Ancient Systems of Sodium/Potassium Homeostasis as Predecessors of Membrane Bioenergetics.

Cell cytoplasm of archaea, bacteria, and eukaryotes contains substantially more potassium than sodium, and potassium cations are specifically required for many key cellular processes, including protein synthesis. This distinct ionic composition and requirements have been attributed to the emergence of the first cells in potassium-rich habitats. Different, albeit complementary, scenarios have been proposed for the primordial potassium-rich environments based on experimental data and theoretical considerations. Specifically, building on the observation that potassium prevails over sodium in the vapor of inland geothermal systems, we have argued that the first cells could emerge in the pools and puddles at the periphery of primordial anoxic geothermal fields, where the elementary composition of the condensed vapor would resemble the internal milieu of modern cells. Marine and freshwater environments generally contain more sodium than potassium. Therefore, to invade such environments, while maintaining excess of potassium over sodium in the cytoplasm, primordial cells needed means to extrude sodium ions. The foray into new, sodium-rich habitats was the likely driving force behind the evolution of diverse redox-, light-, chemically-, or osmotically-dependent sodium export pumps and the increase of membrane tightness. Here we present a scenario that details how the interplay between several, initially independent sodium pumps might have triggered the evolution of sodium-dependent membrane bioenergetics, followed by the separate emergence of the proton-dependent bioenergetics in archaea and bacteria. We also discuss the development of systems that utilize the sodium/potassium gradient across the cell membranes.

Host Preference of Mating Type in Pseudoperonospora cubensis, the Downy Mildew Causal Agent of Cucurbits.

The A2 mating type of Pseudoperonospora cubensis was first discovered in Israel in May 2010 on butternut gourd (Cucurbita moschata) (1). We monitored the occurrence of the A2 mating type of P. cubensis in isolates collected during May 2010 through September 2012 from downy mildew-infected cucurbit crops growing along the coastal plain of Israel. Mating type was determined by oospore production in melon leaf discs co-inoculated with sporangia of a test isolate mixed with sporangia of A1 or A2 tester isolates (2). The A1 and A2 tester isolates were maintained at 14°C (14 h light/day) by repeated inoculation of detached leaves of cucumber and pumpkin, respectively. The 29 isolates that were collected from cucumber (Cucumis sativum) were all A1. Of the 33 isolates collected from pumpkin (Cucurbita maxima), squash (C. pepo), or butternut gourd (C. moschata), 88% were A2 and 12% were A1. The host preference of mating type in P. cubensis was monitored at Bar-Ilan University farm during April to July 2012, among about 800 plants of eight cucurbit species (~100 plants per species) that were grown side-by-side in three adjacent net-houses (two 6 × 50 m and one 6 × 100 m) and exposed to natural infection. Downy mildew developed on cucumber, melon, pumpkin, squash, and butternut gourd, but not on watermelon, sponge gourd (Luffa cylindrica), or Momordica balsamina. Three-hundred and three isolates of P. cubensis were collected and tested for mating type: 123 from cucumber, 53 from melon, 30 from pumpkin, 48 from butternut gourd, and 41 from squash. The cucumber isolates expressed A1, A2, and A1A2 at a ratio of 94.3%, 3.3%, and 2.4%, respectively; the melon isolates 58.5%, 26.4%, and 15.1%; the pumpkin isolates 0%, 96.7%, and 3.3%; the butternut isolate 7.3%, 87.3%, and 5.5%; and the squash isolates 2.4%, 97.6%, and 0%, respectively. A1A2 isolates produce oospores when crossed with either A1 or A2 tester isolates. This is the first evidence suggesting a preference of A1 isolates to Cucumis spp. and of A2 isolates to Cucurbita spp. similar preference was recently observed among Chinese isolates of this pathogen (unpublished data). The mechanism(s) controlling this preference is not known. Classical genetics is currently employed to P. cubensis in order to understand if it derives from true linkage. The practical implication for downy mildew management is that growing cucumber/melon in close proximity to pumpkin/squash/butternut gourd should be avoided as it may enhance oospore production in nature. Oospores in soil were recently shown to serve as a primary source of downy mildew infection in cucumber (3). References: (1) Y. Cohen, A. E. Rubin, and M.Galperin. Plant Dis. 95:874, 2011; (2) Y. Cohen and A. E. Rubin. Eur. J. Plant Pathol. 132:577, 2012; (3) Y. J. Zhang et al. J. Phytopathol. 160:469, 2012.

First Report of the Occurrence and Resistance to Mefenoxam of Peronospora belbahrii, Causal Agent of Downy Mildew of Basil (Ocimum basilicum) in Israel.

Downy mildew in basil was first reported from Uganda in 1933 (4). In 2004, it was reported from Italy (3) and, thereafter, from other countries around the world. In Israel, the disease was first observed in November 2011 in two greenhouses located in the northern part of the Jordan Valley. Within a month, second and third outbreaks of the disease occurred simultaneously near the southwest and southeast borders of Israel, 250 km from the initial disease outbreak. By the summer of 2012, the disease had appeared throughout the country, causing major economic damage. The causal agent, identified as Peronospora belbahrii (see below), produced chlorotic lesions on leaf blades with sporangia developing on the lower leaf surfaces. Lesions gradually turn necrotic, and infected leaves abscised. Sporangia were dark purple, oval, 30.4 ± 2.9 µm long × 21.4 ± 1.7 µm wide. Sporangiophores emerged from stomatal openings in a saturated atmosphere, were hyaline, 400 to 600 µm long, dichotomously branched, with three to five branches per sporangiophore, and bore a single sporangium on each branchlet tip. Oospores, seldom seen, were brown, round, and 46.2 ± 2.8 µm in diameter. Sporangia germinated directly, each producing a single germ tube that penetrated the periclinal wall of epidermal cells. PCR assays using sporangia and infected leaves as the template, and specific BAZ primers (1), produced a 134-bp band typical of P. belbahrii (1,2). Twenty isolates, collected from 12 locations in Israel from December 2011 to September 2012, were all sensitive to mefenoxam as the isolates did not cause symptoms on 15-leaf, potted basil plants (cv. Peri, Volcani Center, Israel) that were sprayed with 10 µg mefenoxam/ml (Ridomil Gold 48%, Syngenta, Basel, Switzerland) prior to inoculation. However, one isolate collected in early October 2012 from a severely infected plant in a greenhouse at Rehov in Bet-Shaan Valley, in which the plants had been treated with mefenoxam, was resistant to mefenoxan, showing abundant sporulation on leaves of potted basil plants that had been sprayed with 1,000 µg of mefenoxam/ml prior to inoculation. To our knowledge, this is the first report of the occurrence of downy mildew in basil in Israel. This is also the first global report of resistance to mefenoxam in P. belbahrii. References: (1) L. Belbahri et al. Mycol. Res. 109:1276, 2005. (2) R. Djalali et al. Mycol. Progress 11:961, 2012. (3) A. Garibaldi et al., Plant Dis. 89:683, 2004. (4) C. G. Hansford. Rev. Appl. Mycol. 12:421, 1933.

Formation and Infectivity of Oospores of Pseudoperonospora cubensis, the Causal Agent of Downy Mildew in Cucurbits.

The oomycete Pseudoperonospora cubensis attacks members of the Cucurbitaceae, causing severe foliage damage especially to cucumber and melon. Recently, new pathotypes of this oomycete appeared in Israel (2) and Italy (1) and highly aggressive isolates appeared in the United States (3). Since oospores of P. cubensis were rarely seen and sexual propagation by oospores was never reported (4), it is assumed that it propagates clonally by sporangia. Here we report on sexual reproduction of P. cubensis under controlled conditions in the laboratory. We found that field isolates belonging to the old pathotype 3 or to the new pathotype 6 (2) inoculated singly onto detached leaves of cucurbits in growth chambers at 15 or 20°C produced no oospores, even after prolonged incubation periods. However, when sporangia of some paired field isolates were mixed together at a 1:1 ratio, similarly inoculated onto detached leaves, and incubated at 15 or 20°C, numerous oospores (up to ~300/cm2) were formed in the mesophyll within 6 to 11 days, depending on the isolates pair, the host inoculated, and temperature. Oospores were also formed at 12.5°C but not at 25°C. Oospores developed in intact plants when kept at 15 or 20°C under a humidity-saturated atmosphere during disease development. Oospores were round, light brown to brown with an average diameter of ~40 µm. Oospores were produced in Cucumis sativum (cvs. Nadiojni and Dalila) and Cucumis melo (cvs. Ananas-Yokneam and Ein-Dor) but not in Cucurbita pepo (cv. Arlika, Beiruti), C. moschata (cv. Dalorit), or C. maxima (cv. Tripoli). To verify that oospores are infective, cucumber or melon leaves containing oospores were homogenized in water. The homogenate was twice brought to dryness at 25 to 30°C in petri dishes to differentially kill the vegetative structures of the pathogen (sporangia, cystospores, zoospores, and mycelia), resuspended in water, and inoculated onto detached leaves of various cucurbits in growth chambers at 15 or 20°C. Downy mildew lesions carrying sporangia appeared within 7 to 20 days in leaves of Cucumis sativum, Cucumis melo, and C. moschata but not in C. pepo or C. maxima. The recombinant origin of the F1 offspring isolates was confirmed by mefenoxam sensitivity tests, random amplified polymorphic DNA, and simple sequence repeat analyses. F1 progeny isolates of some crosses lost pathogenicity to C. moschata or C. maxima, toward which one of their parents was pathogenic, while others gained pathogenicity to Luffa cylindrica or Citrullus lanatus toward which neither parent was pathogenic. Data confirmed that isolates of P. cubensis can mate to produce oospores, especially under constant humidity conditions; such oospores are infective to cucurbits and F1 progeny isolates show altered sensitivity to fungicides or altered host range relative to their parents. To our knowledge, this is the first report of oospore formation by P. cubensis in the laboratory and on their pathogenicity to cucurbits. Reasons for the parallel appearance of new pathotypes of P. cubensis in Israel in 2002 (2) and Italy in 2003 (1) and the reemergence of highly aggressive isolates of the pathogen in the United States in 2004 (3) are not known. They may be related to oospore production and sexual recombination in P. cubensis. References: (1) C. Cappelli et al. Plant Dis. 87:449, 2003. (2) Y. Cohen et al. Phytoparasitica 31:458, 2003. (3) G. J. Holmes et al. Am. Veg. Grower. February, 14-15, 2006. (4) A. Lebeda and Y. Cohen. Eur. J. Plant Pathol.129:157, 2011.

Sodium ion cycle in bacterial pathogens: evidence from cross-genome comparisons.

Analysis of the bacterial genome sequences shows that many human and animal pathogens encode primary membrane Na+ pumps, Na+-transporting dicarboxylate decarboxylases or Na+ translocating NADH:ubiquinone oxidoreductase, and a number of Na+ -dependent permeases. This indicates that these bacteria can utilize Na+ as a coupling ion instead of or in addition to the H+ cycle. This capability to use a Na+ cycle might be an important virulence factor for such pathogens as Vibrio cholerae, Neisseria meningitidis, Salmonella enterica serovar Typhi, and Yersinia pestis. In Treponema pallidum, Chlamydia trachomatis, and Chlamydia pneumoniae, the Na+ gradient may well be the only energy source for secondary transport. A survey of preliminary genome sequences of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Treponema denticola indicates that these oral pathogens also rely on the Na+ cycle for at least part of their energy metabolism. The possible roles of the Na+ cycling in the energy metabolism and pathogenicity of these organisms are reviewed. The recent discovery of an effective natural antibiotic, korormicin, targeted against the Na+ -translocating NADH:ubiquinone oxidoreductase, suggests a potential use of Na+ pumps as drug targets and/or vaccine candidates. The antimicrobial potential of other inhibitors of the Na+ cycle, such as monensin, Li+ and Ag+ ions, and amiloride derivatives, is discussed.

Prokaryotic genomes: the emerging paradigm of genome-based microbiology.

Comparative analysis of the complete sequences of seven bacterial and three archaeal genomes leads to the first generalizations of emerging genome-based microbiology. Protein sequences are, generally, highly conserved, with -70% of the gene products in bacteria and archaea containing ancient conserved regions. In contrast, there is little conservation of genome organization, except for a few essential operons. The most striking conclusions derived by comparison of multiple genomes from phylogenetically distant species are that the number of universally conserved gene families is very small and that multiple events of horizontal gene transfer and genome fusion are major forces in evolution.

A High Frequency of HIV-Specific Circulating Follicular Helper T Cells Is Associated with Preserved Memory B Cell Responses in HIV Controllers.

Follicular helper T cells (Tfh) play an essential role in the affinity maturation of the antibody response by providing help to B cells. To determine whether this CD4+ T cell subset may contribute to the spontaneous control of HIV infection, we analyzed the phenotype and function of circulating Tfh (cTfh) in patients from the ANRS CO21 CODEX cohort who naturally controlled HIV-1 replication to undetectable levels and compared them to treated patients with similarly low viral loads. HIV-specific cTfh (Tet+), detected by Gag-major histocompatibility complex class II (MHC-II) tetramer labeling in the CD45RA- CXCR5+ CD4+ T cell population, proved more frequent in the controller group (P = 0.002). The frequency of PD-1 expression in Tet+ cTfh was increased in both groups (median, >75%) compared to total cTfh (<30%), but the intensity of PD-1 expression per cell remained higher in the treated patient group (P = 0.02), pointing to the persistence of abnormal immune activation in treated patients. The function of cTfh, analyzed by the capacity to promote IgG secretion in cocultures with autologous memory B cells, did not show major differences between groups in terms of total IgG production but proved significantly more efficient in the controller group when measuring HIV-specific IgG production. The frequency of Tet+ cTfh correlated with HIV-specific IgG production (R = 0.71 for Gag-specific and R = 0.79 for Env-specific IgG, respectively). Taken together, our findings indicate that key cTfh-B cell interactions are preserved in controlled HIV infection, resulting in potent memory B cell responses that may play an underappreciated role in HIV control.IMPORTANCE The rare patients who spontaneously control HIV replication in the absence of therapy provide a unique model to identify determinants of an effective anti-HIV immune response. HIV controllers show signs of particularly efficient antiviral T cell responses, while their humoral response was until recently considered to play only a minor role in viral control. However, emerging evidence suggests that HIV controllers maintain a significant but "silent" antiviral memory B cell population that can be reactivated upon antigenic stimulation. We report that cTfh help likely contributes to the persistence of controller memory B cell responses, as the frequency of HIV-specific cTfh correlated with the induction of HIV-specific antibodies in functional assays. These findings suggest that T follicular help may contribute to HIV control and highlight the need for inducing such help in HIV vaccine strategies that aim at eliciting persistent B cell responses.

Beyond complete genomes: from sequence to structure and function.

Computer analysis of complete prokaryotic genomes shows that microbial proteins are in general highly conserved--approximately 70% of them contain ancient conserved regions. This allows us to delineate families of orthologs across a wide phylogenetic range and, in many cases, predict protein functions with considerable precision. Sequence database searches using newly developed, sensitive algorithms result in the unification of such orthologous families into larger superfamilies sharing common sequence motifs. For many of these superfamilies, prediction of the structural fold and specific amino acid residues involved in enzymatic catalysis is possible. Taken together, sequence and structure comparisons provide a powerful methodology that can successfully complement traditional experimental approaches.

Who's your neighbor? New computational approaches for functional genomics.

Several recently developed computational approaches in comparative genomics go beyond sequence comparison. By analyzing phylogenetic profiles of protein families, domain fusions, gene adjacency in genomes, and expression patterns, these methods predict many functional interactions between proteins and help deduce specific functions for numerous proteins. Although some of the resultant predictions may not be highly specific, these developments herald a new era in genomics in which the benefits of comparative analysis of the rapidly growing collection of complete genomes will become increasingly obvious.

The COG database: new developments in phylogenetic classification of proteins from complete genomes.

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

Simulation of scanning tunneling microscope images of 1,3-cyclohexadiene bound to a silicon surface.

Scanning tunneling microscope (STM) images of 1,3-cyclohexadiene bound to silicon are interpreted using a nonequilibrium Green's function method. The resolution of the carbon-carbon double bond for positive bias voltages but not for negative bias voltages is explained using a quasiprobability density analysis. The asymmetry in the images arises from the system's voltage dependent electronic structure. A pi* orbital is found to be responsible for the empty state STM images of the carbon-carbon double bond, which is observed experimentally. The pi orbital relevant for the opposite bias does not produce an STM image sharply localized in the bond region because the molecule induces a Si-surface dipole dependent on the bias. The dipole voltage dependence arises from molecular charging. This result emphasizes the importance of simulating the molecule as an element in an open quantum system.

Searching for drug targets in microbial genomes.

Comparative analysis of the complete genome sequences of 10 bacterial pathogens available in the public databases offers the first insights into the drug discovery approaches of the near future. Genes that are conserved in different genomes often turn out to be essential, which makes them attractive targets for new broad-spectrum antibiotics. Subtractive genome analysis reveals the genes that are conserved in all or most of the pathogenic bacteria but not in eukaryotes; these are the most obvious candidates for drug targets. Species-specific genes, on the other hand, may offer the possibility to design drugs against a particular, narrow group of pathogens.

A diverse superfamily of enzymes with ATP-dependent carboxylate-amine/thiol ligase activity.

The recently developed PSI-BLAST method for sequence database search and methods for motif analysis were used to define and expand a superfamily of enzymes with an unusual nucleotide-binding fold, referred to as palmate, or ATP-grasp fold. In addition to D-alanine-D-alanine ligase, glutathione synthetase, biotin carboxylase, and carbamoyl phosphate synthetase, enzymes with known three-dimensional structures, the ATP-grasp domain is predicted in the ribosomal protein S6 modification enzyme (RimK), urea amidolyase, tubulin-tyrosine ligase, and three enzymes of purine biosynthesis. All these enzymes possess ATP-dependent carboxylate-amine ligase activity, and their catalytic mechanisms are likely to include acylphosphate intermediates. The ATP-grasp superfamily also includes succinate-CoA ligase (both ADP-forming and GDP-forming variants), malate-CoA ligase, and ATP-citrate lyase, enzymes with a carboxylate-thiol ligase activity, and several uncharacterized proteins. These findings significantly extend the variety of the substrates of ATP-grasp enzymes and the range of biochemical pathways in which they are involved, and demonstrate the complementarity between structural comparison and powerful methods for sequence analysis.

A superfamily of metalloenzymes unifies phosphopentomutase and cofactor-independent phosphoglycerate mutase with alkaline phosphatases and sulfatases.

Sequence analysis of the probable archaeal phosphoglycerate mutase resulted in the identification of a superfamily of metalloenzymes with similar metal-binding sites and predicted conserved structural fold. This superfamily unites alkaline phosphatase, N-acetylgalactosamine-4-sulfatase, and cerebroside sulfatase, enzymes with known three-dimensional structures, with phosphopentomutase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, phosphoglycerol transferase, phosphonate monoesterase, streptomycin-6-phosphate phosphatase, alkaline phosphodiesterase/nucleotide pyrophosphatase PC-1, and several closely related sulfatases. In addition to the metal-binding motifs, all these enzymes contain a set of conserved amino acid residues that are likely to be required for the enzymatic activity. Mutational changes in the vicinity of these residues in several sulfatases cause mucopolysaccharidosis (Hunter, Maroteaux-Lamy, Morquio, and Sanfilippo syndromes) and metachromatic leucodystrophy.

Aldolases of the DhnA family: a possible solution to the problem of pentose and hexose biosynthesis in archaea.

Sequence analysis of the recently identified class I aldolase of Escherichia coli (dhnA gene product) helped to identify its homologs in Chlamydia trachomatis, Chlamydiophyla pneumoniae and in each of the completely sequenced archaeal genomes. Iterative database searches revealed sequence similarities between the DhnA-family enzymes, deoxyribose phosphate aldolases and bacterial (class II) fructose bisphosphate aldolases and allowed prediction of similar three-dimensional structures (TIM-barrel fold) in all these enzymes. The Schiff base-forming lysyl residues of DhnA and deoxyribose phosphate aldolase are conserved in all members of the DhnA and deoxyribose phosphate aldolase families, indicating that these enzymes share common features with both class I and class II aldolases. The DhnA-family enzymes are predicted to possess an aldolase activity and to play a critical role in sugar biosynthesis in archaea.

Novel domains of the prokaryotic two-component signal transduction systems.

The archetypal two-component signal transduction systems include a sensor histidine kinase and a response regulator, which consists of a receiver CheY-like domain and a DNA-binding domain. Sequence analysis of the sensor kinases and response regulators encoded in complete bacterial and archaeal genomes revealed complex domain architectures for many of them and allowed the identification of several novel conserved domains, such as PAS, GAF, HAMP, GGDEF, EAL, and HD-GYP. All of these domains are widely represented in bacteria, including 19 copies of the GGDEF domain and 17 copies of the EAL domain encoded in the Escherichia coli genome. In contrast, these novel signaling domains are much less abundant in bacterial parasites and in archaea, with none at all found in some archaeal species. This skewed phyletic distribution suggests that the newly discovered complexity of signal transduction systems emerged early in the evolution of bacteria, with subsequent massive loss in parasites and some horizontal dissemination among archaea. Only a few proteins containing these domains have been studied experimentally, and their exact biochemical functions remain obscure; they may include transformations of novel signal molecules, such as the recently identified cyclic diguanylate. Recent experimental data provide the first direct evidence of the participation of these domains in signal transduction pathways, including regulation of virulence genes and extracellular enzyme production in the human pathogens Bordetella pertussis and Borrelia burgdorferi and the plant pathogen Xanthomonas campestris. Gene-neighborhood analysis of these new domains suggests their participation in a variety of processes, from mercury and phage resistance to maintenance of virulence plasmids. It appears that the real picture of the complexity of phosphorelay signal transduction in prokaryotes is only beginning to unfold.

MHYT, a new integral membrane sensor domain.

MHYT, a new conserved protein domain with a likely signaling function, is described. This domain consists of six transmembrane segments, three of which contain conserved methionine, histidine, and tyrosine residues that are projected to lie near the outer face of the cytoplasmic membrane. In Synechocystis sp. PCC6803, this domain forms the N-terminus of the sensor histidine kinase Slr2098. In Pseudomonas aeruginosa and several other organisms, the MHYT domain forms the N-terminal part of a three-domain protein together with previously described GGDEF and EAL domains, both of which have been associated with signal transduction due to their presence in likely signaling proteins. In Bacillus subtilis YkoW protein, an additional PAS domain is found between the MHYT and GGDEF domains. A ykoW null mutant of B. subtilis did not exhibit any growth alterations, consistent with a non-essential, signaling role of this protein. A model of the membrane topology of the MHYT domain indicates that its conserved residues could coordinate one or two copper ions, suggesting a role in sensing oxygen, CO, or NO.

Dehydroepiandrosterone selectively inhibits production of tumor necrosis factor alpha and interleukin-6 [correction of interlukin-6] in astrocytes.

Dehydroepiandrosterone (DHEA) is a native neurosteroid with immunomodulating activity. DHEA effectively protects animals from several viral, bacterial and parasitic infections and it was suggested that its age-associated decline is related with immunosenescence. In the present study we examined the ability of DHEA to inhibit the production of inflammatory mediators by mycoplasma-stimulated glial cells and to change the course of acute central nervous system (CNS) inflammatory disease in vivo. Addition of DHEA (10 microg/ml) markedly inhibited tumor necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6) production (98 and 95%, respectively), whereas nitric oxide (NO) and prostaglandin E2 (PGE2) production was not affected. However, daily administration of 0.5 mg DHEA to mice or 5 mg to rats did not change the clinical outcome of experimental autoimmune encephalomyelitis (EAE).

Mycoplasma fermentans-induced inflammatory response of astrocytes: selective modulation by aminoguanidine, thalidomide, pentoxifylline and IL-10.

Exposure of primary rat glial cells, mostly astrocytes, to heat-inactivated Mycoplasma fermentans triggers the production of tumor necrosis factor alpha (TNFalpha) nitric oxide (NO) and prostaglandin E2 (PGE2). To attenuate the production of these proinflammatory mediators, four agents: aminoguanidine, pentoxifylline, thalidomide and IL-10 were added to astrocyte cultures. Aminoguanidine (1 and 3 mM), an inhibitor of inducible nitric oxide synthase (iNOS), suppressed the production of the three mediators. TNFalpha was the most sensitive to thalidomide, showing dose-response inhibition at concentrations of 20 microg/ml, 50 microg/ml and 250 microg/ml. PGE2 was affected only by concentrations of 50 microg/ml and 250 microg/ml, whereas NO responded solely to the highest amount of this inhibitor. The cytokine IL-10, at 10 U and 50 U, inhibited only TNFalpha production. Our results imply that selective suppression of proinflammatory mediators by various agents may prove feasible for amelioration of central nervous system inflammatory diseases.

[Sore throat: an analysis of medical management in a community clinic].

The diagnostic approach to sore throat and its treatment by 7 doctors was studied in 692 community clinic patients. Redness of the throat, the commonest clinical feature in all age groups, was present in 88%. The prevalence of other clinical findings varied in the different age groups. 54% of the patients, most of whom were less than 13 years old, were treated initially with antibiotics. Most of the physicians prescribed penicillin V (average, 8.4 days). From 11 (13%) of the throat cultures taken from children under the age of 4, beta hemolytic streptococci (BHS) were grown. Based on throat cultures, antibiotics had been given unnecessarily in 53% of those in whom it was initially prescribed, and wrongly withheld in 10% of those who initially did not receive them. This study reveals an urgent need for a rapid and accurate method of detecting BHS in patients with sore throat.