The origin of Borneo's elephants is controversial. Two competing hypotheses argue that they are either indigenous, tracing back to the Pleistocene, or were introduced, descending from elephants imported in the 16th–18th centuries. Taxonomically, they have either been classified as a unique subspecies or placed under the Indian or Sumatran subspecies. If shown to be a unique indigenous population, this would extend the natural species range of the Asian elephant by 1300 km, and therefore Borneo elephants would have much greater conservation importance than if they were a feral population. We compared DNA of Borneo elephants to that of elephants from across the range of the Asian elephant, using a fragment of mitochondrial DNA, including part of the hypervariable d-loop, and five autosomal microsatellite loci. We find that Borneo's elephants are genetically distinct, with molecular divergence indicative of a Pleistocene colonisation of Borneo and subsequent isolation. We reject the hypothesis that Borneo's elephants were introduced. The genetic divergence of Borneo elephants warrants their recognition as a separate evolutionary significant unit. Thus, interbreeding Borneo elephants with those from other populations would be contraindicated in ex situ conservation, and their genetic distinctiveness makes them one of the highest priority populations for Asian elephant conservation. Comparison between DNA sequences of Borneo elephants with those of other Asian elephants settles a longstanding dispute about the origins of these endangered animals Elephants have a very limited distribution in Borneo, being restricted to approximately 5% of the island in the extreme northeast . There aPopular belief holds that elephants presented to the Sultan of Sulu in 1750 by the East India Trading Company and subsequently transported to Borneo founded the current population . These aConversely, if elephants occurred naturally on Borneo, they would have colonised the island during Pleistocene glaciations, when much of the Sunda shelf was exposed  and the Elephas maximus borneensis) based on morphological differences from other populations  to 0.020 (haplotype BE), with a mean of 0.014. Assuming a nucleotide substitution rate of 3.5% per million years for the elephant mtDNA d-loop , including the hypervariable left domain of the d-loop , from 20A d-loop , the obsEMX-2) to 11.0 (LafMS03) ; the average number of alleles across loci, per population (excluding Borneo), from 2.0 (Sumatra) to 3.6 (Sri Lanka) ; the observed heterozygosity H0 across all populations (excluding Borneo) from 0.38 (EMX-4) to 0.63 (LafMS03) ; and gene diversity from 0.39 (EMX-4) to 0.69 (LafMS03) . Comparatively, all indices demonstrated very low genetic diversity in the Borneo population: proportion of polymorphic loci, 0.4; number of alleles per locus, 1–2 ; gene diversity, 0–0.13 ; heterozygosity H0 = 0–0.07 . The number of alleles, observed heterozygosity, and gene diversity, averaged across Asian elephant populations, were all higher than those in Borneo, at all loci  , and maximum-likelihood ratios of the most-likely (Borneo) to the next-most-likely population ranged from 2.97 to 48.20 . Borneo was significantly more likely to be the source than any other population for all five genotypes, since each of the assignment likelihoods to Borneo fell outside the upper end of the corresponding distribution of assignment likelihoods to the other populations. Assignment likelihoods to the putative Indian, Sumatran, and peninsular Malaysian source populations were very small , indicating that Borneo's genotypes were highly unlikely to have originated from any of these populations.We also genotyped 15 Borneo elephants for five polymorphic autosomal microsatellite loci  and compall loci . Similarall loci . Five un, 0.034) . In test, 0.034)  or mode-, 0.034) , charactmtDNA evidence supports an indigenous hypothesis in three ways. First, this hypothesis assumes an ancient, independent evolution of Borneo's elephants, resulting in the unique, divergent Borneo haplotype(s), as we observed. Conversely, the introduction hypothesis assumes an introduction at 500 years ago or less, which approximates zero time on a scale of mtDNA d-loop evolution, and hence requires Borneo and source population haplotypes to be identical. This was not observed. Second, the estimated divergence time between the Borneo haplotype and other Asian elephant haplotypes is concordant with a mid- to late-Pleistocene isolation of elephants on Borneo and the vicariant history of the island . Third, Microsatellite data also support the indigenous hypothesis. If the Borneo population originated from animals introduced in the 16th–18th centuries, it would have reached its mid-20th-century size of approximately 2,000 individuals  in fewerF statistics  sequences from zoo animals in the United States were used as an out-group. Genetic distances among sequences were calculated using uncorrected p distances. Maximum-parsimony analysis was conducted using a heuristic search with random stepwise addition of taxa, tree bisection/reconnection branch swapping, and equal weighting; neighbour joining, with Kimura two-parameter distances; and maximum likelihood, using empirical base frequencies and estimated values for the shape parameter for among-site rate variation and transition/transversion ratios. A network of haplotypes was created using statistical parsimony in the software TCS version 1.13 . Sequences were deposited in GenBank (accession numbers AY245538 and AY245802 to AY245827). Phylogenetic analyses were conducted using PAUP* version 4.0 . Three Aion 1.13 .EMX-1 to EMX-4 (LafMS03 (Samples were screened with five published microsatellite loci, to EMX-4  and LafM(LafMS03 . Forward(LafMS03 , and 1 μ(LafMS03 .STF, using the program Arlequin version 2 (Deviations from Hardy–Weinberg equilibrium for each locus and population were tested using the exact Hardy–Weinberg test as implemented in GENEPOP 3.2 , with thersion 2 .Evidence for a recent bottleneck in the Borneo population in terms of a heterozygote excess  or a modAssignment tests were performed using WHICHRUN version 4.1 . AssuminThe GenBank accession numbers for the sequences reported in this paper are AY245538 and AY245802 to AY245827.


However, the significance of an increase in cAMP produced by α1A-AR stimulation on proliferation, hypertrophy and differentiation in these cells is not known.Fibroblasts, as connective tissue cells, are able to transform into another cell type including smooth muscle cells. α1A-AR in rat-1 fibroblasts by phenylephrine (PE) inhibited DNA synthesis by 67% and blocked the re-entry of 81% of the cells into S phase of the cell cycle. This cell cycle blockage was associated with hypertrophy characterized by an increase in protein synthesis (64%) and cell size. Elevation of cAMP levels decreased both DNA and protein synthesis. Inhibition of adenylyl cyclase or protein kinase A reversed the antiproliferative effect of cAMP analogs but not PE; the hypertrophic effect of PE was also not altered. The functional response of rat-1 cells to PE was accompanied by increased expression of cyclin-dependent kinase (Cdk) inhibitors p27kip1 and p21cip1/waf1, which function as negative regulators of the cell cycle. Stimulation of α1A-AR also upregulated the cell cycle regulatory proteins pRb, cyclin D1, Cdk 2, Cdk 4, and proliferating cell nuclear antigen. The antiproliferative effect of PE was blocked by p27kip1 antisense but not sense oligonucleotide. PE also promoted expression of smooth muscle cell differentiation markers  as well as the muscle development marker MyoD.Activation of the α1A-AR promotes cell cycle arrest, hypertrophy and differentiation of rat-1 fibroblasts into smooth muscle-like cells and expression of negative cell cycle regulators by a mechanism independent of the cAMP/PKA signaling pathway.Stimulation of α Both pharmacological and molecular cloning studies have indicated the existence of multiple subtypes of α1-ARs including α1A/C-AR, α1B-AR, and α1D-AR thymidine. PE decreased basal [3H]thymidine incorporation in a concentration-dependent manner with a maximum effect at 10 μM PE , even after 48 h of serum-deprivation , a β-AR antagonist , had no effect on the decrease in basal [3H]thymidine or the increase in [3H]leucine incorporation in rat-1 cells caused by exposure to PE. These results show that PE stimulates protein synthesis and inhibits DNA synthesis in rat-1 cells through activation of α1A-ARs. The hypertrophy index, an indicator of cellular hypertrophy, is defined as the ratio of protein synthesis ([3H]leucine incorporation) to DNA synthesis ([3H]thymidine incorporation) thymidine (20 Ci/mmol) was purchased from NEN Life Science Products, Inc. . L- leucine from Amercham Pharmacia Biotech. . L-phenylephrine hydrochloride, penicillin, streptomycin, prazosin, propranolol, epidermal growth factor (EGF), and propidium iodide were obtained from Sigma . Forskolin (FN), 8-cpt-cAMP and SQ 22536 were purchased from Calbiochem . 2'-5'dideoxyadenosine and 2'-deoxyadenosine 3'-monophosphates were purchased from Biomol . G418 sulfate from Invitrogen . Hanks' balanced salt solution and fetal bovine serum were from Mediatech, Inc. . Dulbecco's Modified Eagle's Medium (DMEM) and trypsin/EDTA were obtained from Life Technologies Inc. .thymidine incorporation. Inhibition of [3H]thymidine incorporation was used as a quantitative measure of reduction in DNA synthesis. Cells were serum-starved in the presence or absence of agonists for the indicated time period, and [3H]thymidine (0.5 μCi/ml) was added for the last 4 h of the incubation. This time period was chosen because it gave the maximum incorporation of [3H]thymidine into DNA after addition of agonists. The medium was then removed, and the cells were washed twice with phosphate-buffered saline and three times with 10% ice-cold trichloroacetic acid. Cells were kept in contact with trichloroacetic acid for 10 min during each wash. The precipitated material was dissolved in 1 M NaOH containing 0.1% SDS, and radioactivity was determined by liquid scintillation spectrometry.Cells at a density of 150,000 cells/well were seeded in DMEM containing 10% FBS on 24-well plates until they reached 80–90% confluency and were serum-deprived for 48 h prior to the addition of agonists. Rat-1 cells, even after serum-deprivation for 2 days, exhibit a measurable level of [3H]leucine was added for the last 4 h of each incubation with or without the indicated concentrations of the agonists. Cells were fixed and washed three times with ice-cold trichloroacetic acid (10%). The precipitated material was solubilized with 0.1 M NaOH and the incorporated radioactivity was counted by liquid spectrometry.Rat-1 cells were seeded at a density of 150,000 cells/well in 24-well plates and just prior to confluency were serum-deprived for 48 h. Cells were rinsed with DMEM and 1 μCi/ml  × 100.Rat-1 cells resuspended in culture medium were seeded on six-well plates . Preconfluent cells were serum-deprived for 2 days, pre-incubated with inhibitor (s) for 30 min, and then stimulated with 5 μM PE and /or 1 μM FN for the indicated time. Cells were rinsed twice in PBS to remove non-adherent cells and then fixed for 10 min in [1:1] methanol-acetone mixture at room temperature. Cells were washed once in distilled water and stained in hematoxylin solution  for 15 min at room temperature. Cells were again washed three times in water, air dried and were finally observed under a phase contrast microscope. Images were captured and saved as TIFF files. For each condition, data were collected by random observation. Hypertrophic phenotype was defined as both enlarged, elongated and spindle-shaped whereas rat-1 fibroblasts are round and/or polygonal-shaped cells.kip1 experiments, phosphorothionate oligonucleotides (ODN)  were used. The antisense ODN sequence used in the experiments was 5'-CACTCTCACGTTTGACAT-3' (nuc 1–18 of rat p27 kip1); the sense ODN sequence was 5'-ATGTCAAACGTGAGAGTG. ODN transfection was performed with oligofectamine in Opti-MEM according to the manufacturer's protocol . Cells were left in the transfection mixture for 48 h and then incubated for 24 h with or without agonists and harvested for Western blot, or labeled with [3H]thymidine to determine DNA synthesis.To determine whether protein kinase A (PKA) mediates the antiproliferative effect of PE, rat-1 cells were transiently transfected with πLXX-PKI ,24, a plkip1, p21waf1/cip1, pRb ; caldesmon (smooth muscle), myosin (smooth muscle), α-smooth muscle actin, β-actin (Sigma Biosciences); MyoD, cyclin D1, Cdk 2, Cdk 4, PCNA, lamin A/C or vimentin (Santa Cruz Biotechnology). Following incubation, the membranes were washed three times 10 min each in TBST and incubated with a secondary antibody coupled to peroxidase for 1 h at room temperature. Then the membranes were washed three times 10 min each in TBST. Specific proteins were detected by enhanced chemiluminescence  according to the manufacturer's instructions and analyzed with an Alpha Innotech Fluorochem imaging system (Packard Canberra). A lysate from cultured aortic vascular smooth muscle cell (VSMC) was used as a control for the expression of smooth muscle markers.Cells were rinsed twice with PBS and lysed in ice-cold lysis buffer . Lysates were centrifuged at 4°C for 15 min in a microfuge at maximum speed, and the supernatant was collected for Western blot analysis. Equal amounts of protein were separated on denaturing SDS/polyacrylamide gel and transferred on nitrocellulose blots  by electrophoresis. Blots were blocked for 1 h in 5% nonfat dry milk in TBST, washed three times 5 min each in TBST and then incubated with the indicated specific primary antibody overnight at 4°C: p273H]thymidine and/or leucine were variable in different batches of cells. However, the effect of various agents on the incorporation of [3H]thymidine and [3H]leucine in rat-1 cells was consistent within each batch of cells. The results are expressed as mean ± SEM. The data were analyzed by one-way analysis of variance; the Newman-Keuls multiple range test was applied to determine the differences among multiple groups, the unpaired Student's t-test was applied to determine the difference between two groups. The null hypothesis was rejected at p < 0.05. The protein level were estimated by densitometric analysis of the Western blots and performed on the indicated number of blots using NIH Image software, and expressed as a percentage of the control, arbitrarily chosen as 100%.The basal values of incorporation of [AR, adrenergic receptor; Cdk, cyclin-dependent kinase; FN, forskolin; MHC, myosin heavy chain; PCNA, proliferating cell nuclear antigen; PE, phenylephrine; pRb, retinoblastoma protein; SMC, smooth muscle cellAES performed thymidine/leucine incorporation, morphological studies, cell viability, flow cytometry and Western blot analysis. JHP carried out the antisense design, transfection experiments, statistical analysis and some thymidine/leucine incorporation and Western blot analysis. KUM conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Globally ICUs are encountering emergence and spread of antibiotic-resistant pathogens and for some pathogens there are few therapeutic options available.Antibiotic in vitro susceptibility data of predominant ICU pathogens during 2000–2 were analyzed using data from The Surveillance Network (TSN) Databases in Europe , Canada, and the United States (US).Staphylococcus aureus isolates ranged from 19.7% to 59.4%. Penicillin resistance rates among Streptococcus pneumoniae varied from 2.0% in Germany to as high as 20.2% in the US; however, ceftriaxone resistance rates were comparably lower, ranging from 0% in Germany to 3.4% in Italy. Vancomycin resistance rates among Enterococcus faecalis were ≤ 4.5%; however, among Enterococcus faecium vancomycin resistance rates were more frequent ranging from 0.8% in France to 76.3% in the United States. Putative rates of extended-spectrum β-lactamase (ESBL) production among Enterobacteriaceae were low, <6% among Escherichia coli in the five countries studied. Ceftriaxone resistance rates were generally lower than or similar to piperacillin-tazobactam for most of the Enterobacteriaceae species examined. Fluoroquinolone resistance rates were generally higher for E. coli (6.5% – 13.9%), Proteus mirabilis (0–34.7%), and Morganella morganii (1.6–20.7%) than other Enterobacteriaceae spp (1.5–21.3%). P. aeruginosa demonstrated marked variation in β-lactam resistance rates among countries. Imipenem was the most active compound tested against Acinetobacter spp., based on resistance rates.Oxacillin resistance rates among Enterococcus spp., which were highest in the US. This data highlights the differences in resistance encountered in intensive care units in Europe and North America and the need to determine current local resistance patterns by which to guide empiric antimicrobial therapy for intensive care infections.There was a wide distribution in resistance patterns among the five countries. Compared with other countries, Italy showed the highest resistance rates to all the organisms with the exception of  Enterococcus faecium. Awareness of these problems has been underscored with data from a number of surveillance studies aimed at improving the use of empiric therapy. In the United States there have been several national programs, which have focused on both the etiology of infections and resistance patterns of nosocomial or ICU infections including the National Nosocomial Infections Surveillance (NNIS) [Antimicrobial resistance has emerged as an important factor in predicting outcomes and overall resource use after infections in intensive care units (ICU) . Globalle (NNIS)  and moree (NNIS) ,4. Stephe (NNIS) .European data on the antimicrobial resistance of ICU pathogens has also been collected in several recent surveillance studies. A large prevalence survey of nosocomial infections in ICUs in 17 countries was published in 1995 , and morThis study using TSN program reports the antimicrobial resistance profiles of bacterial isolates from ICU patients in five countries during the period 2000–2002. The relevance of these recent nation-specific data will be discussed on a country-by-country basis, as part of improving and updating empiric therapeutic approaches to specific pathogens causing infections in the ICU setting according to each country. These surveillance programs help to maintain current knowledge of susceptibilities and relevant treatment options.TSN is a queriable, real-time database that electronically assimilates daily antimicrobial susceptibility testing and patient demographic data from a network of geographically dispersed laboratories in the United States , France , Germany , Italy  and Canada  .Laboratories included in TSN include those servicing university, community, and private hospitals with bed sizes ranging from 100 to >1000 beds. Routine diagnostic susceptibility testing results are collected daily from each participating laboratory. The methods used by these laboratories include VITEK , MicroScan , Sceptor and Pasco MIC/ID  and Etest , as well as manual broth microdilution MIC, disk diffusion and agar dilution. TSN reflects current testing in participant laboratories and represents the data reported to physicians from the respective laboratories .Although some European countries have alternate breakpoints, all data forwarded to TSN Databases are derived from hospitals that utilized NCCLS standards and definitions   or the CS. aureus, coagulase negative staphylococci, Enterococcus faecalis,Enterococcus faecium, Streptococcus pyogenes, Streptococcus pneumoniae and viridans group streptococci. Gram-negative species studied comprised the predominantly encountered enteric species , and Pseudomonas aeruginosa and Acinetobacter spp.For this study, data from TSN results for each individual database from January 1, 2000 through to December 31, 2002 were included in the analysis to determine the proportion of species and their susceptibility to antimicrobial agents commonly tested in clinical laboratories throughout the participating regions. Only isolates derived from patients located in hospital ICUs were considered in the analysis. Gram-positive species included in the analysis were comprised of E. coli, putative ESBL production was defined as those isolates that were intermediate or resistant (non-susceptible) to ceftazidime [P <0.001).The antibiotics studied are listed in Tables tazidime . Given tS. aureus, being most common in three of the five countries  and P. aeruginosa (10.8%–22.3%) were the most frequent Gram-negative organisms encountered. The Gram-positive genus Enterococcus, either as E. faecalis, E. faecium or non-speciated isolates accounted for <10% of isolates in most countries with E. faecalis being the most common species <4.3%. Community-acquired respiratory pathogens such as Streptococcus pneumoniae and Haemophilus influenzae were relatively uncommon in all five countries.In vitro susceptibility data from over 220,000 isolates from ICUs in five countries over the period 2000–2002 were assimilated. The most frequent species isolated from infections in the ICU was es Table . The oxaTables E. faecalis <5%, whereas vancomycin-resistant E. faecium was more prevalent ranging from 0.8% in France to 76.3% in the United States, with a wide inter-country variation  compared with most agents having 85%–94% susceptibility in 1996–1997. Changes of 15–20% have been reported with ceftazidime, imipenem, ciprofloxacin and meropenem, while piperacillin-tazobactam has shown the smallest decrease in susceptibility with <6% over the 4-year period. Piperacillin plus or minus tazobactam and cefepime were the most active agents, based on susceptibility, against P. aeruginosa in Germany. Conversely, ceftriaxone and imipenem were the most active agents, based on susceptibility, against Klebsiella spp., which account for almost 8% of ICU isolates.In Germany the prevalence of resistance among gram-positive organisms remained comparatively low with an incidence of 21% MRSA. In 2000, Frank et al. reported that 96% of German isolates of S. aureus and coagulase-negative staphylococci spp., respectively. S. pneumoniae showed penicillin resistance of 17.9%, higher than the other four countries, although the activity of third-generation cephalosporins, ceftriaxone and cefotaxime, showed only 0.6% and 0.8% resistance, respectively. Despite a lower ceftazidime susceptibility breakpoint compared to NCCLS standards (MIC 4 μg/ml instead of 8 μg/ml) putative ESBL expression were slightly lower in France than in Germany in 2000–2002. Ceftazidime non-susceptibility rates among E. coli, K. oxytoca, and P. mirabilis were ≤ 2.2%; however, ceftazidime non-susceptibility rates among K. pneumoniae, M. morganii and S. marcescens were 7.5%, 21.4%, and 5.3%, respectively. Imipenem was active against all Enterobacteriaceae. Against P. aeruginosa and Acinetobacter spp., imipenem resistance rates were 21.4% and 3.8%, respectively. Previously, a lower imipenem resistance of 24% among French isolates of P. aeruginosa was reported [Staphylococcal species from French ICU isolates showed a high proportion of oxacillin resistance, 40.6% and 69. 9% of S. aureus and 84.8% of coagulase-negative isolates. This MRSA rate was similar to that reported by Frank et al. from bacteremic isolates in Italy; however, they reported an increase in MRSA from 25% to 55% over the period 1997 to 2001 [E. faecalis and 24.2% for E. faecium are some of the highest rates recorded in Europe, although still modest compared to rates experienced in the United States; however, teicoplanin was more active with 2.4% and 13.7% of strains being resistant, respectively. Pneumococcal resistance to penicillin and erythromycin was 7.6% and 28.1%, respectively. The impact of alterations in penicillin-binding protein that reduce penicillin susceptibility have less effect on the activity of third-generation cephalosporins such as ceftriaxone with 3.4% and cefotaxime with 4.6% resistance, respectively. S. pyogenes was fully susceptible to penicillin; however, 11.8% of isolates were resistant to clarithromycin and 23.7% were resistant to erythromycin.Among the Italian isolates of staphylococci, oxacillin resistance occurred in 59.4% of  to 2001 . VancomyE. coli showing ceftazidime non-susceptibility of 5.3%, whereas K. pneumoniae and K. oxytoca demonstrated 30.2% and 16.6% ceftazidime non-susceptibility, respectively. Fluoroquinolone resistance rates among the Enterobacteriaceae, using ciprofloxacin as a marker, varied from 3.0% for K. oxytoca to 22.7% for P. mirabilis, and 12.7% for E. coli. Thus, among Enterobacteriaceae, ciprofloxacin was generally less active than the third-generation cephalosporin, ceftriaxone. P. aeruginosa and Acinetobacter spp. strains from Italian ICUs demonstrated significant resistance rates. Isolates of P. aeruginosa showed resistance rates of >28% for all agents tested except piperacillin-tazobactam. Thus empiric therapy for possible pseudomonal infections will require combination therapy. Acinetobacter spp. showed a similar lack of susceptibility except to imipenem and meropenem (19.0% and 13.6% resistant). An increase in fluoroquinolone resistance in E. coli and K. pneumoniae in bacteremic isolates from Italy was observed during 1997–2001, with rates of 26.7% and 24%, respectively [P. aeruginosa isolates in Italy from 30% to 37% in a 4-year period [P. aeruginosa isolates.The proportion of ESBLs was slightly higher in Italy with S. aureus was noted in 19.7% and coagulase-negative staphylococci in 79.4%. Vancomycin resistance was reported among 0.9% and 14.5% of E. faecalis and E. faecium, respectively. The lowest rate of penicillin resistance in S. pneumoniae in this study was noted from Canada at 7.1%; however, clarithromycin resistance was 30.4%. Ceftriaxone showed 0.7% resistance whereas cefepime exhibited 12.0% resistance among pneumococci from the ICU.In Canada oxacillin-resistance among Klebsiella spp. and S. marcescens tested. The rate of fluoroquinolone resistance was similar to those of other countries with E. coli showing 13.9% levofloxacin resistance. Among Enterobacteriaceae, <10% of most species were resistant to third-generation cephalosporins tested with the exception of ceftazidime and M. morganii. Resistance among P. aeruginosa and Acinetobacter spp. was generally lower than in other countries apart from Germany. Only piperacillin-tazobactam showed reliable activity against P. aeruginosa (9% resistant), while resistance to all other agents was >19%. Acinetobacter spp. remained susceptible to only the carbapenems, imipenem and meropenem.Overall the susceptibility rates for Gram-negative isolates from Canadian ICUs were higher than those in the other four countries examined. A low rate of ESBLs was reported, but there was variable activity of piperacillin-tazobactam which showed >9% resistance among S. aureus and coagulase-negative species, respectively.Comparison of the data from Canadian isolates with those from the United States shows some significant differences. This demonstrates the limitations of pooling Canadian and United States data since the differences between the two regions, such as the rate of MRSA, may have some impact on empiric therapy. Data from the NNIS system has previously reported an increasing trend towards resistance within ICUs in the United States . OxacillS. aureus and very similar to the CNS data reported by the 1999 NNIS system. The NNIS highlighted a 37% increase in MRSA over the period 1994–98 to 1999, but only a 2% increase among CNS strains [E. faecalis; however, over 76% E. faecium were vancomycin non-susceptible.This value is identical to that of S. pneumoniae in the United States has acquired a range of resistance mechanisms with resistance to penicillin and the macrolides, clarithromycin and erythromycin, being common, 20.2% and 25.5%–30.5% respectively. The newer generation cephalosporins, ceftriaxone, cefotaxime and cefepime showed good activity against pneumococci, 3.2%, 6.3% and 4.5% resistant, respectively. Less than 1.0% of isolates were resistant to levofloxacin. These data are similar to other recent reports [Although streptococci are uncommon ICU pathogens they can be rapidly invasive and possibly fatal unless adequate therapeutic approaches are adopted. Enterobacteriaceae which account for approximately 30% of all isolates from ICU infections, the incidence of putative ESBLs was low in E. coli, 4.7% but ceftazidime non-susceptibility was higher in K. oxytoca 8.3%,K. pneumoniae 11.5%,S. marcescens 10.3% and M. morganii 26.4%. These data are consistent with other recent reports [Enterobacteriaceae tested, in the US for example, resistance rates were as follows, using ciprofloxacin as a marker: E. coli 10.7%, K. oxytoca 5.9%, K. pneumoniae 8.4%, M. morganii 20.7%, P. mirabilis 12.7% and S. marcescens 6.7%. These data show increased fluoroquinolone resistance compared with recent reports [For  reports . Fluoroq reports . Jones e reports .E. coli isolates from 3.3–5.5% to 10.8–11.4% [Klebsiella spp.,P. aeruginosa, and Acinetobacter spp. present significant therapeutic challenges in ICUs in the United States. With the exception of cefepime, all other tested antimicrobials demonstrated >12% resistance to P. aeruginosa, many considerably higher. Piperacillin-tazobactam showed the next lowest resistance rate, 14.4%, with all other agents having rates of 17% or higher. Non-susceptibility to ciprofloxacin among P. aeruginosa was 37.2%, higher than in the Neuberger report. Sahm et al. reported a 10% increase in fluoroquinolone resistance among P. aeruginosa in the United States, whereas resistance emerged more slowly with the other classes of antimicrobials tested [Acinetobacter infections continue to present significant therapeutic challenges due to the extensive resistance mechanisms demonstrated by the >25% resistance shown in Table Acinetobacter spp. with an 87% susceptibility rate.Specifically, enteric bacteria showed changes over this time. Fluoroquinolone resistance doubled among .8–11.4% . This st.8–11.4% , with ceEnterobacteriaceae and some staphylococci. Ceftazidime has lost potency due to the emergence of ESBL enzymes and also has diminished activity against P. aeruginosa. Piperacillin-tazobactam is generally active against P. aeruginosa in ICUs. The aminoglycoside, gentamicin has shown continued activity against most Enterobacteriaceae in all five countries, and modest activity against S. aureus but not against CNS strains. The gentamicin susceptibility of P. aeruginosa ranged from 44.0% in France to 74.0% in Germany, whereas Acinetobacter spp . showed more variable gentamicin susceptibility varying from 23.3% in Italy to 82.0% in Germany. These local data should be considered when treating infections in the ICU.There are several implications of these data. It is essential that local surveillance programs be maintained in each country's ICU setting. The local data are vital to the formulary committees as they select appropriate agents to treat infections. There are clear differences among the five countries studied in this report. Although the predominant pathogens are similar, ongoing surveillance is essential to detect the emergence of resistant species. It is clear that certain classes of compounds are losing activity against the ICU pathogens tested. For example, the fluoroquinolones have reduced susceptibility among many Gram-negative species as well as staphylococci; however, the newer class members have enhanced activity against pneumococci. Advanced-generation cephalosporins have variable activity, with ceftriaxone showing consistently good activity against the Use of agents with anti-pseudomonal activity such as cefepime, piperacillin-tazobactam or the carbapenems should preferably be reserved for patient types or infections where this pathogen is present or risk factors exist, as per the ATS Community acquired-pneumonia guidelines . A combiEnterobacteriaceae was lower than noted in other studies and appeared to be stable. The prevalence of MRSA, perhaps the most significant resistant hospital pathogen, varied among the five countries and appeared to be increasing. Parenteral cephalosporins such as ceftriaxone and cefotaxime remained quite active against Enterobacteriaceae. Up-to-date susceptibility data should be made available as rapidly as possible to physicians so that appropriate targeted empirical therapy can be instituted, this approach can assist in maintaining the activity of the current antimicrobials. While local surveillance studies remain crucial, national surveillance studies such as this can provide an invaluable data source to provide guidance in formulary decision-making.The current study confirmed the emergence of fluoroquinolone resistance among various Gram-negative species and staphylococci, which may be increasing due to the heightened use of these drugs; however the reported ESBL rates among MJ conceived the study, provided data interpretation and drafted the manuscript. DD analyzed the study data; JK and DS provided expert microbiological analysis and interpretation of study data; RW provided clinical expertise in interpretation of data and drafting manuscript. All authors read and approved the final manuscript.


To maintain the integrity of their genetic content, cells closely monitor the state of their chromosomal DNA. Any break or pairing anomaly in the double helix is perceived as damage that must be repaired. Mechanisms that sense DNA damage enlist the help of cell cycle checkpoint proteins, which stall cell division until the damage has been repaired. The exposed ends of linear chromosomes of eukaryotic cells (cells with nuclei) resemble the double-strand breaks of damaged DNA, which should make them vulnerable to unnecessary manipulations by repair enzymes. But chromosomes have special DNA sequences at their tips, called telomeres, that are coated with telomere-binding proteins that appear to protect ends from the unwanted attentions of repair enzymes. For instance, removing a telomere-specific DNA-binding protein, called TRF2, from telomeres leads to a rapid attack on chromosome tips, associated with the activation of the DNA damage pathway. The biochemical basis for TRF2's shielding effects remains obscure.PLoS Biology, Jan Karlseder et al. propose that TRF2 is a direct inhibitor of an early mediator of the DNA damage signal. Their observations offer new insights into how telomeres resist the inappropriate interventions of the DNA repair machinery.In this issue of Ataxia telangiectasia mutated (ATM) kinase is an enzyme that induces the activation of DNA repair enzymes as well as regulators of the cell cycle and apoptosis. Its enzymatic function is triggered by DNA damage sensors. Several observations suggest an antagonism between TRF2 and ATM: ATM is activated in aging cells with shortened telomeres and participates in ushering the cell into replicative senescence.  In contrast, TRF2 overexpression protects shortened telomeres from decay and delays cell entry into senescence.Here the authors examine the effect of TRF2 overexpression in cells subjected to radiation-induced DNA damage. This treatment is expected to lead to cell cycle arrest, an outcome mediated for the most part by ATM activation. Irradiated cells that overexpress TRF2, however, continue to enter cell division. Known targets of ATM's enzymatic activity are activated to a lesser degree in these cells, which harbor only about half the amount of active ATM detected in controls. This suggests that TRF2 may interfere with the DNA damage pathway early on, when ATM is activated.Do these findings reflect a direct interaction between ATM and TRF2? Karlseder et al. suggest they might. Though only a small fraction of TRF2 associates with ATM in normal cells, ATM and TRF2 proteins can form a complex in a test tube. The region of the ATM protein that interacts with TRF2 is the very region required for ATM's activation by DNA damage sensors. The authors propose that TRF2 binds to inactive ATM, and in so doing, prevents ATM's transition to an active state.The authors integrate their results in an elegant proposal that relates a cell's health to the length of its telomeres. They have previously shown that TRF2 binds another protein called Mre11, a DNA damage sensor known to activate ATM. Thus, by inhibiting ATM, TRF2 may nip in the bud any misguided attempts by Mre11 to “repair” DNA breaks in telomeres. But in aging cells, whose shortened telomeres no longer retain large amounts of TRF2, ATM activation, no longer muffled, eventually allows the onset of senescence. While TRF2 is abundant in normal cells, the authors note that its strict association with telomeres should locally limit its effect on ATM, leaving the DNA damage pathway intact at other chromosomal locations.


SLC1A2 with schizophrenia.The glutamatergic dysfunction hypothesis of schizophrenia suggests that genes involved in glutametergic transmission are candidates for schizophrenic susceptibility genes. We have been performing systematic association studies of schizophrenia with the glutamate receptor and transporter genes. In this study we report an association study of the excitatory amino acid transporter 2 gene, SLC1A2 region using the direct sequencing and pyrosequencing methods, and examined allele, genotype and haplotype association with schizophrenia.The positive finding observed in the Kyushu samples was re-examined using 100 Japanese schizophrenics and 100 controls recruited from the Aichi area.We genotyped 100 Japanese schizophrenics and 100 controls recruited from the Kyushu area for 11 single nucleotide polymorphism (SNP) markers distributed in the P = 0.013 and 0.008, respectively). After Bonferroni corrections, the two significant differences disappeared. We tested haplotype associations for all possible combinations of SNP pairs. SNP2 showed significant haplotype associations with the disease  in 8 combinations. Moreover, the significant haplotype association of SNP2-SNP7 was replicated in the cumulative analysis of our two sample sets.We found significant differences in genotype and allele frequencies of SNP2 between cases and controls (SLC1A2 in the Japanese population.We concluded that at least one susceptibility locus for schizophrenia is probably located within or nearby  G72 and NRG1 [Schizophrenia is a severe mental disorder characterized by hallucinations, delusions, disorganized thoughts, and various cognitive impairments. The life-time prevalence is about 1%, and genetic factors were known to play a critical role in its pathogenesis -4. This and NRG1 -10.In addition, other synaptic elements related to glutamate, such as excitatory amino acid transporters (EAATs), also potentially affect glutamatergic neurotransmission. EAATs maintain extracellular glutamate concentrations within physiological levels by reuptaking the synaptically released glutamate. A deficient uptake has been implicated in the pathogenesis of ischemic brain damage  and may SLC1A2) in association studies of schizophrenia. SLC1A2 has been mapped to 11p13-p12 [SLC1A2 with an average interval of 15.9 kb. To enhance the detection power of the study, we also examined the haplotype associations of the SNPs with the disease.The EAATs family consists of five members (EAAT1-EAAT5). Their cellular localizations are different: EAAT1 and EAAT2 are astroglial, whereas EAAT3 EAAT4 and EAAT5 are neuronal . Since E1p13-p12  and consBlood samples were obtained from unrelated Japanese individuals who had provided written informed consent. We used two Japanese sample sets in this study. In the first one, Kyushu samples, 100 schizophrenia patients  were recruited from hospital in the Fukuoka and Oita areas and 100 healthy unrelated controls  were recruited from the Fukuoka area. In the initial SNP selection process, we used another 16 Japanese samples which are recruited in the Fukuoka area and informed in the same way. In the second one, Aichi samples, 100 schizophrenia patients  and 100 healthy unrelated controls  were collected in the Aichi area about 600 km east of Fukuoka. All patients fulfilled the DSM-IV criteria for schizophrenia . All of . Assuming the same size of the half length of linkage disequilibrium (LD) (60 kb) as reported in Caucasians [SLC1A2. We tested 22 candidate SNPs including all of the exonic SNPs, in the 16 healthy Japanese samples by the direct sequencing method. Out of the 22 SNPs we selected the following 7 common SNPs with minor allele frequencies over 10% for further analyses: SNP1, rs1923295; SNP3, rs4534557; SNP6, rs1885343; SNP8, rs752949; SNP9, rs1042113; SNP10, rs3838796; SNP11, rs1570216. We also identified a novel SNP, SNP7, in intron 1 (conting location: 34105026). After the LD analyses described below, we noticed LD gaps (D' < 0.3) of the initial SNP set and examined additional 20 candidate SNPs. Out of the 20 SNPs, we selected the following 3 SNPs to fill the LD gaps: SNP2, rs4755404; SNP4, rs4756224; SNP5, rs1923298. The locations of the total 11 SNPs are shown in Figure We retrieved the primary SNP information from the dbSNP database ucasians , we init2, 0.2 mM of each dNTP and 0.25 U of Taq DNA polymerase. An initial denaturing step of 1 min at 94°C was followed by 30, 35 or 40 cycles of 94°C for 30 sec, appropriate annealing temperature for 30 sec and 72°C for 30 sec. A final extension step was carried out at 72°C for 7 min. The nucleotide sequences of each primer, PCR conditions and genotyping methods for each SNP are shown in Table 1 - Eleven SNPs were amplified as 11 individual fragments by PCR using the primers shown in Table 1 - 2-test (d.f. = 1). We evaluated the statistical differences in genotype and allele frequencies between cases and controls by the χ2-test (d.f. = 2) and the Fisher's exact probability test (d.f. = 1), respectively. The magnitude of LD was evaluated in D' and r2 using the haplotype frequencies estimated by the EH program, version 1.14 [To control genotyping errors, Hardy-Weinberg equilibrium (HWE) was checked in the control samples by the χion 1.14 . Statistion 1.14 . The sigSLC1A2 region with LD as described in Materials and Methods. Table 2 - P = 0.013) and allele (P = 0.008) frequencies between cases and controls. After Bonferroni corrections, these two P values became non-significance levels .We selected 11 SNPs at average interval of 15.9 kb to cover the entire D' and r2 (Table 3 - D' > 0.8) in the seven combinations: SNP4-SNP5 (D' = 0.800), SNP7-SNP8 (D' = 0.877), SNP8-SNP9 (D' = 0.925), SNP4-SNP11 (D' = 0.838), SNP5-SNP11(D' = 0.999), SNP7-SNP11 (D' = 0.816), SNP9-SNP11 (D' = 0.819). Modest LD (D' > 0.4) was observed in the combinations of adjacent SNPs except for SNP5-SNP6 (D' = 0.286) in the control samples. However, modest LD was detected in cases in the SNP5-SNP6 combination (D' = 0.497).We compared the magnitude of LD for all possible pairs of the 11 SNPs in controls by calculating P = 0.0021), SNP2-SNP4 (P = 0.0274), SNP2-SNP5 (P = 0.0054), SNP2-SNP6 (P = 0.0178), SNP2-SNP7 (P = 9.4 × 10-5), SNP2-SNP9 (P = 0.0354), SNP2-SNP10 (P = 0.0089) and SNP2-SNP11 (P = 0.0216). The combination of SNP2-SNP7 was the only one remained significant after Bonferroni correction (Pcorr = 0.0052).We constructed pairwise haplotypes for all of the 55 possible SNP pairs , nor haplotypes of SNP2-SNP7 (P = 0.084) in the second sample set, the significant haplotype association of SNP2-SNP7 was replicated in the cumulative analysis including the two sample sets (P = 5.0 × 10-4)  was observed in the combinations of neighboring SNPs except for SNP5-SNP6 (D' = 0.286) in the control samples, suggesting that there may be a recombination hot spot present in the small region (7.8 kb) between the two SNPs (Table 3 - Modest LD D' = 0.95 ~ 0.409P = 0.013) and allele (P = 0.008) frequencies of SNP2 (rs4755404) were detected . In our second sample set, the Aichi sample, no significant association of SNP2 was observed in any of the analyses of genotypes, alleles and haplotypes. Cumulative analyses of the two sample sets, however, provide the replication of the significant haplotype association of SNP2-SNP7 with schizophrenia (P = 5.0 × 10-4). The frequency of the G-C haplotype in schizophrenics (26.6%) was notably higher than in controls (5.6%), suggesting that the G-C haplotype may be a risk haplotype for schizophrenia. We observed that the G-C haplotype frequency of schizophrenics (20.0%) was only slightly higher than controls (14.2%) in the Aichi sample, suggesting a less contribution of this locus on schizophrenia pathogenesis in the Aichi sample, although no apparent difference in clinical subtypes between both sample sets studied in this paper. The positive association reported here needs to be validated in larger sample sets, and it would also be worthwhile to search for functional SNPs in the region spanning SNP2-SNP7.In pairwise haplotype association analyses, SNP2 consistently showed significant haplotype associations. The SLC1A2 in the Japanese population.We concluded that at least one susceptibility locus for schizophrenia is probably located within or nearby None declared.SNP; single nucleotide polymorphismth ednDSM-IV; dianostic and statistical manual of mental disorders, 4PCR; polymerase chain reactionHWE; Hardy-Weinberg equilibriumLD; linkage disequilibriumEAAT; excitatory amino acid transporterXD carried out genotyping, statistical analyses and drafted the manuscript: HS participated in design of this study and statistical analyses: HN, NT, NI and NO participated in collecting specimens and clinical data: YF conceived of the study and participated in its design and coordination.The pre-publication history for this paper can be accessed here: PCR primers for genotyping of SNPs in SLC1A2.Click here for fileGenotype and allele frequencies of SNPs in SLC1A2 in Kyushu samples.Click here for filePairwise linkage disequilibrium and haplotype association in SLC1A2.Click here for fileAssociation analysis of the SNP2-SNP7 haplotype using two sample sets.Click here for file


The reovirus M1 genome segment encodes the μ2 protein, a structurally minor component of the viral core, which has been identified as a transcriptase cofactor, nucleoside and RNA triphosphatase, and microtubule-binding protein. The μ2 protein is the most poorly understood of the reovirus structural proteins. Genome segment sequences have been reported for 9 of the 10 genome segments for the 3 prototypic reoviruses type 1 Lang (T1L), type 2 Jones (T2J), and type 3 Dearing (T3D), but the M1 genome segment sequences for only T1L and T3D have been previously reported. For this study, we determined the M1 nucleotide and deduced μ2 amino acid sequences for T2J, nine other reovirus field isolates, and various T3D plaque-isolated clones from different laboratories.Determination of the T2J M1 sequence completes the analysis of all ten genome segments of that prototype. The T2J M1 sequence contained a 1 base pair deletion in the 3' non-translated region, compared to the T1L and T3D M1 sequences. The T2J M1 gene showed ~80% nucleotide homology, and the encoded μ2 protein showed ~71% amino acid identity, with the T1L and T3D M1 and μ2 sequences, respectively, making the T2J M1 gene and μ2 proteins amongst the most divergent of all reovirus genes and proteins. Comparisons of these newly determined M1 and μ2 sequences with newly determined M1 and μ2 sequences from nine additional field isolates and a variety of laboratory T3D clones identified conserved features and/or regions that provide clues about μ2 structure and function.The findings suggest a model for the domain organization of μ2 and provide further evidence for a role of μ2 in viral RNA synthesis. The new sequences were also used to explore the basis for M1/μ2-determined differences in the morphology of viral factories in infected cells. The findings confirm the key role of Ser/Pro208 as a prevalent determinant of differences in factory morphology among reovirus isolates and trace the divergence of this residue and its associated phenotype among the different laboratory-specific clones of type 3 Dearing. RNA viruses represent the most significant and diverse group of infectious agents for eukaryotic organisms on earth ,2. VirtuReoviridae, which includes segmented double-stranded RNA (dsRNA) viruses of both medical (rotavirus) and economic (orbivirus) importance  are prototype members of the family iewed in -9). Reov.g., see ,11). Eac.g., see ).Previous studies have identified the reovirus λ3 protein, encoded by the L1 genome segment, as the viral RdRp ,12-14. PThe reovirus μ2 protein has been proposed as a transcriptase cofactor, but it remains the most functionally and structurally enigmatic of the eight proteins found in virions. Like λ3, μ2 is a minor component of the inner capsid, present in only 20–24 copies per particle . It is tFor this study, we performed nucleotide-sequence determinations of the M1 genome segments of reovirus T2J, nine other reovirus field isolates, and reovirus T3D clones obtained from several different laboratories. The determination of the T2J M1 sequence completes the sequence determination of all ten genome segments of that prototype strain. We reasoned that comparisons of additional M1 and μ2 sequences may reveal conserved features and/or regions that provide clues about μ2 structure and function. The findings provide further evidence for a role of μ2 in viral RNA synthesis. We also took advantage of the newly available sequences to explore the basis for M1/μ2-determined strain differences in the morphology of viral factories in reovirus-infected cells.We determined the nucleotide sequence of the M1 genome segment of reovirus T2J to complete the sequencing of that isolate's genome. T2J M1 was found to be 2303 base pairs in length (GenBank accession no. AF124519) . However, several of the T3C12 genome segments show distinguishable mobilities in polyacrylamide gels (data not shown), confirming that T3C12 is indeed a distinct isolate.The M1 genome segments of the nine other reovirus isolates examined in this study are much more closely related to those of T1L and T3D than to that of T2J  mutant tsH11.2 (Pro414→His) [e.g., [Comparisons of the twelve μ2 sequences showed eight highly conserved regions, each containing ≥ 15 consecutive residues that are identical in all of the isolates Fig. 414→His) . The pat) [e.g., ). In add14→His . 2 domains are also "α-β " proteins by this designation. The T1L and T3D μ2 hydropathy profiles were identical to each other. Both show numerous regions of similarity to the hydropathy profile of the T2J μ2. However, there also are several distinct differences between the T1L and T2J profiles . The YAD motif is located in the exonuclease region of DNA polymerase A, a region which also functions as an NTPase and enhances the rate of DNA polymerization [2 domain of tyrosine kinases was the highest score hit for the conserved regions of μ2 with FingerPRINTScan. Four of the five motifs in the 100 amino acid SH2 domain matched the μ2 sequence. The SH2 domain mediates protein-protein interactions through its capacity to bind phosphotyrosine [The MOTIF and FingerPRINTScan programs were used to compare the highly conserved regions of μ2 with other sequences in protein data banks . The results revealed that several of the conserved regions in μ2 share limited similarities with members of the DNA polymerase A family and with the SHrization . The SH2tyrosine . The protyrosine ,31 attesIn an effort to provide biochemical confirmation of the predicted variation in the different isolates' μ2 proteins, we analyzed the T1L, T2J, and T3D proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Despite the slightly larger molecular mass calculated from its sequence Table . This abWe took advantage of the new M1/μ2 sequences to extend analysis of the role of μ2 in determining differences in viral factory morphology among reovirus isolates . Sequencts mutants tsC447, tsE320, and tsG453 [ts mutants were not examined; however, [tsF556 [T3D clones from the Nibert and Cashdollar laboratories have been shown to exhibit different factory morphologies based on differences in the microtubule-binding capacities of their μ2 proteins and the presence of either serine or proline at μ2 residue 208 . We tookhowever,  have shots T3D clones newly tested for factory morphology. All clones with globular factories have a serine at μ2 position 208 whereas all those with filamentous factories have a proline there .All wild-type T3D clones with globular factories were recently derived from a Fields laboratory parent whereas all wild-type or s e.g., ,48) and  and ts T, [e.g., ,50), theboratory ). This cboratory , severaly [e.g., ,52), foly [e.g., . Since my [e.g., , but noty [e.g., , the μ2 y [e.g.,  containsSeveral types of genome-wide comparisons of T1L, T2J, and T3D have been reported previously . For thiThe types of amino acid substitutions within each of the prototype isolates' proteins were also examined. Pairwise analyses showed that most substitutions in most proteins were conservative Table . NonconsThe frequencies with which different redundant codons are used to encode certain mammalian amino acids are non-random (reviewed in ). This pReoviruses T1L, T2J, T3D, and T3C12 were Coombs and/or Nibert laboratory stocks. Other reovirus isolates were provided by Dr. T. S. Dermody (Vanderbilt University). Virus clones were amplified to the second passage in murine L929 cell monolayers in Joklik's modified minimal essential medium (Gibco) supplemented to contain 2.5% fetal calf serum (Intergen), 2.5% neonatal bovine serum (Biocell), 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 μg/ml amphotericin B, and large amounts of virus were grown in spinner culture, extracted with Freon (DuPont) or Vertrel-XF (DuPont), and purified in CsCl gradients, all as previously described ,58.All oligonucleotide primers were obtained from Gibco/BRL. Genomic dsRNA was extracted from gradient-purified virions with phenol/chloroform . Strain Sequences at the termini of each M1 segment were determined by one or both of two methods. For some isolates, sequences near the ends of the segment were determined by modified procedures for rapid amplification of cDNA ends (RACE) as previously described ,65. In a. DNAPARS (parsimony)  Fig.  and DNAMProtein sequence analyses were performed using the GCG programs in SeqWeb version 2 (Accelrys). Multiple sequence alignments were done with PRETTY. Determinations of molecular weights, isoelectric points, and residue counts were done with PEPTIDESORT. Determinations of percent identities in pairwise comparisons were done with GAP. Plots of sequence identity over running windows of different numbers of amino acids , heated to 95°C for 3–5 min, and resolved in a 5–15% SDS-PAGE gradient gel (16.0 × 12.0 × 0.1 cm)  at 5 mA Gradient-purified viral and core proteins were resolved by SDS-PAGE as described above, and sets of resolved proteins were transferred to nitrocellulose membranes with a Semi-Dry Transblot manifold (Bio-Rad Laboratories) according to the manufacturer's instructions. Transfer of all proteins was confirmed by Ponceau S staining. Nonspecific binding was blocked in TBS-T  supplemented with 5% milk proteins, and the membranes probed with polyvalent anti-μ2 antibody . Membranes were washed with TBS-T, reacted with horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories), and immune complexes detected with the enhanced chemiluminescence system (Amersham Life Sciences) according to the manufacturer's instructions.4/cm2 in 6-well plates (9.6 cm2/well) containing round glass cover slips (18 mm). Cells on cover slips were inoculated with 5 PFU/cell in phosphate-buffered saline (PBS)  containing 2 mM MgCl2. Virus was adsorbed for 1 h at room temperature before fresh medium was added. Cells were further incubated for 18–24 h at 37°C before fixation for 10 min at room temperature in 2% paraformaldehyde in PBS or 3 min at -20°C in ice-cold methanol. Fixed cells were washed with PBS three times and permeabilized and blocked in PBS containing 1% bovine serum albumin and 0.1% Triton X-100. Antibody was diluted in the blocking solution and incubated with cells for 25–40 min at room temperature. After three washes in PBS, cover slips were mounted on glass slides with Prolong (Molecular Probes). Samples were examined using a Nikon TE-300 inverted microscope equipped with phase and fluorescence optics, and images were collected digitally as described elsewhere [CV-1 cells were maintained in Dulbecco's modified Eagles medium (Invitrogen) containing 10% fetal bovine serum (HyClone Laboratories) and 10 μg/ml Gentamycin solution (Invitrogen). Rabbit polyclonal IgG against μNS  was purilsewhere . All imats mutants.PY and NDK participated equally in designing primers and determining the T2J M1 sequence; TJB, MMA, and JSLP determined the M1 sequences of the T3C12 clone and other labs' T3D clones, as well as factory morphologies of all clones; and all authors participated in writing the manuscript. MLN and KMC are the principal investigators and KMC determined the M1 sequences of the other field isolates and


Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca2+ concentration ([Ca2+]i). The [Ca2+]i-change is composed of liberation of stored Ca2+ and extracellular Ca2+-entry. The significance of the [Ca2+]i-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca2+-buffers in the cytosol indicates that a [Ca2+]i-increase is required for chemotaxis. Yet, the iplA- mutant disrupted in a gene bearing similarity to IP3-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca2+]i-transient which favours the view that [Ca2+]i-changes are insignificant for chemotaxis.Stimulation of 2+-fluxes and the effect of their disturbance on chemotaxis and development of iplA- cells. Differentiation was altered as compared to wild type amoebae and sensitive towards manipulation of the level of stored Ca2+. Chemotaxis was impaired when [Ca2+]i-transients were suppressed by the presence of a Ca2+-chelator in the cytosol of the cells. Analysis of ion fluxes revealed that capacitative Ca2+-entry was fully operative in the mutant. In suspensions of intact and permeabilized cells cAMP elicited extracellular Ca2+-influx and liberation of stored Ca2+, respectively, yet to a lesser extent than in wild type. In suspensions of partially purified storage vesicles ATP-induced Ca2+-uptake and Ca2+-release activated by fatty acids or Ca2+-ATPase inhibitors were similar to wild type. Mn2+-quenching of fura2 fluorescence allows to study Ca2+-influx indirectly and revealed that the responsiveness of mutant cells was shifted to higher concentrations: roughly 100 times more Mn2+ was necessary to observe agonist-induced Mn2+-influx. cAMP evoked a [Ca2+]i-elevation when stores were strongly loaded with Ca2+, again with a similar shift in sensitivity in the mutant. In addition, basal [Ca2+]i was significantly lower in iplA- than in wild type amoebae.We investigated Ca2+]i-transients are essential for chemotaxis and differentiation. Moreover, capacitative and agonist-activated ion fluxes are regulated by separate pathways that are mediated either by two types of channels in the plasma membrane or by distinct mechanisms coupling Ca2+-release from stores to Ca2+-entry in Dictyostelium. The iplA- strain retains the capacitative Ca2+-entry pathway and an impaired agonist-activated pathway that operates with reduced efficiency or at higher ionic pressure.These results support the view that [Ca Dictyostelium discoideum proceeds by an oriented migration of the amoebae towards a source of the attractant cAMP which is synthesized and released periodically by cells in the center of the aggregate. Stimulation with cAMP activates liberation of stored Ca2+ and extracellular Ca2+-entry [2+]i-transient i-elevation is necessary and release of stored Ca2+ is sufficient for oriented migration [2+]i-increase is essential for chemotaxis was called into question by analysis of a cell line where the iplA gene was disrupted by homologous recombination i-elevation were detected [iplA gene product is central to the regulation of [Ca2+]i and that its presence and thus the presence of an agonist-activated [Ca2+]i-increase is not necessary for proper chemotaxis and development. However, agents that interfere with IP3-receptor mediated signaling such as XestosponginC i was not detectable resulted in the conclusion that [Ca2+]i-changes were not necessary to accomplish chemotaxis [2+]i-gradient for a given response. If in Dictyostelium a [Ca2+]i-increase was necessary for chemotaxis, the presence of a Ca2+-chelator in the cytosol should impair orientation and/or migration. In a previous study, we had introduced the Ca2+-chelator BAPTA and its derivatives into the cytosol of wild type amoebae which indeed had inhibited chemotactic migration and reduced chemotactic orientation [2+-indicator Fura2-dextran to clamp [Ca2+]i and loaded the indicator into wild type and mutant cells in the absence of external CaCl2. The treatment affected chemotactic performance of wild type as well as iplA- amoebae. Lack of extracellular CaCl2 during the loading process induced strong rounding of the amoebae and loss of migration. Figure iplA- cells . These results show that not only in wild type but also in the iplA- cell line the ability to orient and to migrate in fact depends on an agonist-activated [Ca2+]i-elevation.The observation that aggregation occurred in the mutant cell line although a cAMP-activated increase in e thus reflects efflux of Ca2+ from storage compartments. Both, cAMP and arachidonic acid activated release of stored Ca2+ . The amount of Ca2+-efflux from stores after cAMP stimulation was 61% of that found in wild type cells  by thapsigargin. XestosponginC that inhibits Ca2+-uptake and activates Ca2+-release in wild type i level in iplA- amoebae was significantly lower than in wild type and amounted to an average of 36 ± 3 nM (mean ± s.e.m. of 8 determinations in 3 independent experiments) as compared to 50 ± 2 nM in wild type .In wild type cells treatment with EGTA augments responsiveness and cAMP-elicited [Car [Ca2+] . Howeverobserved . In accoobserved  this con2+-stores, we preincubated cells with 1 mM CaCl2 for 4 h. After this treatment a [Ca2+]i-elevation upon addition of cAMP was detected in 60% of wild type amoebae even at low extracellular [Ca2+] levels, i.e. when the buffer used to wash the cells had been supplemented with only 1 μM CaCl2 . Again, these conditions were not effective in iplA- cells; rather, the sensitivity of the mutant was shifted to higher Ca2+ concentrations as had been found with Mn2+-quenching experiments. We preincubated iplA- amoebae with 20 mM CaCl2 for 3 h; after washing thoroughly, a cAMP-induced [Ca2+]i-elevation in the presence of 1 mM CaCl2 was observed . Yet, this was a rare event (once in 31 determinations).For stronger loading of CaCl2 Fig. . Startinved Fig.  in 28% o2+]i-changes for chemotaxis has been questioned by Traynor et al. [iplA- mutant cell line favouring insignificance of [Ca2+]i for the chemotactic response. The authors had observed formation of fruiting bodies even though neither 45Ca2+-fluxes nor an agonist induced [Ca2+]i-elevation were detectable. The discrepancy between our view that a [Ca2+]i-elevation is necessary for a proper chemotactic response [2+]-regulation of the iplA- mutant in detail. In particular, we tested not only basal and cAMP-activated ion fluxes but also capacitative Ca2+-entry which is induced by emptying internal Ca2+-stores via preincubation of amoebae with EGTA i it is possible that the continued presence of 10 mM EGTA or CaCl2 for many hours affects basal levels of [Ca2+]i which in turn might mediate effects on gene expression as was shown for prolonged incubation of cells with BHQ i are controlled by the iplA gene product. Besides studying agonist-induced Ca2+-fluxes we also investigated capacitative Ca2+-entry and found that this type of influx was similar in mutant and wild type cell suspensions. We obtained equivalent results by testing Mn2+-quenching of Fura2-dextran fluorescence which showed that capacitative entry is independent of the iplA gene product.By using a Ca2+-sensitive electrode, we found that in the iplA- mutant the agonist cAMP and also AA did activate Ca2+-entry into intact cells. The difference between the data published by Traynor et al. [2+-fluxes that we observed was considerably lower than in wild type cells and detectable at low extracellular [Ca2+] only. The 45Ca2+-flux studies had been performed at 100 μM external CaCl2; so the fraction of 45Ca2+ entering the cells was presumably too low to be detected reliably. Moreover, we found cAMP- and AA-induced Ca2+-release from stores in cells with permeabilized plasma membranes. These data show that cAMP-induced Ca2+-release from stores in iplA- cells is functional. However, much like the Ca2+-influx, agonist-activated liberation from stores was smaller than in wild type amoebae. In line with these results are the findings using Mn2+-quenching to assay ion fluxes in intact single cells: higher doses of Mn2+ were necessary to detect influx.On the other hand, using the Car et al.  and our 2+-influx: one type being activated by emptying of the stores and sustaining capacitative Ca2+-entry which is unaffected in iplA- cells and the other one mediating agonist-induced Ca2+-fluxes, the latter being under the control of the iplA gene product. The view that there are two strictly separated ion channels seems unlikely as under conditions of emptied stores cAMP-activated Mn2+-quenching occurred in the mutant as well. (ii) The same channel(s) mediate capacitative and agonist-activated fluxes but upon stimulation with agonists it cannot be addressed properly when iplA is disrupted. This implies a role of the protein in the liberation of Ca2+ from the stores which is a prerequisite for the triggering of Ca2+-entry [2+-influx and the generation of a full [Ca2+]i-increase is impaired. The results of the experiments where stores were strongly loaded with Ca2+ prior to stimulation support this notion. In this situation release from stores should be augmented. Indeed, in both, wild type and mutant cells, cAMP-activated [Ca2+]i-elevations occurred at an extracellular [Ca2+]  There are two types of channels responsible for Ca2+-entry . In the see Fig.  where wi2+-entry upon liberation of Ca2+ from the stores. From our data we conclude that in Dictyostelium these signals are different when the stores are emptied by EGTA or by agonist-activated signaling cascades. Otherwise one cannot explain normal capacitative Ca2+- and Mn2+-influx induced by EGTA-treatment and a requirement for 100 fold higher ion concentrations to induce Mn2+-entry by cAMP. If indeed the iplA gene product constitutes an IP3-receptor like channel that is located on membranes of stores the physical coupling of the receptor to channels in the plasma membrane as a mechanism to activate extracellular Ca2+-entry [3-sensitive store but also other stores and thus exerts a much more general effect on the cells. Studies using microarrays should reveal whether the expression of other genes is affected by the absence of iplA and thus might give a clue how [Ca2+]i is regulated in the mutant although one type of Ca2+-store is malfunctional.An as yet unresolved issue is the mechanism that induces Ca2+-entry  should b2+ fluxes and regulation of Ca2+ homeostasis take place in the iplA- mutant and that chemotaxis and development of the mutant are sensitive to disturbance of the Ca2+ homeostasis. In wild type cells and in cells lacking the iplA gene changes in [Ca2+]i are necessary to orient and to migrate chemotactically; their abolition causes loss of chemotaxis towards a cAMP source. The iplA gene product exerts a crucial role in the control of basal [Ca2+]i and of agonist induced Ca2+-fluxes. It is not required to activate capacitative Ca2+-influx. Thus the mechanisms responsible for capacitative and agonist-activated Ca2+-fluxes are different.Our results show that CaFura2-dextran and Fura2 were purchased from MobiTec; cAMP was from Boehringer.D. discoideum wild type strain Ax2 and the iplA- cell lines HM1049 and HM1038 (kindly provided by Dr. D. Traynor) were cultured as described e in cell suspensions was recorded as described elsewhere i-imaging was performed at t7–t8 as described  with Fura2.Analysis of vesicular Caescribed . In brie5 cells/ml in H5-buffer were pipetted onto a glass coverslip and allowed to settle for 60 min. Chemotaxis was recorded on a video recorder for 30–45 min. Chemotaxis was also assayed in the presence of EGTA or CaCl2; then amoebae were incubated in the respective agents for 60 min before they were challenged with cAMP. Images were digitized and the behaviour of the cells was analyzed using a computer program written for this purpose. For determination of cell velocity, a square area of interest (AI) of variable size  was placed at the perimeter of the cell in the first image digitized at the beginning of the assay. In the next image  the program analyzed an area larger than the AI (this area was defined by adding a given number of pixels on each side of the AI) for a pattern that resembled that of the AI; when such a pattern was found then the AI was placed on this new spot. The difference between the position of the AI in the first image to that in the second image was expressed as a vector of a given length. The changes in cell shape during migration were compensated by updating the pattern within the AI for every consecutive image analyzed. Calibration of the system allowed to convert the sum of the vector lengths to the distance in μm that the cells had migrated at the end of the experiment and to calculate the velocity of the amoebae. To test the effect of the intracellular presence of a Ca2+-buffer on chemotaxis, cells were loaded with Fura2-dextran (5 mg/ml in the loading solution) by electroporation in the absence of added external Ca2+. The amount of indicator present in the cytosol is in the range of 2–5% of the concentration present during electroporation iCytosolic free Ca2,5-di-(t-butyl)-1,4-hydroquinone: BHQArachidonic acid: AAArea of interest: AI2+] in cell suspensions and participated in the design of the study. DFL participated in the recordings of extracellular [Ca2+] and the design of the study. KBR analyzed chemotaxis and differentiation of wild type and mutant cells. KH performed [Ca2+]i measurements. DM analyzed fluxes in partially purified storage compartments and was involved in the design of the study. CS participated in [Ca2+]i measurements, did Mn2+-flux studies, participated in the design of the study and wrote the manuscript.RS recorded extracellular [CaiplA- cells in H5-buffer. Images of cells at t5 were captured every 15 sec for 20 min.Spontaneous cell motility of wild type and Click here for file4–t5). Images of cells at t5 were captured every 15 sec for 20 min in the continued presence of EGTA. Cells were rounded and extended smaller pseudopods than under control conditions.Basal cell motility after preincubation of wild type and mutant amoebae with 10 mM EGTA for 60 min (tClick here for file2 for 80 min (t4–t5.3) is shown. Images of cells at t5.3 were captured every 15 sec for 20 min in the continued presence of CaCl2. The behaviour of treated cells was not different from control amoebae.Cell motility of wild type and mutant cells after preincubation with 10 mM CaClClick here for file


PLoS Biology, volume 2, issue 5:In Mre11 Assembles Linear DNA Fragments into DNA Damage Signaling ComplexesVincenzo Costanzo, Tanya Paull, Max Gottesman, Jean Gautier10.1371/journal.pbio.0020110DOI: Because of a labeling error, the size of the DNA fragment used throughout the experiments was reported incorrectly (reported as 1 kb). The actual size of the DNA fragment was 131 bp. The fragment corresponds to the M13 DNA sequence from nucleotide 5656 to nucleotide 5787. This difference in fragment size does not affect any of the conclusions of the paper.10.1371/journal.pbio.0020229.This correction note may be found online at DOI:


Anti-Aβ immunotherapy in transgenic mice reduces both diffuse and compact amyloid deposits, improves memory function and clears early-stage phospho-tau aggregates. As most Alzheimer disease cases occur well past midlife, the current study examined adoptive transfer of anti-Aβ antibodies to 19- and 23-month old APP-transgenic mice.We investigated the effects of weekly anti-Aβ antibody treatment on radial-arm water-maze performance, parenchymal and vascular amyloid loads, and the presence of microhemorrhage in the brain. 19-month-old mice were treated for 1, 2 or 3 months while 23-month-old mice were treated for 5 months. Only the 23-month-old mice were subject to radial-arm water-maze testing.After 3 months of weekly injections, this passive immunization protocol completely reversed learning and memory deficits in these mice, a benefit that was undiminished after 5 months of treatment. Dramatic reductions of diffuse Aβ immunostaining and parenchymal Congophilic amyloid deposits were observed after five months, indicating that even well-established amyloid deposits are susceptible to immunotherapy. However, cerebral amyloid angiopathy increased substantially with immunotherapy, and some deposits were associated with microhemorrhage. Reanalysis of results collected from an earlier time-course study demonstrated that these increases in vascular deposits were dependent on the duration of immunotherapy.The cognitive benefits of passive immunotherapy persist in spite of the presence of vascular amyloid and small hemorrhages. These data suggest that clinical trials evaluating such treatments will require precautions to minimize potential adverse events associated with microhemorrhage. The CAA observed in both Alzheimer's disease patients  and some1–42 fibrils progressed to human clinical trials where its administration was suspended due to meningoencephalitits in a subset of patients [Anti-Aβ immunotherapy has been considered as a potential treatment for Alzheimer's disease for some time ,4. Activpatients . To datepatients ,7. Both et al. [Given the adverse reactions to the active immunization, the irreversibility of such procedures and the variable antibody response to vaccines in older individuals , passiveet al.  reported28–40 IgG1; Rinat Neurosciences, Palo Alto, CA) for a period of 5 months (n = 6). The second group received weekly intraperitoneal anti-AMN antibody  injections for a period of 5 months (n = 7). Seven nontransgenic mice were also assigned to one of two groups. The first group received weekly intraperitoneal anti-Aβ antibody injections for a period of 5 months (n = 4). The second group received weekly intraperitoneal anti-AMN antibody injections for a period of 5 months (n = 3).Mice derived from APP Tg2576 mice were obtained from our breeding program at University of South Florida started in 1996 . For theFor the time course study of 1-, 2- or 3-month treatment, 22 APP-transgenic mice aged 19 months were assigned to one of four experimental groups, as described previously . The firFollowing 3 and 5 months of treatment, the mice from the 5-month study were subjected to a two-day radial-arm water-maze paradigm. The apparatus was a 6-arm maze as described previously . On day 4 (pH 7.2) for histopathology. The hemi-brains were then incubated for 24 h in 10%, 20% and 30% sucrose sequentially for cyroprotection. Horizontal sections of 25 μ thickness were collected using a sliding microtome and stored at 4°C in Dulbecco's phosphate-buffered saline with sodium azide (pH 7.2) to prevent microbial growth. A series of 8 equally spaced tissue sections 600 μ apart were randomly selected spanning the entire brain and stained using free-floating immunohistochemistry for total Aβ  as previously described [On the day of sacrifice mice were weighed, overdosed with 100 mg/kg Nembutal , and then intracardially perfused with 25 mL of 0.9% sodium chloride. Brains were rapidly removed, and the left half of the brain was immersion fixed for 24 h in freshly prepared 4% paraformaldehyde in 100 mM KPOescribed . A seconDrosophila-specific protein) and tested them for spatial navigation learning in a two-day version of the radial-arm water maze after 3 months of treatment and, using a new platform location, again after 5 months of treatment. At both testing times we found that APP-transgenic mice treated with the control antibody failed to learn platform location over two days of testing and were significantly impaired compared to the nontransgenic mice treated with either antibody  after 5 months of immunotherapy. We noted that the bulk of what remained was vascular amyloid. We then separately analyzed vascular and parenchymal deposits which revealed a near 90% reduction in parenchymal deposits (p < 0.001) but a 3–4 fold elevation of vascular Congo-red staining  and a corresponding nearly 90% decrease in parenchymal deposits .In a prior experiment examining the effects of passive anti-Aβ immunotherapy for 1, 2 or 3 months in APP-transgenic mice killed at 21 months of age , we founThese differences were readily observed examining micrographs of sections from these mice. Mice treated with control antibodies revealed occasional cortical vascular amyloid deposits . Following this initial increase in Prussian-blue staining, we observed a linear increase in staining associated with increasing duration of anti-Aβ antibody treatment , combined with less spacing of aggregate trials  to facilitate learning of platform location in the nontransgenic mice, with a clear absence of learning in the age-matched transgenic mice.ex vivo studies [A substantial reduction in total Congophilic amyloid deposits was observed in old APP-transgenic mice treated with anti-Aβ antibodies for 2 or more months. This measurement of total Congo-red staining included both parenchymal and vascular amyloid staining. When we analyzed the sections for only vascular amyloid (CAA) we found that this measure was significantly increased following 2, 3 and 5 months of anti-Aβ antibody treatment. The remaining parenchymal amyloid load was almost completely eliminated with this antibody approach. Clearly, because total amyloid load was significantly reduced not all amyloid was shifted into the vessels; but, it appears that at least some of the Congophilic material was redistributed to the vasculature. At the present time the mechanism for this redistribution is unclear. However, one possibility is that the microglia associated with the antibody-opsonized amyloid, either by phagocytosis or surface binding, and transported the material to the vasculature, possibly in an attempt to expel it. We and others have shown evidence for microglial involvement in the removal of amyloid using both intracranial anti-Aβ antibody injections ,21 and s studies ,22. HereCerebral amyloid angiopathy (CAA) is defined as the deposition of congophilic material in meningeal and cerebral arteries and arterioles , and it occurs to some extent in nearly all Alzheimer's disease patients . Severe et al. [et al. [et al. [et al. [In the current study we found a significantly increased number of microhemorrhages in the brain as detected by Prussian-blue staining, associated with the increase in CAA following passive immunization. Another transgenic mouse model of amyloid deposition, the APP23 mice, have been shown to deposit amyloid in both brain parenchyma and blood vessels and show a CAA associated increase in spontaneous cerebral hemorrhages . Moreoveet al.  showed tet al. ,28,29. W [et al.  showed t [et al. . Importa [et al.  noted th [et al.  noted thIt remains to be determined whether these observations regarding increased CAA and microhemorrhage in transgenic mice are relevant to trials of passive immunotherapy in humans. It should be noted that, in spite of extending the period of immunotherapy to 5 months, there was no discernable loss of the cognitive benefits of immunotherapy in the transgenic mice, all of whom showed increased microhemorrhage. While the observation that antibody 266 does not result in vascular leakage encourages testing of this idiotype, data from the Zurich cohort of the Aβ vaccine trial argue that brain-reactive antibodies may be important for cognitive benefits .Our opinion is that these results suggest that passive immunotherapy against Aβ should proceed with appropriate precautions taken to minimize the risk of hemorrhage  and instituting measures to detect such hemorrhages if they do occur, irrespective of the antibody specificity or proclivity for microhemorrhage in aged APP-transgenic mice.Aβ : Amyloid-beta.APP: Amyloid precursor proteinCAA: Cerebral amyloid angiopathy.IgG1: Immunoglobulin G type 1.The authors declare that they have no competing interests.DMW treated the mice, performed the behavioral analysis, processed the tissue and performed pathological analyses, and drafted the manuscript. ARojiani evaluated slides and provided expert opinion regarding CAA and microhemorrhage. ARosenthal and SS developed, produced and purified the antibodies used in the studies. MJF performed DNA extraction and PCR for genotyping of the mice. MNG oversees the breeding colony generating mice for the studies, collected samples from the mice and assisted in editing the manuscript. DM conceived the design of the study, guided data interpretation and assisted in editing the manuscript.


PLoS Biology content is used and a request for feedback from creative usersSome examples of how the  Time magazine to Al-Jazeera and Die Zeit), which demonstrates a thirst for the original scientific paper and makes a strong argument for open access. We were even more surprised by the popularity of the “issue PDF,” a whopping 72 MB file that contains the entire journal, cover to cover. The inaugural issue PDF was downloaded thousands of times within the first few weeks, even though we had printed 30,000 hard copies and distributed them widely.In the first month after our launch, the PDF of the “monkey-robot” article by Miguel Nicolelis received tens of thousands of downloads. We are not sure who downloaded the paper because we do not ask people to register at our site. We suspect, however, that its popularity is in part due to the widespread media coverage of the article  from the United States government's National Library of Medicine, which provides a standard for archiving and exchanging XML versions of published documents.Gerencia Ambiental. We hope that this will catch on—and urge anyone translating our content to let us know so that we can point others to the various language versions. We merely ask that the translators and their publishers acknowledge the authors and the source, by including a statement such as “this is a translation from the original article by Virginia Gewin published in PLoS Biology, DOI: 10.1371/journal.pbio.0000008.” Almost any translation is better than none for those excluded by language barriers, but quality control is a concern, and we are keen to collaborate with individuals or organizations who are interested in providing high-quality translations for some or all of our content on a regular basis.A barrier for many potential users is that all our content—at least for now—is in English. We are delighted to hear that some of it is already being translated into other languages for local use. The feature article on the environmental benefits and risks of genetically modified crops, for example, will be republished (in Spanish) in the Argentinian environmental magazine PLoS Biology content to readers by, for example, increasing the range of formats available at our Web site. Let us know what would help.Internet connectivity is another obstacle. We know that some of the downloads of the issue PDF were transferred onto CD-ROMs that were copied and distributed in Uganda and Cambodia, areas in which Internet access is often slow and expensive. Other copies of the PDF were being used to create local hardcopies of the journal for communal use. We are happy to support these and related efforts to bring PLoS Biology. We'd like to hear from even more. Tell us how you use PLoS Biology: your ideas might inspire others. As a way of building on the work and ideas of others, we have added a page on the PLoS Web site (www.plos.org/creative_uses) where we list some of the more creative and unusual uses of PLoS Biology. Let us know what you do with PLoS Biology, or what you'd like to do, and we'll see what we can do to make it possible.Besides the encouraging Web statistics, we have heard from many individual users since the launch of


Proliferation and apoptosis of mesangial cells (MC) are important mechanisms during nephrogenesis, for the maintenance of glomerular homeostasis as well as in renal disease and glomerular regeneration. Expression of chemokines and chemokine receptors by intrinsic renal cells, e.g. SLC/CCL21 on podocytes and CCR7 on MC is suggested to play a pivotal role during these processes. Therefore the effect of selected chemokines on MC proliferation and apoptosis was studied.Proliferation assays, cell death assays including cell cycle analysis, hoechst stain and measurement of caspase-3 activity were performed.A dose-dependent, mesangioproliferative effect of the chemokine SLC/CCL21, which is constitutively expressed on human podocytes was seen via activation of the chemokine receptor CCR7, which is constitutively expressed on MC. In addition, in cultured MC SLC/CCL21 had a protective effect on cell survival in Fas-mediated apoptosis. The CXCR3 ligands IP-10/CXCL10 and Mig/CXCL9 revealed a proproliferative effect but did not influence apoptosis of MC. Both the CCR1 ligand RANTES/CCL5 and the amino-terminally modified RANTES analogue Met-RANTES which blocks CCR1 signalling had no effect on proliferation and apoptosis.The different effects of chemokines and their respective receptors on proliferation and apoptosis of MC suggest highly regulated, novel biological functions of chemokine/chemokine receptor pairs in processes involved in renal inflammation, regeneration and glomerular homeostasis. This MCescribed .3 cells in 100 μl medium were cultured in 96-well microtitre plates for 24 hours under standard conditions to yield firmly attached and stably growing cells. Subsequently the medium was changed to 100 μl of medium containing test substances and the cells were incubated from 24 to 72 hours. After discarding the supernatants 50 μl of a 1 mg/ml solution of MTT were added. The cells were incubated for 3 hours at 37°C and then formazan crystals were dissolved by addition of 50 μl isopropanol. Absorbance was measured at 550 nm against 630 nm reference using a DYNATECH (Germany) MR7000 ELISA reader. For each experiment at least 6 wells were analyzed per experimental condition and time point.To assess proliferation we performed a MTT -2,5-diphenyl-tetrazolium bromide, Sigma, Germany) assay . AliquotApoptosis of MC was induced by Fas/CD 95 ligation as previously described . Fas/CD t test. Significant differences are indicated for p values < 0.05 (*) or < 0.01 (**), respectively.Values are provided as mean ± SEM. Statistical analysis was performed by unpaired To demonstrate that activation of CCR7 has an influence on the proliferative activity of MC MTT assays were performed as described in Methods. The CCR7 ligand SLC/CCL21 led to a concentration dependent increase of proliferation of human MC in a range from 10 to 250 ng/ml after 24 hours of stimulation Figure . In a tiCXCR3 activation had an influence on the proliferative activity of MC. As shown in Figure Incubation of human MC with various concentrations of the CCR1 ligands RANTES/CCL5  and Met-RANTES  had no effect on the proliferation of MC at an incubation period of 24 hours under standard conditions Figure . To induThe effect of CCR7 activation for MC survival in Fas/CD95-induced cell death was studied with three different methods. Flow cytometric cell cycle analysis using propidium iodide staining showed 8.3 ± 1.1% apoptotic cells under normal conditions. After serum-starvation and incubation with IFN-γ for 48 hours human MC express Fas/CD95 on surface (data not shown). Under these conditions the population with a sub-G1 DNA content was 20.4 ± 3.6% Figure . Serum-sTo investigate the effect of the CXCR3 ligands IP-10/CXCL10 and Mig/CXCL9 on Fas-induced cell death MC were grown without serum and stimulated with IFN-γ. Induction of cell death by an activating anti-Fas-antibody led to significantly increased number of apoptotic cells. Coincubation with IP-10/CXCL10 or Mig/CXCL9 had no effect on Fas-induced apoptosis of MC Figure . The datTo induce expression of chemokine receptor CCR1 cells were pretreated with a combination of IFN-γ (20 ng/ml), TNF-α (25 ng/ml) and IL-1β (10 ng/ml) for 24 hours, followed by serum-starvation and incubation with IFN-γ for 48 hours Figure . Cell deProliferation and apoptosis play a pivotal role in a variety of biological processes, such as morphogenesis during the embryonic stage, cell selection during lymphoid development, tissue repair after injury, regression of inflammation, elimination of cells at risk of developing into a tumor and lymphocyte-mediated killing . A balanApoptosis as mechanism of controlled cell death is a well-controlled process and progresses through a series of morphological and biochemical phases, including chromatin condensation and activation of proteolytic enzymes ,23. A nuIn the kidney beside the damage induced by infiltrating inflammatory cells the relationship among resident cell proliferation and apoptosis in glomeruli determines the outcome in various glomerulonephritides . SeveralIn the glomerulus a balance between endothelial, mesangial and visceral epithelial cells and their extracellular matrix is essential for structural and functional integrity. During glomerular injury function and morphology of these cells are altered. Intrinsic cell proliferation in the glomerulus is regulated by a large number of mediators and growth factors like IL-10 , insulinThe basis for the experiments performed in this work was the hypothesis that chemokines and chemokine receptors expressed by intrinsic renal cells may be involved both in the maintenance of glomerular homeostasis in normal adult human kidney and regulation of glomerular cell numbers during disease states. We previously showed constitutive expression of CCR7 protein and its ligand secondary lymphoid tissue chemokine (SLC/CCL21) in human renal tissue. In immunohistochemistry we found a clear staining pattern for SLC/CCL21 on podocytes and CCR7 on MC during nephrogenesis and in adult kidney. Also constitutive mRNA expression has been shown for CCR7 in cultured MC and for SLC/CCL21 in isolated human glomeruli. Furthermore it was demonstrated that mesangial CCR7 is functionally active since for example SCL/CCL21 induced a dose-dependent migration of MC . We therA role of the chemokine receptor CXCR3 in inflammatory glomerular disease has been proposed before by the group of Romagnani et al. since a mesangial expression of CXCR3 in biopsies from patients with mesangioproliferative glomerulonephritis could be demonstrated . We therin vitro and in vivo. The humoral response and the Th1/Th2 balance in HAF-glomerulonephritis and mesangial cell proliferation in vitro were not affected by the CCL5/RANTES analogs [The third chemokine receptor of interest was CCR1 since our group has demonstrated functionally active expression of CCR1 on human MC, inducible after stimulation with a combination of the proinflammatory cytokines TNF-α, IL-1β and IFN-γ . Futherm analogs . TherefoIn summary it is tempting to speculate that the different effects of SLC/CCL21 and IP-10/CXCL10 and Mig/CXCL9 on proliferation and apoptosis of MC represent specialized functions of chemokine receptos on non-immune cells. CCR7 could be a chemokine receptor important for the development of the glomerular architecture during ontogenesis and for maintaining glomerular homeostasis in adult human kidney. CXCR3 may have its main functions important for mesangial expansion in mesangioproliferative disease. In contrast, the chemokine receptor CCR1 and its ligands RANTES/CCL5 and Met-RANTES seems not to have a special impact for the regulation of proliferation and apoptosis on MC. CCR1 may be important for local immunomodulation especially in glomerular inflammation. The chemokines and their receptors we have analyzed seem to be part of a complex system of factors which regulate proliferation and apoptosis in kidney and therefore play a pivotal role in regulation of glomerulogenesis, during glomerular injury and in homeostatic balance in the glomerulum. Studying these locally synthesized chemokines and their interaction with corresponding receptors on non-immune cells deserves further investigation and will reveal novel chemokine/chemokine receptor functions far beyond their orignal functions in guiding inflammatory cells to sites of tissue injury.None declared.All authors were involved in experimental procedures and manuscript preparation.mesangial cell (MC); chemokines monocyte chemoattractant protein-1 (MCP-1); regulated upon activation, normal T cell expressed and secreted (RANTES/CCL5); interferon-γ (IFN-γ)-inducible protein of 10 kD (IP-10/CXCL10); monokine induced by IFN-γ (Mig/CXCL9); -2,5-diphenyl-tetrazolium bromide (MTT)The pre-publication history for this paper can be accessed here: