinesin KIF5B mediates insulin-stimulated GLUT4 movements on microtubules; Semiz S et al.; Insulin stimulates glucose uptake in muscle and adipose cells by mobilizing intracellular membrane vesicles containing GLUT4 glucose transporter proteins to the plasma membrane . Here we show in live cultured adipocytes that intracellular membranes containing GLUT4-yellow fluorescent protein (YFP) move along tubulin-cyan fluorescent protein-labeled microtubules in response to insulin by a mechanism that is insensitive to the phosphatidylinositol 3 (PI3)-kinase inhibitor wortmannin . Insulin increased by several fold the observed frequencies, but not velocities, of long-range movements of GLUT4-YFP on microtubules, both away from and towards the perinuclear region . Genomics screens show conventional kinesin KIF5B is highly expressed in adipocytes and this kinesin is partially co-localized with perinuclear GLUT4 . Dominant-negative mutants of conventional kinesin light chain blocked outward GLUT4 vesicle movements and translocation of exofacial Myc-tagged GLUT4-green fluorescent protein to the plasma membrane in response to insulin . These data reveal that insulin signaling targets the engagement or initiates the movement of GLUT4-containing membranes on microtubules via conventional kinesin through a PI3-kinase-independent mechanism . This insulin signaling pathway regulating KIF5B function appears to be required for GLUT4 translocation to the plasma membrane. Arch Intern Med, 2003 May 12, 163(9), 1099 - 104 Risk of cardiovascular disease-related and all-cause death according to serum concentrations of enterolactone: Kuopio Ischaemic Heart Disease Risk Factor Study; Vanharanta M et al.; BACKGROUND: Enterolactone is a plant-derived compound that has been associated with a reduced risk of acute coronary events and cancer . Several studies have suggested that serum enterolactone concentration may play a role as a biomarker of a diet high in fiber and vegetables . Owing to its phenolic structure, enterolactone and its plant lignan precursors, which are converted by intestinal bacteria to enterolactone, are potential antioxidants . METHODS: The associations between serum enterolactone level and the risk of coronary heart disease (CHD)-related, cardiovascular disease (CVD)-related, and all-cause mortality were investigated in the Kuopio Ischaemic Heart Disease Risk Factor Study, which is a prospective population-based study of middle-aged Finnish men . The serum enterolactone concentration and cardiovascular risk factors were determined in 1889 men aged 42 to 60 years . In an average follow-up of 12.2 years, 70 CHD-related, 103 CVD-related, and 242 all-cause deaths occurred in participants free of prior CVD . RESULTS: Multivariate analyses showed significant associations between elevated serum enterolactone concentration and reduced risk of CHD- and CVD-related mortality, but weaker associations in relation to all-cause mortality . In the Cox proportional hazards regression model adjusting for the most potent confounding factors, the risk of CHD-related (P =.03 for trend) and CVD-related (P =.04 for trend) death decreased linearly across quartiles of serum enterolactone concentration . CONCLUSIONS: Our data suggest that a high serum enterolactone level is associated with reduced CHD- and CVD-related mortality in middle-aged Finnish men . These results add to the evidence supporting the importance of whole grain foods, fruits, and vegetables in the prevention of premature death from CVD. Ecotoxicol Environ Saf, 2003 Jun, 55(2), 157 - 61 Biogenic arsenopyrite in holocene peat sediment, India; Farooqui A et al.; The plant organic matter in the peat layer deposited around 6650+/-110 and 4800+/-80 years BP, during the transgressive and regressive phases of sea level changes, respectively in the dried part (playa) of the present Pulicat lagoon in Palar Basin (southeast coast of India) was studied to elucidate the biogenic pyrite generation and associated trace elements . The scanning electron microscopic (SEM) observations show strongly curved unique C-shaped bacteria of uniform coccoidal shape and size (1 microm) freely scattered on the plant epidermal microfragments . These form spheroidal microcolonies 8-15 microm in diameter attached to the epidermis in a linear fashion or haphazardly enclosed in the translucent sheath as observed in surface view . These colonies are dense with a number of toxic trace elements as analyzed under an SEM-EDX spectrometer indicating its affinity toward metal binding . On maturity the sheath ruptures disseminating tetrad form of bacteria along with some C-shaped bacteria and few rhombohedral crystals of framboidal pyrite . The pyrite crystals intact in spheroidal shape in cavities on the epidermal fragments indicate its biogenic precipitation mediated by the bacteria . The pyrite crystals are rich in sulfur, iron, and traces of arsenic when analyzed by the SEM-EDX spectrometer . It is suggested that cubic and pyritohedran forms of crystals are perhaps arsenopyrite precipitated biogenically in saturated coastal wetland ecosystem in the past mediated by tetrad strain of the observed coccoid bacteria . Thus, the strata-bound peat layer could be the potent residing place for arsenopyrite or other toxic trace elements that may contaminate the groundwater aquifer. J Microbiol Immunol Infect, 2003 Mar, 36(1), 47 - 50 Early diagnosis of ventriculoperitoneal shunt infections and malfunctions in children with hydrocephalus; Lan CC et al.; This retrospective study assessed the parameters of ventriculoperitoneal shunt infection and malfunction found in 129 children with hydrocephalus who had undergone a ventriculoperitoneal shunt placement at Taipei Veterans General Hospital from January 1997 to June 2001 . The clinical characteristics and differences among these patients were reviewed to determine the potential predictors of shunt infection and malfunction . Fever (60%, 6/ 10) and seizure (40%, 4/10) occurred significantly more often in the infection group than in the malfunction and control groups (p < 0.05) . A higher blood C-reactive protein level was noted in the infection group than in the malfunction and control groups (p < 0.05) . Examination of the cerebrospinal fluid of infected patients showed significantly higher white blood cell count and neutrophil count, higher protein concentration, and lower glucose levels compared with the other 2 groups (p < 0.05) . However, the number of patients with cerebrospinal fluid eosinophilia was significantly higher in the malfunction group (p < 0.05) . The diagnostic usefulness of laboratory parameters in patients with ventriculoperitoneal shunt infection is as follow: cerebrospinal fluid white blood cell count over 100/mm3, 96% specificity and a positive predictive value of 0.55; and cerebrospinal fluid neutrophils over 10%, 90% sensitivity and a negative predictive value of 0.99 . In conclusion, Fever, seizure, high blood C-reactive protein, combined with leukocytosis, neutrophil over 10%, low glucose level, and high protein level in the ventricular fluid are factors that may help in distinguishing shunt infection from shunt malfunction. Vestn Ross Akad Med Nauk, 2003, (4), 3 - 10 {Role of viral-herpetic infections in the etiology of atherosclerosis: clinical, virological and immunological evidence}; Vorob'ev AA et al.; The article describes the clinical, virological and immunological data confirming the etiological role of herpes virus in the initiation of atherosclerosis . 226 patients with atherosclerosis of the predominantly coronary localization were examined; hypertension and stenocardia were found in a part of them, while myocardial infarction was diagnosed in 22% of the patients . The control group consisted of patients with other diseases related with infections (bronchial asthma, rheumatism etc.) as well as of healthy persons . A total of 558 patients were examined and it was established that there is a reliable relation between atherosclerosis and the infection of patients with, mainly, herpes virus . The correlation was of the seasonal nature, it was linked to the specific features of an infection process and it was confirmed by the condition of the cholesterol supply and by immunodeficiency in patients . The infectious nature of atherosclerosis demands further research for the sake of finding proof of the etiological role of viruses and bacteria and for the sake of working out the means of prophylaxis and treatment of atherosclerosis aimed at removing the infectious etiological factor. C R Biol, 2003 Jan, 326(1), 23 - 35 {Contribution of cell and molecular biology and genetics to plant protection}; Durand-Tardif M et al.; Plants resist to the majority of their potential aggressors by opposing physical and chemical barriers: cell walls, secondary metabolites... . Phenomena of specific recognition between a plant variety and a pathovar induce on the one hand, a local (hypersensitive) reaction that tends to limit pathogen growth and, on the other hand, a cascade of signals that allows the activation of a non-specific general (systemic) resistance . The contribution of genetics to the fight against pathogens depends on the natural variability that comes from the co-evolution between plants and their aggressors . Many plant varieties resistant to one or several pathogens have been obtained and are cultivated . The use of biotechnology will facilitate the rapid generation of new, resistant cultivars and cultivars with multiple resistances . New methods in order to increase the efficiency and the durability of resistance are envisaged. Chemistry, 2003 May 9, 9(9), 2034 - 8 1H and 13C NMR characterization of hemiamidal isoniazid-NAD(H) adducts as possible inhibitors of InhA reductase of Mycobacterium tuberculosis; Broussy S et al.; Isoniazid (INH) is easily oxidized with manganese(III) pyrophosphate, a chemical model of the KatG protein involved in activation of INH inside the bacteria Mycobacterium tuberculosis . Performed in the presence of NAD(+), this oxidation generates a family of isomeric INH-NAD(H) adducts, which have been shown to be effective inhibitors of InhA, an enzyme essential in mycolic acid biosynthesis . In this work, we fully characterized by (1)H and (13)C NMR spectroscopy four main species of INH-NAD(H) adducts that coexist in solution . Two of them are open diastereoisomers consisting of the covalent attachment of the isonicotinoyl radical at position four of the nicotinamide coenzyme . The other two result from a cyclization involving the amide group from the nicotinamide and the carbonyl group from the isonicotinoyl radical to give diastereoisomeric hemiamidals . Although an INH-NAD(H) adduct with a 4S configuration has been characterized within the active site of InhA from Xray crystallography and this bound adduct interpreted as an open form (Rozwarski et al., Science 1998, 279, 98-102), it is legitimate to raise the question about the effective active form(s), open or cyclic, of INH-NAD(H) adduct(s) . Is there a single active form or are several forms able to inhibit the InhA activity with different levels of inhibitory potency? Proc Nutr Soc, 2003 Feb, 62(1), 95 - 9 Motor effects of short-chain fatty acids and lactate in the gastrointestinal tract; Cherbut C; Short-chain fatty acids (SCFA) affect local and remote motility of the gastrointestinal tract by mechanisms that are not completely understood . In the large intestine where they are produced, they inhibit peristaltic activity and may stimulate tonic activity . When present in the terminal ileum as a result of reflux of colon contents, they elicit propulsive contractions . These local motor effects could involve a neuro-hormonal sensory mechanism located in the mucosa of the terminal ileum and proximal colon . Finally, through a humoral pathway probably involving polypeptide YY release, ileal and colonic SCFA modify upper motility by inducing relaxation of the proximal stomach and lower oesophageal sphincter and reducing gastric emptying . One characteristic feature of the SCFA effects is the dose-dependency of the gastrointestinal motor responses . Indeed, the effects occur only below or above a threshold of SCFA concentration in lumen contents . One putative physiological role of the motor effects of SCFA might be to maintain the physico-chemical balance of the lumen environment in the terminal ileum and proximal colon . Another role might be to co-regulate motility of the upper intestine . The clinical relevance of these effects is unclear . However, some recent findings suggest that excessive SCFA concentrations might induce adverse effects on gastrointestinal and colonic motility and sensitivity in certain diseases such as inflammatory bowel disease and gastro-oesophageal reflux disease. Pharm Res, 2003 Apr, 20(4), 674 - 83 The stenlying effect of high hydrostatic pressure on thermally and hydrolytically labile nanosized carriers; Brigger I et al.; PURPOSE: To investigate whether high hydrostatic pressure (HHP) treatment allows the sterilization of thermosensitive polymer nanoparticle suspensions without jeopardizing their physicochemical integrity . METHODS: Application of HHP was explored on a wide variety of thermosensitive poly(cyanoacrylate) nanoparticles, varying by their type (nanospheres or nanocapsules), by their preparation method (nanoprecipitation or emulsion/solvent evaporation), as well as by their surface characteristics . Physicochemical characterization before and after pressurization included turbidimetry, size measurement, zeta potential, scanning electron microscopy and infrared analysis . A sterility test also conducted according to pharmacopoeial requirements on an importantly contaminated nanoparticle suspension . RESULTS: Poly(cyanoacrylate) nanoparticles appeared to be extremely baroresistant . Continuous or oscillatory HHP treatment up to 500 MPa during 30 min induced generally neither physical, nor chemical damage . However, precautions should be taken when surface modifiers are adsorbed onto nanoparticles, as a layer destabilization may occur . Finally, this process allowed the successful inactivation of vegetative bacteria, yeast, and fungi . CONCLUSIONS: This work proposes HHP as a new method for polymer drug carriers sterilization, taking into account that further exploration in this area is needed to propose novel protocols for spores inactivation. Pathologe, 2003 May, 24(3), 192 - 5 Epub 2003 Apr 03. {Human intestinal spirochetosis}; Knopf B et al.; Whenever biopsy material obtained from endoscopically normal colorectal mucosa reveals the blue haematoxyphilic line between the microvilli of the covering epithelium, the rare condition of intestinal spirochetosis is diagnosed . The classification of the bacteria detected with the aid of special stains (e.g . the Warthin Starry silver stain) and in the electron microscope, continues to be something of a problem . A further point of contention is the question whether this spirochetal infection is of pathological significance or not . A point mitigating against pathogenicity is the fact that no histological signs of an inflammatory reaction are to be seen . Also, the symptoms of patients with intestinal spirochetosis are such that they provide no basis for a pronouncement on whether the infection is of a pathological or a pathological nature . On the other hand, however, a number of studies do seem to indicate that the spirochetes might be the cause of such symptoms as diarrhoea, constipation and abdominal pain . A point that would appear to support this view is the fact that such symptoms may disappear after successful treatment with metronidazole . The histological diagnosis is easily established when, faced by an apparently normal histological appearance of the colorectal mucosa, the pathologist considers the possibility of spirochetosis, and undertakes a specific search for the blue haematoxyphilic line in the covering epithelium of the colorectal mucosa. Science, 2003 May 9, 300(5621), 984 - 7 Recombinant antibodies to the small GTPase Rab6 as conformation sensors; Nizak C et al.; Here we report an approach, based on antibody phage display, to generate molecular conformation sensors . Recombinant antibodies specific to the guanosine triphosphate (GTP)-bound conformation of the small guanosine triphosphatase (GTPase) Rab6, a regulator of membrane traffic, were generated and used to locate Rab6.GTP in fixed cells, and, after green fluorescent protein (GFP) tagging and intracellular expression, to follow Rab6.GTP in vivo . Rab6 was in its GTP-bound conformation on the Golgi apparatus and transport intermediates, and the geometry of transport intermediates was modulated by Rab6 activity . More generally, the same approach could be applied to other molecules that can be locked in a particular conformation in vitro. Science, 2003 May 9, 300(5621), 931 - 6 Transition metal speciation in the cell: insights from the chemistry of metal ion receptors; Finney LA et al.; The essential transition metal ions are avidly accumulated by cells, yet they have two faces: They are put to use as required cofactors, but they also can catalyze cytotoxic reactions . Several families of proteins are emerging that control the activity of intracellular metal ions and help confine them to vital roles . These include integral transmembrane transporters, metalloregulatory sensors, and diffusible cytoplasmic metallochaperone proteins that protect and guide metal ions to targets . It is becoming clear that many of these proteins use atypical coordination chemistry to accomplish their unique goals . The different coordination numbers, types of coordinating residues, and solvent accessibilities of these sites are providing insight into the inorganic chemistry of the cytoplasm. FASEB J, 2003 Jul, 17(10), 1310 - 2 Epub 2003 May 08. Hydrogen sulfide induces serum-independent cell cycle entry in nontransformed rat intestinal epithelial cells; Deplancke B et al.; Hydrogen sulfide (H2S), produced by commensal sulfate-reducing bacteria, is an environmental insult that potentially contributes to chronic intestinal epithelial disorders . We tested the hypothesis that exposure of nontransformed intestinal epithelial cells (IEC-18) to the reducing agent sodium hydrogen sulfide (NaHS) activates molecular pathways that underlie epithelial hyperplasia, a phenotype common to both ulcerative colitis (UC) and colorectal cancer . Exposure of IEC-18 cells to NaHS rapidly increased the NADPH/NADP ratio, reduced the intracellular redox environment, and inhibited mitochondrial respiratory activity . The addition of 0.2-5 mM NaHS for 4 h increased the IEC-18 proliferative cell fraction (P<0.05), as evidenced by analysis of the cell cycle and proliferating cell nuclear antigen expression, while apoptosis occurred only at the highest concentration of NaHS . Thirty minutes of NaHS exposure increased (P<0.05) c-Jun mRNA concentrations, consistent with the observed activation of mitogen activated protein kinases (MAPK) . Microarray analysis confirmed an increase (P<0.05) in MAPK-mediated proliferative activity, likely reflecting the reduced redox environment of NaHS-treated cells . These data identify functional pathways by which H2S may initiate epithelial dysregulation and thereby contribute to UC or colorectal cancer . Thus, it becomes crucial to understand how genetic background may affect epithelial responsiveness to this bacterial-derived environmental insult. Chemosphere, 2003 Jul, 52(2), 289 - 97 The diversity of naturally produced organohalogens; Gribble GW; More than 3800 organohalogen compounds, mainly containing chlorine or bromine but a few with iodine and fluorine, are produced by living organisms or are formed during natural abiogenic processes, such as volcanoes, forest fires, and other geothermal processes . The oceans are the single largest source of biogenic organohalogens, which are biosynthesized by myriad seaweeds, sponges, corals, tunicates, bacteria, and other marine life . Terrestrial plants, fungi, lichen, bacteria, insects, some higher animals, and even humans also account for a diverse collection of organohalogens. Mar Environ Res, 2003 Sep, 56(3), 367 - 85 Vegetable oil spills on salt marsh sediments; comparison between sunflower and linseed oils; Pereira MG et al.; The effects of a simulated spill of sunflower oil in salt marsh sediments were compared with an experiment with linseed oil . Sunflower and linseed oil penetrated the sediments at the same rates but different adsorption of the oils onto sediment particles resulted in the establishment of anaerobic conditions at shallower depths in sediments contaminated with linseed oil than with sunflower oil . The total lipid content of sunflower oil contaminated sediments remained almost stable for 6 months, whilst only 40% of linseed oil remained in the sediment after 2 months . Numbers of culturable heterotrophic bacteria and aerobic oil degrading bacteria in muddy sediment increased rapidly in response to the presence of the oils but bacterial numbers in sandy sediments increased more slowly for sunflower oil . Changes in fatty acid composition indicate similar degradation pathways for both oils but sunflower oil degraded more slowly than linseed oil and thus has the potential for longer lasting effects in marine environments. Microbes Infect, 2003 Apr, 5(5), 397 - 403 Francisella novicida LPS has greater immunobiological activity in mice than F . tularensis LPS, and contributes to F . novicida murine pathogenesis; Kieffer TL et al.; To further understand the role of LPS in the pathogenesis of Francisella infection, we characterized murine infection with F . novicida, and compared immunobiological activities of F . novicida LPS and the LPS from F . tularensis live vaccine strain (LVS) . F . novicida had a lower intradermal LD(50) in BALB/cByJ mice than F . tularensis LVS, and mice given a lethal F . novicida dose intraperitoneally died faster than those given the same lethal F . tularensis LVS dose . However, the pattern of in vivo dissemination was similar, and in vitro growth of both bacteria in bone marrow-derived macrophages was comparable . F . novicida LPS stimulated very modest in vitro proliferation of mouse splenocytes at high doses, but F . tularensis LVS LPS did not . Murine bone marrow macrophages treated in vitro with F . novicida LPS produced IL12 and TNF-alpha, but did not produce detectable interferon-gamma, IL10, or nitric oxide; in contrast, murine macrophages treated with F . tularensis LVS LPS produced none of these mediators . In contrast to clear differences in stimulation of proliferation and especially cytokines, both types of purified LPS stimulated early protection against lethal challenge of mice with F . tularensis LVS, but not against lethal challenge with F . novicida . Thus, although LPS recognition may not be a major factor in engendering protection, the ability of F . novicida LPS to stimulate the production of proinflammatory cytokines including TNF-alpha likely contributes to the increased virulence for mice of F . novicida compared to F . tularensis LVS. Waste Manag, 2003, 23(3), 261 - 72 Air stripping of ammonia from pig slurry: characterisation and feasibility as a pre- or post-treatment to mesophilic anaerobic digestion; Bonmati A et al.; The objective of the present paper has been to study the effect of pig slurry waste type, fresh or anaerobically digested, and the effect of initial pH on ammonia air stripping from pig slurry waste at high temperature (80 degrees C) . Stripping process as pre- or post-treatment to anaerobic digestion has been also evaluated . Treatment performances differ according to pig slurry type . When fresh pig slurry is used, despite working at 80 degrees C, a high initial pH (11.5) is required for complete ammonia removal . On the other hand, for digested pig slurry, complete ammonia removal without pH modification is possible and organic matter significantly less contaminates recovered ammonia salt . Batch anaerobic tests showed that ammonia air stripping is not an advisable pre-treatment to pig slurry anaerobic digestion. Cell Mol Life Sci, 2003 Mar, 60(3), 474 - 94 ATP synthases: structure, function and evolution of unique energy converters; Muller V et al.; A-, F- and V-adenosine 5'-triphosphatases (ATPases) consist of a mosaic of globular structural units which serve as functional units . These ion-translocating ATPases are thought to use a common mechanism to couple energy of ATP hydrolysis to ion transport and thus create an electrochemical ion gradient across the membrane . In vitro, all of these large protein complexes are able to use an ion gradient and the associated membrane potential to synthesize ATP . A-/F-/V-type ATPases are composed of two distinct segments: a catalytic sector, A1/F1/V1, whose three-dimensional structural relationship will be reviewed, and the membrane-embedded sector, Ao/Fo/Vo, which functions in ion conduction . Recent studies on the molecular biology of the Ao/Fo/Vo domains revealed surprising findings about duplicated and triplicated versions of the proteolipid subunit and shed new light on the evolution of these ion pumps. New Microbiol, 2003 Apr, 26(2), 157 - 62 A molecular method for the recovery and identification of enteric virus in shellfish; Zanetti S et al.; In this paper we report the results of an investigation into the presence of enteric viruses in shellfish from the waters around Sardinia . Twenty two samples of shellfish were examined using a rapid and sensitive technique to concentrate and detect viral RNA in shellfish tissues . After recovery of viral particles, RNA was extracted, transcribed into cDNA and amplified using "nested PCR" . Testing with enterovirus-specific RT-PCR produced positive results in over 13% of specimens . The virus detection procedure appears to be effective . In some circumstances it could be a better test of water quality than conventional monitoring techniques. Acta Vet Hung, 2003, 51(2), 165 - 70 Seroprevalence of Lawsonia intracellularis in large pig production units; Hagen B et al.; In 11 'farrow-to-finish' outdoor or indoor production units, blood samples from late pregnant gilts were tested by indirect immunofluorescence antibody (IFA) serum assay for Lawsonia intracellularis . The offspring of positively tested gilts were tested at 2, 7, 12, 17, 22 and 27 weeks of age for seroprevalence of Lawsonia intracellularis . All offspring of IFA positive gilts were seronegative at 2 and 7 weeks of age . At 12 weeks of age 81.0% of indoor and 51.0% of outdoor pigs were tested positive . While at 17 weeks of age 82.5% of indoor-raised pigs showed seropositivity, in outdoor units the seropositivity declined to 31.3% . At weeks 22 and 27 indoor-raised pigs still showed marked seropositivity (17.7% and 11.5%) but their outdoor-raised counterparts revealed declining values (7.4% and 0%). Nature, 2003 May 8, 423(6936), 185 - 90 Computational design of receptor and sensor proteins with novel functions; Looger LL et al.; The formation of complexes between proteins and ligands is fundamental to biological processes at the molecular level . Manipulation of molecular recognition between ligands and proteins is therefore important for basic biological studies and has many biotechnological applications, including the construction of enzymes, biosensors, genetic circuits, signal transduction pathways and chiral separations . The systematic manipulation of binding sites remains a major challenge . Computational design offers enormous generality for engineering protein structure and function . Here we present a structure-based computational method that can drastically redesign protein ligand-binding specificities . This method was used to construct soluble receptors that bind trinitrotoluene, l-lactate or serotonin with high selectivity and affinity . These engineered receptors can function as biosensors for their new ligands; we also incorporated them into synthetic bacterial signal transduction pathways, regulating gene expression in response to extracellular trinitrotoluene or l-lactate . The use of various ligands and proteins shows that a high degree of control over biomolecular recognition has been established computationally . The biological and biosensing activities of the designed receptors illustrate potential applications of computational design. J Biol Chem, 2003 Jul 18, 278(29), 27241 - 50 Epub 2003 May 07. Kinetic modulation in carbonmonoxy derivatives of truncated hemoglobins: the role of distal heme pocket residues and extended apolar tunnel; Samuni U et al.; Truncated hemoglobins (trHbs), are a distinct and newly characterized class of small myoglobin-like proteins that are widely distributed in bacteria, unicellular eukaryotes, and higher plants . Notable and distinctive features associated with trHbs include a hydrogen-bonding network within the distal heme pocket and a long apolar tunnel linking the external solvent to the distal heme pocket . The present work compares the geminate and solvent phase rebinding kinetics from two trHbs, one from the ciliated protozoan Paramecium caudatum (P-trHb) and the other from the green alga Chlamydomonas eugametos (C-trHb) . Unusual kinetic patterns are observed including indications of ultrafast (picosecond) geminate rebinding of CO to C-trHb, very fast solvent phase rebinding of CO for both trHbs, time-dependent biphasic CO rebinding kinetics for P-trHb at low CO partial pressures, and for P-trHb, an increase in the geminate yield from a few percent to nearly 100% under high viscosity conditions . Species-specific differences in both the 8-ns photodissociation quantum yield and the rebinding kinetics, point to a pivotal functional role for the E11 residue . The response of the rebinding kinetics to temperature, ligand concentration, and viscosity (glycerol, trehalose) and the viscosity-dependent changes in the resonance Raman spectrum of the liganded photoproduct, together implicate both the apolar tunnel and the static and dynamic properties of the hydrogen-bonding network within the distal heme pocket in generating the unusual kinetic patterns observed for these trHbs. J Clin Microbiol, 2003 May, 41(5), 1991 - 5 Simple and fast lateral flow test for classification of leprosy patients and identification of contacts with high risk of developing leprosy; Buhrer-Sekula S et al.; The interruption of leprosy transmission is one of the main challenges for leprosy control programs since no consistent evidence exists that transmission has been reduced after the introduction of multidrug therapy . Sources of infection are primarily people with high loads of bacteria with or without clinical signs of leprosy . The availability of a simple test system for the detection of antibodies to phenolic glycolipid-I (PGL-I) of Mycobacterium leprae to identify these individuals may be important in the prevention of transmission . We have developed a lateral flow assay, the ML Flow test, for the detection of antibodies to PGL-I which takes only 10 min to perform . An agreement of 91% was observed between enzyme-linked immunosorbent assay and our test; the agreement beyond chance (kappa value) was 0.77 . We evaluated the use of whole blood by comparing 539 blood and serum samples from an area of high endemicity . The observed agreement was 85.9% (kappa = 0.70) . Storage of the lateral flow test and the running buffer at 28 degrees C for up to 1 year did not influence the results of the assay . The sensitivity of the ML Flow test in correctly classifying MB patients was 97.4% . The specificity of the ML Flow test, based on the results of the control group, was 90.2% . The ML Flow test is a fast and easy-to-perform method for the detection of immunoglobulin M antibodies to PGL-I of M . leprae . It does not require any special equipment, and the highly stable reagents make the test robust and suitable for use in tropical countries. J Clin Microbiol, 2003 May, 41(5), 1819 - 26 Characterization of a tandem repeat polymorphism in Legionella pneumophila and its use for genotyping; Pourcel C et al.; We have analyzed the variability of minisatellite sequences (also called variable-number tandem repeats {VNTRs}) in the genome of Legionella pneumophila . Based upon the genome sequence of the Philadelphia-1 strain (serogroup 1), 25 minisatellites were selected and their polymorphisms were analyzed by PCR with the DNA of serogroup 1 to 14 reference strains . For 22 markers, a PCR product of the expected size was found with the DNA of the Philadelphia-1 strain . Most of these markers did not amplify the DNA of other Legionella species or other bacteria used as controls . A polymorphism was observed for seven markers among the L . pneumophila strains tested . To check whether these markers could be used to compare strains of L . pneumophila, we analyzed two groups of isolates from clinical and environmental samples which had been independently genotyped by other methods . The results showed that, for the isolates in these two sets of samples, VNTR typing is as informative as pulsed-field gel electrophoresis for comparison of strains . Sequencing of one minisatellite from 14 reference strains was performed . Comparison of the sequences allowed a classification and confirmed the existence of subspecies of L . pneumophila . We also tested the usefulness of one very polymorphic marker as a tool for the rapid screening of colonies grown from water samples . This allowed the rapid identification of the L . pneumophila colonies and gave a first hint as to the presence of several strains in a single sample. J Biol Chem, 2003 Jul 18, 278(29), 26604 - 11 Epub 2003 May 06. Activation of the leptin receptor by a ligand-induced conformational change of constitutive receptor dimers; Couturier C et al.; Binding of leptin to the leptin receptor is crucial for body weight and bone mass regulation in mammals . Leptin receptors were shown to exist as dimers, but the role of dimerization in receptor activation remains unknown . Using a quantitative Bioluminescence Resonance Energy Transfer approach, we show here in living cells that approximately 60% of the leptin receptor exists as constitutive dimers at physiological expression levels in the absence of leptin . No further increase in leptin receptor dimerization was detected in the presence of leptin . Importantly, in cells expressing the short leptin receptor isoform, leptin promoted a robust enhancement of energy transfer signals that reflect specific conformational changes of pre-existing leptin receptor dimers and that may be used as read-out in screening assays for leptin receptor ligands . Both leptin receptor dimerization and the leptin-induced energy transfer were Janus kinase 2-independent . Taken together, our data support a receptor activation model based on ligand-induced conformational changes rather than ligand-induced dimerization. Bioresour Technol, 2003 Mar, 87(1), 87 - 91 Study on anaerobic and aerobic degradation of different non-ionic surfactants; Mezzanotte V et al.; Six alcohol ethoxylates (C5E2, C6E4, C7E4, C8E2, C8E4, C10E4) and two fatty acid esters were tested at lab-scale for degradation in anaerobic and aerobic conditions and oxygen uptake rate (OUR) . Anaerobic removal of C5E2, C6E4 and C7E4 improved with increasing number of ethoxy groups (E) and decreasing length of the alkyl chain (C) . Their aerobic removal was also great but lower than the anaerobic values . C8E2, C8E4 and C10E4 were adsorbed on sludge but not degraded in anaerobic conditions, while they were efficiently removed under aerobiosis . The fatty acid esters were removed to a level between the two alcohol ethoxylates groups in both anaerobiosis and aerobiosis . The measured OUR confirmed the different behaviours of the three groups of compounds. Curr Microbiol, 2003 Jun, 46(6), 443 - 7 Detection of Actinobacillus pleuropneumoniae by PCR on field strains from healthy and diseased pigs; Klein CS et al.; We investigated whether primers able to specifically amplify a 0.7-kb DNA fragment from the conserved cpx genes could be applied to analyze A . pleuropneumoniae field isolates . The specific cpx primers were tested on 120 strains of A . pleuropneumoniae and other NAD-dependent field isolates from healthy and diseased animals to analyze A . pleuropneumoniae isolates from pigs in Brazil . We found that PCR and hybridization were able to discriminate between isolates of A . pleuropneumoniae and other bacteria . The 0.7-kb cpx DNA fragments were amplified from all 63 A . pleuropneumoniae isolates from herds with clinical symptoms and were isolated from lesions of acute cases of swine pleuropneumonia, both serotypable and nonserotypable . The PCR was also applied to 57 field isolates obtained from animals of apparently healthy herds, and the amplified cpx product was present in four serotypable and only two out of eleven A . pleuropneumoniae nonserotypable isolates . All nonserotypable A . pleuropneumoniae isolates revealed the apxA amplification pattern compatible with previously known serotypes . Some nonserotypable isolates might represent a population of isolates that originally were serotypable but lost the ability to react with serotype-specific antisera or might belong to novel serotypes . The PCR method applied is highly sensitive for serotypable A . pleuropneumoniae strains and for nonserotypable strains isolated from acute cases of swine pleuropneumoniae in Brazil. Appl Environ Microbiol, 2003 May, 69(5), 2423 - 9 Quantitative detection of methanotrophs in soil by novel pmoA-targeted real-time PCR assays; Kolb S et al.; Methane oxidation in soils is mostly accomplished by methanotrophic bacteria . Little is known about the abundance of methanotrophs in soils, since quantification by cultivation and microscopic techniques is cumbersome . Comparison of 16S ribosomal DNA and pmoA (alpha subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation and revealed five distinct groups of methanotrophs within the alpha and gamma subclasses of Proteobacteria: the Methylococcus group, the Methylobacter/Methylosarcina group, the Methylosinus group, the Methylocapsa group, and the forest clones group (a cluster of pmoA sequences retrieved from forest soils) . We developed quantitative real-time PCR assays with SybrGreen for each of these five groups and for all methanotrophic bacteria by targeting the pmoA gene . Detection limits were between 10(1) and 10(2) target molecules per reaction for all assays . Real-time PCR analysis of soil samples spiked with cells of Methylococcus capsulatus, Methylomicrobium album, and Methylosinus trichosporium recovered almost all the added bacteria . Only the Methylosinus-specific assay recovered only 20% of added cells, possibly due to a lower lysis efficiency of type II methanotrophs . Analysis of the methanotrophic community structure in a flooded rice field soil showed (5.0 +/- 1.4) x 10(6) pmoA molecules g(-1) for all methanotrophs . The Methylosinus group was predominant (2.7 x 10(6) +/- 1.1 x 10(6) target molecules g(-1)) . In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0 x 10(6) +/- 0.9 x 10(6) target molecules g of soil(-1)) . On the other hand, pmoA affiliated with the forest clones and the Methylocapsa group was below the detection limit of 1.9 x 10(4) target molecules g of soil(-1) . Our results showed that pmoA-targeted real-time PCR allowed fast and sensitive quantification of the five major groups of methanotrophs in soil . This approach will thus be useful for quantitative analysis of the community structure of methanotrophs in nature. J Theor Biol, 2003 Jun 7, 222(3), 307 - 22 The dynamics of single-substrate continuous cultures: the role of transport enzymes; Shoemaker J et al.; A chemostat limited by a single growth-limiting substrate displays a rich spectrum of dynamics . Depending on the flow rate and feed concentration, the chemostat settles into a steady state or executes sustained oscillations . The transients in response to abrupt increases in the flow rate or the feed concentration are also quite complex . For example, if the increase in the flow rate is small, there is no perceptible change in the substrate concentration . If the increase in the flow rate is large, there is a large increase in the substrate concentration lasting several hours or days before the culture adjusts to a new steady state . In the latter case, the substrate concentration and cell density frequently undergo damped oscillations during their approach to the steady state . In this work, we formulate a simple structured model containing the inducible transport enzyme as the key intracellular variable . The model displays the foregoing dynamics under conditions similar to those employed in the experiments . The model suggests that long recovery times (on the order of several hours to several days) can occur because the initial transport enzyme level is too small to cope with the increased substrate supply . The substrate concentration, therefore, increases until the enzyme level is built up to a sufficiently high level by the slow process of enzyme induction . Damped and sustained oscillations can occur because transport enzyme synthesis is autocatalytic, and hence, destabilizing . At low dilution rates, the response of stabilizing processes, such as enzyme dilution and substrate consumption, becomes very slow, leading to damped and sustained oscillations. J Microbiol Methods, 2003 Jul, 54(1), 37 - 45 A quantitative analysis of DNA extraction and purification from compost; Howeler M et al.; We quantified both DNA and humic acid concentrations during the extraction and purification of DNA from compost . The DNA extraction method consisted of bead-beating with SDS for cell lysis, poly(ethylene glycol)-8000 precipitation for preliminary DNA purification, and chromatography on a 10-ml Sephadex G-200 column for final DNA purification . Direct microscopic observation of pre- and post-lysis samples revealed that 95.3+/-2.3% of native cells was lysed . Sixty-three percent of the original DNA was lost during purification, resulting in a final DNA yield of 18.2+/-3.8 microg DNA/g of wet compost . The humic acid content was reduced by 97% during the purification steps resulting in a final humic acid concentration of 27+/-4.7 ng humic acid/microl . The purified DNA fragments were up to 14 kbp in size and were sufficiently free of contaminants to allow both restriction enzyme digestion by four different enzymes and PCR amplification of 16S rDNA. Curr Opin Biotechnol, 2003 Apr, 14(2), 177 - 93 Deciphering plant-pathogen communication: fresh perspectives for molecular resistance breeding; Hammond-Kosack KE et al.; Activation of local and systemic plant defences in response to pathogen attack involves dramatic cellular reprogramming . Over the past 10 years many novel genes, proteins and molecules have been discovered as a result of investigating plant-pathogen interactions . Most attempts to harness this knowledge to engineer improved disease resistance in crops have failed . Although gene efficacy in transgenic plants has often been good, commercial exploitation has not been possible because of the detrimental effects on plant growth, development and crop yield . Biotechnology approaches have now shifted emphasis towards marker-assisted breeding and the construction of vectors containing highly regulated transgenes that confer resistance in several distinct ways. Curr Opin Microbiol, 2003 Apr, 6(2), 191 - 7 LuxS quorum sensing: more than just a numbers game; Xavier KB et al.; Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers . Quorum sensing allows populations of bacteria to collectively control gene expression, and thus synchronize group behavior . Processes controlled by quorum sensing are typically ones that are unproductive unless many bacteria act together . Most autoinducers enable intraspecies communication; however, a recently discovered autoinducer AI-2 has been proposed to serve as a 'universal signal' for interspecies communication . Studies suggest that AI-2 encodes information in addition to specifics about cell number. Curr Opin Microbiol, 2003 Apr, 6(2), 114 - 9 Uses and pitfalls of microarrays for studying transcriptional regulation; Rhodius VA et al.; Microarrays provide a powerful new tool for understanding the regulation of gene expression in bacteria . Many recent publications have used microarrays for identifying regulon members and stimulons that describe the complex organismal responses to environmental perturbations . The use of bioinformatics to identify DNA binding sites of transcription factors greatly facilitates the interpretation of these experiments . Understanding the transcriptome of an organism includes identifying all transcripts and mapping their 5' and 3' ends . High-density oligonucleotide arrays have enabled the identification of many new transcripts, including small RNAs and antisense RNAs. Curr Opin Microbiol, 2003 Apr, 6(2), 93 - 100 RNA polymerase holoenzyme: structure, function and biological implications; Borukhov S et al.; The past three years have marked the breakthrough in our understanding of the structural and functional organization of RNA polymerase . The latest major advance was the high-resolution structures of bacterial RNA polymerase holoenzyme and the holoenzyme in complex with promoter DNA . Together with an array of genetic, biochemical and biophysical data accumulated to date, the structures provide a comprehensive view of dynamic interactions between the major components of transcription machinery during the early stages of the transcription cycle . They include the binding of sigma factor to the core enzyme, and the recognition of promoter sequences and DNA melting by holoenzyme, transcription initiation and promoter clearance. Prog Biophys Mol Biol, 2003 May-Jul, 82(1-3), 197 - 206 Passive ventricular constraint; Power JM et al.; Heart failure (HF) is a progressive degenerative and malignant syndrome with a large number of aetiologies including coronary artery disease, chronic hypertension, exposure to toxins, bacteria and viruses and in a significant percentage of HF patients, the causal mechanism is unclear . The HF trail of morbidity and mortality is well documented and is characterised by step-like periods of relative symptomatic stability, compensation, separated by decompensatory episodes . The homeostatic response to the decline in cardiac function is diverse and involves most organs . There is an increase in resting rate, intra-cardiac hormone production (catecholamines, aldosterone, etc.) and in particular structural changes occur with increased mass and dilatation (dilated cardiomyopathy, DCM) . DCM is associated with decreased cardiac output, contractility and energy efficiency and an increase in pro-arrhythmia and conduction defects . Kass et al . (Circulation 91(9) (1995) 2314) first demonstrated in patients who had undergone a dynamic cardio-myoplasty procedure, that, preventing further dilatation in DCM was beneficial and that the improved cardiovascular status was largely independent of muscle stimulation . We hypothesised that this outcome could be achieved by implanting a fabric cardiac support device around both ventricles to the AV junction . Subsequently, it was shown by us and others (Kass et al., 1995) (Cardiovasc . Res . 44(3) (1999) 549); (Ann . Thorac . Surg . 70(4) (2000) 1275) (in different animal models of DCM) that passive ventricular constraint prevented further dilatation, initiated left ventricular volume reduction and reversed the decline in ejection fraction, mitral valve integrity and left ventricular contractility, when compared with untreated controls . Subsequent European and North American clinical trials in patients with DCM of varying aetiologies have shown equal promise and an absence of device related complications (Circulation 104(12 Suppl . 1) (2001) I270); (Ann . Thorac . Cardiovasc . Surg . 7(5) (2001) 278) . The mechanisms behind this improvement have yet to be fully clarified however the support generated by the device upon the right and ventricular freewall would lower wall tension . Not only is passive ventricular constraint a very promising treatment modality for heart failure and DCM it should provide a useful research tool for the study of the role of ventricular dilatation in the progression of heart failure. Gastroenterology, 2003 May, 124(5), 1265 - 76 Impaired expression of peroxisome proliferator-activated receptor gamma in ulcerative colitis; Dubuquoy L et al.; BACKGROUND & AIMS: The peroxisome proliferator-activated receptor gamma (PPAR gamma) has been proposed as a key inhibitor of colitis through attenuation of nuclear factor kappa B (NF-kappa B) activity . In inflammatory bowel disease, activators of NF-kappa B, including the bacterial receptor toll-like receptor (TLR)4, are elevated . We aimed to determine the role of bacteria and their signaling effects on PPAR gamma regulation during inflammatory bowel disease (IBD) . METHODS: TLR4-transfected Caco-2 cells, germ-free mice, and mice devoid of functional TLR4 (Lps(d)/Lps(d) mice) were assessed for their expression of PPAR gamma in colonic tissues in the presence or absence of bacteria . This nuclear receptor expression and the polymorphisms of gene also were assessed in patients with Crohn's disease (CD) and ulcerative colitis (UC), 2 inflammatory bowel diseases resulting from an abnormal immune response to bacterial antigens . RESULTS: TLR4-transfected Caco-2 cells showed that the TLR4 signaling pathway elevated PPAR gamma expression and a PPAR gamma-dependent reporter in an I kappa kappa beta dependent fashion . Murine and human intestinal flora induced PPAR gamma expression in colonic epithelial cells of control mice . PPAR gamma expression was significantly higher in the colon of control compared with Lps(d)/Lps(d) mice . Although PPAR gamma levels appeared normal in patients with CD and controls, UC patients displayed a reduced expression of PPAR gamma confined to colonic epithelial cells, without any mutation in the PPAR gamma gene . CONCLUSIONS: These data showed that the commensal intestinal flora affects the expression of PPAR gamma and that PPAR gamma expression is considerably impaired in patients with UC. Gastroenterology, 2003 May, 124(5), 1202 - 9 Prophylaxis of pouchitis onset with probiotic therapy: a double-blind, placebo-controlled trial; Gionchetti P et al.; BACKGROUND & AIMS: We have recently documented the efficacy of a highly concentrated probiotic preparation (VSL#3) in the prevention of flare-up in patients with chronic pouchitis . The aim of this study was to compare probiotic therapy with VSL#3 versus placebo in the ability to prevent the onset of acute pouchitis during the first year after ileal pouch-anal anastomosis . METHODS: Forty consecutive patients who underwent ileal pouch-anal anastomosis for ulcerative colitis were randomized to receive either VSL#3 (1 packet containing 900 billion bacteria/day) (n = 20) or an identical placebo (n = 20) immediately after ileostomy closure for 1 year . The patients were assessed clinically, endoscopically, and histologically after 1, 3, 6, 9, and 12 months . Health-related quality of life was assessed using the Inflammatory Bowel Disease Questionnaire . RESULTS: Two of the 20 patients (10%) treated with VSL#3 had an episode of acute pouchitis compared with 8 of the 20 patients (40%) treated with placebo (log-rank test, z = 2.273; P < 0.05) . Treatment with VSL#3 determined a significant improvement in Inflammatory Bowel Disease Questionnaire score, whereas this was not the case with placebo . CONCLUSIONS: Treatment with VSL#3 is effective in the prevention of the onset of acute pouchitis and improves quality of life of patients with ileal pouch-anal anastomosis. Proc Natl Acad Sci U S A, 2003 May 13, 100(10), 5891 - 5 Epub 2003 May 01. Controlling integration specificity of a yeast retrotransposon; Zhu Y et al.; Retrotransposons and retroviruses integrate nonrandomly into eukaryotic genomes . For the yeast retrotransposon Ty5, integration preferentially occurs within domains of heterochromatin . Targeting to these locations is determined by interactions between an amino acid sequence motif at the C terminus of Ty5 integrase (IN) called the targeting domain, and the heterochromatin protein Sir4p . Here we show that new Ty5 integration hot spots are created when Sir4p is tethered to ectopic DNA sites . Targeting to sites of tethered Sir4p is abrogated by single amino acid substitutions in either IN or Sir4p that prevent their interaction . Ty5 target specificity can be altered by replacing the IN-targeting domain with other peptide motifs that interact with known protein partners . Integration occurs at high efficiency and in close proximity to DNA sites where the protein partners are tethered . These findings define a mechanism by which retrotransposons shape their host genomes and suggest ways in which retroviral integration can be controlled. Proc Natl Acad Sci U S A, 2003 May 13, 100(10), 6133 - 8 Epub 2003 May 02. The two-component PhoR-PhoP system controls both primary metabolism and secondary metabolite biosynthesis in Streptomyces lividans; Sola-Landa A et al.; The biosynthesis of most secondary metabolites in different bacteria is strongly depressed by inorganic phosphate . The two-component phoR-phoP system of Streptomyces lividans has been cloned and characterized . PhoR showed all of the characteristics of the membrane-bound sensor proteins, whereas PhoP is a member of the DNA-binding OmpR family . Deletion mutants lacking phoP or phoR-phoP, were unable to grow in minimal medium at low phosphate concentration (10 microM) . Growth was fully restored by complementation with the phoR-phoP genes . Both S . lividans DeltaphoP and DeltaphoR-phoP deletion mutants were unable to synthesize extracellular alkaline phosphatase (AP) as shown by immunodetection with anti-AP antibodies and by enzymatic analysis, suggesting that the PhoR-PhoP system is required for expression of the AP gene (phoA) . Synthesis of AP was restored by complementation of the deletion mutants with phoR-phoP . The biosynthesis of two secondary metabolites, actinorhodin and undecylprodigiosin, was significantly increased in both solid and liquid medium in the DeltaphoP or DeltaphoR-phoP deletion mutants . Negative phosphate control of both secondary metabolites was restored by complementation with the phoR-phoP cluster . These results prove that expression of both phoA and genes implicated in the biosynthesis of secondary metabolites in S . lividans is regulated by a mechanism involving the two-component PhoR-PhoP system. Proc Natl Acad Sci U S A, 2003 May 13, 100(10), 5980 - 5 Epub 2003 May 02. A novel class of gene controlling virulence in plant pathogenic ascomycete fungi; Lu SW et al.; Insertional mutants of the fungal maize pathogen Cochliobolus heterostrophus were screened for altered virulence . One mutant had 60% reduction in lesion size relative to WT but no other detectable change in phenotype . Analysis of sequence at the insertion site revealed a gene (CPS1) encoding a protein with two AMP-binding domains . CPS1 orthologs were detected in all Cochliobolus spp . examined, in several other classes of ascomycete fungi, and in animals but not in basidiomycete fungi, bacteria, or plants . Phylogenetic analysis suggested that CPS1 represents a previously undescribed subset of adenylate-forming enzymes that have diverged from certain acyl-CoA ligases, which in bacteria are involved in biosynthesis of nonribosomal peptides or polyketidepeptide hybrids . Disruption of CPS1 caused reduced virulence of both race T and race O of C . heterostrophus on maize, of Cochliobolus victoriae on oats, and of Gibberella zeae on wheat . These results suggest that CPS1 functions as a general fungal virulence factor in plant pathogenic ascomycetes. J Pharmacol Exp Ther, 2003 Aug, 306(2), 718 - 25 Epub 2003 May 02. Sigma-1 receptors (sigma(1) binding sites) form raft-like microdomains and target lipid droplets on the endoplasmic reticulum: roles in endoplasmic reticulum lipid compartmentalization and export; Hayashi T et al.; The brain sigma-1 receptors can bind neurosteroids and psychotropic drugs, including neuroleptics and cocaine and are implicated in schizophrenia, depression, and drug dependence . In this study, we found that sigma-1 receptors specifically target lipid storage sites (lipid droplets) on the endoplasmic reticulum by forming a distinct class of lipid microdomains . Both endogenously expressing sigma-1 receptors and transfected C-terminally enhanced yellow fluorescent protein (EYFP)-tagged sigma-1 receptors (Sig-1R-EYFP) target unique "ring-like" structures associated with endoplasmic reticulum reticular networks in NG108-15 cells . The ring-like structures contain neutral lipids and are enlarged by the oleate treatment, indicating that they are endoplasmic reticulum-associated lipid droplets (ER-LDs) . sigma-1 receptors colocalize with caveolin-2, a cholesterol-binding protein in lipid rafts on the ER-LDs, but not with adipocyte differentiation-related protein (ADRP), a cytosolic lipid droplet (c-LD)-specific protein . When the double-arginine ER retention signal on the N terminus of sigma-1 receptors is truncated, sigma-1 receptors no longer exist on ER-LDs, but predominantly target c-LDs, which contain ADRP . sigma-1 receptors on ER-LDs form detergent-resistant raft-like lipid microdomains, the buoyancy of which is different from that of plasma membrane lipid rafts . (+)-Pentazocine causes sigma-1 receptors to disappear from the microdomains . N-Terminally EYFP-tagged sigma-1 receptors (EYFP-Sig-1R) failed to target ER-LDs . EYFP-Sig-1R-transfected cells showed an unrestricted distribution of neutral lipids all over the endoplasmic reticulum network, decreases in c-LDs and cholesterol in plasma membranes, and the bulbous aggregation of endoplasmic reticulum . Thus, sigma-1 receptors are unique endoplasmic reticulum proteins that regulate the compartmentalization of lipids on the endoplasmic reticulum and their export from the endoplasmic reticulum to plasma membrane and c-LDs. Annu Rev Microbiol, 2003, 57, 57 - 76 Epub 2003 May 01. Brucella stationary-phase gene expression and virulence; Roop RM 2nd et al.; The capacity of the Brucella spp . to establish and maintain long-term residence in the phagosomal compartment of host macrophages is critical to their ability to produce chronic infections in their mammalian hosts . The RNA binding protein host factor I (HF-I) encoded by the hfq gene is required for the efficient translation of the stationary-phase sigma factor RpoS in many bacteria, and a Brucella abortus hfq mutant displays a phenotype in vitro, which suggests that it has a generalized defect in stationary-phase physiology . The inability of the B . abortus hfq mutant to survive and replicate in a wild-type manner in cultured murine macrophages, and the profound attenuation displayed by this strain and its B . melitensis counterpart in experimentally infected animals indicate that stationary-phase physiology plays an essential role in the capacity of the brucellae to establish and maintain long-term intracellular residence in host macrophages . The nature of the Brucella HF-I-regulated genes that have been identified to date suggests that the corresponding gene products contribute to the remarkable capacity of the brucellae to resist the harsh environmental conditions they encounter during their prolonged residence in the phagosomal compartment. J Bacteriol, 2003 May, 185(10), 3068 - 75 Cell-cycle-regulated expression and subcellular localization of the Caulobacter crescentus SMC chromosome structural protein; Jensen RB et al.; Structural maintenance of chromosomes proteins (SMCs) bind to DNA and function to ensure proper chromosome organization in both eukaryotes and bacteria . Caulobacter crescentus possesses a single SMC homolog that plays a role in organizing and segregating daughter chromosomes . Approximately 1,500 to 2,000 SMC molecules are present per cell during active growth, corresponding to one SMC complex per 6,000 to 8,000 bp of chromosomal DNA . Although transcription from the smc promoter is induced during early S phase, a cell cycle transcription pattern previously observed with multiple DNA replication and repair genes, the SMC protein is present throughout the entire cell cycle . Examination of the intracellular location of SMC showed that in swarmer cells, which do not replicate DNA, the protein forms two or three foci . Stalked cells, which are actively engaged in DNA replication, have three or four SMC foci per cell . The SMC foci appear randomly distributed in the cell . Many predivisional cells have bright polar SMC foci, which are lost upon cell division . Thus, chromosome compaction likely involves dynamic aggregates of SMC bound to DNA . The aggregation pattern changes as a function of the cell cycle both during and upon completion of chromosome replication. J Bacteriol, 2003 May, 185(10), 3036 - 41 Leucines 193 and 194 at the N-terminal domain of the XylS protein, the positive transcriptional regulator of the TOL meta-cleavage pathway, are involved in dimerization; Ruiz R et al.; Members of the AraC/XylS family of transcriptional regulators are usually organized in two domains: a conserved domain made up of 100 amino acids and frequently located at the C-terminal end, involved in DNA binding; and an N-terminal nonconserved domain involved in signal recognition, as is the case for regulators involved in the control of carbon metabolism (R . Tobes and J . L . Ramos, Nucleic Acids Res . 30:318-321, 2002) . The XylS protein, which is extremely insoluble, controls expression of the meta-cleavage pathway for alkylbenzoate metabolism . We fused the N-terminal end of XylS to the maltose-binding protein (MBP) in vitro and found in glutaraldehyde cross-linking assays that the protein dimerized . Experiments with a chimeric N-terminal XylS linked to a 'LexA protein showed that the dimer was stabilized in the presence of alkylbenzoates . Sequence alignments with AraC and UreR allowed us to identify three residues, Leu193, Leu194, and Ile205, as potentially being involved in dimerization . Site-directed mutagenesis of XylS in which each of the above residues was replaced with Ala revealed that Leu193 and Leu194 were critical for activity and that a chimera in which LexA was linked to the N terminus of XylSLeu193Ala or XylSLeu194Ala was not functional . Dimerization of the chimeras MBP-N-XylSLeu193Ala and MBP-N-XylSLeu194Ala was not observed in cross-linking assays with glutaraldehyde. Genes Dev, 2003 May 1, 17(9), 1084 - 9 Latent ClpX-recognition signals ensure LexA destruction after DNA damage; Neher SB et al.; The DNA-damage response genes in bacteria are up-regulated when LexA repressor undergoes autocatalytic cleavage stimulated by activated RecA protein . Intact LexA is stable to intracellular degradation but its auto-cleavage fragments are degraded rapidly . Here, both fragments of LexA are shown to be substrates for the ClpXP protease . ClpXP recognizes these fragments using sequence motifs that flank the auto-cleavage site but are dormant in intact LexA . Furthermore, ClpXP degradation of the LexA-DNA-binding fragment is important to cell survival after DNA damage . These results demonstrate how one protein-processing event can activate latent protease recognition signals, triggering a cascade of protein turnover in response to environmental stress. Chemosphere, 2003 Jul, 52(1), 203 - 12 Use of oxytetracycline and tylosin in intensive calf farming: evaluation of transfer to manure and soil; De Liguoro M et al.; Antibiotics may enter soils with manure from treated animals . Because of their biological effects, antibiotics are regarded as potential micropollutants . The levels of oxytetracycline and tylosin over time were followed in faeces, bedding and manure, and then in the soil of a manured field and surrounding drainage courses, after oral treatment of calves . Fifty Simmental calves were treated for 5 days with 60 mg/kg/day of oxytetracycline . After 15 days the animals were treated for 5 days with 20 mg/kg/day of tylosin . Tylosin degraded rapidly, and was no longer detected in manure 45 days after cessation of treatment and no trace of the compound was detected in soil or surrounding water (detection limits 10 microg/l) . The half-life of oxytetracycline in manure was 30 days and the compound was still detectable in this matrix (820 microg/kg) after 5 months maturation . In the manured soil oxytetracycline was detected at concentrations at least 10 times lower than the European Agency for the Evaluation of Medicinal Products threshold (100 microg/kg) requiring phase II environmental risk assessment . Oxytetracycline was not detected in the water courses (detection limit 1 microg/l) . These results demonstrate that the processes occurring between faeces production and application of manure to the soil are very effective in reducing the load of TYL and OTC in the environment . For both drugs a toxicity test was performed using the alga Selenastrum capricornutum . The EC50 was 4.18 mg/l for oxytetracycline and 0.95 mg/l for tylosin . A worst-case hazard assessment for the aquatic environment was performed comparing the ratio between the measured concentrations (LOD) and effect data from previous work (OTC) or from this work (TYL) . This showed ratio between toxicity levels (bacteria) (EC50=0.14 mg/l) and measured concentrations (LOD=1 microg/l) for OTC to be 140 . The corresponding value for TYL (LOD=10 microg/l) was 95. Biochem Biophys Res Commun, 2003 May 9, 304(3), 481 - 6 Scission, spores, and apoptosis: a proposal for the evolutionary origin of mitochondria in cell death induction; Frank S et al.; Mitochondria fragment prior to caspase activation during many pathways of apoptosis . Inhibition of the machinery that normally regulates mitochondrial morphology in healthy cells inhibits the fission that occurs during apoptosis and actually delays the process of cell death . Interestingly, there are certain parallels between mitochondrial fission and bacterial sporulation . As bacterial sporulation can be considered a stress response we suggest that a primordial stress response of endosymbiont mitochondrial progenitors may have been adopted for the stress response of early eukaryotes . Thus, the mitochondrial fission process may represent an early stress response of primitive mitochondria that could have integrated the stress signals and acted as an initial sensor for the eukaryotic response system . The fact that mitochondria fragment during apoptosis using the machinery descended from or that superceded the bacterial stress response of sporulation is consistent with this hypothesis . This hypothesis would explain why what is generally considered the "power house" of the cell came to integrate the cell death response and regulate apoptosis. Epidemiol Infect, 2003 Apr, 130(2), 239 - 49 Molecular study on human tuberculosis in three geographically distinct and time delineated populations from ancient Egypt; Zink AR et al.; We describe the molecular identification of human tuberculosis (TB) from vertebral bone tissue samples from three different populations of ancient Egypt . The specimens were obtained from the predynastic to early dynastic necropolis of Abydos (7 individuals, c . 3500-2650 B.C.), from a Middle Kingdom to Second Intermediate Period tomb of the necropolis of Thebes-West (37 . c . 2100-1550 B.C.) and from five further Theban tombs used in the New Kingdom and the Late Period (39, c . 1450-500 B.C.) . A total of 18 cases tested positive for the presence of ancient DNA (aDNA) of the M . tuberculosis complex . Out of the 9 cases with typical macromorphological signs of tuberculous spondylitis, 6 were positive for mycobacterial aDNA (66.7%) . Of 24 cases with non-specific pathological alterations, 5 provided a positive result (20.8%) . In 50 cases of normally appearing vertebral bones 7 tested positive (14.0%) . There were only minor differences in the frequencies between the three populations . These data strongly support the notion that tuberculosis was present and prevalent in ancient Egypt since very early periods of this civilization . The unexpectedly high rate of mycobacterial aDNA in normal bone samples is presumably due to a pre- to perimortal systemic spread of the bacteria and indicates a generalized infection by M . tuberculosis. Cutis, 2003 Apr, 71(4), 271 - 5 Epidemiology of skin diseases in people of color; Taylor SC; The epidemiology of skin diseases in people of color has not been extensively studied . Many skin diseases (eg, acne vulgaris; eczematous dermatitis; infections caused by bacteria, fungi, or viruses) are common to most people of color--blacks, Asians, Hispanics/Latinos, and Native Americans . Diseases of more cosmetic concern (eg, melasma, postinflammatory pigmentation, acne keloidalis nuchae, scalp and facial folliculitis, keloids, alopecias) occur more in skin of color than in white skin. Rev Esp Salud Publica, 2003 Mar-Apr, 77(2), 287 - 95 {Outbreak of gastroenteritis caused by Norwalk virus at a senior citizens assisted living facility in Granada, Spain}; Almagro Nievas D et al.; BACKGROUND: The outbreaks of acute gastroenteritis caused by a virus are currently one of the health problems having the greatest impact on those living in senior citizen facilities . The objective of this study is that of investigating the causes of the start and contagion of an outbreak of acute gastroenteritis caused by a Norwalk-Like virus . METHOD: At a senior citizen living facility with 141 residents and 71 employees exposed, the relations between the disease in question and different independent variables: personal traits, features of location, date of onset of symptoms, clinical pattern of the disease and causal factors (food and drinking water intake) . A descriptive study, a contingency table and a hypothesis ?2 test, design of cases and controls with the calculation of the unprocessed and subsequently adjusted Odds Ratio (IC 95%) were carried out using logic regression . RESULTS: No significant differences were found in the personal trait and features of location variables . The attack rate among the residents was 30.1%, being 21.12% among the employees . In the adjusted Odds Ratio calculation, solely the whipped cream mousse showed causal relationship values (adjusted Odds Ratio = 4.66; IC-95% 1.15-18.91; p = 0.031) . Ten patient stool samples were sent for analysis, no disease-causing bacteria having been detected, Norwalk-Like virus however having been isolated in three samples . CONCLUSIONS: The whipped cream mousse having been found to possibly have been responsible for the outbreak is a first involving Norwalk virus, but this result is subject to limitations, such as the possible bias of classification due to failing to recall having eaten foods . Even so, this is an element to be taken into account in the control of acute viral gastroenteritis. EMBO J, 2003 May 1, 22(9), 2091 - 103 Cdc2-mediated phosphorylation of Kid controls its distribution to spindle and chromosomes; Ohsugi M et al.; The chromokinesin Kid is important in chromosome alignment at the metaphase plate . Here, we report that Kid function is regulated by phosphorylation . We identify Ser427 and Thr463 as M phase-specific phosphorylation sites and Cdc2-cyclin B as a Thr463 kinase . Kid with a Thr463 to alanine mutation fails to be localized on chromosomes and is only detected along spindles, although it retains the ability to bind DNA or chromosomes . Localization of rigor-type mutant Kid, which shows nucleotide-independent microtubule association, is also confined to the spindle, implying that strong association of Kid with the spindle can sequester it from chromosomes . T463A substitution in DNA-binding domain-truncated Kid consistently enhances its spindle localization . At physiological ionic strength, unphosphorylated Kid shows ATP-independent microtubule association, whereas Thr463-phosphorylated Kid shows ATP dependency . Moreover, the stalk region of unphosphorylated Kid interacts with microtubules and the interaction is weakened when Thr463 is phosphorylated . Our data suggest that phosphorylation on Thr463 of Kid downregulates its affinity for microtubules to ensure reversible association with spindles, allowing Kid to bind chromosomes and exhibit its function. EMBO J, 2003 May 1, 22(9), 1959 - 68 Ferritins, iron uptake and storage from the bacterioferritin viewpoint; Carrondo MA; Ferritins constitute a broad superfamily of iron storage proteins, widespread in all domains of life, in aerobic or anaerobic organisms . Ferritins isolated from bacteria may be haem-free or contain a haem . In the latter case they are called bacterioferritins . The primary function of ferritins inside cells is to store iron in the ferric form . A secondary function may be detoxification of iron or protection against O(2) and its radical products . Indeed, for bacterioferritins this is likely to be their primary function . Ferritins and bacteroferritins have essentially the same architecture, assembling in a 24mer cluster to form a hollow, roughly spherical construction . In this review, special emphasis is given to the structure of the ferroxidase centres with native iron-containing sites, since oxidation of ferrous iron by molecular oxygen takes place in these sites . Although present in other ferritins, a specific entry route for iron, coupled with the ferroxidase reaction, has been proposed and described in some structural studies . Electrostatic calculations on a few selected proteins indicate further ion channels assumed to be an entry route in the later mineralization processes of core formation. FEMS Immunol Med Microbiol, 2003 May 15, 36(1-2), 63 - 9 The CD14 receptor does not mediate entry of Mycobacterium tuberculosis into human mononuclear phagocytes; Shams H et al.; Prior reports have suggested that CD14 mediates uptake of Mycobacterium tuberculosis into porcine alveolar macrophages and human fetal microglia, but the contribution of CD14 to cell entry in human macrophages has not been studied . To address this question, we used flow cytometry to quantify uptake by human monocytes and alveolar macrophages of M . tuberculosis expressing green fluorescent protein . Neutralizing anti-CD14 antibodies did not affect bacillary uptake and the efficiency of bacillary entry was similar in THP-1 cells expressing low and high levels of CD14 . However, most internalized bacteria were found in CD14+ but not in CD14- monocytes because M . tuberculosis infection upregulated CD14 expression . We conclude that: (1) CD14 does not mediate cellular entry by M . tuberculosis; (2) M . tuberculosis infection upregulates CD14 expression on mononuclear phagocytes, and this may facilitate the pathogen's capacity to modulate the immune response. Water Res, 2003 May, 37(10), 2449 - 57 Effect of temperature on removal of heavy metals from contaminated river sediments via bioleaching; Tsai LJ et al.; The aim of this study was to determine the effect of temperature on the solubilization of heavy metals from contaminated river sediment by sulfur oxidizing bacteria taken from Ell-Ren River sediment . Of three temperatures tested (25 degrees C, 37 degrees C and 55 degrees C), pH decrease was greatest at 37 degrees C, indicating that, after acclimation, bacterial oxidizing activity is greatest at this temperature . At 55 degrees C, pH change was similar to that which occurred with no inoculum added . The increase in sulfates and high pH at 55 degrees C indicate that the indirect mechanism was not initiated at this temperature . Solubilization efficiency of total extractable Ni, Zn, Cu and Cr was high (>90%) at 37 degrees C, whilst that of Pb was only 60.4% . Except for Pb, the optimal temperature for solubilization of total extractable heavy metal was 37 degrees C . The order of average solubilization efficiency of total extractable heavy metals was Ni, Zn, Cu>Cr>Co, Pb . The solubilization efficiency of Pb and Co was markedly less than that of other heavy metals . Transfer of heavy metals between binding fractions was most apparent at 55 degrees C before and after bioleaching. Life Sci, 2003 May 23, 73(1), 19 - 26 Melatonin in Chinese medicinal herbs; Chen G et al.; Melatonin is a highly conserved molecule that not only exists in animals, but also is present in bacteria, unicellular organisms and in plants . Since melatonin is an antioxidant, in plants melatonin was speculated to protect them from intrinsic and environmental oxidative stress . More importantly, melatonin in edible plants inevitably enters animals and human through feed and food . In this study, more than 100 Chinese medicinal herbs were analyzed using the methods of solid phase extraction and HPLC-FD on-line with MS to determine whether melatonin is present in these commonly used herbs . Melatonin was detected in majority of these plants . Sixty-four of them contain melatonin in excess of 10 ng per gram dry mass . Melatonin levels in several herbs are in excess of 1000 ng/g . It is well known that normal average physiological plasma levels of melatonin are only 10-60 pg/mL . These high level-melatonin containing plants are traditionally used to treat diseases which presumably involve free radical damage . The current study provides new information concerning one potentially effective constituent present in a large number of medicinal herbs . The results suggest that these herbs should be reevaluated in reference to their nutritional and medicinal value. FEMS Microbiol Lett, 2003 Apr 25, 221(2), 299 - 304 Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment; Hongoh Y et al.; We investigated biases occurring in the polymerase chain reaction (PCR) amplification of 16S rRNA genes from an environmental sample, by comparing the clone libraries that we had previously prepared from the gut homogenate of the termite Reticulitermes speratus . We detected a significant increase in the expected number of phylotypes by lowering the annealing temperature, and a significant decrease in the proportion of clones belonging to the predominant group by raising the number of PCR cycles . We also found that the Bacteria-universal primer, 63F, introduced a seriously biased amplification caused by primer mismatches, in contrast to a previous report . These results, together with suggestions from previous studies using simplified model samples, will help us to recognize the limitations of PCR-based analysis. FEMS Microbiol Lett, 2003 Apr 25, 221(2), 293 - 8 Phylogenetic analysis of the family Thermaceae with an emphasis on signature position and secondary structure of 16S rRNA; Chen C et al.; The sequences of the 16S rRNA genes from 38 strains of the family Thermaceae were compared by alignment analysis . The genus-specific and species-specific base substitutions or base deletions (signature positions) were found in three hypervariable regions (in the helices 6, 10 and 17) . The differentiation of secondary structures of the high variable regions in the 5' end (38-497) containing several signature positions further supported the concept . Based on the comparisons of the secondary structures in the segments of 16S rRNAs, a key to the species of the family Thermaceae was proposed. FEMS Microbiol Lett, 2003 Apr 25, 221(2), 269 - 75 Detecting pathogenicity islands and anomalous gene clusters by iterative discriminant analysis; Tu Q et al.; We present a simple method to detect pathogenicity islands and anomalous gene clusters in bacterial genomes . The method uses iterative discriminant analysis to define genomic regions that deviate most from the rest of the genome in three compositional criteria: G+C content, dinucleotide frequency and codon usage . Using this method, we identify many virulence-related gene islands, e.g . encoding protein secretion systems, adhesins, toxins, and other anomalous gene clusters, such as prophages . The program and the whole dataset, including the catalogs of genes in the detected anomalous segments, are publicly available at This program can be used in searching for virulence-related factors in newly sequenced bacterial genomes. Isotopes Environ Health Stud, 2002 Dec, 38(4), 227 - 43 Feeding and growth of apple snail Pomacea lineata in the Pantanal wetland, Brazil--a stable isotope approach; Fellerhoff C; Apple snails Pomacea lineata (SPIX 1827) are widespread in the tropical regions of Brazil as well as in the Pantanal wetland of Mato Grosso in the western part of the country . They have a key position in the Pantanal food web and serve as food for many animals e.g . fishes, birds, and caimans . However, little is known about their feeding preferences and growth rates . Stable isotopes have been used successfully on numerous studies as food source indicator . Therefore, the delta15N and delta13C values of snails from 0.45 to 3.03 cm in length, which were collected in the rainy season from March through May, were analyzed . Snails signatures revealed ambiguous evidence for food preferences . Delta15N and delta13C values ranged between -2.8 and 12.4 per thousand and between -24.2 and -16.4, per thousand respectively . This range of values mirrors the highly variable isotope values of possible food sources comprising C3 and C4 macrophytes . To test whether all common food sources were similarly assimilated, feeding experiments with different diets were conducted . Snail eggs were reared in tanks and offered different but single plants . Snails fed different diets and delta13C values of the food were reflected in the animal tissue . Growth varied considerably in experiments with different diets indicating the preference for certain food sources . Also, the fractionation of nitrogen isotopes between food and animal varied from 0.1 to 17.0 per thousand . The results are explained by different feeding habits, and it is supposed that animals fed either on the plant itself or on bacteria mats growing in the tanks . In an additional experiment juvenile snails were offered one single food with a distinctive C4 grass signature . These snails did not grow detectably, but nevertheless isotope signatures approached to values of the diet. Anal Sci, 2003 Apr, 19(4), 499 - 504 A screening method for estrogens using an array-type DNA glass slide; Kim SB et al.; A new screening assay was described for the determination of endocrine disrupting chemicals (EDCs), such as synthetic estrogens, with an array-type DNA glass slide having characteristics of 1) a high sample throughput, 2) a compact size allowing a small sample volume, and 3) a sensitive determination based on the estrogen-dependent binding of the human estrogen receptor a (hERalpha) with its estrogen responsive element (ERE; Vit . A2 gene promoter) . We devised a glass slide on which a thin agarose gel was mounted . Avidin was then covalently immobilized on each well of the glass slide after the gel was activated by a NaIO4 solution . Also, the biotinylated ERE as a DNA probe was immobilized on the gel layer through avidin-biotin binding . After the estrogen-dependent binding of a yellow fluorescent protein-fused hERalpha (YFP-hERalpha) to ERE on the gel layer, the fluorescence intensity of YFP-hERalpha quantitatively extracted into the gel was directly determined with a fluorescence microplate reader . Pre-incubation of YFP-hERalpha with estrogen at 37 degrees C for 30 min enhanced the estrogen-dependent hERalpha-ERE binding . The determined hormonal activities of estrogens on the interaction of YFP-hERalpha with ERE were as follows in their decreasing order: diethylstilbestrol (DES) > 17beta-estradiol (E2) <==> ethynylestradiol (EE2) > 4-hydroxy tamoxifen (OHT) > clomiphene (Clo) . The present method provides a sensitive estrogen-dependent dose-response curve down to approximately 10(-13) M in the case of DES . This method will become a competitive alternative to the conventional in vitro assays, such as a DNA-binding assay using radioisotopes. Microbiology, 2003 May, 149(Pt 5), 1139 - 46 Diurnal expression of hetR and diazocyte development in the filamentous non-heterocystous cyanobacterium Trichodesmium erythraeum; El-Shehawy R et al.; The marine non-heterocystous cyanobacterium Trichodesmium fixes atmospheric N(2) aerobically in light . In situ immunolocalization/light microscopy of NifH revealed that lighter, non-granulated cell regions observed correspond to the nitrogenase-containing diazocyte clusters in Trichodesmium IMS101 . The number of diazocyte clusters per trichome varied from 0 to 4 depending on trichome length . The constant percentage of diazocytes (approx . 15 %) in cultured strains and five natural populations suggests a developmentally regulated differentiation process . Real-time RT-PCR showed that ntcA, encoding the global nitrogen regulator in cyanobacteria, and hetR, the key regulatory gene in heterocyst differentiation, are both constitutively expressed during a 12 h/12 h light/dark cycle . hetR in addition showed a distinct peak in the dark (close to midnight) while nifH expression commenced 6-8 h later . The expression of all three genes was negatively affected by addition of ammonia . Some early heterocyst differentiation genes were also identified in the genome of Trichodesmium . The data suggest that hetR and ntcA may be required for development and function of diazocytes in Trichodesmium. Microbiology, 2003 May, 149(Pt 5), 1113 - 25 Immunological and genetic characterization of Borrelia burgdorferi BapA and EppA proteins; Miller JC et al.; A large majority of examined Lyme disease spirochaete isolates were demonstrated to contain one or both of the paralogous genes bapA and eppA . Immunological analyses of serum samples collected from infected patients coupled with comparative sequence analyses indicated that bapA gene sequences are quite stable but the encoded proteins do not provoke a strong immune response in most individuals . Conversely, EppA proteins are much more antigenic but vary widely in sequence between different bacteria . Considerable evidence of insertion, deletion and other mutations within eppA genes was observed . A number of significant recombination events were also found to have occurred in regions flanking bapA genes, while the genes themselves rarely exhibited evidence of mutation, suggesting strong selective pressure to maintain BapA sequences within narrow limits . Data from these and other studies suggest important roles for BapA and EppA during the Borrelia burgdorferi infectious cycle. Am J Physiol Gastrointest Liver Physiol, 2003 Aug, 285(2), G404 - 13 Epub 2003 Apr 30. Enterocyte apoptosis after enterectomy in mice is activated independent of the extrinsic death receptor pathway; Knott AW et al.; Intestinal adaptation following small bowel resection (SBR) is associated with greater rates of enterocyte apoptosis by unknown mechanism(s) . Because postresection adaptation is associated with increased translocation of luminal bacteria, we sought to characterize the role for the extrinsic, death receptor pathway for the activation of enterocyte apoptosis after massive SBR . We first performed SBR or sham operations in mice, and the temporal expression of caspases 8, 9, and 3, death receptors tumor necrosis factor receptor-1 (TNFR1) and Fas and corresponding ligands (TNF and Fas ligand) was determined in the remnant intestine at various postoperative time points . Ileal TNFR1 and Fas expression were then measured after SBR in the setting of increased (waved-2 mice) or decreased (exogenous EGF administration) apoptosis . Finally, intestinal adaptation and apoptosis were recorded in the remnant ileum after SBR in TNFR1-null and Fas-null mice . The expression of death receptor family proteins and caspases demonstrated only modest changes after SBR and did not correlate with the histological appearance of apoptosis . In the setting of accelerated apoptosis, TNFR1 and Fas expression were paradoxically decreased . Apoptotic and adaptive responses were preserved in both TNFR1-null and Fas-null mice . These results suggest that the mechanism for increased enterocyte apoptosis following massive SBR does not appear to involve the extrinsic, death receptor-mediated pathway. Pediatr Dent, 2003 Mar-Apr, 25(2), 143 - 8 The detection of Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans in the supragingival plaque of children with and without caries; Tanaka S et al.; PURPOSE: The present study was undertaken to determine the presence of 3 periodontopathic bacteria in the supragingival plaques of 3- to 16-year-old children with different oral health conditions . METHODS: DMFT and dft, PMA index (P=papillary gingivitis, M=marginal gingivitis, and A=attached gingivitis), OHI (oral hygiene index), and oral malodor of each subject were determined prior to the collection of supragingival plaques . Periodontopathic bacteria (P . gingivalis, P . intermedia, and A . actinomycetemcomitans) in supragingival plaques were detected using an immunoslot blot assay with monoclonal 3 periodontopathic bacteria in the 2 subject groups (children with and without caries) . P . gingivalis-positive subjects, but not their P . intermedia or A . actinomycetemcomitans counterparts, were correlated to oral malodor . Oral malodor was also correlated to debris index, a component of OHI . RESULTS: The group with the higher OHI showed a higher prevalence of periodontopathic bacteria . For the 3 periodontopathic bacteria in the subjects tested, P . gingivalis-, P . intermedia-, and A . actinomycetemcomitans-positive plaques were not age related . CONCLUSIONS: The supragingival plaques in children can harbor periodontopathic bacteria such as P . gingivalis, P . intermedia, and A . actinomycetemcomitans. Am J Chin Med, 2003, 31(1), 111 - 7 Hepatoprotective activity of reduohanxiao-tang (yuldahanso-tang) is related to the inhibition of beta-glucuronidase; Bae HS et al.; Beta-glucuronidase-inhibitory and hepatoprotective effects of Reduohanxiao-tang (Yuldahanso-tang), which has been used for liver diseases and stroke, on carbon tetrachloride (CCl4)-induced hepatotoxicity of rats were investigated . Reduohanxiao-tang potently inhibited beta-glucuronidases . Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactic acid dehydrogenase (LDH) levels of the CCl4 group orally treated with Reduohanxiao-tang (100 mg/kg) were lowered to 54%, 71.5% and 66.1% of the CCl4-treated control group, respectively . Among the ingredients of the Reduohanxiao-tang, the rhizomes of Pueraria thunbergiana and Scutellaria baicalensis potently inhibited beta-glucuronidases and protected against CCl4-induced liver injury . Orally administered puerarin, which is a main component of Pueraria thunbergiana, showed potent hepatoprotective activity, but did not inhibit beta-glucuronidase . However, daidzein, which is produced from puerarin by human intestinal bacteria, potently inhibited beta-glucuronidase . These results suggest that beta-glucuronidase inhibition by herbal medicines may protect against CCl4-induced liver injury. Mol Biol (Mosk), 2003 Mar-Apr, 37(2), 266 - 76 {Definitive role of polar residue clusters in B-DNA major groove recognition by protein factors}; Chirgadze IuN et al.; The 3D structural data for a number of protein-DNA complexes were used to analyze the regions of specific contact with the major groove of B-DNA double helix . The set included seven nonhomologous complexes featuring 12 DNA-binding domains of transcription factors and regulatory factors . The protein domains differed in structure, contained different motifs in the binding region, and broadly varied in chain length, from 30 to 200 residues . Protein-DNA interaction was assessed as hydrogen bonding between polar atoms and van der Waals contacts between nonpolar atoms . The binding sites were formed mainly through polar side chain interactions . On average, the recognition site comprises seven residues, six of them polar . The contact residues nearly always belong to a largest polar cluster of the protein . Thus one can think that the protein polar residue clusters play an important role in forming the protein-DNA recognition module. Zh Obshch Biol, 2003 Mar-Apr, 64(2), 160 - 5 {The possible contribution of late pleistocene biota to biodiversity in present permafrost zone}; Gubin SV et al.; During the last decade a wide range of biological objects, which have preserved their viability for tens and hundreds of thousands of years, was found in the samples of permafrost sediments from North-East Eurasia . Among them are bacteria, fungi, algae, moss spores, seeds of higher plants, protists . Along with physiological mechanisms of cryoconservation and constant low temperature of great importance for long-term preservation of biological objects in permafrost layers are ways of burying the organisms and conditions that prevail before the transition of sediments to the permafrost state . The analysis of viability showed by preserved biological objects gives reasons to suppose that some representatives of Pleistocene biota buried in permafrost thickness may contribute to the biodiversity of present cryolite zone. Zh Obshch Biol, 2003 Mar-Apr, 64(2), 128 - 45 {The budget model of ecosystem of a shallow highly eutrophic lake}; Kazantseva TI; A model of energy budget of Lake Bolshoi Okunenok ecosystem was based on the data received during field studies from May through November 1986 . The model takes into account 36 components including dissolved organic matter, bacteria, phytoplankton, zooplankton, meiobenthos, macrobenthos, fish, suspended and sediment detritus . The growing season has been divided into 16 intervals according to the number of observations . The balance equation for each live component describes the change in its biomass for a time interval between two successive sampling dates . The change is considered as a balance of energy input with assimilation or feeding, and energy loss due to respiration, excretion, predation, natural mortality, fishery catchment or and emergence of imago insects . For non-live components we estimate an increase and a decrease in their mass due to the activity of living organisms, as well as organic matter exchange between water and sediments . Seasonal value of balance elements for each component are equal to sums of appropriate interval value . Comparison of energy flows through different links of a trophic web has shown that the role of a bacterial-detrial link was extremely important in Lake Bolshoi Okunenok for the growth season of 1986 . Detritus constituted 58% of seasonal diet of non-predatory zooplankton, 39% of diet of predatory zooplankton, 50% of diet of planktivorous fish (fry of whitefish) and 92% of diet of benthivorous fish (fry of carp) . The contribution of bacteria to the total seasonal decomposition amounted to 46% . Approximately 57% of the forage phytoplankton production, 86% of non-predatory benthos production, and 23-38% of the other trophic groups production were consumed by all grazers . "Coefficient of energy transformation" is proposed . It is calculated as: CET(s, k) = Ps(k)/Pk, where Ps(k) is production of consumers "s", built due to consumption of source "k"; Pk is production of source "k" itself . In Lake Bolshoi Okunenok only 14% of energy built by phytoplankton were accumulated in organic matter of zooplankton due to direct consumption. Biofizika, 2003 Mar-Apr, 48(2), 221 - 6 {Calculation of spectra of absorption and circular dichroism of Rhodopseudomonas acidophila light harvesting complexes based on roentgen structural data}; Pishchal'nikov RIu et al.; Absorption and circular dichroism spectra of two forms of the peripheral light-harvesting complex from photosynthetic purple bacteria Rhodopseudomonas acidophila were calculated . Calculations were carried out on the basis of exciton theory for circular aggregates of bacteriochlorophyll molecules and X-ray data for these forms of the complex . It was shown that theoretical spectra fit well experimental ones at the same values of excitation energy, homogeneous and inhomogeneous broadening, and bandwidth for all bacteriochlorophyll molecules of complexes . To approximate the circular dichroism spectra of complexes, it was necessary to change the orientations and the values of the moments of transition of Qy molecules relative to their orientation determined on the basis of X-ray structure analysis data. Prikl Biokhim Mikrobiol, 2003 Mar-Apr, 39(2), 173 - 9 {Identification of a new steroid transforming strain of mycobacteria as Mycobacterium neoaurum}; Voishvillo NE et al.; The ability to utilize sterols as a sole source of carbon was studied in 80 strains and consortia of hydrocarbon-oxidizing bacteria . One of the strains, which efficiently transformed both individual sterols and their mixtures, was identified as Mycobacterium neoaurum, based on the analysis of the sequence of the 16S rRNA gene. J Infect Dis, 2003 May 15, 187(10), 1646 - 53 Epub 2003 Apr 30. Phagocytosis of Bordetella pertussis incubated with convalescent serum; Mobberley-Schuman PS et al.; Convalescent serum samples were examined for the ability to promote phagocytosis of Bordetella pertussis by human neutrophils . One sample promoted phagocytosis, and 11 of the 51 samples caused a statistically significant reduction in phagocytosis, compared with that of bacteria not incubated with serum . Phagocytosis was influenced by interactions between antibodies that promoted phagocytosis and antibodies that inhibited phagocytosis . Adenylate cyclase toxin (ACT) has been shown to block phagocytosis by neutrophils . Antibodies to ACT were removed from the sample that promoted phagocytosis, by incubation with ACT-coated paramagnetic beads, and the depleted serum no longer enhanced phagocytosis . The adhesin filamentous hemagglutinin (FHA) has been shown to mediate attachment of B . pertussis to neutrophils in a way that promotes phagocytosis . Depletion of antibodies to FHA from samples that blocked phagocytosis improved phagocytosis, compared with the no-antibody control . These results suggest that antibodies to ACT can promote phagocytosis, whereas antibodies to FHA can counteract beneficial opsonins. Proc Natl Acad Sci U S A, 2003 May 13, 100(10), 5885 - 90 Epub 2003 Apr 29. The loss of the hemoglobin H2S-binding function in annelids from sulfide-free habitats reveals molecular adaptation driven by Darwinian positive selection; Bailly X et al.; The hemoglobin of the deep-sea hydrothermal vent vestimentiferan Riftia pachyptila (annelid) is able to bind toxic hydrogen sulfide (H(2)S) to free cysteine residues and to transport it to fuel endosymbiotic sulfide-oxidising bacteria . The cysteine residues are conserved key amino acids in annelid globins living in sulfide-rich environments, but are absent in annelid globins from sulfide-free environments . Synonymous and nonsynonymous substitution analysis from two different sets of orthologous annelid globin genes from sulfide rich and sulfide free environments have been performed to understand how the sulfide-binding function of hemoglobin appeared and has been maintained during the course of evolution . This study reveals that the sites occupied by free-cysteine residues in annelids living in sulfide-rich environments and occupied by other amino acids in annelids from sulfide-free environments, have undergone positive selection in annelids from sulfide-free environments . We assumed that the high reactivity of cysteine residues became a disadvantage when H(2)S disappeared because free cysteines without their natural ligand had the capacity to interact with other blood components, disturb homeostasis, reduce fitness and thus could have been counterselected . To our knowledge, we pointed out for the first time a case of function loss driven by molecular adaptation rather than genetic drift . If constraint relaxation (H(2)S disappearance) led to the loss of the sulfide-binding function in modern annelids from sulfide-free environments, our work suggests that adaptation to sulfide-rich environments is a plesiomorphic feature, and thus that the annelid ancestor could have emerged in a sulfide-rich environment. Br J Pharmacol, 2003 Apr, 138(8), 1495 - 504 Characterization of a fluorescent conjugate of the rabbit angiotensin AT(1) receptor; Fortin JP et al.; 1 . The rabbit AT(1) receptor (AT(1)R) for angiotensin II (A(II)) has been conjugated to the yellow fluorescent protein (YFP) in order to establish the pharmacological profile of such a fusion protein and to facilitate the study of ligand-induced regulation . 2 . A(II) bound AT(1)R-YFP (K(D) 8.1 nM in transiently transfected cells) and stimulated HEK 293 cells expressing the fusion protein at concentration ranges similar to the ones that stimulate the contraction of the isolated rabbit aorta . Antagonists found to be insurmountable in the latter assay (candesartan and EXP-3174 being the most extreme cases) were also insurmountable in the phospholipase A(2) assay applied to cells expressing AT(1)R-YFP, whereas losartan appeared to be surmountable in both assays . 3 . Cells expressing AT(1)R-YFP exhibited a membrane-associated fluorescence that was partly and reversibly translocated into intracellular structures upon A(II) stimulation (confocal microscopy); the nonpeptide antagonists were not active in this respect, but prevented the effect of the agonist . 4 . A(II) treatment increased the quantity of the fusion protein in cells, and phorbol 12-myristate 13-acetate (PMA) treatment even more so (immunoblot, confocal microscopy) but, unlike the agonist, the latter drug did not induce receptor endocytosis . A protein kinase C (PKC) inhibitor prevented the effect of either A(II) or PMA on AT(1)R-YFP abundance . 5 . The conjugate AT(1)R-YFP retains the pharmacological properties of the parent rabbit AT(1)R . Agonist-induced downregulation was not documented using this system; to the contrary, we have observed a PKC-mediated increased expression AT(1)R-YFP likely to be the result of a decreased breakdown rate of the fusion protein. J Clin Pathol, 2003 May, 56(5), 348 - 51 Helicobacter pylori water soluble surface proteins prime human neutrophils for enhanced production of reactive oxygen species and stimulate chemokine production; Shimoyama T et al.; BACKGROUNDS/AIMS: Chronic gastritis induced by Helicobacter pylori is characterised by considerable neutrophil infiltration into the gastric mucosa without mucosal invasion of bacteria . Bacteria have different characteristics with respect to their ability to stimulate human neutrophils to produce reactive oxygen species and chemokines . The aim of this study was to examine the effects of H pylori water extracts on the oxidative burst and chemokine production of human neutrophils . METHODS: Helicobacter pylori cells were extracted by harvesting into distilled water and centrifugation . Neutrophils were incubated with H pylori water extracts and the production of reactive oxygen species was measured using luminol dependent chemiluminescence (LmCL) . In addition, the concentrations of chemokines (interleukin 8 (IL-8), macrophage inflammatory protein 1-alpha (MIP1-alpha), and MIP1-beta) were measured by enzyme linked immunosorbent assay . Neutrophils were also stimulated by opsonised zymosan (OZ) after preincubation with H pylori water extracts . RESULTS: Helicobacter pylori water extracts alone induced only a weak oxidative burst but preincubation of neutrophils with water extracts dose dependently enhanced the LmCL response stimulated by OZ . Helicobacter pylori water extracts also stimulated neutrophil IL-8 production, although MIP-1beta production was only stimulated weakly, and MIP-1alpha was not stimulated at all . CONCLUSIONS: Helicobacter pylori products in water extracts may have a role in the activation and migration of neutrophils, which results in enhanced oxidative damage to gastric mucosa . These findings may explain the pathology of H pylori induced gastritis, in which there is little invasion of bacteria into the gastric mucosa. Biophys J, 2003 May, 84(5), 3353 - 63 Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching; Wachsmuth M et al.; Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells . For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP) . Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other . For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained . In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species . Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP . The cells show fluorescent nucleoli, and binding is transient . CP yields residence times for mTTF-I-EGFP of approximately 13 s. Prev Vet Med, 2003 May 30, 59(1-2), 95 - 102 A clinical trial of the effect of a mouth-rinse prepared with Lippia sidoides Cham essential oil in dogs with mild gingival disease; Girao VC et al.; We evaluated the effect of a mouth-rinse prepared using Lippia sidoides essential oil (EO) in dogs with marginal gingivitis . German Shepherd dogs were distributed in two groups: control (control mouth-rinse) and EO (EO mouth-rinse) . Both mouth-rinses were applied on the dogs' teeth every 2 days for 2 weeks . At day 0 and day 15, the scores for plaque-bacteria (P), calculus (C), gingivitis (G) and the inflammatory infiltrate (INF) were evaluated blindly . The results were analyzed by the Wilcoxon signed-rank and Mann-Whitney tests (P</=0.05) . P, C, G, and INF did not show any alteration in the control group, while in the EO group there were significant reductions in these scores. Neuron, 2003 Apr 24, 38(2), 225 - 35 Conformational rearrangements associated with the gating of the G protein-coupled potassium channel revealed by FRET microscopy; Riven I et al.; G protein-coupled potassium channels (GIRK/Kir3.x) are key determinants that translate inhibitory chemical neurotransmission into changes in cellular excitability . To understand the mechanism of channel activation by G proteins, it is necessary to define the structural rearrangements in the channel that result from interaction with Gbetagamma subunits . In this study we used a combination of fluorescence spectroscopy and through-the-objective total internal reflection microscopy to monitor the conformational rearrangements associated with the activation of GIRK channels in single intact cells . We detect activation-induced changes in FRET consistent with a rotation and expansion of the termini along the central axis of the channel . We propose that this rotation and expansion of the termini drives the channel to open by bending and possibly rotating the second transmembrane segment. Neuron, 2003 Apr 24, 38(2), 145 - 7 New turf for CFP/YFP FRET imaging of membrane signaling molecules; Evans J et al.; TIRF microscopy can be used in conjunction with CFP/YFP FRET to detect movements of the cytoplasmic tails of GIRK channels (Riven et al., this issue of Neuron) . This innovative combination of techniques allows molecular resolution of small motions underlying ion channel activation by G proteins and will likely find widespread use for study of membrane-associated molecules. Environ Monit Assess, 2003 Apr, 83(3), 229 - 53 The water quality of the Vrgorska Matica River; Stambuk-Giljanovic N; The article presents the results of investigations carried out on the 42 km long Vrgorska Matica River, which flows through the 15 km long Vrgorsko polje (polje = field) which covers an area of 3000 ha, and is at 24 m a.s.l., located in Southern Croatia . It covers the years 1997-2000 after this field had been reclaimed for agricultural use . The purpose of the investigations was to evaluate the influence of the Vrgorska Matica River which is part of the catchment area of the Trebizat River, on the water quality in Modro Oko Lake and Prud Spring, which are used for water supply and are located downstream of the Vrgorska Matica River on the right bank of the Neretva River . The water quality was evaluated by using the quality index based on the following nine parameters: temperature, mineralization, corrosion coefficient, K = (Cl + SO4)/HCO3, dissolved oxygen, BOD5, total N, protein N, total phosphorus and total coliform bacteria (100 mL)-1 (MPN coli (100 mL)-1) for which concentrations C95 are calculated . After completing the nine parameters the results of C95 were recorded and transferred to the score table to obtain the q-value . The q-value used is an attempt to quantify environmental factors which would otherwise be qualitative . For each parameter the q-value was multiplied by a weighting factor based upon the relative significance of the parameter . The nine resulting scores values were then added to arrive at an overall water quality index (sigmaS95) . According to this index the water can be classified into four categories . The first category, according to the Croatian Water Classification Act (Official Bulletin No . 77,1998), includes ground and surface waters used for drinking or in the food industry either in its natural state or after disinfection, and surface water used for raising high-quality species of fish, ranging from 85-100 scores; the second category includes water used in its natural state for swimming and recreation, sports or for other species of fish and the treated water used for drinking and other industrial purposes ranging between 70-85 scores . The third category includes water used in industry with no specific requests upon water quality and in agriculture ranging from 50-70 scores; the fourth category includes water used only after being treated in areas with water shortage which is less than 50 scores . According to results obtained by investigations, the water of the Vrgorska Matica River and Prud Spring falls into the second (II) category, while water from the Modro Oko Lake belongs to the first (I) category . This means that the Matica River water does not influence the water quality of the Modro Oko Lake . This lake water quality is influenced by the Rastocka Matica River from the Rastocko polje which is located upstream from the Vrgorska Matica River . This has been proved by dyeing tests. J Commun Dis, 2002 Mar, 34(1), 1 - 14 Management of disability in lymphatic filariasis--an update; Shenoy RK; The global lymphatic filariasis elimination programme incorporates disability management along with transmission control, to ensure 'a visible impact' on those who are already affected by the disease . The common manifestations of lymphatic filariasis like lymphoedema; elephantiasis and hydrocele result from irreversible damage caused to the lymphatics by the adult worms . Only palliative treatment in the form of physical methods and surgery is available for lymphoedema and elephantiasis . Hydrocele can be corrected by surgery . The most distressing aspect of lymphatic filariasis is the attacks of acute adenolymphangitis, which cause considerable short-term and also long-term disability by worsening the lymphoedema . Since each episode prevents the person from attending his work for several days, the economic loss is substantial . The precipitating cause of these attacks is secondary infection, the bacteria entering the tissues through 'entry lesions' in the skin . These episodes can very well be prevented by proper 'local-hygiene' of the affected limbs, which is a simple, effective, cheap and sustainable method that can be carried out even in the patient's house . These subjects and the providers of 'home care' should be trained in foot-hygiene programme, so that the message percolates to various levels in the affected communities, ultimately benefiting the patient. Zh Mikrobiol Epidemiol Immunobiol, 2001 Nov-Dec, (6 Suppl), 55 - 8 {New preparations for the rapid diagnostics of dangerous infectious diseases and their use in emergency situations}; Efremenko VI et al.; The use of new diagnostic preparations (magnetic immunosorbents, polyvalent fluorescent immunoglobulins and immunoperoxidase conjugates), as well as the combination of magnetic immunosorbents with the polymerase chain reaction, for the rapid diagnostics of dangerous infectious diseases has been shown to have good prospects . Information on their successful use for laboratory diagnostics in emergency situations is presented. World J Gastroenterol, 2003 May, 9(5), 1038 - 44 The mRNA expression patterns of tumor necrosis factor-alpha and TNFR-I in some vital organs after thermal injury; Fang WH et al.; AIM: To investigate changes of tumor necrosis factor-alpha (TNF-alpha) and TNFR-I expression in vital organs and their significance in the pathogenesis of multiple organ damage associated with endogenous endotoxin following major burns . METHODS: Wistar rats subjected to a 35 % full-thickness scald injury were sacrificed at 12 h, 24 h, 48 h, and 72 h postburn, respectively . Meanwhile, eight rats were taken as normal controls . Tissue samples from liver, spleen, kidney, lung and intestine were collected to assay tissue endotoxin levels and measure TNF-alpha and TNFR-I expression . In addition, blood samples were obtained for the determination of organ function parameters . RESULTS: Endotoxin levels in liver, spleen and lung increased markedly after thermal injury, with the highest level in liver . The gene expression of TNF-alpha in liver, lung and kidney was up-regulated after thermal injury, while the TNFR-I mRNA expression in liver, lung, kidney and intestine was shown decreased throughout the observation period . Thus, the mRNA expression ratio of TNF-alpha to TNFR-I was significantly increased postburn, particularly in pulmonary tissue (67-fold) . In addition, the significant correlations between the expression of TNFR-I or the expression ratio of TNF-alpha/TNFR mRNA in liver tissue and serum aspartate aminotransferase levels were noted (P<0.05-0.01) . Similar results were also obtained between pulmonary TNF-alpha mRNA expression and myeloperoxidase activities (P<0.01), whereas there was a highly negative correlation between levels of renal TNFR-I mRNA expression and serum creatinine . CONCLUSION: Burn injury could result in the translocation of gut-derived endotoxin that was mainly distributed in the liver, spleen and lung . The translocated endotoxin then made the expression of TNF-alpha and TNFR-I mRNA up-regulated and down-regulated respectively in various organs, which might be involved in the pathogenesis of multiple organ damage following burns. Nat Mater, 2003 Jun, 2(6), 391 - 5 Functionalized silicon membranes for selective bio-organism capture; Letant SE et al.; Membranes with various pore size, length, morphology and density have been synthesized from diverse materials for size-exclusion-based separation . An example is the sterilization of intravenous lines by exclusion of bacteria and viruses using polyvinylidene fluoride membranes with 0.1-microm-diameter pores . Chemically specific filtration has recently been addressed for small molecules . Nevertheless, specific bio-organism immobilization and detection remains a great technical challenge in many biomedical applications, such as decontamination or analysis of air and liquids such as drinking water and body fluids . To achieve this goal, materials with controlled pore diameter, length and surface chemistry are required . In this letter, we present the first functionalized silicon membranes and demonstrate their ability to selectively capture simulated bio-organisms . These extremely versatile and rigid devices open the door to a new class of materials that are able to recognize the external fingerprints of bio-organisms-such as size and outer membrane proteins-for specific capture and detection applications. Pancreas, 2003 May, 26(4), 357 - 62 The role of inducible nitric oxide synthase inhibitor, meropenem, and taurine in experimental acute necrotizing pancreatitis; Ozturk M et al.; INTRODUCTION: Translocation of bacteria from the gut is one of the most important factors in the development of septic complications and mortality in acute pancreatitis . AIMS: To investigate whether S-methylisothiourea (SMT), taurine (TAU), and meropenem (MER) could effect bacterial translocation and the course of acute necrotizing pancreatitis . METHODOLOGY: Seventy male Sprague-Dawley rats were studied . Rats were randomly allocated into seven groups . Acute pancreatitis was induced in group II (MER), group III (TAU), group IV (TAU + MER), group V (TAU + SMT), group VI (TAU + MER + SMT), and group VII (positive control) by retrograde injection of taurocholate into the common biliopancreatic duct . Group I rats (sham) received normal saline infusion into the common biliopancreatic duct as negative control . Rats were treated with drug combinations intraperitoneally for 48 hours after induction of pancreatitis . At the 48th hour of induction, all animals were killed, and specimens were collected . RESULTS: Bacterial translocation to peritoneum and pancreas in groups treated with MER were lower than in the other groups . Pancreatic tissue GSHpx and SOD levels were higher in all groups in comparison with levels in group VII . Pancreatic tissue MDA levels were also lower in all treatment groups except group II . The most favorable results were obtained in group VI (TAU + MER + SMT) . Also, the lowest pathologic score between the groups in which acute pancreatitis developed was obtained in group VI . CONCLUSIONS: Addition of TAU and SMT to the treatment protocol for acute pancreatitis seems to improve the pathologic score and oxidative stress parameters . Also, antibiotherapy with MER decreases the risk of bacterial translocation. J Genet, 2002 Dec, 81(3), 105 - 10 An orphan gyrB in the Mycobacterium smegmatis genome uncovered by comparative genomics; Jain P et al.; DNA gyrase is an essential topoisomerase found in all bacteria . It is encoded by gyrB and gyrA genes . These genes are organized differently in different bacteria . Direct comparison of Mycobacterium tuberculosis and Mycobacterium smegmatis genomes reveals presence of an additional gyrB in M . smegmatis flanked by novel genes . Analysis of the amino acid sequence of GyrB from different organisms suggests that the orphan GyrB in M . smegmatis may have an important cellular role. Mol Biol Evol, 2003 Jun, 20(6), 907 - 13 Epub 2003 Apr 25. Evidence for cocladogenesis between diverse dictyopteran lineages and their intracellular endosymbionts; Lo N et al.; Bacteria of the genus Blattabacterium are intracellular symbionts that reside in specialized cells of cockroaches and the termite Mastotermes darwiniensis . They appear to be obligate mutualists, and are transmitted vertically in the eggs . Such characteristics are expected to lead to equivalent phylogenies for host and symbiont, and we tested this hypothesis using recently accumulated data on relationships among termites and cockroaches and their Blattabacterium spp . Host and symbiont topologies were found to be highly similar, and various tests indicated that they were not statistically different . A close relationship between endosymbionts from termites and members of the wood-feeding cockroach genus Cryptocercus was found, supporting the hypothesis that the former evolved from subsocial, wood-dwelling cockroaches . The majority of the Blattabacterium spp . sequences appear to have undergone similar rates of evolution since their divergence from a common ancestor, and an estimate of this rate was determined based on early Cretaceous host fossils . The results support the idea that the stem group of modern cockroaches radiated sometime between the late Jurassic and early Cretaceous-not the Carboniferous, as has been suggested on the basis of roach-like fossils from this epoch. Mol Biol Evol, 2003 Jun, 20(6), 979 - 87 Epub 2003 Apr 25. Genome-wide survey for genes horizontally transferred from cellular organisms to baculoviruses; Hughes AL et al.; The phylogeny of 13 viral species in the genera Granulovirus and Nucleopolyhedrovirus (family Baculoviridae) was reconstructed on the basis of 22 conserved protein families shared by all species, and a comprehensive homology search and phylogenetic analysis of the complete genomes of these viruses was used to test for horizontal gene transfer from cellular organisms . Statistically significant evidence of horizontal transfer was found in the case of six protein families (DNA ligase, ribonucleotide reductase 1, SNF2 global transactivator, inhibitor of apoptosis, chitinase, and UDP-glucosyltransferase) . Three of these families are known to play key roles in the infection of insect hosts by these viruses . There was evidence that two of these (inhibitor of apoptosis and UDP-glucosyltransferase) were derived from the insect host . By contrast, the gene encoding chitinase in these viruses was evidently derived from a group of bacteria (the gamma subdivision of proteobacteria), which use chitinase to break down fungal chitins. Med Clin (Barc), 2003 Apr 12, 120(13), 494 - 5 {Kinetics of Bartonella spp . IgG antibodies in cat-scratch disease and in asymptomatic HIV-infected patients}; Garcia-Garcia JA et al.; BACKGROUND AND OBJECTIVE: Our goal was to investigate the kinetics of serum IgG antibodies against Bartonella spp . in patients with cat-scratch disease and in HIV-infected indiviuals . PATIENTS AND METHOD: Sequential determinations were made in seven patients with cat scratch disease and in seven HIV-infected individuals who were seropositive for this bacteria . RESULTS: In five patients from each group, serum antibodies fell under the positivity threshold within two years . Two HIV coinfected patients harbored Bartonella spp . DNA in bone marrow . CONCLUSIONS: These results suggest that the finding of a positive titer of serum IgG against Bartonella spp . is a marker of acute or recent infection. J Neurochem, 2003 May, 85(4), 1064 - 74 Differential subcellular localization of two dopamine D2 receptor isoforms in transfected NG108-15 cells; Takeuchi Y et al.; The dopamine D2 receptor (D2R) is target for antipsychotic drugs and associated with several neuropsychiatric disorders . D2R has a long third cytoplasmic loop and a short carboxyl-terminal cytoplasmic tail . It exists as two alternatively spliced isoforms, termed D2LR and D2SR, which differ in the presence and absence, respectively, of a 29 amino acid insert in the third cytoplasmic loop . To evaluate the differential roles of the two D2R isoforms, we transfected both isoforms into NG108-15 cells and observed their subcellular localization by a confocal laser scanning light microscope . D2SR was predominantly localized at the plasma membrane, whereas D2LR was mostly retained in the perinuclear region around the Golgi apparatus . Using a yeast two hybrid system with a mouse brain cDNA library and coimmunoprecipitation assay, we found that heart-type fatty acid binding protein (H-FABP) interacts with D2LR but not with D2SR . H-FABP is a cytosolic protein involved in binding and transport of fatty acids . Overexpressed H-FABP and endogenous H-FABP were colocalized with the intracellular D2LR in NG108-15 cells . Furthermore, in the rat striatum, H-FABP was detected in the D2R-expressing neurons . From these results, H-FABP is associated with D2LR, and may thereby modulate the subcellular localization and function of D2LR. IUBMB Life, 2003 Jan, 55(1), 23 - 7 FHA domains as phospho-threonine binding modules in cell signaling; Hammet A et al.; Forkhead-associated (FHA) domains are present in >200 diverse proteins in all phyla from bacteria to mammals and seem to be particularly prevalent in proteins with cell cycle control functions . Recent work from several laboratories has considerably improved our understanding of the structure and function of these domains that were virtually unknown a few years ago, and the first disease associations of FHA domains have now emerged . FHA domains form 11-stranded beta-sandwiches that contain some 100-180 amino acid residues with a high degree of sequence diversity . FHA domains act as phosphorylation-dependent protein-protein interaction modules that preferentially bind to phospho-threonine residues in their targets . Interestingly, point mutations in the human CHK2 gene that lead to single-residue amino acid substitutions in the FHA domain of this cell cycle checkpoint kinase have been found to cause a subset of cases of the Li-Fraumeni multi-cancer syndrome. J Antibiot (Tokyo), 2003 Feb, 56(2), 154 - 9 Action mechanism of a selective anti-cyanobacterial compound, argimicin A; Hibayashi R et al.; Argimicin A is a potent anti-cyanobacterial compound produced by one of algae-lysing bacteria, Sphingomonas sp . M-17 . Since the compound seemed to exhibit selective activities against cyanobacteria and such selectivity were considered to be quite rare, the mode of action of argimicin A was investigated . Argimicin A showed a unique delayed action, i.e., the cyanobacterial cell division continued until at least 36 hours treatment even though the decrement of oxygen evolution has been observed at 24 hours treatment . The compound is concluded to be a photosynthetic inhibitor which interrupts electron transport chain prior to photosystem II . From the preliminary fluorescent spectrum of argimicin A treated cyanobacterial cells, the site of action was speculated to be photo energy transfer from a cyanobacterial specific complex of accessory protein pigments, phycobilisome, to photosystem II. Science, 2003 Apr 25, 300(5619), 650 - 3 Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling; Wood ZA et al.; Eukaryotic 2-Cys peroxiredoxins (2-Cys Prxs) not only act as antioxidants, but also appear to regulate hydrogen peroxide-mediated signal transduction . We show that bacterial 2-Cys Prxs are much less sensitive to oxidative inactivation than are eukaryotic 2-Cys Prxs . By identifying two sequence motifs unique to the sensitive 2-Cys Prxs and comparing the crystal structure of a bacterial 2-Cys Prx at 2.2 angstrom resolution with other Prx structures, we define the structural origins of sensitivity . We suggest this adaptation allows 2-Cys Prxs to act as floodgates, keeping resting levels of hydrogen peroxide low, while permitting higher levels during signal transduction. DNA Repair (Amst), 2003 May 13, 2(5), 629, 637 - 8 Photoreactivation; Cleaver JE; Kelner and Dulbecco first reported in the 1940s and 1950s the reversal of ultraviolet damage in bacteria and phage by illumination with visible light . The first publications, reprinted here, represented the discovery of a widespread repair mechanism that was named "photoreactivation" (PHR), that directly reversed photoproducts to their individual pyrimidine components . Between them, these pioneers demonstrated that photoreactivation had a cellular basis, could be defined by wavelength optima indicating specific molecular photoreceptors, and had a widespread phyletic distribution except for its absence from placental mammals. Cell Microbiol, 2003 May, 5(5), 315 - 22 Expression and translocation of chlamydial protease during acute and persistent infection of the epithelial HEp-2 cells with Chlamydophila (Chlamydia) pneumoniae; Heuer D et al.; Chlamydial protease-like activity factor (CPAF) is secreted to the cytoplasm of the infected cells where it proteolytically cleaves eukaryotic transcription factor RFX5 . Here, we determined the localization pattern of CPAF during the course of an acute and persistent in vitro infection of the epithelial cell line HEp-2 with Chlamydophila pneumoniae strain VR1310 . Using immunoblotting, confocal microscopy and electron microscopy, we found CPAF in the inclusion lumen or associated with bacteria during the first 48 h of an acute infection . Seventy-two hours and later, CPAF was present predominantly in the cytoplasm of the infected cells . Translocation of CPAF into cytoplasm correlated in time with degradation of the transcription factor RFX5, as confirmed by immunoblotting . Interestingly, during the persistent infection induced by either IFN-gamma or iron limitation CPAF translocation to the cytoplasm was inhibited resulting in unaffected or only partially reduced levels of RFX5 . Based on presented findings, we propose that CPAF translocation to the cytoplasm is separated from its production . The translocation mechanism appears to be fully active during an acute infection; however, it is fully or partially inhibited during persistent infection induced by IFN-gamma or by iron limitation respectively . Consequently, this work demonstrates the importance of subcellular localization of CPAF for the characteristics of chlamydial acute and persistent infection in epithelial HEp-2 cells. J Anat, 2003 Mar, 202(Pt 3), 315 - 25 Histological and immunohistological investigation of the lymphoid tissue in normal and mycobacteria-affected specimens of the Rufous Hare-wallaby (Lagorchestes hirsutus); Young LJ et al.; The histology of the spleen, lymph nodes, Gut-associated lymphoid tissue (GALT) and Bronchus-associated lymphoid tissue (BALT) are described for samples collected opportunistically from healthy and mycobacteria-affected specimens of the endangered marsupial Lagorchestes hirsutus, the Rufous Hare-wallaby . The structural elements, organization and distribution of T and B lymphocytes determined by immunohistological techniques using species cross-reactive antibodies in the lymph nodes, spleen and GALT of this species demonstrated lymphoid cell distributions that were consistent with other marsupial and eutherian mammals . The tissues of animals identified as acid-fast positive displayed immunopathology consistent with the responses to intracellular bacteria displayed in some eutherian mammals and included the presence of focal lesions, giant cells in the lung and lymphoid aggregations situated adjacent to blood and airway vessels . This is the first study to describe the lymphoid tissue of this rare macropod species and the first to document the tissue bed response to mycobacteria. Avian Dis, 2003 Jan-Mar, 47(1), 234 - 9 Focal transmural necrotic tracheitis in commercial meat turkeys; Senties-Cue G et al.; This report describes an unusual presentation of severe focal necrotic tracheitis in a flock of 8-wk-old commercial turkeys . The flock was kept on a range that is located near a cotton field . The cotton field had been chemically defoliated 2 wk before the birds were submitted for necropsy . At necropsy, most of the birds had a 1-cm, yellow-white constricture in the upper third of the trachea at which the lumen was partially occluded by necrotic tissue . Microscopically, there was severe, transmural necrosis with an accumulation of inflammatory exudate in the tracheal lumen and numerous bacteria within the necrotic debris, mucosa, and lamina propria . Mixed bacteria were isolated from the trachea . No viruses were detected . Neither abnormal heavy metal concentrations in the liver nor paraquat in the respiratory tract were detected . The exact cause of this severe, necrotic tracheitis was not determined . Based on the clinical history and laboratory findings, it was concluded that a combination of a toxic irritant, possibly an aerosolized cotton defoliant, and bacterial infections were likely the cause of this lesion. Anal Chem, 2003 Apr 15, 75(8), 1924 - 30 Multiplexed liquid arrays for simultaneous detection of simulants of biological warfare agents; McBride MT et al.; Liquid array-based multiplexed immunoassays designed for rapid, sensitive, specific, and simultaneous detection of multiple simulants of biological warfare agents have been developed . In both blind and standard laboratory trials, we demonstrate the simultaneous detection of four simulant agents from a single sample . The challenge agents comprise broad classes of pathogens (virus, protein toxins, bacterial spores, vegetative cells) . Assay performance of each analyte was optimized, and dose-response curves and the limits of detection (LODs) for individual analytes are presented . Assay performance, including dynamic range, sensitivity, and LODs for liquid arrays and enzyme-linked immunosorbant assay were compared and are shown to be similar . Maximum assay sensitivity is obtained in approximately 1 h, and good sensitivity is achieved in as little as 30 min . Although the sample matrixes are very complex, even for highly multiplexed assays the samples do not exhibit evidence of nonspecific binding, demonstrating that the assays also have high specificity. AIDS Treat News . 2003 Mar 14;(389):8. Warning, counterfeit Procrit (epoetin alfa); Sequence analysis corresponding to the PPE and PE proteins in Mycobacterium tuberculosis and other genomes; School of Chemistry, University of Hyderabad, Hyderabad 500 046, IndiaAmino acid sequence analysis corresponding to the PPE proteins in H37Rv and CDC1551 strains of the Mycobacterium tuberculosis genomes resulted in the identification of a previously uncharacterized 225 amino acid-residue common region in 22 proteins . The pairwise sequence identities were as low as 18% . Conservation of amino acid residues was observed at fifteen positions that were distributed over the whole length of the region . The secondary structure corresponding to this region is predicted to be a mixture of a-helices and b-strands . Although the function is not known, proteins with this region specific to mycobacterial species may be associated with a common function . We further observed another group of 20 PPE proteins corresponding to the conserved C-terminal region comprising 44 amino acid residues with GFxGT and PxxPxxW sequence motifs . This region is preceded by a hydrophobic region, comprising 40-100 amino acid residues, that is flanked by charged amino acid residues . Identification of conserved regions described above may be useful to detect related proteins from other genomes and assist the design of suitable experiments to test their corresponding functions . Amino acid sequence analysis corresponding to the PE proteins resulted in the identification of tandem repeats comprising 41-43 amino acid residues in the C-terminal variable regions in two PE proteins (Rv0978 and Rv0980) . These correspond to the AB repeats that were first identified in some proteins of the Methanosarcina mazei genome, and were demonstrated as surface antigens . We observed the AB repeats also in several other proteins of hitherto uncharacterized function in Archaea and Bacteria genomes . Some of these proteins are also associated with another repeat called the C-repeat or the PKD-domain comprising 85 amino acid residues . The secondary structure corresponding to the AB repeat is predicted mainly as 4 b-strands . We suggest that proteins with AB repeats in Mycobacterium tuberculosis and other genomes may be associated as surface antigens . The M . leprae genome, however, does not contain either the AB or C-repeats and different proteins may therefore be recruited as surface antigens in the M . leprae genome compared to the M . tuberculosis genome. J Biol Chem, 2003 Jul 11, 278(28), 25481 - 9 Epub 2003 Apr 23. Functional characterization of human receptors for short chain fatty acids and their role in polymorphonuclear cell activation; Le Poul E et al.; Short chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are produced at high concentration by bacteria in the gut and subsequently released in the bloodstream . Basal acetate concentrations in the blood (about 100 microm) can further increase to millimolar concentrations following alcohol intake . It was known previously that SCFAs can activate leukocytes, particularly neutrophils . In the present work, we have identified two previously orphan G protein-coupled receptors, GPR41 and GPR43, as receptors for SCFAs . Propionate was the most potent agonist for both GPR41 and GPR43 . Acetate was more selective for GPR43, whereas butyrate and isobutyrate were more active on GPR41 . The two receptors were coupled to inositol 1,4,5-trisphosphate formation, intracellular Ca2+ release, ERK1/2 activation, and inhibition of cAMP accumulation . They exhibited, however, a differential coupling to G proteins; GPR41 coupled exclusively though the Pertussis toxin-sensitive Gi/o family, whereas GPR43 displayed a dual coupling through Gi/o and Pertussis toxin-insensitive Gq protein families . The broad expression profile of GPR41 in a number of tissues does not allow us to infer clear hypotheses regarding its biological functions . In contrast, the highly selective expression of GPR43 in leukocytes, particularly polymorphonuclear cells, suggests a role in the recruitment of these cell populations toward sites of bacterial infection . The pharmacology of GPR43 matches indeed the effects of SCFAs on neutrophils, in terms of intracellular Ca2+ release and chemotaxis . Such a neutrophil-specific SCFA receptor is potentially involved in the development of a variety of diseases characterized by either excessive or inefficient neutrophil recruitment and activation, such as inflammatory bowel diseases or alcoholism-associated immune depression . GPR43 might therefore constitute a target allowing us to modulate immune responses in these pathological situations. Sci Total Environ, 2003 May 20, 307(1-3), 19 - 33 Formation of alkylated aromatic acids in groundwater by anaerobic degradation of alkylbenzenes; Martus P et al.; Evidence for the presence of previously unreported alkylated aromatic acids in anaerobic groundwater samples from a jet fuel-contaminated site is presented . The redox conditions are dominated by sulfate reduction . A gas chromatography/mass spectrometry screening showed a composition of aromatic hydrocarbons ranging from BTEX (C(0)-C(2)-substituted benzenes) up to C(7)-benzenes . Known metabolites like methylbenzoates (C(1)-BA), C(2)-BA, C(3)-BA as well as methylbenzylsuccinates (C(1)-BSA) were detected . Additionally, previously unreported metabolites of higher alkylated benzenes have been identified in the extracts: those are C(2)-BSA to C(5)-BSA as well as C(4)-BA to C(6)-BA, which were detected in the microg/l-concentration range . The formation of C(1)- and C(2)-BSA as well as the C(1)- and C(2)-BA can be linked to structurally corresponding parent contaminants like the xylene isomers and the trimethylbenzene isomers . The C(3)-BSA and C(3)-BA represent metabolites of C(4)-benzenes . The occurrence of C(4)-C(5)-BSA and C(4)-C(6)-BA is attributed to C(5)-C(7)-benzenes acting as parent compounds . The concentration of total aromatic contaminants decreased along the plume centre line . In contrast, benzoates and benzylsuccinates (BA and BSA) showed constant concentrations over a distance of 84 m . BA concentrations were up to 162 times higher compared to BSA, as indicated by the ratio f(BA/BSA) . A pronounced transient behaviour of BSA or an overall persistent behaviour of BA can explain this . Hence, along the plume centre line, f(BA/BSA) was constant over a distance of 128 m . The degradation products detected in a monitoring well are not necessarily tied to contaminants detected in that well . Therefore, the metabolite plume is partly located in front of the contaminant plume, but does not significantly migrate downstream the plume toe. Biochem Biophys Res Commun, 2003 May 2, 304(2), 217 - 22 Measuring dynamics of caspase-8 activation in a single living HeLa cell during TNFalpha-induced apoptosis; Luo KQ et al.; In this study, we reported the first measurement of the dynamics of activation of caspase-8 in a single living cell . This measurement was conducted using a specially developed molecular sensor based on the FRET (fluorescence resonance energy transfer) technique . This sensor was constructed by fusing a CFP (cyan fluorescent protein) and a YFP (yellow fluorescent protein) with a linker containing a tandem caspase-8-specific cleavage site . The change of the FRET ratio upon cleavage was larger than 4-fold . Using this sensor, we found that during TNFalpha-induced apoptosis, the activation of caspase-8 was a slower process than that of caspase-3, and it was initiated much earlier than the caspase-3 activation . Inhibition of caspase-9 delayed the full activation of caspase-3 but did not affect the dynamics of caspase-8 . Results of these single-cell measurements suggested that caspase-3 was activated by caspase-8 through two parallel pathways during TNFalpha-induced apoptosis in HeLa cells. Trends Genet, 2003 May, 19(5), 269 - 77 The methyl-CpG binding domain and the evolving role of DNA methylation in animals; Hendrich B et al.; DNA methylation occurs in bacteria, fungi, plants and animals, however its role varies widely among different organisms . Even within animal genomes, methylation patterns vary substantially from undetectable in nematodes, to global methylation in vertebrate genomes . The number and variety of proteins containing methyl-CpG binding domains (MBDs) that are encoded in animal genomes also varies, with a general correlation between the extent of genomic methylation and the number of MBD proteins . We describe here the evolution of the MBD proteins and argue that the vertebrate MBD complement evolved to exploit the benefits and protect against the dangers of a globally methylated genome. Med Hypotheses, 2003 May, 60(5), 711 - 5 What is, mutatis mutandis, the sequence of plasmid DNAs used in gene therapy? Ratel D, Wion D. Mutation is a fundamental biological process occurring in each living organism . Plasmid DNA which is used in gene therapy protocols or DNA vaccination passes through two different living cells which are, respectively, the producing cell (bacterial) and the target cell (eukaryotic) . Hence, modifications in the nucleotide sequence of plasmids are likely to occur both in bacteria during the amplification step of plasmid DNA and in eukaryotic cells following gene transfer . In addition to these biological modifications resulting from the physical passage of the plasmid into two different living organisms, an additional source of sequence alteration resides in our mode of representation of the nucleotide sequence of plasmid DNA which uses a four letters code, whereas, bacterial DNA is made of six different nucleosides . Indeed, the therapeutic DNA paradigm seems to have neglected the qualitative importance of these DNA sequence alterations . In this review we discuss the importance and the role of these DNA sequence modifications in the context of non-viral gene therapy approaches. J Periodontol, 2003 Mar, 74(3), 385 - 90 Clinical and histologic aspects of dental implants removed due to mobility; Piattelli A et al.; BACKGROUND: Implant failures are rare but do occur . Mechanical factors are certainly important in implant failures; so are biologic, iatrogenic, and functional factors . Mobility is the cardinal sign of implant failure . Three major etiologic factors have been suggested for implant failures: infection, impaired healing, and overload . The aim of the present study was to histologically and histochemically evaluate the fibrous connective tissue found around failed dental implants removed due to mobility . METHODS: In a 5-year period, 51 root-form implants were retrieved because of mobility and underwent histological examination . Thin ground sections were obtained from each implant . RESULTS: Almost all implants had been inserted in posterior (premolar-molar) regions of both jaws . In all specimens, there was the presence of a 600 to 1,100 microm thick connective tissue between implants and surrounding bone . In every case, the surrounding bone was compact and highly mineralized, with well-structured Haversian canals and few areas of remodeling . No bacteria were found in the most coronal portion of the implants . A scarce inflammatory cell infiltrate was present in the connective tissue of some specimens . In about 10% of specimens, the epithelium tended to surround the perimeter of the implant . CONCLUSION: Our histological results are consistent with the hypothesis that late failures of osseointegrated implants can be caused by a combination of poor bone quality, mechanical trauma to bone, and overloading forces. Sci STKE . 2003 Apr 22;2003(179):RE7. PDZ domain proteins: plug and play! Nourry C, Grant SG, Borg JP. Protein-protein interactions are key elements in building functional protein complexes . Among the plethora of domains identified during the last 10 years, PDZ domains are one of the most commonly found protein-protein interaction domains in organisms from bacteria to humans . Although they may be the sole protein interaction domain within a cytoplasmic protein, they are most often found in combination with other protein interaction domains (for instance, SH3, PTB, WW) participating in complexes that facilitate signaling or determine the localization of receptors . Diversity of PDZ-containing protein function is provided by the large number of PDZ proteins that Mother Nature has distributed in the genome and implicates this protein family in the wiring of a huge number of molecules in molecular networks from the plasma membrane to the nucleus . Although at first sight their binding specificity appeared rather monotonous, involving only binding to the carboxyl-terminus of various proteins, it is now recognized that PDZ domains interact with greater versatility through PDZ-PDZ domain interaction; they bind to internal peptide sequences and even to lipids . Furthermore, PDZ domain-mediated interactions can sometimes be modulated in a dynamic way through target phosphorylation . In this review, we attempt to describe the structural basis of PDZ domain recognition and to give some functional insights into their role in the scaffolding of protein complexes implicated in normal and pathological biological processes. Antimicrob Agents Chemother, 2003 May, 47(5), 1503 - 8 Prevention of human rhinovirus infection by multivalent fab molecules directed against ICAM-1; Charles CH et al.; We have developed a technology for improving avidity by making bivalent, trivalent, or tetravalent recombinant polypeptides . We designed tripartite proteins consisting of the Fab fragment of an antibody fused with a hinge derived from human immunoglobulin D that was further linked to polymerization domains derived from human coiled-coil proteins . We report here on the application of this method with a Fab domain directed against the major human rhinovirus receptor, intercellular adhesion molecule 1 (ICAM-1) . Multivalent anti-ICAM-1 molecules were produced in bacteria and purified as soluble preassembled homogeneous proteins at high yield . These proteins successfully blocked rhinovirus infection in vitro, with the efficiency increasing from monomer to dimer, trimer, and tetramer . The diminished dissociation rate of these multivalent antibodies and their improved efficacy in preventing rhinovirus infection provide a foundation for producing prophylactic and therapeutic molecules against human rhinovirus, the causative agent of the majority of common colds. Eur J Biochem, 2003 May, 270(9), 1900 - 15 Open questions in ferredoxin-NADP+ reductase catalytic mechanism; Carrillo N et al.; Ferredoxin (flavodoxin)-NADP(H) reductases (FNR) are ubiquitous flavoenzymes that deliver NADPH or low potential one-electron donors (ferredoxin, flavodoxin) to redox-based metabolisms in plastids, mitochondria and bacteria . The plant-type reductase is also the basic prototype for one of the major families of flavin-containing electron transferases that display common functional and structural properties . Many aspects of FNR biochemistry have been extensively characterized in recent years using a combination of site-directed mutagenesis, steady-state and transient kinetic experiments, spectroscopy and X-ray crystallography . Despite these considerable advances, various key features in the enzymology of these important reductases remain yet to be explained in molecular terms . This article reviews the current status of these open questions . Measurements of electron transfer rates and binding equilibria indicate that NADP(H) and ferredoxin interactions with FNR result in a reciprocal decrease of affinity, and that this induced-fit step is a mandatory requisite for catalytic turnover . However, the expected conformational movements are not apparent in the reported atomic structures of these flavoenzymes in the free state or in complex with their substrates . The overall reaction catalysed by FNR is freely reversible, but the pathways leading to NADP+ or ferredoxin reduction proceed through entirely different kinetic mechanisms . Also, the reductases isolated from various sources undergo inactivating denaturation on exposure to NADPH and other electron donors that reduce the FAD prosthetic group, a phenomenon that might have profound consequences for FNR function in vivo . The mechanisms underlying this reductive inhibition are so far unknown . Finally, we provide here a rationale to interpret FNR evolution in terms of catalytic efficiency . Using the formalism of the Albery-Knowles theory, we identified which parameter(s) have to be modified to make these reductases even more proficient under a variety of conditions, natural or artificial . Flavoenzymes with FNR activity catalyse a number of reactions with potential importance for biotechnological processes, so that modification of their catalytic competence is relevant on both scientific and technical grounds. Immunology, 2003 May, 109(1), 127 - 36 TRAF6-NF-kappaB pathway is essential for interleukin-1-induced TLR2 expression and its functional response to TLR2 ligand in murine hepatocytes; Matsumura T et al.; We have previously reported that the expressions of TLR2 and TLR4 mRNA are differentially regulated in mouse liver and in the parenchymal cells . In the present study, we investigated the mechanism of the up-regulatory effects of interleukin-1alpha (IL-1alpha), tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), or bacterial lipoprotein (BLP) on TLR2 mRNA expression in primary cultured murine hepatocytes . Although TLR2 mRNA stability was not affected, these treatments enhanced NF-kappaB activity and TLR2 gene transcription simultaneously . The up-regulation of TLR2 transcription in response to these reagents was completely inhibited by blocking the NF-kappaB activation pathway, demonstrating a pivotal role of NF-kappaB activation in the regulation of hepatocyte TLR2 transcription . The expression of TLR2 protein by hepatocytes was also remarkably up-regulated by IL-1alpha and, to a lesser extent, by TNF-alpha as well, but not by LPS or BLP . In addition, pretreatment of mice with IL-1alpha markedly increased the BLP (a ligand for TLR2)-induced serum level of serum amyloid A (SAA), an acute-phase protein predominantly produced by hepatocytes, indicating that IL-1alpha may also up-regulate functional TLR2 in vivo . These results demonstrate that IL-1alpha, through activating the TRAF6-NF-kappaB pathway, serves as the most potent inducer for TLR2 up-regulation, and plays an important role in the regulation of hepatocyte functions by augmenting the hepatocyte response to bacteria or bacterial products. J Environ Qual, 2003 Mar-Apr, 32(2), 406 - 16 Odor and gas release from anaerobic treatment lagoons for swine manure; Lim TT et al.; Odor and gas release from anaerobic lagoons for treating swine waste affect air quality in neighboring communities but rates of release are not well documented . A buoyant convective flux chamber (BCFC) was used to determine the effect of lagoon loading rate on measured odor and gas releases from two primary lagoons at a simulated wind speed of 1.0 m s(-1) . Concentrations of ammonia (NH3), hydrogen sulfide (H2S), carbon dioxide (CO2), sulfur dioxide (SO2), and nitric oxide (NO) in 50-L air samples were measured . A panel of human subjects, whose sensitivity was verified with a certified reference odorant, evaluated odor concentration, intensity, and hedonic tone . Geometric mean odor concentrations of BCFC inlet and outlet samples and of downwind berm samples were 168 +/- 44 (mean +/- 95% confidence interval), 262 +/- 60, and 114 +/- 38 OU(E) m(-3) (OU(E), European odor unit, equivalent to 123 microg n-butanol), respectively . The overall geometric mean odor release was 2.3 +/- 1.5 OU(E) s(-1) m(-2) (1.5 +/- 0.9 OU s(-1) m(-2)) . The live mass specific geometric mean odor release was 13.5 OU(E) s(-1) AU(-1) (animal unit = 500 kg live body mass) . Overall mean NH3, H2S, CO2 and SO2 releases were 101 +/- 24, 5.7 +/- 2.0, 852 +/- 307, and 0.5 +/- 0.4 microg s(-1) m(-2), respectively . Nitric oxide was not detected . Odor concentrations were directly proportional to H2S and CO2 concentrations and odor intensity, and inversely proportional to hedonic tone and SO2 concentration (P < 0.05) . Releases of NH3, H2S, and CO2 were directly proportional (P < 0.05) to volatile solids loading rate (VSLR). J Air Waste Manag Assoc, 2003 Apr, 53(4), 417 - 24 Sulfur and carbon cycling in a flue gas desulfurization sludge disposal site; Eastoe C et al.; Products of a power plant flue gas desulfurization scrubber are discharged into a pond as sludge consisting of calcite (initial delta13C 3.2-3.8 per thousand), gypsum (initial delta34S 7.6-8.6 per thousand), and aqueous solution . Reducing conditions exist below a boundary that appears to move vertically as a function of changes in pond water level . Under reducing conditions, bacteria partially reduce aqueous sulfate to low-delta34S sulfide, consuming organic carbon and generating low-delta13C bicarbonate . Under oxidizing conditions, sulfide is converted to sulfate, leading to calcite dissolution, gypsum precipitation, and isotopic re-equilibration of remaining calcite with dissolved bicarbonate near the pond surface . The gypsum has delta34S near 6 per thousand, and calcite has delta13C as low as -1.7 per thousand; the changes from initial values correspond to predictions based on isotopic balance and reaction stoichiometry . The pond largely contains the products of bacterial reduction . After the pond is abandoned, these products may adversely affect attempts to revegetate the site . Future bacterial reduction may be best controlled by dewatering and limiting the supply of organic matter in percolating surface water. J Occup Environ Med, 2003 Apr, 45(4), 385 - 92 Occupational lung disease related to cytophaga endotoxin exposure in a nylon plant; Nordness ME et al.; Workers at a nylon plant developed pulmonary disease with systemic symptoms . Differentiating between humidifier fever and hypersensitivity pneumonitis (HP) is challenging . Cytophaga, an endotoxin-producing bacteria, was isolated from the plant air-conditioning system . A number of workers had systemic and pulmonary symptoms . Precipitins to Cytophaga endotoxin were identified . Several workers underwent lung biopsies demonstrating HP . Inhalation challenges with purified Cytophaga endotoxin were performed on three pairs of subjects: group 1, or employees with clinical features and biopsy consistent with HP; group 2, asymptomatic exposed workers with precipitins; and group 3, nonexposed healthy individuals . All subjects had fever and leukocytosis after inhalation challenge . Acute and/or late pulmonary function changes occurred in groups 1 and 2 . Group 3 only had acute and transient pulmonary function changes . Cytophaga bacterial endotoxin is capable of inducing HP as well as humidifier fever. J Mol Biol, 2003 May 2, 328(3), 669 - 81 The X-ray crystal structure of human beta-hexosaminidase B provides new insights into Sandhoff disease; Maier T et al.; Human lysosomal beta-hexosaminidases are dimeric enzymes composed of alpha and beta-chains, encoded by the genes HEXA and HEXB . They occur in three isoforms, the homodimeric hexosaminidases B (betabeta) and S (alphaalpha), and the heterodimeric hexosaminidase A (alphabeta), where dimerization is required for catalytic activity . Allelic variations in the HEXA and HEXB genes cause the fatal inborn errors of metabolism Tay-Sachs disease and Sandhoff disease, respectively . Here, we present the crystal structure of a complex of human beta-hexosaminidase B with a transition state analogue inhibitor at 2.3A resolution (pdb 1o7a) . On the basis of this structure and previous studies on related enzymes, a retaining double-displacement mechanism for glycosyl hydrolysis by beta-hexosaminidase B is proposed . In the dimer structure, which is derived from an analysis of crystal packing, most of the mutations causing late-onset Sandhoff disease reside near the dimer interface and are proposed to interfere with correct dimer formation . The structure reported here is a valid template also for the dimeric structures of beta-hexosaminidase A and S. Burns, 2003 May, 29(3), 280 - 3 Using a plastic sheet to prevent the risk of contamination of the burn wound during the shower; Akin S et al.; The shower technique for hydrotherapy was used in the treatment of the burn patients in our burn center . Before showering, the shower trolley (stretcher) was draped with a sterilized disposable plastic sheet to decrease the potential for seeding surface bacteria to open burn wounds . The patients were washed on the draped shower trolley . Samples for culture were taken from the surface of the shower trolley (stretcher) before it was draped with the sterilized disposable plastic sheet following the treatment of a patient . The samples were taken once a week for the first 6 months and only once a month for the last 42 months . At the period of 48 months (4 years), a total of 67 samples were obtained . Results of cultures of all the samples were negative . No patients were contaminated from the surface of the shower trolley during the shower . The use of the sterilized disposable plastic sheet proved to be a most effective procedure to decrease the risk of contamination between patients during the shower. Cell . 2003 Apr 18;113(2):141. Global phage diversity; Rohwer F; Ten new mycobacteriophage genomes presented by show that most phage diversity remains uncharacterized . Extrapolation suggests that less than 0.0002% of the global phage metagenome has been sampled . The new genomes also contain a number of potential virulence factors that may be important in pathogenesis. Dent Update, 2003 Mar, 30(2), 70 - 4, 76 The role of lasers in dentistry: present and future; Pearson GJ et al.; Lasers have been used for hard tissue cutting in dentistry for a number of years . The quality of the cavity preparation and the surface finish achievable is variable and is dependent on operating wavelength . The collateral damage, which may be produced at differing wavelengths, is quite marked . Lasers in current form are now able to remove tissue in bulk at a similar rate to conventional methods such as bur and turbine handpiece . Some lasers may, however, provide precision cutting, which may be developed further in the future . Alternative uses of laser light are potentially more beneficial in the shorter term . The use of diode lasers as a means of activating a photosensitizer to carry out photo-activated disinfection appears to be beneficial. Int J Cancer, 2003 Jun 20, 105(3), 291 - 9 Marine pharmacology in 2000: antitumor and cytotoxic compounds; Mayer AM et al.; During 2000, marine antitumor pharmacology research aimed at the discovery of novel antitumor agents was published in 85 peer-reviewed articles . The purpose of this article is to present a structured review of the antitumor and cytotoxic properties of 143 marine natural products, many of them novel compounds that belong to diverse structural classes, including polyketides, terpenes, steroids and peptides . The organisms yielding these bioactive compounds comprised a taxonomically diverse group of marine invertebrate animals, algae, fungi and bacteria . Antitumor pharmacological studies were conducted with 19 marine natural products in a number of experimental and clinical models that defined or further characterized their mechanisms of action . Potentially promising in vitro cytotoxicity data generated with murine and human tumor cell lines were reported for 124 novel marine chemicals with as yet undetermined mechanisms of action . Noteworthy is the fact that marine anticancer research clearly remains a multinational effort, involving researchers from Austria, Australia, Brazil, Canada, England, France, Germany, Greece, Indonesia, Italy, Japan, New Zealand, Russia, Spain, South Korea, Switzerland, Taiwan, the Netherlands and the United States . Finally, this 2000 overview of the marine pharmacology literature highlights the fact that the discovery of novel marine antitumor agents continued at the same high level of research activity as during 1998 and 1999 . Microb Ecol, 2003 May, 45(4), 433 - 43 Epub 2003 Apr 22. Consumption of dissolved amino acids and carbohydrates by limnetic bacterioplankton according to molecular weight fractions and proportions bound to humic matter; Rosenstock B et al.; We investigated the bacterial decomposition of dissolved amino acids (DAA) and carbohydrates (DCHO) bound to humic substances and in the nonhumic fraction, and of low (< 3 KDA) AND HIGH (> 3 kDa) molecular weight (MW) . Experiments were conducted in mesotrophic Lake Constance, Germany, in October and November 1997 and June 1998 during periods of low phytoplankton biomass . Dilution cultures, inoculated with bacterioplankton of the < 1 mm size fraction from 3 m, were run over 5 to 8 days to determine bacterial consumption of the various fractions of DAA and DCHO . In all experiments consumption rates of DCHO were higher than that of DAA . On average, 70% of the DAA and DCHO consumed were associated with the humic fraction, which, however, also consisted of a substantial recalcitrant component . In contrast, there was no preferential consumption of DAA and DCHO in either of the two MW fractions . Overall, we found a highly significant positive correlation between the consumption of the various fractions of DAA and DCHO and their initial concentrations . The consumed DAA, i.e, their labile pool, was dominated by serine and glutamate and that of DCHO by arabinose . In contrast, the residual DAA, i.e., the recalcitrant pool, comprised highest proportions of glycine + threonine and that of DCHO of mannose + xylose . These results indicate that the bacterial consumption of DAA and DCHO was mainly a function of their concentrations, irrespective of the molecular weight and whether they are bound to humic substances or not and despite the fact that they consist of a more labile and a more refractory pool. Infect Immun, 2003 May, 71(5), 2976 - 80 Glycoconjugate binding of gastric and enterohepatic Helicobacter spp; Hynes SO et al.; Helicobacter pylori is able to utilize several lectin-like, protein-carbohydrate interactions for binding to mucins, cell surfaces, and extracellular matrix proteins . As determined by hemagglutination assays and binding of radiolabeled bacteria to glycosphingolipids on thin-layer chromatograms, strains of gastric helicobacters and enterohepatic helicobacters, including Helicobacter canis, Helicobacter hepaticus, and Helicobacter bilis, also demonstrated evidence for the presence of lectin-hemagglutinin adhesins . In addition, in H . hepaticus and H . bilis, binding may be sialic acid dependent . The presence or absence and differences in the levels of activity of lectin adhesins may reflect the species' ecological niche. Infect Immun, 2003 May, 71(5), 2591 - 7 Unexpected similarities between Bordetella avium and other pathogenic Bordetellae; Spears PA et al.; Bordetella avium causes an upper respiratory tract disease (bordetellosis) in avian species . Commercially raised turkeys are particularly susceptible . Like other pathogenic members of the genus Bordetella (B . pertussis and B . bronchiseptica) that infect mammals, B . avium binds preferentially to ciliated tracheal epithelial cells and produces similar signs of disease . These similarities prompted us to study bordetellosis in turkeys as a possible nonmammalian model for whooping cough, the exclusively human childhood disease caused by B . pertussis . One impediment to accepting such a host-pathogen model as relevant to the human situation is evidence suggesting that B . avium does not express a number of the factors known to be associated with virulence in the other two Bordetella species . Nevertheless, with signature-tagged mutagenesis, four avirulent mutants that had lesions in genes orthologous to those associated with virulence in B . pertussis and B . bronchiseptica (bvgS, fhaB, fhaC, and fimC) were identified . None of the four B . avium genes had been previously identified as encoding factors associated with virulence, and three of the insertions (in fhaB, bvgS, and fimC) were in genes or gene clusters inferred as being absent or incomplete in B . avium, based upon the lack of DNA sequence similarities in hybridization studies and/or the lack of immunological cross-reactivity of the putative products . We further found that the genotypic arrangements of most of the B . avium orthologues were very similar in all three Bordetella species . In vitro tests, including hemagglutination, tracheal ring binding, and serum sensitivity, helped further define the phenotypes conferred by the mutations . Our findings strengthen the connection between the causative agents and the pathogenesis of bordetellosis in all hosts and may help explain the striking similarities of the histopathologic characteristics of this upper airway disease in avian and mammalian species. Infect Immun, 2003 May, 71(5), 2555 - 62 Temporal expression of type III secretion genes of Chlamydia pneumoniae; Slepenkin A et al.; Chlamydia pneumoniae has been shown to possess at least 13 genes that are homologous with other known type III secretion (TTS) systems . Upon infection of HEp-2 cells with C . pneumoniae, the expression of these genes was followed by reverse transcriptase PCR throughout the developmental cycle of this obligate intracellular pathogen . In addition, expression was analyzed when C . pneumoniae was grown in the presence of human gamma interferon (IFN-gamma) . The groEL-1, ompA, and omcB genes were used as markers for the early, middle, and late stages of the developmental cycle, respectively, and the inhibition of expression of the fstK gene was used as a marker for the effect of IFN-gamma on the maturation of C . pneumoniae . In the absence of IFN-gamma, the TTS genes were expressed as follows: early stage (1.5 to 8 h), yscC, yscS, yscL, yscJ and lcrH-2; middle stage (by 12 to 18 h), lcrD, yscN, and yscR; and late stage (by 24 h), lcrE, sycE, lcrH-1, and yscT . Of the genes expressed early, the lcrH-2 gene was detected the earliest, at 1.5 h . Expression of the yscU gene was not detected at any of the time points examined . Under the influence of IFN-gamma, the cluster of TTS genes that were normally not expressed until the middle to late stages of the developmental cycle, namely, lcrD, lcrE, and sycE, as well as lcrH-1, were down-regulated, and expression could not be detected up to 48 h . In contrast, the expression of the other TTS genes appeared to be unchanged in the presence of IFN-gamma . The lcrH-1 and lcrH-2 genes differed from one another in both their temporal expression and response to IFN-gamma . In other TTS systems, these genes code for proteins that function in regulation of effector protein synthesis as well as serve as chaperones for proteins that provide for the translocation of the effector proteins into the host cell . In summary, the expression pattern of the TTS genes of C . pneumoniae examined suggests that they are temporally regulated throughout the developmental cycle . Furthermore, paralleling the inhibition of the maturation of the reticulate body to the elementary body, TTS genes expressed in the later stages of the cycle appear to be down-regulated when the organism is grown in the presence of IFN-gamma. Infect Immun, 2003 May, 71(5), 2534 - 41 Development of an interleukin-12-deficient mouse model that is permissive for colonization by a motile KE26695 strain of Helicobacter pylori; Hoffman PS et al.; The identification of genes associated with colonization and persistence of Helicobacter pylori in the gastric mucosa has been limited by the lack of robust animal models that support infection by strains whose genomes have been completely sequenced . Here we report that an interleukin-12 (IL-12)-deficient mouse (IL-12(-/-) p40 subunit knockout in C57BL/6 mouse) is permissive for infection by a motile variant (KE88-3887) of The Institute For Genomic Research-sequenced strain (KE26695) of H . pylori . The IL-12-deficient mouse was also more permissive for colonization by the mouse-colonizing Sydney 1 strain of H . pylori than were wild-type C57BL/6 mice . Differences in colonization efficiency were demonstrated by mouse challenge with SS1 strains containing loss-of-function mutations in two genes (hspR and hrcA), whose products negatively regulate several heat shock genes . At 5 weeks postinfection, double-knockout mutants (SS1 hspR hrcA) efficiently colonized IL-12-deficient mice (5 of 5 animals compared to 4 of 10 for C57BL6 mice) and bacterial counts were higher in stomachs of IL-12-deficient mice (10(6) versus 10(5) CFU/g of stomach, respectively) . IL-12-deficient mice were efficiently colonized by KE88-3887 (29 of 30), but not by nonmotile KE26695, and bacterial numbers (10(4) to 10(5) CFU/g of stomach) were unchanged over an 8-week period postinfection . In contrast, C57BL/6 mice were inefficiently colonized by KE88-3887 (8 of 20 animals with bacterial loads at the limit of detection, approximately 10(3) CFU/g), and infection did not persist much beyond 5 weeks . Cytokine responses (tumor necrosis factor alpha and gamma interferon), pathology, and antral-predominant infection were indistinguishable between IL-12-deficient and C57BL/6 mice . The increased permissiveness of the IL-12-deficient mouse for infection with H . pylori should facilitate whole-genome-based strategies to study genes associated with virulence and immune modulation. Infect Immun, 2003 May, 71(5), 2350 - 5 Failure of surface ring mutant strains of Helicobacter mustelae to persistently infect the ferret stomach; Patterson MM et al.; Helicobacter mustelae, the gastric pathogen of ferrets, produces an array of surface ring structures which have not been described for any other member of the genus Helicobacter, including H . pylori . The unique ring structures are composed of a protein named Hsr . To investigate whether the Hsr rings are important for colonization of the ferret stomach, ferrets specific pathogen free for H . mustelae were inoculated with an Hsr-deficient mutant strain or the wild-type H . mustelae strain . Quantitative cultures from antral biopsy specimens obtained at 3, 6, and 9 weeks postinoculation demonstrated no significant difference in the levels of bacteria in the ferrets that received the Hsr-negative strain and the ferrets infected with the parent strain . However, when the ferrets were biopsied at 12 and 15 weeks and necropsied at 18 weeks after infection, the levels of bacteria of the Hsr-negative strain in the stomach antrum were significantly reduced . This decline contrasted the robust antral colonization by the wild-type strain . The Hsr-negative strain did not efficiently colonize the gastric body of the study ferrets . Histological examination at 18 weeks postinoculation revealed minimal gastric inflammation in the animals that received the mutant H . mustelae strain, a finding consistent with its waning infection status, whereas lesions characteristic of helicobacter infection were present in ferrets infected with the wild-type strain . Scant colonization by the Hsr-negative H . mustelae strain at the end of the 18-week study, despite initial successful colonization, indicates an inability of the mutant to persist, perhaps due to a specific host response. Proc Natl Acad Sci U S A, 2003 Apr 29, 100(9), 5136 - 41 Epub 2003 Apr 17. On schemes of combinatorial transcription logic; Buchler NE et al.; Cells receive a wide variety of cellular and environmental signals, which are often processed combinatorially to generate specific genetic responses . Here we explore theoretically the potentials and limitations of combinatorial signal integration at the level of cis-regulatory transcription control . Our analysis suggests that many complex transcription-control functions of the type encountered in higher eukaryotes are already implementable within the much simpler bacterial transcription system . Using a quantitative model of bacterial transcription and invoking only specific protein-DNA interaction and weak glue-like interaction between regulatory proteins, we show explicit schemes to implement regulatory logic functions of increasing complexity by appropriately selecting the strengths and arranging the relative positions of the relevant protein-binding DNA sequences in the cis-regulatory region . The architectures that emerge are naturally modular and evolvable . Our results suggest that the transcription regulatory apparatus is a "programmable" computing machine, belonging formally to the class of Boltzmann machines . Crucial to our results is the ability to regulate gene expression at a distance . In bacteria, this can be achieved for isolated genes via DNA looping controlled by the dimerization of DNA-bound proteins . However, if adopted extensively in the genome, long-distance interaction can cause unintentional intergenic cross talk, a detrimental side effect difficult to overcome by the known bacterial transcription-regulation systems . This may be a key factor limiting the genome-wide adoption of complex transcription control in bacteria . Implications of our findings for combinatorial transcription control in eukaryotes are discussed. J Clin Periodontol, 2003 Jan, 30(1), 26 - 34 Clinical evaluation of an Er:YAG laser combined with scaling and root planing for non-surgical periodontal treatment . A controlled, prospective clinical study; Schwarz F et al.; OBJECTIVES: The purpose of the present controlled clinical trial was to compare the treatment of advanced periodontal disease with a combination of an Er:YAG laser (KEY II, KaVo, Germany) and scaling and root planing with hand instruments (SRP) to laser alone . MATERIAL AND METHODS: Twenty healthy patients with moderate to advanced periodontal destruction were randomly treated in a split-mouth design with a combination of an Er:YAG laser and SRP (test) or with laser (control) alone . The used energy setting for laser treatment was 160 mJ/pulse at a repetition rate of 10 Hz . Prior to treatment and 3, 6 and 12 months later the following parameters were evaluated by a blinded examiner: Plaque index (PI), gingival index (GI), bleeding on probing (BOP), probing depth (PD), gingival recession (GR) and clinical attachment level (CAL) . Subgingival plaque samples were taken at each appointment and analysed using darkfield microscopy for the presence of cocci,-non-motile rods, motile rods and spirochetes . No statistical significant differences in any of the investigated parameters between both groups were observed at baseline . RESULTS: Initially, the plaque index was 1.0 +/- 0.6 in both groups . At the 3-month examination the plaque scores were markedly reduced and remained low throughout the study . A significant reduction of the GI and BOP occurred in both groups after 3, 6 and 12 months (P < 0.05, P < 0.05, respectively) . The mean PD decreased in the test group from 5.2 +/- 0.8 mm at baseline to 3.2 +/- 0.8 mm after 12 months (P < 0.05) and in the control group from 5.0 +/- 0.7 mm at baseline to 3.3 +/- 0.7 mm after 12 months (P < 0.05) . The mean CAL decreased in the test group from 6.9 +/- 1.0 mm at baseline to 5.3 +/- 1.0 mm after 12 months (P < 0.05) and in the control group from 6.6 +/- 1.1 mm at baseline to 5.0 +/- 0.7 after 12 months (P < 0.05) . Both groups showed a significant increase of cocci and-non-motile rods and a decrease in the amount of motile rods and spirochetes . Conclusion: In conclusion, the present results have indicated that: (i) non-surgical periodontal therapy with both an Er:YAG laser + SRP and an Er:YAG laser alone may lead to significant improvements in all clinical parameters investigated, and (ii) the combined treatment Er:YAG laser + SRP did not seem to additionally improve the outcome of the therapy compared to Er:YAG laser alone. Water Sci Technol, 2003, 47(5), 77 - 82 Monitoring cell detachment by surfactants in a parallel plate flow chamber; Azeredo L et al.; The efficacy of the surfactants SDS and CTAB in detaching P . fluorescens from glass surface was evaluated in a parallel plate flow chamber . This device enables "in situ" determinations of cells detachment following the application of surfactants under well controlled hydrodynamic conditions . The results showed that SDS was able to remove almost all adhering bacteria in a short period of time, whereas CTAB did not promote much cell desorption . On the contrary, this surfactant increased the adhesion strength between cells and glass . Both surfactants promoted different alterations of cell surface properties, which explain their dissimilar effectiveness as cleansing agents. Pathology, 2003 Feb, 35(1), 65 - 9 Legionella pneumophila mip gene sequencing to investigate a cluster of pneumonia cases; Ratcliff RM et al.; AIMS: To use fluorescent antibody (FA) and PCR studies on fixed lung tissue to investigate whether Legionella pneumophila was the cause of pneumonia in a cluster of three haematology patients . METHODS: Cut sections of paraffin blocks of lung tissue were examined by direct FA (DFA) using fluorescently labelled antibody to serogroup 1 and Pontiac strains of L . pneumophila . In addition, a single tube 'hanging drop' nested PCR targeting the mip gene of Legionella was performed on DNA extracted from the lung sections . Products were sequenced using dye terminator chemistry . RESULTS: Numerous fluorescing bacteria were seen on staining with both antibodies in lung tissue from two of the patients . Identical L . pneumophila mip gene sequences were amplified from both DFA-positive lung sections . Two differing L . pneumophila mip sequences were obtained on three separate occasions from the tissue sections from the third patient negative by DFA . These sequences differed slightly from those obtained from the two DFA positive lung tissues . CONCLUSIONS: There is good epidemiological evidence to link the first two cases who had been treated in the same ward prior to development of fever within two days of each other . The significance of results is controversial for the third patient. Parassitologia, 2002 Dec, 44(3-4), 179 - 87 Different rates of nucleotide substitutions in Wolbachia endosymbionts of arthropods and nematodes: arms race or host shifts? Baldo L, Bartos JD, Werren JH, Bazzocchi C, Casiraghi M, Panelli S. The genus Wolbachia encompasses intracellular bacteria found in arthropods and in filarial nematodes . In arthropods, Wolbachia is primarily a reproductive parasite and shows relatively frequent horizontal transfer between host species, while in nematodes it appears to be a mutualist and is strictly vertically transmitted . We can expect that different selective pressures are acting on their genomes . Here we present an analysis of three Wolbachia genes, wsp, ftsZ and dnaA . In wsp of arthropod Wolbachia, an excess of non-synonymous substitutions was observed, providing evidence for positive selection . In nematode Wolbachia, no evidence for positive selection was found . Pressure for amino acid variation in wsp of arthropod Wolbachia could derive either from an arms race with the host or from the occurrence of more frequent hosts shifts due to horizontal transmission . In nematode Wolbachia, the lack of positively selected sites could result from the absence of an arms race, or from the homogeneity of the biochemical environment they exist in (ensured by strict vertical transmission) . In ftsZ minor differences in substitution patterns were observed between arthropod and nematode Wolbachia, only in the 3'-portion of the gene . dnaA showed comparable patterns of variation in both lineages, with evidence for strong conservation. Clin Exp Pharmacol Physiol, 2003 Apr, 30(4), 249 - 53 Effects of hypochlorite and hydrogen peroxide on cardiac autonomic receptors and vascular endothelial function; Sand C et al.; 1 . Reactive oxygen species (ROS) are known to be involved in the progression of various cardiovascular diseases . One source of ROS is activated neutrophils, which can release superoxide anion radicals and hydrogen peroxide by membrane-bound NAD(P)H oxidases . These ROS not only destroy bacteria, but may also affect mammalian tissue . In addition, hydrogen peroxide serves as a substrate for myeloperoxidase, an enzyme that is released by activated neutrophils during inflammatory processes, as seen, for instance, in reperfusion injury and atherosclerosis . Myeloperoxidase catalyses the oxidation of chloride by hydrogen peroxide, yielding hypochlorite, an extremely potent oxidant . 2 . The purpose of the present study was to evaluate the effects of hypochlorite on a variety of receptor-dependent processes in rat isolated left atria and rat thoracic aorta and to compare these results with the phenomena observed after incubation with hydrogen peroxide . 3 . In the presence of hypochlorite (300 micro mol/L), the positive inotropic response of alpha1-adrenoceptor stimulation by methoxamine (300 micro mol/L) was converted into a negative inotropic response . In contrast, the positive inotropic effects of the beta1/beta2-adrenoceptor agonist isoprenaline (3 micro mol/L) and endothelin (ET)-1 (100 nmol/L) remained largely unaffected . 4 . The inversion of alpha1-adrenoceptor-mediated inotropy was not obtained in the presence of hydrogen peroxide (500 micro mol/L) . Hydrogen peroxide did not affect the positive inotropic response of isoprenaline, but it completely abolished the inotropic effect of ET-1 . 5 . The effect of cardiac M2-receptor stimulation was studied in the presence of hypochlorite and hydrogen peroxide . The negative inotropic response to acetylcholine (ACh) was significantly enhanced after hypochlorite incubation compared with control . 6 . In the rat thoracic aorta, endothelial function, evaluated by means of ACh-induced vasodilation, was completely abolished in the presence of hypochlorite (100 micro mol/L), but remained unaffected by treatment with the same concentration of hydrogen peroxide . 7 . From these data, we conclude that hypochlorite exerts more toxic properties than its precursor hydrogen peroxide, leading to substantial physiological alterations in cardiac and vascular tissue. Biochemistry, 2003 Apr 15, 42(14), 4226 - 34 Recombinant Lhca2 and Lhca3 subunits of the photosystem I antenna system; Castelletti S et al.; In this study, two gene products (Lhca2 and Lhca3), encoding higher plants (Arabidopsis thaliana) Photosystem I antenna complexes, were overexpressed in bacteria and reconstituted in vitro with purified chloroplast pigments . The chlorophyll-xanthophyll proteins thus obtained were characterized by biochemical and spectroscopic methods . Both complexes were shown to bind 10 chlorophyll (a and b) molecules per polypeptide, Lhca2 having higher chlorophyll b content as compared to Lhca3 . The two proteins differed for the number of carotenoid binding sites: two and three for Lhca2 and Lhca3, respectively . beta-carotene was specifically bound to Lhca3 in addition to the xanthophylls violaxanthin and lutein, indicating a peculiar structure of carotenoid binding sites in this protein since it is the only one so far identified with the ability of binding beta-carotene . Analysis of the spectroscopic properties of the two pigment proteins showed the presence of low energy absorption forms (red forms) in both complexes, albeit with different energies and amplitudes . The fluorescence emission maximum at 77 K of Lhca2 was found at 701 nm, while in Lhca3 the major emission was at 725 nm . Reconstitution of Lhca3 without Chl b reveals that Chl b is not involved in originating the low energy absorption forms of this complex . The present data are discussed in comparison to the properties of the recombinant Lhca1 and Lhca4 complexes and of the native LHCI preparation, previously analyzed, thus showing a comprehensive description of the gene products composing the Photosystem I light harvesting system of A . thaliana. Rev Gastroenterol Peru, 1999 Jul, 19(3), 196 - 201 {HELICOBACTER PYLORI, CHRONIC GASTRITIS, GASTRIC ANDDUODENAL ULCER: STUDY OF 1638 PATIENTS}; Ramirez Ramos A et al.; We anaylize in patients from high socio economic level the histologic findings of biopsies from the gastric mucosa taken endoscopically . Patients with upper gastrointestinal symptoms were examined with esophagogastroduodenoscopy . In the histologic study we considered: presence of Helicobacter pylori, mucinous damage, displasia, atrophy and intestinal metaplasia . Patients were divided in two groups: those who endoscocopically did not present gastric or duodenal active ulcer, gastric neoplasia or were not gastrectomized or vagotomized and those in which active gastric or duodenal ulcer was found . The first group was comprised of 1406 patients (647 women and 769 men) . 57.33% (806) showed presence of Helicobacter pylori in the histologic study . Males presented higher prevalence of the bacteria (57.32% vs 42.68%, p=0.021873) . Atrophys was found in 8.25% and was more frequent in patients with H . pylori (78.45% vs 21.55% p=0.0000026) / Intestinal metaplasla was found in 11.24% being related more with the presence of H.pylori (74.68% vs 25.32%, p=0.0000043) . In the second group comprised of 232 patients: 192 males (82.7%) 82.75% showed presence of the bacteria . There was not a statistical difference by comparing sex and age vs presence of ulcer. Rev Gastroenterol Peru, 1999 Jul, 19(3), 179 - 194 {HELICOBACTER PYLORI INFECTION ERRADICATION IN DISPEPTIC PATIENTS WITH AND WITHOUT PEPTIC ULCER}; De Idiaquez D et al.; BACKGROUND: Helicobacter pylori (HP) infection is very prevalent worldwide, and has been associated with the presence of duodenal ulcer, gastric ulcer and chronic active gastritis . It is also speculated that HP may have a role in gastric cancer development . Triple drug schemes have been shown to be the most effective approach to erradicate HP infection . Nevetheless, high rates of resistance against some antibiotics as well as high costs affect the effectiveness of these therapies . The goal of the present study is to assess the effectiveness of the combination of tetracycline, furazolidone and bismuth in erradicating HP, as well as the changes in the histology.METHODS: Patients with diagnosis of HP infection, found in their antral gastric biopsies (hematoxylin and eosin staining (H-E)), were included . They received the following scheme for 10 days: tetracycline 500 mg qid., furazolidone 100 mg qid., and colloidal bismuth subcitrate 120 mg qid . Patients were instructed to come back for follow-up 6 to 8 weeks after starting the therapy . At that time a control upper endoscopy was performed and 3 antral biopsies were taken . Biopsies were stained with H-E and read by experienced pathologists . In both, the biopsy before treatment and the control biopsy, the following parameters were looked for: presence and density of HP; presence, depth and grade of chronic gastritis (lymphoplasmocytic infiltrate); presence and grade of inflammatory activity (polymorphonuclear inflitrate); presence of glandular atrophy; presence, grade (partial or total) and extent (focal or multifocal) of mucinous damage (epithelial damage); presence of intestinal metaplasia; and presence of lymphoid follicles.RESULTS: Fifty-nine patients (30 men and 29 women) completed per protocol . Mean age was 43 +/- 18 (range: 14-73) . HP erradication was achieved in 54 patients (91.5%) . Control biopsies showed improvement in the following parameters: presence and density of HP (p<0.001); presence, depth and grade of chronic gastritis (p<0.001); presence and grade of inflammatory activity (p<0.001); presence, grade and extent of mucinous damage (p<0.001); and presence of lymphoid follicles (p<0.001) . Neither the presence of glandular atrophy nor the presence of intestinal metaplasia showed any significant change . Patients who did not erradicate HP showed no significant difference in any of the parameters.CONCLUSIONS: The triple drug scheme including tetracycline, furazolidone and bismuth is effective in HP erradication . Erradication of HP is followed by an improvement in the following histologic parameters: presence, depth and grade of chronic gastritis (LMN infiltrate); presence and grade of inflammatory activity (PMN infiltrate); presence, grade and extent of mucinous damage; and presence of lymphoid follicles . This scheme is a cost-effective alternative for the therapy of HP infection in low income populations with a high prevalence of infection with this bacteria. Cytometry A, 2003 May, 53(1), 39 - 54 Flow cytometric measurement of fluorescence (Förster) resonance energy transfer from cyan fluorescent protein to yellow fluorescent protein using single-laser excitation at 458 nm; He L et al.; BACKGROUND: Use of distinct green fluorescent protein (GFP) variants permits the study of protein-protein interactions and colocalization in viable transfected cells by fluorescence (Forster) resonance energy transfer (FRET) . Flow cytometry is a sensitive method to detect FRET . However, the typical dual-laser methods used in flow cytometric FRET assays are not generally applicable because they require a specialized krypton ultraviolet (UV) laser . The purpose of this work was to develop a flow cytometric method to detect FRET between cyan fluorescent protein (CFP; donor) and yellow fluorescent protein (YFP; acceptor) by using the 458-nm excitation from a single tunable argon-ion laser . METHODS: FUSE-binding protein (FBP) interacting repressor (FIR) and FBP are c-myc transcription factors and are known to interact physically . To examine their interaction within viable cells, FIR and the binding motif of FBP, the FBP central domain (FBPcd), were fused with CFP and YFP, respectively, and this pair of fluorescently-tagged proteins was used to detect FRET in vivo . Cells transfected with expression plasmids encoding a CFP-FIR fusion protein and YFP as a negative control, a CFP-YFP fusion protein as a positive control, or CFP-FIR and YFP-FBPcd fusion proteins were examined for FRET after excitation with a 458-nm line from a tunable argon-ion laser . FRET was measured as the ratio of YFP:CFP emission or as YFP emission at 564-606 nm . Conventional FRET using the 413-nm UV line from a krypton laser was examined for comparison . Fluorescence signals were separated with a customized optical filter configuration using 530-nm shortpass, 500-nm longpass, and 560-nm shortpass dichroics in addition to 488/30 nm (CFP), 530/30 nm (YFP), and 585/42 nm (FRET) bandpass filters . Further, a laser-scanning confocal microscopic photobleach technique was used to document that FRET occurred by showing that the intensity of donor CFP fluorescence increased after its acceptor YFP was photobleached . Steady-state spectrofluorometry was used to confirm and validate the results detected by flow cytometry . RESULTS: Upon excitation with the 458-nm line of the argon-ion laser, the enhancement of the acceptor YFP signal and the decrease of the CFP signal were easily detected in cells transfected with the CFP-YFP construct or CFP-FIR and YFP-FBPcd . Similarly, FRET was detected under these conditions when the YFP emission was assessed at 564-606 nm . A strong correlation was observed between the increase in the YFP:CFP ratio and the YFP emission detected at 564-606 nm, consistent with the conclusion that FRET was detected comparably by both methods . A conventional flow cytometric krypton UV-laser technique was also used to confirm that FRET occurred with the CFP-YFP fusion protein and from CFP-FIR --> YFP-FBPcd . FRET also was confirmed by a confocal photobleaching technique, in which donor CFP intensity was enhanced after its acceptor YFP was photobleached . The flow cytometric and confocal microscopic results were confirmed by spectrofluorometry . CONCLUSION: These results demonstrated the feasibility of flow cytometric detection of FRET signals from CFP to YFP by excitation with the 458-nm line from the tunable argon-ion laser . The method was as efficient as excitation with the krypton UV laser and therefore should make FRET a more generally available flow cytometric technique . Published 2003 Wiley-Liss, Inc. Nature, 2003 Apr 17, 422(6933), 766 - 74 Adaptation of core mechanisms to generate cell polarity; Nelson WJ; Cell polarity is defined as asymmetry in cell shape, protein distributions and cell functions . It is characteristic of single-cell organisms, including yeast and bacteria, and cells in tissues of multi-cell organisms such as epithelia in worms, flies and mammals . This diversity raises several questions: do different cell types use different mechanisms to generate polarity, how is polarity signalled, how do cells react to that signal, and how is structural polarity translated into specialized functions? Analysis of evolutionarily diverse cell types reveals that cell-surface landmarks adapt core pathways for cytoskeleton assembly and protein transport to generate cell polarity. Front Biosci, 2003 May 01, 8, s544 - 56 DNA transport during transformation; Chen I et al.; The ability to take up exogenous DNA is a requisite for genetic competence and transformation . Here we review the process of DNA transport in competent bacteria . Two kinds of machineries have been described . In the first one, the components show similarities to the proteins involved in biogenesis of type IV pili and type II secretion systems . The second one is related to the type IV secretion system and conjugation apparatus. Bioresour Technol, 2003 Sep, 89(2), 191 - 7 The inhibitory effects and removal of dieldrin in continuous upflow anaerobic sludge blanket reactors; Erguder TH et al.; The inhibitory effects and removal efficiency of dieldrin (DLD) in anaerobic reactors were investigated . Anaerobic toxicity assay (ATA) experiments conducted in batch reactors revealed that 30 mg/l DLD had inhibitory effects on the unacclimated mixed anaerobic cultures . Continuous reactor experiments performed in a lab-scale two-stage upflow anaerobic sludge blanket (UASB) reactor system which was fed with ethanol as the sole carbon source, indicated that anaerobic granular cultures could be successfully acclimated to DLD . Chemical oxygen demand (COD) removal efficiencies were 88-92% for the two-stage system . The influent DLD concentration of 10 mg/l was removed by 44-86% and 86-94% in the second stage and overall UASB system, respectively . Biosorption of DLD on granular anaerobic biomass was found to be a significant mechanism for DLD removal in the UASB system . The maximum DLD loading rate and minimum HRT achievable for the first stage UASB reactor were 0.5 mg/lday (76 microg DLD/g VSS.day) and 10 h, respectively, which resulted in the overall COD removal efficiency of 85%. Protein Expr Purif, 2003 Apr, 28(2), 211 - 9 Secretion of functional anti-CD30-angiogenin immunotoxins into the supernatant of transfected 293T-cells; Stocker M et al.; Immunotoxins consist of a target-cell-specific binding moiety, chemically or recombinantly linked to a cytotoxic component . A number of different immunotoxins (IT) have increasingly been evaluated for immunotherapy . Since these foreign proteins are highly immunogenic in human, we have developed recombinant IT using the human ribonuclease angiogenin . Due to their potential toxic effects on eucaryotic cells, these IT are usually expressed in bacteria . Depending on the structure, size, and sequence of the desired IT, bacterial expression can be limited and the yield after purification be unsatisfactory . Therefore, the expression of IT in eucaryotic cells could provide a promising alternative . For this purpose we genetically fused the anti-CD30 single-chain variable fragment (scFv) Ki4 to the N- and C-termini of recombinant angiogenin . Both IT possess leader sequences, which mediate their secretion into the cell culture supernatant . Using a bicistronic mRNA the IT were simultaneously expressed together with enhanced green fluorescent protein (EGFP) . This allows direct monitoring of transfected cells . An additional plasmid encoded Zeocin resistance enhances the cultivation of transfected cells under selection pressure . Three days after transfection of 293T-cells, unpurified IT were analyzed by flow cytometry and competitive cell proliferation assays . This is the first report on the use of eucaryotic cells for the secretion of functionally active IT with a human effector domain. Curr Biol, 2003 Apr 15, 13(8), 691 - 7 A novel mechanism for activation of the protein kinase Aurora A; Eyers PA et al.; Segregation of chromosomes during mitosis requires interplay between several classes of protein on the spindle, including protein kinases, protein phosphatases, and microtubule binding motor proteins {1-4} . Aurora A is an oncogenic cell cycle-regulated protein kinase that is subject to phosphorylation-dependent activation {5-11} . Aurora A localization to the mitotic spindle depends on the motor binding protein TPX2 (Targeting Protein for Xenopus kinesin-like protein 2), but the protein(s) involved in Aurora A activation are unknown {11-13} . Here, we purify an activator of Aurora A from Xenopus eggs and identify it as TPX2 . Remarkably, Aurora A that has been fully deactivated by Protein Phosphatase 2A (PP2A) becomes phosphorylated and reactivated by recombinant TPX2 in an ATP-dependent manner . Increased phosphorylation and activation of Aurora A requires its own kinase activity, suggesting that TPX2 stimulates autophosphorylation and autoactivation of the enzyme . Consistently, wild-type Aurora A, but not a kinase inactive mutant, becomes autophosphorylated on the regulatory T loop residue (Thr 295) after TPX2 treatment . Active Aurora A from bacteria is further activated at least 7-fold by recombinant TPX2, and TPX2 also impairs the ability of protein phosphatases to inactivate Aurora A in vitro . This concerted mechanism of stimulation of activation and inhibition of deactivation implies that TPX2 is the likely regulator of Aurora A activity at the mitotic spindle and may explain why loss of TPX2 in model systems perturbs spindle assembly {14-16} . Our finding that a known binding protein, and not a conventional protein kinase, is the relevant activator for Aurora A suggests a biochemical model in which the dynamic localization of TPX2 on mitotic structures directly modulates the activity of Aurora A for spindle assembly. J Med Chem, 2003 Apr 24, 46(9), 1645 - 60 Construction and assessment of models of CYP2E1: predictions of metabolism from docking, molecular dynamics, and density functional theoretical calculations; Park JY et al.; 3D models of CYP2E1 were constructed for the purpose of structure-based prediction of 2E1 metabolism of diverse substrates based on configuration sampling of ligand-atom-oxyferryl center distances and quantum chemical criteria . Models were constructed on the basis of sequence alignments of 2E1 with templates of known structure, including rabbit CYP2C5 (3LVdH) and bacterial CYP450s . Following geometric and energetic assessments, the utility of the model was tested in structure-based predictions of metabolism . Autodock was used to dock chlorzoxazone, p-nitrophenol, N-nitrosodimethylamine, acetominophen, caffeine, theophylline, and methoxyflurane into the model CYP2E1 employing a model oxyferryl heme with charges based on density functional theoretical parametrization . In all cases, the lowest energy bound docked configurations corresponded to ones with the substrate intimately associated with the oxyferryl center . Configurations among the lowest energy docked forms of each of the ligands had orientations relative to the oxyferryl center consistent with the experimentally observed metabolites . Docking of long-chain dialkylnitrosoamines revealed no heme binding site bound configurations, in agreement with the negligible metabolism of these ligands . The lowest energy docked configurations of chlorzoxazone, p-nitrophenol, and N-nitrosodimethylamine, high-affinity substrates of 2E1, were used to initiate 300 ps molecular dynamics (MD) trajectories . The MD-sampled ligand-oxyferryl heme reactant configurations were in good accord with density functional theoretical (DFT) optimized oxyferryl-heme-ligand geometries . Analysis of the MD-sampled ligand-2E1 configurations from multiple docked orientations indicates the configurations with closest exposure of reactive centers to the oxyferryl heme to be correlated with observed metabolites with proper consideration of H-abstraction energetics . DFT assessment of relative radical energetics is directly compared with differential H-abstraction activation energetics by compound I and by a p-nitrosophenoxy radical compound I surrogate for the specific case of methoxyflurane and is shown to be in good agreement. Curr Mol Med, 2003 May, 3(3), 273 - 84 Live recombinant vectors for AIDS vaccine development; Voltan R et al.; Live recombinant vectors entered the AIDS vaccine field with the realization that live attenuated HIV vaccines posed too great a safety risk, and that subunit vaccines elicited antibodies which lacked the breadth or potency needed to induce sterilizing immunity . Vectored vaccines provided a means to bring the cellular arm of the immune system into play by mimicking natural viral infection . By delivering antigens within host cells, processing and presentation could occur for induction of cellular immune responses . This recombinant vector approach, either alone or combined with other strategies, has produced impressive results . Recombinants have been generated from DNA and RNA viruses and bacteria . With few exceptions, each vector poses some risk, yet each possesses unique features that make it attractive . In addition to safety, key considerations in vector selection have included previous success as a vaccine against the wild-type agent or other pathogens; ability to induce potent, persistent immune responses; ability to target mucosal inductive sites and antigen presenting cells; lack of integration into the host genome; presence of pre-existing immunity in people; ease of mucosal administration; cloning capacity; ease of engineering and production; and stability of the final product . Here we up-date the status of several live recombinant vectors that have shown good potential in pre-clinical studies . Some have progressed to human clinical trials, and others will shortly . The abundance of vectors, coupled with the complexity arising from use of combination regimens with other vaccine types and heterologous vectors, will necessitate selection of the most promising candidates for large-scale efficacy trials in people . The sooner comparative studies can be designed and implemented in which live recombinant vectors containing the same inserted genes are evaluated head-to-head, the closer we will be to an eventual vaccine. Mikrobiologiia, 2003 Jan-Feb, 72(1), 76 - 83 {Formation of nonculturable Mycobacterium tuberculosis and their regeneration}; Shleeva MO et al.; Nonculturable cells were found to occur in populations of Mycobacterium tuberculosis cells during the long post-stationary phase . These cells were small (0.6-0.8 micron) ovoid and coccoid forms with intact cell walls and negligible respiratory activity, which allows them to be regarded as dormant cells . Nonculturable cells were characterized by low viability after plating onto solid medium; a minor part of the population of these cells could be cultivated in liquid medium . Cell-free culture liquid of an exponential-phase Mycobacterium tuberculosis culture or the bacterial growth factor Rpf exerted a resuscitating effect, increasing substantially the growth capacity of the nonculturable cells in liquid medium . During resuscitation of nonculturable cells, a transition from ovoid to rodlike cell shape occurred . At early stages of resuscitation, ovoid cells formed small aggregates . The recovery of culturability was associated with the formation of rod-shaped cells in the culture . The data obtained demonstrate the in vitro formation of dormant cells of Mycobacterium tuberculosis, which do not grow on solid media but can be resuscitated in liquid medium under the effect of substance(s) secreted by actively growing cells. Vopr Med Khim, 2002 Nov-Dec, 48(6), 541 - 52 {Peptide hydrolases with catalytic dyad Ser-Lys . Similarity and distinctions of the active centers of ATP-dependent Lon proteases, LexA repressors, signal peptidases and C-terminal processing proteases}; Rotanova TV; It is established that ATP-dependent protease Lon family belongs to the serine-lysine peptide hydrolases clan . Significant similarity of amino acid sequences of proteases Lon and repressors LexA in the regions including the catalytic serine and lysine residues is revealed by comparing primary structures of different families of the enzymes with Ser-Lys catalytic dyad . The both Lon and LexA families are shown to be divided into two subfamilies in accordance with the nature of amino acids in the catalytically active serine environment . Putative DNA binding sites are revealed in proteolytic domains of Lon A subfamily . Similarities and distinctions of the all families peptide hydrolases of the clan in the regions of their active centers are discussed. Appl Microbiol Biotechnol, 2003 Aug, 62(2-3), 168 - 73 Epub 2003 Apr 16. Influence of residual ethanol concentration on the growth of Gluconacetobacter xylinus I 2281; Kornmann H et al.; The influence of residual ethanol on metabolism of food grade Gluconacetobacter xylinus I 2281 was investigated during controlled cultivations on 35 g/l glucose and 5 g/l ethanol . Bacterial growth was strongly reduced in the presence of ethanol, which is unusual for acetic acid bacteria . Biomass accumulated only after complete oxidation of ethanol to acetate and carbon dioxide . In contrast, bacterial growth initiated without delay on 35 g/l glucose and 5 g/l acetate . It was found that acetyl CoA was activated by the acetyl coenzyme A synthetase (Acs) pathway in parallel with the phosphotransacetylase (Pta)-acetate kinase (Ack) pathway . The presence of ethanol in the culture medium strongly reduced Pta activity while Acs and Ack remained active . A carbon balance calculation showed that the overall catabolism could be divided into two independent parts: upper glycolysis linked to glucose catabolism and lower glycolysis liked to ethanol catabolism . This calculation showed that the carbon flux through the tricarboxylic cycle is lower on ethanol than on acetate . This corroborated the diminution of carbon flux through the Pta-Ack pathway due to the inhibition of Pta activity on ethanol. Mol Cell Biol, 2003 May, 23(9), 3265 - 73 Decreased expression of the DNA mismatch repair gene Mlh1 under hypoxic stress in mammalian cells; Mihaylova VT et al.; The hypoxic tumor microenvironment has been shown to contribute to genetic instability . As one possible mechanism for this effect, we report that expression of the DNA mismatch repair (MMR) gene Mlh1 is specifically reduced in mammalian cells under hypoxia, whereas expression of other MMR genes, including Msh2, Msh6, and Pms2, is not altered at the mRNA level . However, levels of the PMS2 protein are reduced, consistent with destabilization of PMS2 in the absence of its heterodimer partner, MLH1 . The hypoxia-induced reduction in Mlh1 mRNA was prevented by the histone deacetylase inhibitor trichostatin A, suggesting that hypoxia causes decreased Mlh1 transcription via histone deacetylation . In addition, treatment of cells with the iron chelator desferrioxamine also reduced MLH1 and PMS2 levels, in keeping with low oxygen tension being the stress signal that provokes the altered MMR gene expression . Functional MMR deficiency under hypoxia was detected as induced instability of a (CA)(29) dinucleotide repeat and by increased mutagenesis in a chromosomal reporter gene . These results identify a potential new pathway of genetic instability in cancer: hypoxia-induced reduction in the expression of key MMR proteins . In addition, this stress-induced genetic instability may represent a conceptual parallel to the pathway of stationary-phase mutagenesis seen in bacteria. J Gravit Physiol, 2000 Jul, 7(2), P79 - 80 Microgravity mediated changes in phytoferritin accumulation in soybean root cap cells; Klymchuk DO et al.; Phytoferritin is an iron-protein complex analogous to the ferritin found in mammalian, bacteria and fungi cells . Phytoferritin molecules are large proteins, about 10.5 nm in diameter, visualised in an electron microscope as discrete, electron dense particles with iron-containing core, where several thousand atoms of iron lie within the proteinaceous shell (apoferritin) . In higher plants, a plastid stroma is the site of phytoferritin storage . Phytoferritin is seen in all types of plastids . It is considered to be a mechanism used by cells to store iron in a non-toxic form . Phytoferritin-bound iron may subsequently be used to form iron-containing components . It was shown that low levels of phytoferritin are synthesised in normal green leaves, whereas chlorotic leaves do not have a measurable amount of phytoferritin and leaves of iron-loaded seedlings contain a high level of total iron, and phytoferritin well-filled by iron . Phytoferritin accumulation was observed in photosynthetic inactivity chloroplasts during senescence and disease . In this study we analised the effects of microgravity and ethylene on production of phytoferritin in the root cap columella cells of soybean seedlings. Dev Comp Immunol, 2003 Jun-Jul, 27(6-7), 603 - 10 Differential nitric oxide production by chicken immune cells; Crippen TL et al.; Nitric oxide is a rapidly reacting free radical which has cytotoxic effects during inflammatory responses and regulatory effects as a component of signal transduction cascades . We quantified the production of nitrite, a stable metabolite of nitric oxide, in chicken heterophils, monocytes and macrophages after stimulation by IFNgamma, LPS and killed bacteria . Our results demonstrate a differential production of nitrite over 72 h by chicken peripheral blood heterophils, monocytes and the chicken macrophage cell line (HD11) . HD11 cells produced an average of 10 fold more nitrite in comparison to monocytes and 30 fold more than heterophils upon stimulation . This production could be inhibited by S-methylisothiourea indicating that the inducible nitric oxide synthase enzyme was participating in the pathway leading to nitrite production. Water Res, 2003 Apr, 37(8), 1737 - 48 Recent water quality trends in the Schuylkill River, Pennsylvania, USA: a preliminary assessment of the relative influences of climate, river discharge and suburban development; Interlandi SJ et al.; Climate, flow rate and land use are all known drivers of water quality in river systems, but determining the relative influences of these factors remains a significant challenge for aquatic science and management . Long-term data from the Schuylkill River at Philadelphia is assessed here in an attempt to ascertain the separate and combined influence of these major drivers on water quality in a developed watershed . Water quality measures including nutrients, conservative solutes and bacteria all elicited distinct seasonal patterns driven primarily by river discharge . Mass transport rates of sodium and chloride have increased with time, and were elevated in winter, presumably as a function of road salt deposition . A steady increase in developed land area in the watershed has occurred in recent decades, which allowed the use of time as a surrogate parameter for regional development in the construction of multiple factor linear models predicting the relative influences of precipitation, river discharge and developed land area on river water quality . Linear models predicting annually averaged water quality measures showed the effects of precipitation, discharge and developed land area to be of nearly equal importance in regulating levels of conductivity, alkalinity, sodium, and chloride in the river . Models predicting water quality variables for discrete samples demonstrated that river flow was the major determinant of daily variability in alkalinity, conductivity, hardness and calcium levels, while still resolving the highly significant influence of watershed development on water quality . Increases in solute transport in the Schuylkill River in recent decades appear to be the direct result of modern suburban development in the watershed. Soc Sci Med, 2003 May, 56(10), 2089 - 96 Searching for an optimum solution to the Bangladesh arsenic crisis; Caldwell BK et al.; Searching for an optimum solution to the Bangladesh arsenic crisis: Thirty years ago Bangladesh experienced very high levels of infant and child mortality, much of it due to water-borne disease in deltaic conditions where surface water was highly polluted . In what appeared to be one of the great public health achievements, 95% of the population were converted to drinking bacteria-free tubewell water from underground aquifers . Recently, it has been shown that perhaps 20% of this water is arsenic contaminated and alternatives to tubewell water have been sought . This paper reports on two national surveys collaboratively carried out in 2000 by the Health Transition Centre, Australian National University and Mitra and Associates, Dhaka: A census of tubewells and a household survey of tubewell use and arseniosis . The study found that the tubewell revolution has been promoted not only by health considerations but also by the demand for a household water facility and the desire by women to reduce workloads associated with using surface water . Because of this, and because the population had absorbed the message about safe tubewell water, it is argued that the movement away from the use of tubewell water should be as limited as possible, even if this means using safe tubewells which are often found in the neighbourhood . To enable such a move the most urgent need is not changing the source of water but comprehensive national water testing providing essential information to households about which wells are safe and which are not. Bioorg Chem, 2003 Feb, 31(1), 24 - 43 Biosynthesis of the 7-deazaguanosine hypermodified nucleosides of transfer RNA; Iwata-Reuyl D; Transfer RNA (tRNA) is structurally unique among nucleic acids in harboring an astonishing diversity of post-transcriptionally modified nucleoside . Two of the most radically modified nucleosides known to occur in tRNA are queuosine and archaeosine, both of which are characterized by a 7-deazaguanosine core structure . In spite of the phylogenetic segregation observed for these nucleosides (queuosine is present in Eukarya and Bacteria, while archaeosine is present only in Archaea), their structural similarity suggested a common biosynthetic origin, and recent biochemical and genetic studies have provided compelling evidence that a significant portion of their biosynthesis may in fact be identical . This review covers current understanding of the physiology and biosynthesis of these remarkable nucleosides, with particular emphasis on the only two enzymes that have been discovered in the pathways: tRNA-guanine transglycosylase (TGT), which catalyzes the insertion of a modified base into the polynucleotide with the concomitant elimination of the genetically encoded guanine in the biosynthesis of both nucleosides, and S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA), which catalyzes the penultimate step in the biosynthesis of queuosine, the construction of the carbocyclic side chain. J Endotoxin Res, 2002, 8(6), 465 - 8 Innate immune responses in oral mucosa; Sugawara S et al.; It is speculated that more than 400 bacterial species reside in the oral cavity . Some cause inflammation (e.g . periodontitis), understanding of which requires examination of innate immunity in the oral cavity . Oral mucosal cells such as epithelial cells are thought to act as a physical barrier against the invasion of pathogenic organisms, but they have an ability to produce inflammatory cytokines and express adhesion molecules . Oral epithelial cells are refractory to many bacterial components although they express Toll-like receptors/MyD88, and acquire responsiveness after priming with IFN-gamma . When the cells are stimulated with lipopolysaccharide (LPS) and neutrophil protease (PR3) after IFN-gamma priming, the cells produce bio-active IL-18, which is critical to Th1 and Th2 responses . PR3 itself is able to activate the cells through G protein-coupled protease-activated receptor-2 on the cell surface . These results suggest that innate immune responses of oral epithelial cells to bacterial components are regulated in the inflammatory process . In addition, saliva contains abundant bio-active CD14 from salivary glands in a soluble form, although LPS-binding protein was below detectable levels, suggesting that saliva CD14 is important for the maintenance of oral health. Physiol Biochem Zool, 2003 Jan-Feb, 76(1), 1 - 11 Experimental evolution and the Krogh principle: generating biological novelty for functional and genetic analyses; Bennett AF; August Krogh counseled the careful selection of the best subject organism on which to undertake mechanistic physiological research . But what if an organism with the desired properties does not exist? It is now within our power to engineer organisms genetically to achieve novel combinations of traits . I propose that it is a logical extension of the Krogh principle that we use biological methodologies to create novel organisms ideally suited for particular physiological studies . Transgenics may first come to mind as the method for such transformations, but here I suggest that an alternative and complementary technique for generating biological novelty is experimental evolution . The latter has several advantages, including modification of multiple characters in one experiment, the production of advantageous traits, the testing of evolutionary hypotheses, and the identification of previously unsuspected factors involved in adaptation . Three experiments are reviewed, each of which examined the evolution of different physiological characters in different environments and organisms: locomotor performance in mice, desiccation tolerance in fruit flies, and high temperature adaptation in bacteria . While diverse in experimental type and subject, all resulted in the successful production of new variants with enhanced function in their new environments . Each experiment successfully tested hypotheses concerning physiological evolution, and in each case, unanticipated results emerged, which suggests previously unsuspected adaptive pathways and mechanisms . In addition, replicate populations in each experiment adjusted to their common environments by several different means, which indicates that physiological evolution may follow diverse stochastic pathways during adaptation . Experimental evolution can be a valuable method to produce and investigate new physiological variants and traits . The choice of experimental subjects, according to the Krogh principle, is no longer limited to currently existing organisms but is open to our imaginations and our ingenuity. Vet Microbiol, 2003 May 29, 93(3), 185 - 96 Development of a diagnostic PCR assay based on novel DNA sequences for the detection of Mycoplasma suis (Eperythrozoon suis) in porcine blood; Hoelzle LE et al.; An efficient method of control of porcine eperythrozoonosis (PE) caused by Mycoplasma suis is eradication of infection by detection and removal of infected carrier animals . At present, only a few tests are available for the diagnosis of these latent M . suis infections in pigs . The objective of this study was to develop a PCR assay based on novel DNA sequences for the identification of M . suis-infected pigs . A 1.8 kb EcoRI DNA fragment of the M . suis genome was isolated from the blood of pigs experimentally infected with M . suis . Specificity of the DNA fragment was confirmed by DNA sequence analysis and PCR using primers directed against sequences contained in the 1.8 kb fragment . PCR products of 782 bp in size were amplified only from M . suis particles prepared from the blood of experimentally infected pigs but not from any controls, comprising blood from gnotobiotic piglets and a panel of bacteria including other porcine mycoplasmas . PCR results were confirmed by dot blot hybridisation . The applicability of the PCR assay to diagnose M . suis infections in pigs was evaluated by investigating blood samples from 10 symptomatic pigs with clinical signs typical of porcine eperythrozoonosis and blood samples from 10 healthy pigs . The M . suis-specific PCR product was amplified from all samples taken at episodes of acute disease as well as from samples taken during the latent stage of infection, thus demonstrating the suitability of the PCR assay for detecting latent infected carrier animals. Mol Microbiol, 2003 May, 48(3), 605 - 15 Archaea: an archetype for replication initiation studies? Kelman LM, Kelman Z. Whereas the process of DNA replication is fundamentally conserved in the three domains of life, the archaeal system is closer to that of eukarya than bacteria . In the time since the complete genome sequences of several members of the archaeal domain became available, there has been a burst of research on archaeal DNA replication . These studies have led to both expected and surprising findings . This review summarizes the search for origins of replication in archaea, and our current knowledge of initiation, the process by which replication origins are recognized, the DNA molecule is unwound and the replicative helicase is loaded onto the DNA in preparation for DNA synthesis . The similarities and differences of the initiation process in archea, bacteria and eukarya are also summarized. Mol Microbiol, 2003 May, 48(3), 599 - 604 The archaeal cell cycle: current issues; Bernander R; The recently discovered structural similarities between the archaeal Orc1/Cdc6 and bacterial DnaA initiator proteins for chromosome replication have exciting implications for cell cycle regulation . Together with current attempts to identify archaeal chromosome replication origins, the information is likely to yield fundamental insights into replication control in both archaea and eukaryotes within the near future . Several proteins that affect, or are likely to affect, chromatin structure and genome segregation in archaea have been described recently, including Sph1 and 2, ScpA and B, Sir2, Alba and Rio1p . Important insights into the properties of the MinD and FtsZ cell division proteins, and of putative cytoskeletal elements, have recently been gained in bacteria . As these proteins also are present among archaea, it is likely that the new information will also be essential for understanding archaeal genome segregation and cell division . A series of interesting cell cycle issues has been brought to light through the discovery of the novel Nanoarchaeota phylum, and these are outlined briefly . Exciting areas for extended cell cycle investigations of archaea are identified, including termination of chromosome replication, application of in situ cytological techniques for localization of cell cycle proteins and the regulatory roles of GTP-binding proteins and small RNAs. Plant J, 2003 Apr, 34(2), 205 - 16 Reactive electrophile species activate defense gene expression in Arabidopsis; Almeras E et al.; Compounds containing alpha,beta-unsaturated carbonyl groups are increasingly implicated as potent regulators of gene expression; some are powerful cytotoxins known to accumulate at the site of lesion formation in host-pathogen interactions . We used a robust measurement of photosynthetic efficiency to quantify the toxicity of a variety of lipid derivatives in Arabidopsis leaves . Small alpha,beta-unsaturated carbonyl compounds (e.g . acrolein and methyl vinyl ketone) were highly active and proved to be potent stimulators of expression of the pathogenesis-related gene HEL (PR4) . These small volatile electrophiles were far more active than larger alkenal homologs like 2(E)-hexenal, and activated HEL expression in a manner independent of salicylate, ethylene, and jasmonate production/perception . Electrophile treatment massively increased the levels of unesterified cyclopentenone jasmonates, which themselves are electrophiles . Patterns of gene expression in response to electrophile treatment and in response to avirulent bacteria were compared, which revealed strikingly similar transcript profiles . The results broaden the range of known biologic effects of reactive electrophile species to include the activation of a pathogenesis-related gene (HEL) and genes involved in metabolism . Electrophiles can act as mediators of both genetic and biochemical effects on core defense signal transduction. J Appl Microbiol, 2003, 94(5), 767 - 80 From tooth to hoof: treponemes in tissue-destructive diseases; Edwards AM et al.; With the advent of new molecular and immunological tools, there is better understanding of the roles that difficult to cultivate bacteria, and not-yet-cultivated bacteria such as spirochaetes, play in polymicrobial diseases . Only relatively recently have studies implicated Treponema spirochaetes in human periodontal disease, a destructive condition of the tissues supporting the teeth . A number of different Treponema species have been isolated and their surface protein components that mediate adhesion, cytotoxicity, and tissue damage have been characterized . More recently Treponema strains closely related to human oral isolates have been cultivated from active lesions of digital dermatitis, an ulcerative condition affecting the feet of cows and sheep . This condition, like periodontal disease, appears to have a polymicrobial aetiology in which enrichment for Treponema may play a crucial part . This article reviews the known mechanisms by which Treponema interact with eukaryotic host cells and tissue proteins, and how these interactions may contribute to pathogenic diversity. J Oral Pathol Med, 2003 May, 32(5), 310 - 3 Ganglion cyst of the temporomandibular joint; Kim SG et al.; We report a case of cystic lesion in a 37-year-old woman . The patient had an oval-shaped lesion adjacent to the temporomandibular joint . Thick fibrotic tissue and muscle were observed microscopically, but the epithelium lining was not observed . The lesion was diagnosed as a ganglion cyst . The patient's general medical history was non-contributory . High performance liquid chromatography (HPLC) and mass spectrophotometry (MS) revealed some proteins from the fluid in the lesion, such as a filaggrin precursor, dystroglycan, a polyprotein of the hepatitis C virus, and proteins originating from bacteria . The follow-up examinations revealed no recurrence . The probable pathogenesis of the lesion is discussed. Biochem J, 2003 Jul 15, 373(Pt 2), 495 - 503 Characterization of XYN10B, a modular xylanase from the ruminal protozoan Polyplastron multivesiculatum, with a family 22 carbohydrate-binding module that binds to cellulose; Devillard E et al.; A new xylanase gene, xyn10B, was isolated from the ruminal protozoan Polyplastron multivesiculatum and the gene product was characterized . XYN10B is the first protozoan family 10 glycoside hydrolase characterized so far and is a modular enzyme comprising a family 22 carbohydrate-binding module (CBM) preceding the catalytic domain . The CBM22 was shown to be a true CBM . It showed high affinity for soluble arabinoxylan and is the first example of a CBM22 that binds strongly to celluloses of various crystallinities . The enzymic properties of XYN10B were also analysed . Its optimal temperature and pH for activity were 39 degrees C and 7.0 respectively; these values being close to those of the ruminal ecosystem . The phylogenetic relationships between the XYN10B CBM22 or catalytic domain and related sequences from ruminal and non-ruminal bacteria and eukaryotes are reported . The xyn10B gene is shown to lack introns. J Med Entomol, 2003 Mar, 40(2), 230 - 7 Molecular evidence for novel tick-associated spotted fever group rickettsiae from Thailand; Hirunkanokpun S et al.; Ticks are of considerable medical and veterinary importance because they directly harm the host through their feeding action and indirectly through vectoring many bacterial pathogens . Despite many ticks being known from Thailand, very little is known about the bacteria they may harbor . We report here the results of a survey of tick-associated bacteria in Thailand . A total of 334 individuals representing 14 species of ticks in five genera were collected from 10 locations in Thailand and were examined for the human pathogens, Borrelia, Francisella, Rickettsia, and the common arthropod endosymbionts, Wolbachia, by polymerase chain reaction (PCR) assay using specific primers . Rickettsial DNA was detected in 30% (9/30) of Amblyomma testudinarium (Koch, 1844) collected from Khao Yai National Park, Nakhon Nayok Province and 16.84% (16/95) of Hemaphysalis ornithophila (Hoogstraal and Kohls, 1959) collected from Khao Yai National Park, Nakhon Nayok Province and Khao Ang Rue Nai Wildlife Sanctuary, Chachoengsao Province . Rickettsial DNA was not detected in any of the other tick species and no DNA of Borrelia, Francisella, or Wolbachia was detected in any of 14 tick species . Phylogenetic relationships among the rickettsiae detected in this study and those of other rickettsiae were inferred from comparison of sequences of the 17-kDa antigen gene, the citrate synthase gene (gltA), and the 190-kDa outer membrane protein gene (ompA) . Results indicated that the three Thai rickettsiae detected in this study represent new rickettsial genotypes and form a separate cluster among the spotted fever group rickettsiae. Ther Umsch, 2003 Mar, 60(3), 127 - 32 {Pathophysiology of inflammatory bowel disease}; Lehmann FS; Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory bowel diseases (IBDs) of unknown origin . Current data suggest that the interaction of genetic, immunologic and environmental factors leads to chronic inflammation . In UC, the inflammation is limited to the mucosa whereas in CD, the whole bowel wall may be affected . The concept of genetic background is supported by the increased prevalence of CD within certain populations and individual families and by the results of twin studies . It is believed that not only the phenotype (CD or UC), but also the clinical course are influenced by genetic factors . It is unknown which trigger induces chronic immune stimulation . It could be either a nutritional antigen, a self antigen, a component of the normal gut flora or certain bacteria such a mycobacteria . The chronic immune stimulation may be due to a dysbalance of pro-inflammatory and inhibitory cytokines . Besides cytokines, a variety of different inflammatory mediators have a crucial role for the inflammatory process . Smoking has a significant effect on occurrence and outcome of CD and UC. J Gene Med, 2003 Apr, 5(4), 324 - 32 Gene electro-transfer improves transduction by modifying the fate of intramuscular DNA; Cappelletti M et al.; BACKGROUND: Intramuscular gene delivery through injection of plasmid DNA has long been considered a promising approach for safe and simple in vivo gene expression for vaccination and gene therapy purposes . Recently, intramuscular gene delivery has been improved by applying low-voltage electric pulses after plasmid injection, a procedure that has been variably called gene electro-transfer, in vivo electroporation or electrical stimulation . Different types of electrical treatments have been used with excellent results both in terms of transgene expression levels and immunization outcome . This approach, therefore, holds promise for safe gene delivery to animals and humans designed for non-viral gene therapy and DNA-based vaccination . The molecular mechanisms underlying this increment in transduction efficiency are, however, still unclear . METHODS: Plasmid DNA status and kinetics following gene electro-transfer was analyzed by different methods (Southern analysis, Q-PCR and transformation into competent bacteria) . RESULTS: A large amount of plasmid DNA is degraded in the first 4 h post-injection, with or without electroporation; later, the amount of intramuscular plasmid DNA is higher in electroporated samples . On electroporation, plasmid is partially protected from degradation, presumably by its early compartmentalization into the nuclei of muscle cells . CONCLUSIONS: By investigating the intracellular outcome and persistence of plasmid DNA following simple injection or gene electro-transfer we provide useful information on the mechanisms of plasmid entry and expression and underline some of the steps that could be taken to further improve this methodology . Clin Microbiol Rev, 2003 Apr, 16(2), 273 - 307 Acanthamoeba spp . as agents of disease in humans; Marciano-Cabral F et al.; Acanthamoeba spp . are free-living amebae that inhabit a variety of air, soil, and water environments . However, these amebae can also act as opportunistic as well as nonopportunistic pathogens . They are the causative agents of granulomatous amebic encephalitis and amebic keratitis and have been associated with cutaneous lesions and sinusitis . Immuno compromised individuals, including AIDS patients, are particularly susceptible to infections with Acanthamoeba . The immune defense mechanisms that operate against Acanthamoeba have not been well characterized, but it has been proposed that both innate and acquired immunity play a role . The ameba's life cycle includes an active feeding trophozoite stage and a dormant cyst stage . Trophozoites feed on bacteria, yeast, and algae . However, both trophozoites and cysts can retain viable bacteria and may serve as reservoirs for bacteria with human pathogenic potential . Diagnosis of infection includes direct microscopy of wet mounts of cerebrospinal fluid or stained smears of cerebrospinal fluid sediment, light or electron microscopy of tissues, in vitro cultivation of Acanthamoeba, and histological assessment of frozen or paraffin-embedded sections of brain or cutaneous lesion biopsy material . Immunocytochemistry, chemifluorescent dye staining, PCR, and analysis of DNA sequence variation also have been employed for laboratory diagnosis . Treatment of Acanthamoeba infections has met with mixed results . However, chlorhexidine gluconate, alone or in combination with propamidene isethionate, is effective in some patients . Furthermore, effective treatment is complicated since patients may present with underlying disease and Acanthamoeba infection may not be recognized . Since an increase in the number of cases of Acanthamoeba infections has occurred worldwide, these protozoa have become increasingly important as agents of human disease. Clin Microbiol Rev, 2003 Apr, 16(2), 230 - 41 Importance of respiratory viruses in acute otitis media; Heikkinen T et al.; Acute otitis media is usually considered a simple bacterial infection that is treated with antibiotics . However, ample evidence derived from studies ranging from animal experiments to extensive clinical trials supports a crucial role for respiratory viruses in the etiology and pathogenesis of acute otitis media . Viral infection of the upper respiratory mucosa initiates the whole cascade of events that finally leads to the development of acute otitis media as a complication . The pathogenesis of acute otitis media involves a complex interplay between viruses, bacteria, and the host's inflammatory response . In a substantial number of children, viruses can be found in the middle-ear fluid either alone or together with bacteria, and recent studies indicate that at least some viruses actively invade the middle ear . Viruses appear to enhance the inflammatory process in the middle ear, and they may significantly impair the resolution of otitis media . Prevention of the predisposing viral infection by vaccination against the major viruses would probably be the most effective way to prevent acute otitis media . Alternatively, early treatment of the viral infection with specific antiviral agents would also be effective in reducing the occurrence of acute otitis media. Med Microbiol Immunol (Berl), 2003 Nov, 192(4), 183 - 8 Epub 2003 Apr 11. Phagocytosis of Treponema pallidum and reactive oxygen species production by isolated rat Kupffer cells; Marangoni A et al.; The in vitro phagocytosis of viable Treponema pallidum subsp . pallidum by isolated rat Kupffer cells, studied by immunofluorescence staining of Kupffer cells-associated bacteria, showed that ingestion of live, unopsonized treponemes was slow: in fact, Kupffer cells started to be positive 1 h after infection, when only 4% of the cells presented small round fluorescent inclusion-like bodies . Thereafter, the number of positive cells progressively increased with time: 7%, 17%, 36%, and 69% of Kupffer cells were positive, respectively, 2, 4, 6 and 8 h after infection . Opsonization of T . pallidum with human immune serum did not substantially modify the percentage (8%) of Kupffer cells ingesting T . pallidum 1 h after infection, whereas opsonization significantly ( P<0.01) increased phagocytosis after 2, 4 and 6 h of incubation, when 44%, 58%, and 68% of Kupffer cells were positive, respectively . At 8 h after infection of Kupffer cells by opsonized T . pallidum, 75% of the cells were positive by immunofluorescence . Heat-inactivation of T . pallidum slightly enhanced phagocytosis . In contrast, opsonization of heat-inactivated spirochetes with specific antibodies significantly ( P<0.01) increased the phagocytosis of bacteria by Kupffer cells, beginning as early as 30 min after infection, when 65% of the cells were positive by immunofluorescence . The reactive oxygen species (ROS) production by Kupffer cells following incubation with spirochetes was also determined by chemiluminescence . Treponemes induced an oxidative burst in Kupffer cells in a dose-dependent manner and the generation of ROS was already detectable 20 min after the exposure of the Kupffer cells to treponemes and peaked at 35 min of incubation . Live, as well as live and opsonized, and heat-inactivated treponemes, induced an O(2)(-) production lower than that induced by heat-inactivated and opsonized spirochetes. J Microbiol Methods, 2003 Jun, 53(3), 411 - 5 A simplified dehydrogenase enzyme assay in contaminated sediment using 2-(p-Iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride; Mosher JJ et al.; 2-(p-Iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) accepts electrons from dehydrogenase enzymes and is reduced to a red-colored formazan (INTF), which can be quantified by colorimetric analysis . Use of previously published methods for this technique was unsuccessful due to background chemical reactions from high levels of polycyclic aromatic hydrocarbons (PAHs) and metals in the sediments . A modified method using acetonitrile extraction of the INTF was efficient and did not chemically reduce INT . This activity method is simple, quick, inexpensive and precise. J Biochem Mol Biol, 2003 Mar 31, 36(2), 230 - 6 Purification and characterization of complement-activating acidic polysaccharides from the fruits of Capsicum annuum; Paik SY et al.; Hot water-soluble crude polysaccharide (HCAP-0) that was obtained from the fruits of Capsicum annuum showed potent anti-complementary activity . The activity was unchanged by pronase digestion, but decreased by periodate oxidation . The HCAP-0 was fractionated by DEAE ion-exchange chromatography to give two major fractions, HCAP-II and III . These two fractions were finally purified by gel filtration to give HCAP-IIa, HCAPIIIa1, and IIIa2 fractions that had high anti-complementary activities . The HCAP-IIIa1 and IIIa2 consisted of homogeneous polysaccharides . The anti-complementary activities were unaffected by treatment with polymyxin B, indicating that the modes of complement activation were not due to preexisting lipopolysaccharide . The molecular weight and sugar content of HCAP-IIIa2 had potent anti-complementary activity . The highest yields were 55 kDa and 75.9%, and the molar ratio of galactose (Ara:Gal, 1.0:4.6) was higher than other sugars . The crossed immuno-electrophoresis showed that both classical and alternative pathways were activated by HCAP-IIIa2. Water Res, 2003 Feb, 37(3), 627 - 33 Metal speciation and pH effect on Pb, Cu, Zn and Cd biosorption onto Sphaerotilus natans: Langmuir-type empirical model; Pagnanelli F et al.; Biosorption data of lead, copper, zinc and cadmium onto Sphaerotilus natans at different equilibrium pH (3-5 units) were here reported and analysed . Experimental results outlined the positive effect of pH increase on pollutant uptake and also the biomass affinity series (Pb > Cu > Zn > Cd) reflecting the hydrolytic properties of metals . An original empirical model was proposed to represent the effect of pH on heavy metal biosorption inserting q(max) vs . pH empirical functions into the classical Langmuir isotherm. Otolaryngol Clin North Am, 2002 Dec, 35(6), 1263 - 81 Telemedicine in otolaryngology; Holtel MR et al.; More research is needed in otolaryngology telemedicine, but it would be a mistake to stop at only determining if telemedicine is as good as an in-person exam . The digital image recorded in a telemedicine encounter can be manipulated to increase diagnostic information not currently available . Radiologists currently take a chest radiograph in which a chest mass or the tip of an nasogastric tube is difficult to visualize, and by inverting the gray scale or viewing other digital manipulations of that image, the mass or tube tip becomes obvious . Examples in otolaryngology might include images of the larynx manipulated to better demonstrate the inflamed tissue of reflux, or images of the tympanic membrane manipulated to better demonstrate early retraction . Despite dramatic and likely continued decreases, equipment cost is still an issue . Current research points to good consumer acceptance, and certainly with each new generation the technology is more readily accepted . As Nesbitt {4} points out, it is certainly not difficult to look to the future and see ubiquitous broadband with video as common as telephone, or even extreme broadband enabling robotics and virtual reality TV with three-dimensional touch . Robotics and genomics will eventually play a greater role in telemedicine and our lives in general . Applications for remote diagnosis in biologic warfare defense and homeland security are currently raising interest in telemedicine . Telemedicine will be combined with new technological advances such as virtual "fly-through" computerized axial tomography examinations . Instead of performing an exploratory tympanotomy, surgeons will use computer programs to "fly through" and examine all aspects of a patient's middle or even inner ear . Spectral imaging of the eardrum, larynx, or oropharynx will immediately identify bacteria without cultures, or gram stain, and potential malignancy without biopsy . By measuring fluorescence emitted from an oropharynx illuminated with a specific visible or nonvisible light spectrum, spectral imaging will be able to provide instant identification of bacteria or evidence of malignant changes . The underlying principles of a successful business model must continue to be applied, with the most critical ingredient for telemedicine's success being the filling of specific health care needs . As long as the need is there, telemedicine in otolaryngology will advance. Nucleic Acids Res, 2003 Apr 15, 31(8), 2242 - 51 Revisiting the codon adaptation index from a whole-genome perspective: analyzing the relationship between gene expression and codon occurrence in yeast using a variety of models; Jansen R et al.; Highly expressed genes in many bacteria and small eukaryotes often have a strong compositional bias, in terms of codon usage . Two widely used numerical indices, the codon adaptation index (CAI) and the codon usage, use this bias to predict the expression level of genes . When these indices were first introduced, they were based on fairly simple assumptions about which genes are most highly expressed: the CAI was originally based on the codon composition of a set of only 24 highly expressed genes, and the codon usage on assumptions about which functional classes of genes are highly expressed in fast-growing bacteria . Given the recent advent of genome-wide expression data, we should be able to improve on these assumptions . Here, we measure, in yeast, the degree to which consideration of the current genome-wide expression data sets improves the performance of both numerical indices . Indeed, we find that by changing the parameterization of each model its correlation with actual expression levels can be somewhat improved, although both indices are fairly insensitive to the exact way they are parameterized . This insensitivity indicates a consistent codon bias amongst highly expressed genes . We also attempt direct linear regression of codon composition against genome-wide expression levels (and protein abundance data) . This has some similarity with the CAI formalism and yields an alternative model for the prediction of expression levels based on the coding sequences of genes . More information is available at http://bioinfo.mbb.yale.edu/expression/codons. J Clin Microbiol, 2003 Apr, 41(4), 1726 - 9 Quantitative detection of Chlamydia psittaci and C . pecorum by high-sensitivity real-time PCR reveals high prevalence of vaginal infection in cattle; DeGraves FJ et al.; Bovine vaginal cytobrush specimens were analyzed for the presence of Chlamydia spp . by a high-sensitivity, high-specificity quantitative PCR . The 53% prevalence of low-level Chlamydia psittaci and C . pecorum genital infection detected in virgin heifers suggests predominantely extragenital transmission of Chlamydia in cattle and conforms to the high seroprevalence of anti-Chlamydia antibodies. Semin Immunol, 2003 Apr, 15(2), 113 - 9 Regulation of B cell development by Notch/RBP-J signaling; Tanigaki K et al.; RBP-J is an essential signal mediator of all four Notches in nuclei . Loss-of-function analyses clearly show the crucial roles of RBP-J in commitment of T cells versus B cells as well as MZ B cells versus Fo B cells . Such Notch/RBP-J regulation of dichotomic differentiation steps in lymphocyte is reminiscent of the development of sensory organ precursors (SOPs) in Drosophila . Studies on RBP-J conditional knockout mice that have lost MZ B cells without affecting Fo B cell functions have shown that MZ B cells play pivotal roles in immune responses to blood-borne bacteria. Carbohydr Res, 2003 Apr 22, 338(9), 879 - 85 Synthesis and structural analysis of five novel oligosaccharides prepared by glucosyltransfer from beta-D-glucose 1-phosphate to isokestose and nystose using Thermoanaerobacter brockii kojibiose phosphorylase; Okada H et al.; Five novel oligosaccharides (tetra-, penta- and hexa-saccharides) were synthesized by glucosyltransfer from beta-D-glucose 1-phosphate to isokestose (O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside) or nystose (O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-alpha-D-glucopyranoside) using Thermoanaerobacter brockii kojibiose phosphorylase . The oligosaccharides were identified as 2(2-alpha-D-glucopyranosyl)(m)isokestose; {O-alpha-D-glucopyranosyl-(1-->2)}(m)-O-{beta-D-fructofuranosyl-(2-->1)}(2)-alpha-D-glucopyranoside: m=1, 2, and 3, and 2(2-alpha-D-glucopyranosyl)(n)nystose; {O-alpha-D-glucopyranosyl-(1-->2)}(n)-O-{beta-D-fructofuranosyl-(2-->1)}(3)-alpha-D-glucopyranoside: n=1 and 2 using gas liquid chromatography analysis of the methyl derivatives, and MALDI-TOF-MS and NMR measurements of the newly formed oligosaccharides . 1H, 13C NMR signals of each saccharide were assigned using 2D-NMR techniques, including COSY, HSQC, HSQC-TOCSY, HMBC, CH(2)-selected E-HSQC, and CH(2)-selected E-HSQC-TOCSY. FEBS Lett, 2003 Apr 10, 540(1-3), 201 - 5 The products of the mitochondrial orf25 and orfB genes are FO components in the plant F1FO ATP synthase; Heazlewood JL et al.; The F(O) portion of the mitochondrial ATP synthase contains a range of different subunits in bacteria, yeast and mammals . A search of the Arabidopsis genome identified sequence orthologs for only some of these subunits . Blue native polyacrylamide gel electrophoresis separation of Arabidopsis mitochondrial respiratory chain complexes revealed intact F(1)F(O), and separated F(1) and F(O) components . The subunits of each complex were analysed by mass spectrometry and matched to Arabidopsis genes . In the F(1)F(O) complex a series of nine known subunits were identified along with two additional proteins matching the predicted products of the mitochondrial encoded orfB and orf25 genes . The F(1) complex contained the five well-characterised F(1) subunits, while four subunits in the F(O) complex were identified: subunit 9, d subunit, and the orfB and orf25 products . Previously, orfB has been suggested as the plant equivalent of subunit 8 based on structural and sequence similarity . We propose that orf25 is the plant b subunit based on structural similarity and its presence in the F(O) complex . Chimerics of orf25, orfB, subunit 9 and subunit 6 have been associated with cytoplasmic male sterility in a variety of plant species, our additional findings now place all these proteins in the same protein complex . Luminescence, 2003 Mar-Apr, 18(2), 113 - 21 Detection of viable fungal spores contaminant on documents and rapid control of the effectiveness of an ethylene oxide disinfection using ATP assay; Rakotonirainy MS et al.; Filamentous fungi are able to damage and even destroy archival and library materials . Nowadays the conventional method for detecting such micro-organisms is to put them in cultures but such methods are laborious and time-consuming . ATP methodology has been widely applied in other domains and its success on bacteria and yeast has been demonstrated . Several commercial reagent kits are available but they did not give satisfactory results on spores mould . We have elaborated new extraction strategies specific to fungi . A comparison of 42 extraction protocols of ATP from fungal spores was carried out . Extraction at 100 degrees C with DMSO 90% in a Tris-acetate-EDTA buffer proved to be the best method . The viability of cells is estimated by the determination of adenylate energy charge (EC) . We applied our method successfully on well-known species such as Aspergillus flavus, A . niger, A . fumigatus, A . versicolor, Neosartorya fischeri, Eurotium chevalieri, Penicillium chrysogenum, Chaetomium globosum and Ulocladium spp . The results suggest that the ATP bioluminescence assay provides a sensitive and time-saving method for detecting viable fungal spores . The validity of the procedure was also tested on spores killed by steam and on spores treated with ethylene oxide . We showed that EC determination could be used for a rapid control of the effectiveness of a disinfection process performed with ethylene oxide . Russ J Immunol, 1999 Oct, 4(3), 229 - 233 Current Views on the Role of Neutrophil Granulocyte System; Nesterova IV et al.; At present there is no doubt concerning the capability of neutrophil granulocytes for cooperative interaction with other immunocompetent cells and substances of different nature, which as a whole provides a wide variety of forms of interaction and makes it possible for neutrophils to occupy key positions in the regulation of the functions of other cells both by direct contact and by secretion of regulatory mediators - neutrophilokines . Neutrophils realize their functional capabilities on a background of stimulating actions in the form of intensification of migrational, adhesive capability, readjustment of metabolism for activation of phagocytic and secretory function . It is also known that many cytokines support the viability of neutrophils, preventing their apoptosis, progressing proportional to the degree of severity of the purulent-septic process . The cytokine-producing function of the neutrophils is thereby necessary for the autocrine and paracrine regulating interaction of neutrophils with surrounding cells . Together with the established role of neutrophils during inflammatory infectious processes, their capability of expressed cytotoxic action in respect to foreign and pathologically changed cells is also known . Data on a study of the neutrophil system in healthy and sick subjects provides the possibility to consider them as the main effector cells of not only inflammatory protective reactions of the organism, but also as functionally relevant effector and regulatory structural units in the case of many other forms of immune protection of the organism against viruses, bacteria, protozoa, alloantigens, allergens both at the general and at the local level. Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 18 - 23 The structure and mechanism of methanol dehydrogenase; Anthony C et al.; This is a review of recent work on methanol dehydrogenase (MDH), a pyrroloquinoline quinone (PQQ)-containing enzyme catalysing the oxidation of methanol to formaldehyde in methylotrophic bacteria . Although it is the most extensively studied of this class of dehydrogenases, it is only recently that there has been any consensus about its mechanism . This is partly due to recent structural studies on normal and mutant enzymes and partly due to more definitive work on the mechanism of related alcohol and glucose dehydrogenases . This work has also led to conclusions about the subsequent path of electrons and protons during the reoxidation of the reduced quinol form of the prosthetic group. Filaria J . 2003 Mar 12;2(1):4. Lymphoedema: Pathophysiology and management in resource-poor settings - relevance for lymphatic filariasis control programmes; Vaqas B et al.; Low cost reduction of morbidity in lymphoedema is an essential goal in the management of lymphatic filariasis . This review emphasises the role of movement and elevation, and refers to the literature on the effects of these on the venous and lymphatic system . The patient with lymphoedema becomes increasingly immobile and the affected limb is often in a permanently dependent position causing venous hypertension and resultant overloading of the failing lymphatics . The evidence that breathing exercises are important for reducing venous hypertension and inducing lymphatic flow is discussed.The contribution of a damaged epidermis to lymphatic failure is emphasised . Loss of barrier function encourages penetration of bacteria and stimulates repair mechanisms that generate cytokines, which, in turn lead to inflammation . Management programmes that improve the health of the epidermis play a part in reducing lymphatic load.In taking morbidity management of lymphoedema into the general health services there are benefits in promoting skin hygiene and self-help regimes that can ameliorate many diseases along with lymphoedema. Sci Total Environ, 2002 Dec 2, 300(1-3), 143 - 54 Implications of natural organic matter binding heterogeneity on understanding lead(II) complexation in aquatic systems; Town RM et al.; A critical analysis of data published for complexation (conditional stability constants and complexation capacities) of lead(II) by natural organic matter (NOM) is presented . The same patterns are observed for the different types of NOM, i.e . isolated humic-type substances, isolated algae- and bacteria derived ligands and cell walls, and filtered whole water systems: apparent stronger binding sites are utilised at lower metal ion loadings, progressively weaker sites contribute to metal complexation at higher loadings . Continuous binding functions should thus be used to adequately describe lead-NOM complexation in natural waters . Two important observations arising from our analysis are that: (i) the binding curves (binding affinity as a function of metal ion loading) for whole natural water samples lie between those for compounds representative of the two main NOM types, viz., isolated humic compounds and biota, and (ii) binding constants commonly used in speciation codes probably underestimate the actual extent of lead(II) binding by NOM at natural concentration levels. Sci STKE . 2003 Apr 08;2003(177):PL8. High-resolution multicolor imaging of dynamic signaling complexes in T cells stimulated by planar substrates; Bunnell SC et al.; The dynamic visualization of developing immunological synapses has been hindered by the difficulty of imaging the contact between the T cell and the antigen-presenting cell (APC) . Here, we describe a technique in which T cell responses are constrained to a planar stimulatory substrate . This approach, when used in conjunction with immunofluorescent staining procedures or fluorescent protein tags, greatly facilitates detection of the dynamic molecular rearrangements that accompany the formation of contacts and the initiation of signal transduction through the T cell receptor (TCR) . Using this method, we have observed signaling complexes of dynamically varying compositions that possess distinct fates. J Drug Target, 2002 Dec, 10(8), 579 - 84 Design and in vitro evaluation of oral colon targeted drug delivery systems for tinidazole; Krishnaiah YS et al.; The aim of the present investigation is to develop colon targeted drag delivery sytems for tinidazole using guar gum as a carrier in the treatment of amoebiasis . Fast-disintegrating tinidazole core tablets were compression-coated with 55, 65 and 75% of guar gum . All the formulations were evaluated for the hardness, drug content uniformity, and subjected to in vitro drug release studies . The amount of tinidazole released from tablets at different time intervals was estimated by HPLC method . The compression-coated formulations released < 0.5% of tinidazole in the physiological environment of stomach and small intestine . When the dissolution study was continued in simulated colonic fluids, the compression coated tablet with 55% of guar gum coat released 99% of tinidazole after degradation by colonic bacteria at the end of 24 h of the dissolution study . The compression coated tablets with 65 and 75% of guar gum coat released about 67 and 20% of tinidazole, respectively in simulated colonic fluids indicating the susceptibility of the guar gum formulations to the rat caecal contents . The results of the study show that compression coated tinidazole tablets with either 55 or 65% of guar gum coat is most likely to provide targeting of tinidazole for local action in the colon owing to its minimal release of the drug in the first 5 h of physiological environment of stomach and small intestine . The tinidazole compression coated tablets showed no change either in physical appearance, drug content or in dissolution pattern after storage at 40 degrees C/75% RH for 6 months. Tsitologiia, 2003, 45(1), 86 - 93 HIV-1 gag expression is quantitatively dependent on the ratio of native and optimized codons; Kofman A et al.; There is a significant variation of codon usage bias among different species and even among genes within the same organisms . Codon optimization, this is, gene redesigning with the use of codons preferred for the specific expression system, results in improved expression of heterologous genes in bacteria, plants, yeast, mammalian cells, and transgenic animals . The mechanisms preventing expression of genes with rare or low-usage codons at adequate levels are not completely elucidated . Human immunodeficiency virus (HIV) represents an interesting model for studying how differences in codon usage affect gene expression in heterologous systems . Construction of synthetic genes with optimized codons demonstrated that the codon-usage effects might be a major impediment to the efficient expression of HIV gag/pol and env gene products in mammalian cells . According to another hypothesis, the poor expression of HIV structural proteins even without HIV context is attributed to the so-called cis-acting inhibitory elements (INS), which are located within the protein-coding region . They consist of AU-rich sequences and may be inactivated through the introduction of multiple mutations over the large regions of gag gene . In our work, we evaluated expression of hybrid HIV-1 gag mRNAs where wild-type (A-rich) gag sequences were combined with artificial sequences . In such "humanized" gag fragments with adapted codon usage, AT-content was significantly reduced in favor of G and C nucleotides without any changes in protein sequence . We show that wild-type gag sequences negatively influence expression of gag-reporter, and the addition of fragments with optimized codons to gag mRNA partially rescues its expression . The results demonstrate that the expression of HIV-1 gag is determined by the ratio of optimized and rare codons within mRNA . Our data also indicates that some wtgag fragments counteract the influence of the other wtgag sequences, which cause the inhibition of gag expression . The presented data do not contradict the concept of INS; yet, it makes the definition of INS more complex . This supports the idea of a broader role of the selected codon usage in influencing the expression of HIV proteins in mammalian cells. Vet Clin North Am Food Anim Pract, 2003 Mar, 19(1), 93 - 108, vi Diagnosis of mastitis for therapy decisions; Sears PM et al.; Identifying specific groups of mastitis pathogens by their growth on selective agars can help identify the pathogens that are present in mastitic milk samples . This article addresses issues that are essential in making good use of diagnostic procedures to improve udder health on dairies. Appl Microbiol Biotechnol, 2003 Aug, 62(2-3), 174 - 9 Epub 2003 Apr 04. Multi parameter in vitro testing of ratjadone using flow cytometry; Burzlaff A et al.; Ratjadone, isolated from the myxobacterium Sorangium cellulosum, belongs to the family of so-called orphan ligands, which includes leptomycin, callystatin and other compounds . In previous screening tests, ratjadone revealed a growth inhibitory effect against bacteria, yeast and human cancer cells . Following these first results, ratjadone was tested on several human tumour cell lines (Jurkat, HepG2, U87-MG) and, as a control, on a non-tumour cell line (RLC18) for its mode of action . The cell analysis was carried out by flow cytometry . This comprised cell density measurements, live-dead analysis, cell-cycle analysis and detection of apoptosis . First experiments confirmed the growth inhibitory effect on any chosen tumour cell line . Following these results a dose effect relationship was monitored, confirming the high effectiveness of ratjadone against cell growth at nanomolar concentration . Cell cycle analysis has shown that ratjadone intervenes in the cell cycle by arresting the cells in G1-phase . Biological testing of additional ratjadone derivatives with changed configuration and stereochemistry, identified the pharmacophoric site of the molecule. Mol Biol Evol, 2003 May, 20(5), 821 - 30 Epub 2003 Apr 02. "Green-like" and "red-like" RubisCO cbbL genes in Rhodobacter azotoformans; Uchino Y et al.; We found that Rhodobacter azotoformans IFO 16436T contains two different cbbL genes coding form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large subunits . One gene is located within a "green-like" group of the RubisCO phylogenetic tree, and the other is located within a "red-like" group . This is the first report that one organism contains both green-like and red-like RubisCO genes . Moreover, by PCR using primers which amplify two green-like and red-like cbbL genes alternatively and dot blot hybridization, we demonstrated that Rhodobacter blasticus, Rhodobacter capsulatus, and Rhodobacter veldkampii possess only green-like cbbL genes, and Rhodobacter sphaeroides possesses only a red-like cbbL gene . In the cbbL phylogenic analysis, R . spaeroides and R . azotoformans 1 (red-like) formed a cluster within the red-like group, and R . capsulatus, R . azotoformans 2 (green-like), R . blasticus, and R . veldkampii formed a cluster within the green-like group . This suggests that red-like cbbL genes of Rhodobacter species were derived from one ancestor, and green-like cbbL genes were derived from another ancestor . On the other hand, molecular phylogeny of the bacteria indicates that R . veldkampii, which has only a green-like cbbL gene, is the earliest evolved Rhodobacter species and that R . azotoformans and R . sphaeroides, which have red-like cbbL genes, are the latest evolved . Consequently, the following hypothesis is proposed: the common ancestor of Rhodobacter had a green-like cbbL gene, the common ancestor of R . azotoformans and R . sphaeroides subsequently obtained a red-like cbbL gene by a horizontal gene transfer, and the ancestor of R . sphaeroides later lost the green-like cbbL gene. Mol Biol Evol, 2003 May, 20(5), 748 - 53 Epub 2003 Apr 02. High levels of multiple Wolbachia infection and recombination in the ant Formica exsecta; Reuter M et al.; Wolbachia bacteria are intracellular symbionts of many arthropod species . Their spread through host populations is promoted by drastic alterations imposed on their hosts' reproductive physiology . In the present study, we analyzed the association between Wolbachia strains and host mitochondrial haplotypes in a Swiss population of the ant Formica exsecta . In this species, female dispersal is extremely limited and the mitochondrial haplotypes are strongly differentiated between and within subpopulations . Our study revealed exceptionally high levels of multiple infection, with all ants harboring four or five distinct Wolbachia strains . Four of these strains were present in all ants analyzed . A fifth strain was associated with only three of the five mitochondrial haplotypes . An analysis of the Wolbachia gene wsp further revealed an unexpected high rate of recombination, with three of the five Wolbachia strains appearing to have arisen by homologous recombination. J Cell Sci, 2003 May 15, 116(Pt 10), 2039 - 50 Epub 2003 Apr 01. Cajal body proteins SMN and Coilin show differential dynamic behaviour in vivo; Sleeman JE et al.; Analysis of stable cell lines expressing fluorescently tagged survival of motor neurons protein (SMN) and coilin shows striking differences in their dynamic behaviour, both in the nucleus and during mitosis . Cajal bodies labelled with either FP-SMN or FP-coilin show similar behaviour and frequency of movements . However, fluorescence recovery after photobleaching (FRAP) studies show that SMN returns approximately 50-fold more slowly to Cajal bodies than does coilin . Time-lapse studies on cells progressing from prophase through to G1 show further differences between SMN and coilin, both in their localisation in telophase and in the timing of their re-entry into daughter nuclei . The data reveal similarities between Cajal bodies and nucleoli in their behaviour during mitosis . This in vivo study indicates that SMN and coilin interact differentially with Cajal bodies and reveals parallels in the pathway for reassembly of nucleoli and Cajal bodies following mitosis. Zhonghua Liu Xing Bing Xue Za Zhi, 2003 Jan, 24(1), 54 - 7 {Meta-analysis on Helicobacter pylori infection between sex and in family assembles}; Wan Y et al.; OBJECTIVE: To understand and analyze the infection situation of Helicobacter pylori (H . pylori) . METHODS: Extensively reviewing Chinese literature collecting the related with electronic documents in combination with manual retrieve and using Meta-analysis to do a quantitative analysis . RESULTS: Slight difference in the infection rate of H . pylori between men and women (95% CI: 0.0579-0.0963) was noticed . The infection rate of H . pylori in children whose parent was positive with infection of this bacteria was higher than that of children whose parent was negative (95% CI: 0.3378-0.5042) . CONCLUSION: The infection rate of H . pylori showed gender difference with nature of family aggregation . Epidemiological studies of H . pylori was comprehensive and involved many aspects . Further investigation needs to be focused on infection rate and other risk factors. Comb Chem High Throughput Screen, 2003 Mar, 6(2), 147 - 54 Rapid identification of allergen-encoding cDNA clones by phage display and high-density arrays; Kodzius R et al.; We describe a high-throughput, quantitative technology for fast identification of all different clones present in selectively enriched phage surface-displayed cDNA libraries . The strategy is based on a combination of phage display and high-density arrays . To demonstrate the utility of the method cDNAs of Aspergillus fumigatus cloned into phagemid pJuFo were expressed on the tip of filamentous M13 phage and affinity-selected on solid phase-immobilized serum IgE from allergic patients . Enriched phagemid libraries were amplified in bacteria, plated and arrayed into 384-well microtitre plates by robotic colony picking . cDNA inserts were amplified by high-throughput PCR and gridded onto high-density filter membranes . Filters were iteratively probed with randomly-sequenced inserts until all clones were identified . Eighty-one different sequences encoding IgE-binding proteins likely to cover a large part of the allergen repertoire of the mould were found . This approach represents a widely applicable method for rapid high-throughput identification of all individual cDNAs present in selectively enriched libraries. Scand J Gastroenterol, 2003 Feb, 38(2), 147 - 52 Helicobacter pylori inhibits activity of cdc2 kinase and delays G2/M to G1 progression in gastric adenocarcinoma cell line; Chiou CC et al.; BACKGROUND: Helicobacter pylori is a bacterial pathogen strongly associated with ulcer diseases and gastric cancer . The bacterial-induced alteration of cell-cycle control in host cells may play a role in the pathogenetic mechanisms . The aims of this study were to define the effect of H . pylori on the G2/M to G1 transition in a gastric cell line . METHODS: Cultured gastric cells, AGS, were synchronized in the S/early G2 phase and treated with intact H . pylori . The cell-cycle distribution of AGS cells was determined by flow cytometry . The activity of cdc2 kinase, as well as of some parameters that affect the kinase activity, was also examined . RESULTS: H . pylori delays cell-cycle progression at the G2/M phase in AGS cells . The G2/M delay was associated with reduced activity of cdc2 kinase . Both down-regulation of cell-cycle regulators (p34cdc2, cyclin B1 and cdc25C) and decreased association between p34cdc2 and cyclin B1 were found to be associated with the activity of cdc2 kinase abated after the H . pylori infection . In addition, the H . pylori-induced G2/M delay required direct contact between the bacteria and host cells . CONCLUSIONS: H . pylori inhibits G2/M to G1 progression and causes a reduction of cell division in gastric epithelial cells. Dev Biol (Basel), 2002, 111, 259 - 72 Standardisation of cell lines; Stacey GN; Standardisation of cell-based assays is significantly enhanced through the preparation and quality control of master and working cell banks . Validation of the cell substrate for use in an assay must address not only the assay conditions but also aspects such as culture passage level, pre-assay preparation and seeding of cells and pre-use validation of "critical reagents". Drug News Perspect, 2002 Jul, 15(6), 358 - 363 DNA Containing Bioactive CpG Motifs Promote Angiogenesis; Klinman DM et al.; Unmethylated CpG motifs exist at high frequency in the genomes of many bacteria and viruses, including the herpes simplex virus (HSV) . These motifs are intrinsically bioactive, stimulating cells of both the immune and central nervous systems . Researchers explored the notion that DNA containing CpG motifs could mediate additional effects in vivo, focusing on whether CpG DNA could directly or indirectly promote the formation of new blood vessels (angiogenesis) . They found that both purified HSV DNA and synthetic oligodeoxynucleotides (ODN) containing CpG motifs stimulated new blood vessel formation . This effect was associated with the production of vascular endothelial growth factor (VEGF) and was prevented by the co-administration of anti-VEGF antibodies . Results of the study suggest that bioactive CpG motifs induce the production of VEGF, thereby promoting angiogenesis, and raise the possibility that CpG ODN may be of benefit in clinical states that require revascularization . (c) 2002 Prous Science . All rights reserved. Drug News Perspect, 2002 Oct, 15(8), 535 - 542 Tuberculosis: Resurgence of a Historic Scourge; Borchardt JK; Tuberculosis is the most widespread and persistent human infection in the world . There are an estimated 8.5 million new cases annually with an annual death toll of nearly 3 million . Pharmaceutical companies continue to search for effective treatments of tuberculosis, as many strains of the bacteria have developed a resistance to first-line drugs currently used to treat the disease . Historical aspects as well as recent developments of tuberculosis are discussed . (c) 2002 Prous Science . All rights reserved. Science, 2003 Apr 4, 300(5616), 142 - 5 Rapid actin transport during cell protrusion; Zicha D et al.; Transformed rat fibroblasts expressing two variants of green fluorescent protein, each fused to beta-actin, were used to study actin dynamics during cell protrusion . The recently developed FLAP (fluorescence localization after photobleaching) method permits the tracking of one fluorophore after localized photobleaching by using the other as a colocalized reference . Here, by visualizing the ratio of bleached to total molecules, we found that actin was delivered to protruding zones of the leading edge of the cell at speeds that exceeded 5 micrometers per second . Monte Carlo modeling confirmed that this flow cannot be explained by diffusion and may involve active transport.
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