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