Individual heritable differences result in unique cell lymphocyte receptor repertoires of naïve and antigen-experienced cells.

The adaptive immune system's capability to protect the body requires a highly diverse lymphocyte antigen receptor repertoire. However, the influence of individual genetic and epigenetic differences on these repertoires is not typically measured. By leveraging the unique characteristics of B, CD4(+) T and CD8(+) T-lymphocyte subsets from monozygotic twins, we quantify the impact of heritable factors on both the V(D)J recombination process and on thymic selection. We show that the resulting biases in both V(D)J usage and N/P addition lengths, which are found in naïve and antigen experienced cells, contribute to significant variation in the CDR3 region. Moreover, we show that the relative usage of V and J gene segments is chromosomally biased, with ∼1.5 times as many rearrangements originating from a single chromosome. These data refine our understanding of the heritable mechanisms affecting the repertoire, and show that biases are evident on a chromosome-wide level.

Trauma-hemorrhagic shock induces a CD36-dependent RBC endothelial-adhesive phenotype.

OBJECTIVE: Microvascular dysfunction is a key element in the development of the multiple organ dysfunction syndrome. Although the mechanisms for this response are unclear, RBC adhesion to endothelium may initiate intravascular occlusion leading to ischemic tissue injury. Thus, we tested the hypothesis that trauma-hemorrhage induces RBC-endothelial cell adhesion. DESIGN: Prospective in vivo and in vitro animal study and analysis of patient blood samples. SETTING: University research laboratory and hospital emergency and trauma units. INTERVENTION: We initially assayed RBC adhesion to endothelial cells in vitro using RBCs obtained from rats subjected to trauma-hemorrhagic shock or sham shock as well as from severely injured trauma patients. Subsequently, we measured the role of putative RBCs and endothelial cell receptors in the increased RBC-endothelial cell adhesive response. MAIN RESULTS: In both rats and humans, trauma-hemorrhagic shock increased RBC adhesion to endothelium as well as increasing several putative RBC surface adhesion molecules including CD36. The critical factor leading to RBC-endothelial cell adhesion was increased surface RBC CD36 expression. Adhesion of trauma-hemorrhagic shock RBCs was mediated, at least in part, by the binding of RBC CD36 to its cognate endothelial receptors (αVß3 and VCAM-1). Gut-derived factors carried in the intestinal lymphatics triggered these trauma-hemorrhagic shock-induced RBC changes because 1) preventing trauma-hemorrhagic shock intestinal lymph from reaching the systemic circulation abrogated the RBC effects, 2) in vitro incubation of naïve whole blood with trauma-hemorrhagic shock lymph replicated the in vivo trauma-hemorrhagic shock-induced RBC changes while 3) injection of trauma-hemorrhagic shock lymph into naïve animals recreated the RBC changes observed after actual trauma-hemorrhagic shock. CONCLUSIONS: 1) Trauma-hemorrhagic shock induces rapid RBC adhesion to endothelial cells in patients and animals. 2) Increased RBC CD36 expression characterizes the RBC-adhesive phenotype. 3) The RBC phenotypic and functional changes were induced by gut-derived humoral factors. These novel findings may explain the microvascular dysfunction occurring after trauma-hemorrhagic shock, sepsis, and other stress states.

Sequencing Y chromosomes resolves discrepancy in time to common ancestor of males versus females.

The Y chromosome and the mitochondrial genome have been used to estimate when the common patrilineal and matrilineal ancestors of humans lived. We sequenced the genomes of 69 males from nine populations, including two in which we find basal branches of the Y-chromosome tree. We identify ancient phylogenetic structure within African haplogroups and resolve a long-standing ambiguity deep within the tree. Applying equivalent methodologies to the Y chromosome and the mitochondrial genome, we estimate the time to the most recent common ancestor (T(MRCA)) of the Y chromosome to be 120 to 156 thousand years and the mitochondrial genome T(MRCA) to be 99 to 148 thousand years. Our findings suggest that, contrary to previous claims, male lineages do not coalesce significantly more recently than female lineages.

Diverse roles and interactions of the SWI/SNF chromatin remodeling complex revealed using global approaches.

A systems understanding of nuclear organization and events is critical for determining how cells divide, differentiate, and respond to stimuli and for identifying the causes of diseases. Chromatin remodeling complexes such as SWI/SNF have been implicated in a wide variety of cellular processes including gene expression, nuclear organization, centromere function, and chromosomal stability, and mutations in SWI/SNF components have been linked to several types of cancer. To better understand the biological processes in which chromatin remodeling proteins participate, we globally mapped binding regions for several components of the SWI/SNF complex throughout the human genome using ChIP-Seq. SWI/SNF components were found to lie near regulatory elements integral to transcription (e.g. 5' ends, RNA Polymerases II and III, and enhancers) as well as regions critical for chromosome organization (e.g. CTCF, lamins, and DNA replication origins). Interestingly we also find that certain configurations of SWI/SNF subunits are associated with transcripts that have higher levels of expression, whereas other configurations of SWI/SNF factors are associated with transcripts that have lower levels of expression. To further elucidate the association of SWI/SNF subunits with each other as well as with other nuclear proteins, we also analyzed SWI/SNF immunoprecipitated complexes by mass spectrometry. Individual SWI/SNF factors are associated with their own family members, as well as with cellular constituents such as nuclear matrix proteins, key transcription factors, and centromere components, implying a ubiquitous role in gene regulation and nuclear function. We find an overrepresentation of both SWI/SNF-associated regions and proteins in cell cycle and chromosome organization. Taken together the results from our ChIP and immunoprecipitation experiments suggest that SWI/SNF facilitates gene regulation and genome function more broadly and through a greater diversity of interactions than previously appreciated.

Comprehensive annotation of the transcriptome of the human fungal pathogen Candida albicans using RNA-seq.

Candida albicans is the major invasive fungal pathogen of humans, causing diseases ranging from superficial mucosal infections to disseminated, systemic infections that are often lifethreatening. We have used massively parallel high-throughput sequencing of cDNA (RNA-seq) to generate a high-resolution map of the C. albicans transcriptome under several different environmental conditions. We have quantitatively determined all of the regions that are transcribed under these different conditions, and have identified 602 novel transcriptionally active regions (TARs) and numerous novel introns that are not represented in the current genome annotation. Interestingly, the expression of many of these TARs is regulated in a condition-specific manner. This comprehensive transcriptome analysis significantly enhances the current genome annotation of C. albicans, a necessary framework for a complete understanding of the molecular mechanisms of pathogenesis for this important eukaryotic pathogen.

X chromosome-wide analyses of genomic DNA methylation states and gene expression in male and female neutrophils.

The DNA methylation status of human X chromosomes from male and female neutrophils was identified by high-throughput sequencing of HpaII and MspI digested fragments. In the intergenic and intragenic regions on the X chromosome, the sites outside CpG islands were heavily hypermethylated to the same degree in both genders. Nearly half of X chromosome promoters were either hypomethylated or hypermethylated in both females and males. Nearly one third of X chromosome promoters were a mixture of hypomethylated and heterogeneously methylated sites in females and were hypomethylated in males. Thus, a large fraction of genes that are silenced on the inactive X chromosome are hypomethylated in their promoter regions. These genes frequently belong to the evolutionarily younger strata of the X chromosome. The promoters that were hypomethylated at more than two sites contained most of the genes that escaped silencing on the inactive X chromosome. The overall levels of expression of X-linked genes were indistinguishable in females and males, regardless of the methylation state of the inactive X chromosome. Thus, in addition to DNA methylation, other factors are involved in the fine tuning of gene dosage compensation in neutrophils.

Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing.

BACKGROUND: Short-read high-throughput DNA sequencing technologies provide new tools to answer biological questions. However, high cost and low throughput limit their widespread use, particularly in organisms with smaller genomes such as S. cerevisiae. Although ChIP-Seq in mammalian cell lines is replacing array-based ChIP-chip as the standard for transcription factor binding studies, ChIP-Seq in yeast is still underutilized compared to ChIP-chip. We developed a multiplex barcoding system that allows simultaneous sequencing and analysis of multiple samples using Illumina's platform. We applied this method to analyze the chromosomal distributions of three yeast DNA binding proteins (Ste12, Cse4 and RNA PolII) and a reference sample (input DNA) in a single experiment and demonstrate its utility for rapid and accurate results at reduced costs. RESULTS: We developed a barcoding ChIP-Seq method for the concurrent analysis of transcription factor binding sites in yeast. Our multiplex strategy generated high quality data that was indistinguishable from data obtained with non-barcoded libraries. None of the barcoded adapters induced differences relative to a non-barcoded adapter when applied to the same DNA sample. We used this method to map the binding sites for Cse4, Ste12 and Pol II throughout the yeast genome and we found 148 binding targets for Cse4, 823 targets for Ste12 and 2508 targets for PolII. Cse4 was strongly bound to all yeast centromeres as expected and the remaining non-centromeric targets correspond to highly expressed genes in rich media. The presence of Cse4 non-centromeric binding sites was not reported previously. CONCLUSION: We designed a multiplex short-read DNA sequencing method to perform efficient ChIP-Seq in yeast and other small genome model organisms. This method produces accurate results with higher throughput and reduced cost. Given constant improvements in high-throughput sequencing technologies, increasing multiplexing will be possible to further decrease costs per sample and to accelerate the completion of large consortium projects such as modENCODE.

Mapping of transcription factor binding regions in mammalian cells by ChIP: comparison of array- and sequencing-based technologies.

Recent progress in mapping transcription factor (TF) binding regions can largely be credited to chromatin immunoprecipitation (ChIP) technologies. We compared strategies for mapping TF binding regions in mammalian cells using two different ChIP schemes: ChIP with DNA microarray analysis (ChIP-chip) and ChIP with DNA sequencing (ChIP-PET). We first investigated parameters central to obtaining robust ChIP-chip data sets by analyzing STAT1 targets in the ENCODE regions of the human genome, and then compared ChIP-chip to ChIP-PET. We devised methods for scoring and comparing results among various tiling arrays and examined parameters such as DNA microarray format, oligonucleotide length, hybridization conditions, and the use of competitor Cot-1 DNA. The best performance was achieved with high-density oligonucleotide arrays, oligonucleotides >/=50 bases (b), the presence of competitor Cot-1 DNA and hybridizations conducted in microfluidics stations. When target identification was evaluated as a function of array number, 80%-86% of targets were identified with three or more arrays. Comparison of ChIP-chip with ChIP-PET revealed strong agreement for the highest ranked targets with less overlap for the low ranked targets. With advantages and disadvantages unique to each approach, we found that ChIP-chip and ChIP-PET are frequently complementary in their relative abilities to detect STAT1 targets for the lower ranked targets; each method detected validated targets that were missed by the other method. The most comprehensive list of STAT1 binding regions is obtained by merging results from ChIP-chip and ChIP-sequencing. Overall, this study provides information for robust identification, scoring, and validation of TF targets using ChIP-based technologies.

Mapping the chromosomal targets of STAT1 by Sequence Tag Analysis of Genomic Enrichment (STAGE).

Identifying the genome-wide binding sites of transcription factors is important in deciphering transcriptional regulatory networks. ChIP-chip (Chromatin immunoprecipitation combined with microarrays) has been widely used to map transcription factor binding sites in the human genome. However, whole genome ChIP-chip analysis is still technically challenging in vertebrates. We recently developed STAGE as an unbiased method for identifying transcription factor binding sites in the genome. STAGE is conceptually based on SAGE, except that the input is ChIP-enriched DNA. In this study, we implemented an improved sequencing strategy and analysis methods and applied STAGE to map the genomic binding profile of the transcription factor STAT1 after interferon treatment. STAT1 is mainly responsible for mediating the cellular responses to interferons, such as cell proliferation, apoptosis, immune surveillance, and immune responses. We present novel algorithms for STAGE tag analysis to identify enriched loci with high specificity, as verified by quantitative ChIP. STAGE identified several previously unknown STAT1 target genes, many of which are involved in mediating the response to interferon-gamma signaling. STAGE is thus a viable method for identifying the chromosomal targets of transcription factors and generating meaningful biological hypotheses that further our understanding of transcriptional regulatory networks.

Nnf1p, Dsn1p, Mtw1p, and Nsl1p: a new group of proteins important for chromosome segregation in Saccharomyces cerevisiae.

Previously, antibodies were raised against a nuclear envelope-enriched fraction of yeast, and the essential gene NNF1 was cloned by reverse genetics. Here it is shown that the conditional nnf1-17 mutant has decreased stability of a minichromosome in addition to mitotic spindle defects. I have identified the novel essential genes DSN1, DSN3, and NSL1 through genetic interactions with nnf1-17. Dsn3p was found to be equivalent to the kinetochore protein Mtw1p. By indirect immunofluorescence, all four proteins, Nnf1p, Mtw1p, Dsn1p, and Nsl1p, colocalize and are found in the region of the spindle poles. Based on the colocalization of these four proteins, the minichromosome instability and the spindle defects seen in nnf1 mutants, I propose that Nnf1p is part of a new group of proteins necessary for chromosome segregation.

4-Dialkylamino-1-(5-substituted or unsubstituted 1-phenyl-1H-pyrazol-4-yl)butan-1-ols: synthesis and evaluation of analgesic, anti-inflammatory and platelet anti-aggregating activities.

A number of 4-dialkylamino-1-(5-substituted or unsubstituted 1-phenyl-1H-pyrazol-4-yl)butan-1-ols 2a-n were synthesized and tested in vivo for anti-inflammatory and analgesic activities and in vitro for platelet anti-aggregating activity. Dimethylaminoderivatives 2b, e, g showed good analgesic activity; almost all of them had strong platelet anti-aggregating properties at a final concentration of 1 x 10(-3) M; pyrazoles 2c, d, f-h showed weak anti-inflammatory activity.

Evaluation of flutamide genotoxicity in rats and in primary human hepatocytes.

Flutamide, an effective competitive inhibitor of the androgen receptor used orally for palliative treatment of prostatic carcinoma and regulation of prostatic hyperplasia was evaluated for its genotoxic effects in the intact rat and in primary cultures of human hepatocytes. Negative responses were obtained in all the in vivo assays as well as in the in vitro assay. In rats given a single oral dose of 500 mg/kg flutamide, fragmentation and repair of liver DNA were absent, and no increase was observed in the frequency of micronucleated hepatocytes. In the liver of rats given flutamide as initiating agent at the dose of 500 mg/kg/week for 6 successive weeks, gamma-glutamyltraspeptidase-positive foci were detected only in 3 of 10 rats. There was no evidence of a promoting effect on the development of aberrant crypt foci in rats given 100 mg/kg flutamide on alternate days for 8 successive weeks. In primary cultures of human hepatocytes from one male and one female donor DNA fragmentation as measured by the Comet assays, and DNA repair synthesis as revealed by quantitative autoradiography, were absent after a 20 hr exposure to flutamide concentrations ranging from 18 to 56 microM. Taken as a whole, our results seem to indicate that flutamide is a non-genotoxic drug.

1,8-Naphthyridines IV. 9-substituted N,N-dialkyl-5-(alkylamino or cycloalkylamino) [1,2,4]triazolo[4,3-a][1, 8]naphthyridine-6-carboxamides, new compounds with anti-aggressive and potent anti-inflammatory activities.

The title compounds (8) were synthesized through the cyclocondensation of the corresponding N-substituted 4-amino-2-chloro-1,8-naphthyridine-3-carboxamides (4) with the proper hydrazides, in order to evaluate their anti-inflammatory and anti-aggressive properties. Several compounds 8 exhibited high anti-inflammatory activity (carrageenin-induced paw edema assay in the rat) along with appreciable anti-aggressive properties (isolation-induced aggressiveness test in mice). With respect to anti-inflammatory activity, the most active compounds (8n and 8c) produced a 61% edema inhibition at the 25 mg/kg dose, and 50 or 35% inhibition, respectively, at the 12.5 mg/kg dose. The structure-activity relationships are discussed.

Induction of micronuclei and of enzyme-altered foci in the liver of female rats exposed to progesterone and three synthetic progestins.

Progesterone (PG) and three structurally similar synthetic progestins-norethisterone (NE), allylestrenol (AE), and dydrogesterone (DG)-have been compared for their ability to induce the formation of micronuclei and of enzyme-altered foci in the liver of female rats. In the micronucleus assay, carried out in rats given a single p.o. dose of 100 mg kg-1 3 days before partial hepatectomy and sacrificed for cell sampling 2 days later, the frequency of micronucleated hepatocytes was 3.5-fold higher than in controls with PG, 2.8-fold with DG, 2.2-fold with NE and 2.1-fold with AE, but the increase was statistically significant only for PG. In the liver foci assay, performed to evaluate the tumor initiating activity of p. o. dosing with 100 mg kg-1 once a week for 6 successive weeks, the values of the number and area of gamma-glutamyltranspeptidase-positive foci were, as compared to controls, 15.9- and 100-fold higher with NE, and 13.9- and 52-fold higher with AE, but only the increase of area produced by NE was statistically significant; PG and DG did not display in this test any activities. Considered together with previous findings, these results suggest that NE might be biotransformed in the liver into reactive species and thus behave as a weak genotoxic agent.

Acid-catalysed hydrolysis and benzodiazepine-like properties of 5-(dialkylamino)- and 5-(alkylthio)-substituted 8-chloro-6-phenyl-6H-[1,2,4]triazolol[4,3-a][1,5]benzodiazepines in mice.

The in-vitro and in-vivo hydrolysis of two benzodiazepine compounds has been studied to evaluate their in-vivo activity in mice. Compounds RL 218 and RL 236, selected as representative examples of N,N-dialkyl-8-chloro-6-phenyl-6H-[1,2,4]triazolo[4,3-a][1,5]benzodiaz epin-5-amines (1) and of their 5-(alkylthio) substituted analogues (2), were rapidly hydrolysed to the corresponding 8-chloro-6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5(6H )-one 3 (RL 214) in aqueous acidic solution at pH 1.5. This reaction also occurred extensively in mice when compounds RL 218 and RL 236 were given orally but not intraperitoneally. Both compounds were active against pentylenetetrazole-induced lethal convulsions in mice only when administered orally. After administration of pharmacologically effective oral doses (ED50, the dose protecting 50% of mice), at the time of assessment of the anti-pentylenetetrazole activity, mean brain concentrations of RL 218 and RL 236 were below the limits of sensitivity of the analytical procedure whereas brain concentrations of their metabolite RL 214 were comparable with that present after an oral equiactive dose of this compound itself. RL 214 but not RL 218 or RL 236 had in-vitro affinity for brain benzodiazepine receptors. These results indicate that the anticonvulsant activity of RL 218 and RL 236 in mice depends essentially on their in-vivo transformation into the common active metabolite RL 214 which most probably arises as a result of acid catalysed hydrolysis in the gastric juice.

Effects of reduced glutathione and n-acetylcysteine on lidocaine metabolism in cimetidine treated rats.

The aim of this work was to evaluate the effects of exogenous glutathione (GSH) and N-acetylcysteine (NAC) on the formation of monoethylglycinexylidide (MEGX) from lidocaine in rats with and without the administration of cimetidine. GSH and NAC were administered intraperitoneally (i.p.) (1 mmol/kg) 1 hour before treatment with cimetidine (0.5 mmol/kg) or saline, and 1 hr later all rats were injected i.p. with lidocaine (1 mg/kg). Blood samples were drawn 30 min after the lidocaine injection. MEGX and lidocaine serum concentrations were determined by means of fluorescence polarization immuno-assay using the TDX system. Cimetidine produced a decrease in MEGX levels (from 210 +/- 18 to 164 +/- 13 ng/mL) and a parallel increase in lidocaine levels (from 73 +/- 22 to 172 +/- 47 ng/mL), consistent with cytochrome P-450 3A inhibition. Both GSH and NAC produce a significant decrease in MEGX levels (151 +/- 16 and 139 +/- 14 ng/mL, respectively), but no significant increase in lidocaine levels were found. As compared to the cimetidine group, pre-treatment using either GSH or NAC with cimetidine produced a marked decrease in lidocaine levels (37 +/- 27 and 63 +/- 28 ng/mL, respectively) and no modification of MEGX levels (155 +/- 12 and 165 +/- 22 ng/mL, respectively). These results suggest that GSH and NAC might accelerate the lidocaine metabolism while counteracting the inhibitory effect of cimetidine.

1,8-Naphthyridines. III. N,N-dialkyl-5-chloro[1,2,4]-triazolo[4,3-a][1,8]naphthyridine-6-carboxa mides with anti-inflammatory activity.

Fifteen N,N-dialkyl-5-chloro[1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxami des (7a-o) were synthesized through the cyclocondensation of the corresponding N,N-dialkyl-2,4-dichloro-1,8-naphthyridine-3-carboxamides with proper hydrazides, in order to evaluate their anti-inflammatory, antiaggressive, and analgesic properties. Four of the compounds tested showed a statistically significant and dose-dependent anti-inflammatory activity.

Penetration of oral cefuroxime axetil into the human aqueous humor.

The purpose of this study was to investigate the penetration into the aqueous humor of cefuroxime after a single oral dose as cefuroxime axetil. Fourteen patients scheduled for cataract extraction received a single oral dose of cefuroxime axetil corresponding to 500 mg of cefuroxime 2-8 h preoperatively. Aqueous humor samples were obtained at the beginning of the cataract surgery and blood samples were drawn at the time of anesthesia. Cefuroxime levels were determined by high-performance liquid chromatography. The aqueous levels were (mean +/- SEM) 0.48 +/- 0.13 microgram/ml from 3 to 8 h after administration. Serum levels averaged 3.80 +/- 0.58 micrograms/ml. These data indicate that detectable levels of cefuroxime, exceeding the MIC of some bacterial species that frequently cause intraocular infections, may be achieved in uninflamed eyes after a low dose of cefuroxime axetil.

A possible medium-term assay for detecting the effects of liver and colon carcinogens in rats.

The aim of the present study was to verify whether the tumorigenic effect of a rat liver carcinogen, 2-acetylaminofluorene (AAF), and of a promoter of rat colon carcinogenesis, chenodeoxycholic acid (CDCA), could be detected with a single medium-term assay using as markers gamma-glutamyltranspeptidase (GGT)-positive foci in the liver and aberrant crypt foci (ACF) in colon mucosa. In rats given in the first 2 weeks of treatment both N-nitrosodiethylamine (NDEA), as initiator of liver carcinogenesis, and azoxymethane (AOM), as initiator of colon carcinogenesis, the subsequent 6-week feeding on a diet containing AAF (0.01%) produced a significant marked increase of the number and area of GGT-positive foci which is consistent with the results of long term assays. When rats initiated with both NDEA and AOM were fed for 6 weeks on a diet containing CDCA (0.1%) a significant increase of large ACF as well as of crypt multiplicity was observed, consistently with the promoting effect of CDCA in colon carcinogenesis. The results obtained in this preliminary study suggest that this medium-term assay might be able to screen both liver and colon carcinogens in rats.

Inhibition of the development of rat liver preneoplastic lesions by verapamil and dexverapamil.

The effect of verapamil and dexverapamil on the development of liver preneoplastic foci was investigated in male Sprague-Dawley rats initiated with N-nitrosodiethylamine (200 mg/kg i.p.), fed on a diet containing 0.01% 2-acetylaminofluorene and subjected to partial hepatectomy, according to the hepatocarcinogenesis model developed by Solt and Farber. Administration of drinking water containing 0.03% verapamil or dexverapamil resulted in a decrease in the incidence and size of gamma-glutamyl transpeptidase-positive foci. The chemopreventive effect of dexverapamil was more marked than that of verapamil. These findings support the hypothesis that these two calcium channel blockers act by reducing the resistance of initiated hepatocytes to the mitoinhibitory and cytotoxic effects of 2-acetylaminofluorene.