Low dose radiation response curves, networks and pathways in human lymphoblastoid cells exposed from 1 to 10cGy of acute gamma radiation.

We investigated the low dose dependency of the transcriptional response of human cells to characterize the shape and biological functions associated with the dose-response curve and to identify common and conserved functions of low dose expressed genes across cells and tissues. Human lymphoblastoid (HL) cells from two unrelated individuals were exposed to graded doses of radiation spanning the range of 1-10cGy were analyzed by transcriptome profiling, qPCR and bioinformatics, in comparison to sham irradiated samples. A set of ∼80 genes showed consistent responses in both cell lines; these genes were associated with homeostasis mechanisms (e.g., membrane signaling, molecule transport), subcellular locations (e.g., Golgi, and endoplasmic reticulum), and involved diverse signal transduction pathways. The majority of radiation-modulated genes had plateau-like responses across 1-10cGy, some with suggestive evidence that transcription was modulated at doses below 1cGy. MYC, FOS and TP53 were the major network nodes of the low-dose-response in HL cells. Comparison our low dose expression findings in HL cells with those of prior studies in mouse brain after whole body exposure, in human keratinocyte cultures, and in endothelial cells cultures, indicates that certain components of the low dose radiation response are broadly conserved across cell types and tissues, independent of proliferation status.

Evaluation of the Genplex SNP typing system and a 49plex forensic marker panel.

Using a 52 SNP marker set previously developed for forensic analysis, a novel 49plex assay has been developed based on the Genplex typing system, a modification of SNPlex chemistry (both Applied Biosystems) using oligo-ligation of pre-amplified DNA and dye-labeled, mobility modified detection probes. This gives highly predictable electrophoretic mobility of the allelic products generated from the assay to allow detection with standard capillary electrophoresis analyzers. The loci chosen comprise the 48 most informative autosomal SNPs from the SNPforID core discrimination set supplemented with the amelogenin gender marker. These SNPs are evenly distributed across all 22 autosomes, exhibit balanced polymorphisms in three major population groups and have been previously shown to be effective markers for forensic analysis. We tested the accuracy and reproducibility of the Genplex system in three SNPforID laboratories, each using a different Applied Biosystems Genetic Analyzer. Genotyping concordance was measured using replicates of 44 standardized DNA controls and by comparing genotypes for the same samples generated by the TaqMan, SNaPshot and Sequenom iPLEX SNP typing systems. The degree of informativeness of the 48 SNPs for forensic analysis was measured using previously estimated allele frequencies to derive the cumulative match probability and in paternity analysis using 24 trios previously typed with 18 STRs together with three CEPH families with extensive sibships typed with the 15 STRs in the Identifiler kit.

Persistence of HIV-1 transcription in peripheral-blood mononuclear cells in patients receiving potent antiretroviral therapy.

BACKGROUND AND METHODS: Although potent antiretroviral therapy can control infection with human immunodeficiency virus type 1 (HIV-1), a long-lived reservoir of infectious virus persists in CD4+ T cells. We investigated this viral reservoir by measuring the levels of cell-associated viral DNA and messenger RNA (mRNA) that are essential for HIV-1 replication. Approximately every 6 months, we obtained samples of peripheral-blood mononuclear cells from five men with long-standing HIV-1 infection who had had undetectable levels of plasma HIV-1 RNA for 20 months or more during treatment with potent antiretroviral drugs. RESULTS: Before treatment, plasma levels of HIV-1 RNA correlated with the levels of cell-associated unintegrated HIV-1 DNA and unspliced viral mRNA. After treatment, plasma levels of HIV-1 RNA fell by more than 2.7 log to undetectable levels. The decrease in cell-associated integrated and unintegrated HIV-1 DNA and mRNA occurred in two phases. The first phase occurred during the initial 500 days of treatment and was characterized by substantial decreases in the levels of DNA and mRNA, but not to undetectable levels. The concentrations of cell-associated unintegrated viral DNA, integrated proviral DNA, and unspliced viral mRNA decreased by 1.25 to 1.46 log. The second phase occurred during the subsequent 300 days or more of treatment and was characterized by a plateau in the levels of HIV-1 DNA and unspliced mRNA. After an initial rapid decline, the ratio of unspliced to multiply spliced viral mRNA (a measure of active viral transcription) stabilized and remained greater than zero at each measurement. CONCLUSIONS: Despite treatment with potent antiretroviral drugs and the suppression of plasma HIV-1 RNA to undetectable levels for 20 months or more, HIV-1 transcription persists in peripheral-blood mononuclear cells. Unless the quasi-steady state levels of HIV DNA and mRNA eventually disappear with longer periods of therapy, these findings suggest that HIV-1 infection cannot be eradicated with current treatments.

Adaptive evolution of human immunodeficiency virus-type 1 during the natural course of infection.

The rate of progression to disease varies considerably among individuals infected with human immunodeficiency virus-type 1 (HIV-1). Analyses of semiannual blood samples obtained from six infected men showed that a rapid rate of CD4 T cell loss was associated with relative evolutionary stasis of the HIV-1 quasispecies virus population. More moderate rates of CD4 T cell loss correlated with genetic evolution within three of four subjects. Consistent with selection by the immune constraints of these subjects, amino acid changes were apparent within the appropriate epitopes of human leukocyte antigen class I-restricted cytotoxic T lymphocytes. Thus, the evolutionary dynamics exhibited by the HIV-1 quasispecies virus populations under natural selection are compatible with adaptive evolution.

Up-regulation of HOXC6, HOXD1, and HOXD8 homeobox gene expression in human neuroblastoma cells following chemical induction of differentiation.

An early event in the pathogenesis of neuroblastoma (NB), a tumor derived from embryonal neural crest tissue, appears to be the arrested differentiation of neuroblasts. However, NB cells can be induced to differentiate in vitro with numerous chemicals including retinoic acid (RA) and dibutyryl cyclic AMP (db-cAMP). One family of transcription factors, encoded by the homeobox (HOX) genes, plays a crucial role in Drosophila, Xenopus, and mammalian embryonic differentiation and development. We have previously identified six HOX genes (HOXC6, HOXC8, HOXD1, HOXD4, HOXD8, and HOXD9), by a sensitive PCR-based approach, in a cDNA library prepared from the human LA-N-5 NB cell line induced to differentiate with RA. In this report, we studied the regulation of these six HOX genes in a series of NB cell lines chemically induced to differentiate. Untreated NB cells express low or undetectable levels of HOX mRNA, and HOXC8 remains undetectable in the induced cells. However, a significant induction of HOXC6, HOXD1, and HOXD8 expression is seen in the RA-treated NB cell lines, albeit with different patterns and degree of up-regulation. db-cAMP treatment also induced HOXC6 and HOXD8 expression in two of the three NB cell lines analyzed. Low levels of HOXD4 and HOXD9 induction were observed in two and one RA-treated NB cell line, respectively. Up-regulation of HOXC6, HOXD1, and HOXD8 expression in human NB cells, chemically induced to differentiate, appears to be associated with maturation toward a differentiated neuronal phenotype.

Mutational analysis of the adenovirus E2-early promoter in fission yeast Schizosaccharomyces pombe.

The human adenovirus E2-early promoter has a complex architecture consisting of overlapping sequences that constitute the major(+1) and minor(-26) promoters in human cells. In human cells the basal transcription of the major promoter is dependent on 4 cis-acting elements: a TTAAGA motif analogous to the TATA box, two E2F sites that are present as inverted repeats, and an ATF/CREB site. It was also demonstrated that the E2-early promoter was expressed efficiently in the fission yeast Schizosaccharomyces pombe and that the major and minor promoters were differentially utilized with preferential transcription from the -26 promoter. In this report the results of an investigation of the E2-early promoter activity in S. pombe, using an additional group of linkerscan mutants that span the E2 promoter, are presented. The efficient expression of the E2-early promoter in yeast was dependent on all 4 cis-acting elements as monitored by reporter gene expression. However, unlike the situation in human cells, the mutation of the TATA-like element present at -50 bps rendered the -26 promoter inactive and was therefore crucial for the maximal promoter function in S. pombe. As in human cells the wild type promoter activity was seen in S. pombe when the -82 to -92 region was mutated. DNA-protein interaction studies confirmed the presence of ATF and E2F-like transcription factor activities in S. pombe. This report demonstrates the degree of conservation that exists between the transcription apparatus of yeast and man.

Changes in the viral mRNA expression pattern correlate with a rapid rate of CD4+ T-cell number decline in human immunodeficiency virus type 1-infected individuals.

The rate of disease progression varies considerably among human immunodeficiency virus type 1 (HIV-1)-infected individuals. Several cross-sectional studies have shown an association between the stage of HIV-1 disease and the viral burden or the relative levels of viral gene expression. To study the extent of HIV-1 transcription and replication and its correlations with disease progression, we quantified serial, longitudinal samples of blood cells from 10 HIV-1-infected individuals with markedly different rates of CD4+ T-cell number decline following seroconversion. After normalization for the input nucleic acid content, multiply spliced viral mRNA and unspliced viral RNA were quantified by competitive reverse transcription-PCR using oligonucleotide primers which flank the major tat/rev/nef splice junction and span an internal region of the gag open reading frame, respectively. Coamplification of internal cRNA template controls was used to normalize for variation in the efficiency of reverse transcription and in vitro enzymatic amplification. Similarly, proviral DNA was also quantified by competitive PCR performed within the linear range of amplification. Viral RNA was detected in the blood cells of each individual from all time points regardless of the rate of CD4+ T-cell decline. Unspliced genomic viral RNA rapidly increased in the blood cells from HIV-1-infected individuals who had a precipitously declining CD4+ T-cell number. In contrast, both unspliced and multiply spliced viral mRNAs remained relatively stable in the blood cells from HIV-1-infected individuals who have had a relatively benign clinical course. These data demonstrate that the extent of viral transcription and replication correlates with the rate of CD4+ T-cell number decline and that quantifying intracellular viral RNA is of potential prognostic value.

[Use of carvedilol compared to nifedipine in the treatment of mild and moderate essential arterial hypertension]. / O uso do carvedilol comparado à nifedipina no tratamento da hipertensão arterial essencial leve e moderada.

PURPOSE: To compare the efficacy of carvedilol, a new antihypertensive drug that combines vasodilatory and beta-blocker properties, with nifedipine. METHODS: In a multicenter double-blind trial, 106 mild to moderate essential hypertensive patients were treated with either carvedilol (n = 51), or nifedipine (n = 55) as monotherapy. Following 4 weeks of wash-out/run-in period, patients from the carvedilol group received this drug once a day at a dosage of 25 mg/day for 8 consecutive weeks. In order to maintain the double-blind character of the study, a placebo was administered in the carvedilol group at identical dosage intervals as used in the nifedipine s.r. group. Nifedipine was also administered for 8 weeks at a dosage of 40 mg/day given b.i.d. RESULTS: Both treatments were equally efficient in reducing blood pressure in the seated and upright positions. Blood pressure response to treatment was obtained in 79% and 78% of patients treated with carvedilol and nifedipine, respectively. The carvedilol group did not develop reflex tachycardia which is usually seen when prescribing vasodilators. Blood biochemistry remained unchanged with both treatments. Besides similar blood pressure efficacy, side effects by patients taking carvedilol were less frequent than nifedipine group. CONCLUSION: Carvedilol is a safe, efficient, once/day choice as monotherapy for mild to moderate essential hypertensive patients.

In vitro analysis of virus-associated RNA I (VAI RNA): inhibition of the double-stranded RNA-activated protein kinase PKR by VAI RNA mutants correlates with the in vivo phenotype and the structural integrity of the central domain.

Adenoviruses use the virus-encoded virus-associated RNA (VAI RNA) as a defense against cellular antiviral response by blocking the activation of the interferon-induced, double-stranded RNA-activated protein kinase PKR. The structure of VAI RNA consists of two long, imperfectly base-paired duplex regions connected by a complex short stem-loop at the center, referred to as the central domain. By using a series of adenovirus mutants with linker-scan mutations in the VAI RNA gene, we recently showed that the critical elements required for function in the VAI RNA molecule are in the central domain and that these same elements of the central domain are also involved in binding to PKR. In virus-infected cells, VAI RNA interacts with latent kinase, which is bound to ribosomes; this interaction takes place in a complex milieu. To more fully understand the relationship between structure and function and to determine whether the in vivo phenotype of these mutants can be reproduced in vitro, we have now analyzed these mutant VAI alleles for their ability to block the activation of a partially purified PKR from HeLa cells. We have also derived the structure of these mutants experimentally and correlated the structure with function. Without exception, when the structure of the short stem-loop of the central domain was perturbed, the mutants failed to inhibit PKR. Structural disruptions elsewhere in the central domain or in the long duplex regions of the molecule were not deleterious for in vitro function. Thus, these results support our previous findings and underscore the importance of the elements present in the central domain of the VAI RNA for its function. Our results also suggest that the interaction between PKR and VAI RNA involves a precise secondary (and tertiary) structure in the central domain. It has been suggested that VAI RNA does not activate PKR in virus-infected cells because of mismatches in the imperfectly base-paired long duplex regions. We constructed mutant VAI genes in which the imperfectly base-paired duplex regions were converted to perfectly base-paired regions and assayed in vitro for the activation of PKR. As with the wild-type VAI RNA, these mutants failed to activate PKR in vitro, while they were able to block the activation of PKR better than did the wild type. These results suggest that the failure of VAI RNA to activate PKR is not the result of mismatches in the long duplex regions.(ABSTRACT TRUNCATED AT 400 WORDS)

Hox gene expression in differentiating human neuroblastoma cells.

Neuroblastoma, a malignancy of early childhood arises in the embryonal neural crest. Neuroblastoma cells are in a state of arrested differentiation; however, they can be induced to differentiate in vitro by retinoic acid. As a first step toward understanding the molecular mechanisms of neuroblastoma differentiation we analyzed the expression pattern of the developmentally important Homeobox genes in cells treated with retinoic acid. The strategy employed involved rapid screening of a cDNA library prepared from retinoic acid treated human LA-N-5 neuroblastoma cells for Homeobox genes by the polymerase chain reaction. Multiple Homeobox genes were amplified from recombinant phage DNA using degenerate primers directed against the conserved homeobox. To date 6 Homeobox genes (HoxC6, HoxC8, HoxD1, HoxD4, HoxD8, and HoxD9) have been identified in the cDNA library prepared from LA-N-5 cells treated with retinoic acid. HoxD1 and HoxC8 are being reported for the first time to be expressed in neuroblastoma cells. Preliminary studies indicate that there is an induction of Homeobox gene expression in differentiating LA-N-5 neuroblastoma cells.

Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry.

Human immunodeficiency virus type-1 (HIV-1) DNA and messenger RNA sequences in both cell lines and blood obtained directly from HIV-1-infected patients were amplified by polymerase chain reaction and hybridized to fluorescein-labeled probes in situ, and the individually labeled cells were analyzed by flow cytometry. After flow cytometric analysis, heterogeneous cell populations were reproducibly resolved into HIV-1-positive and -negative distributions. Fluorescence microscopy showed that the cellular morphology was preserved and intracellular localization of amplified product DNA was maintained. Retention of nonspecific probe was not observed. Analysis of proviral DNA and viral messenger RNA in cells in the blood of HIV-1-infected patients showed that the HIV-1 genome persists in a large reservoir of latently infected cells. With the use of this technique it is now possible to detect single-copy DNA or low-abundance messenger RNA rapidly and reproducibly in a minor subpopulation of cells in suspension at single-cell resolution and to sort those cells for further characterization.

Quantification of human immunodeficiency virus type 1 tat mRNA as a marker for assessing the efficacy of antiretroviral therapy.

To evaluate the use of human immunodeficiency virus type 1 (HIV-1) tat mRNA quantification as a marker for antiretroviral therapy, 10 zidovudine-naive, p24 antibody-positive subjects (Centers for Disease Control classes III and IV) were studied at the start of zidovudine treatment. HIV-1 proviral DNA content and tat mRNA levels were monitored for 20 weeks from the initiation of therapy. Levels of tat mRNA were quantified using an engineered tat cRNA internal control under conditions of linear amplification. Proviral DNA levels increased in 2 patients and decreased in 8 during 20 weeks of therapy. In each case, tat mRNA levels exhibited similar but much more pronounced changes. Results indicate that quantitative measurement of tat mRNA levels is extremely useful for monitoring antiretroviral therapy.

Selective transmission of human immunodeficiency virus type-1 variants from mothers to infants.

Multiple human immunodeficiency virus type-1 sequences from the V3 and V4-V5 regions of the envelope gene were analyzed from three mother-infant pairs. The infants' viral sequences were less diverse than those of their mothers. In two pairs, a proviral form infrequently found in the mother predominated in her infant. A conserved N-linked glycosylation site within the V3 region, present in each mother's sequence set, was absent in all of the infants' sequence sets. These findings demonstrate that a minor subset of maternal virus is transmitted to the infant.

Bradykinin modulation of the hydroosmotic effect of the antidiuretic hormone in water-transporting epithelia.

Rocha e Silva's utmost contribution to science was the isolation of bradykinin, a naturally occurring nonapeptide known to have a broad spectrum of actions. Amongst them, considerable evidence suggests that the diuretic effects of endogenous bradykinin are, in part, mediated by inhibition of vasopressin-stimulated water transport. This is true for both the mammalian renal cortical collecting tubule and the urinary bladder of the toad (functionally analogous to the tubule). A review of the main contributions that led to that knowledge is presented.

Renal sodium handling after noradrenergic stimulation of the lateral hypothalamic area in rats.

1. The participation of special nephron segments in the renal control of sodium handling after adrenergic stimulation was investigated by determining lithium clearance in groups of 5-12 male Wistar rats (230-300 g) microinjected with noradrenaline into the lateral hypothalamic area (LHA). 2. Microinjection of noradrenaline (12.5 to 100.0 nmol/microliters) into the LHA promoted a significant decrease in proximal sodium reabsorption (controls, 86.5 +/- 1.3; 12.5, 81.4 +/- 2.0; 25.0, 72.6 +/- 2.4; 50.0, 75.4 +/- 1.8 and 100.0, 77.2 +/- 1.7%) and a dose-related increase in distal sodium reabsorption (control, 13.4 +/- 1.6; 12.5, 18.4 +/- 1,25.0, 26.9 +/- 2.9; 50.0, 24.1 +/- 2.7; 100.0, 22.1 +/- 1.9%) with no significant changes in creatinine clearance. Fractional sodium reabsorption after different noradrenaline concentrations was significantly reduced in the proximal nephron sites up to the concentration of 25.0 nmol/microliter. Beyond this concentration, a smaller but progressive increase in fractional sodium reabsorption was observed in the post-proximal segment. 3. These findings suggest an effective participation of proximal and post-proximal nephrons in natriuresis after lateral hypothalamic noradrenergic stimulation.

Renal sodium handling after noradrenergic stimulation of the lateral hypothalamic area in rats

The participation of specific of special nephron segments in the renal control of sodium handling after adrenergic stimulation was investigated by determining lithium clearance in groups of 5-12 male Wistar rats (230-300 g) microinjected with noradrenaline into the lateral hypothalamic area (LHA). Microinjection of noradrenaline (12.5 to 100.0 nmol/ul) into the LHA promoted a significant decrease in proximal sodium reabsorption (control, 86.5 ñ 1.3; 12.5,81.4 ñ 2.4; 50.0, 75.4 ñ 1.8 and 100.0,77.2 ñ 1.7%) and a dose-related increase in distal sodium reabsorption (control, 13.4 ñ 1.6; 12.5, 18.4 ñ 1.25.0,26.9 ñ 2.9; 50.0,24.1 ñ 2.7; 100.0,22.1 ñ 1.9%) with no significannt changes inm creatinine clearance. Fractional sodium reabsorption after different noradrenaline concentrations was significantly reduced in the proximal nephron sites up to the concentration of 25.0 nmol/ul. Beyond this concentration, a smaller but progressive increase in fraqctional sodium reabsorption was observed in the post-proximal segment. These findings suggest an effective participation of proximal and post-proximal nephrons in natriuresis after lateral hypothalamic noradrenergic stimulation

Analysis of alternatively spliced human immunodeficiency virus type-1 mRNA species, one of which encodes a novel tat-env fusion protein.

A polymerase chain reaction-based analysis was used to define the structures of the mRNAs that encode human immunodeficiency virus type-1 (HIV-1) regulatory and structural proteins in infected H9 cells. Twenty alternatively spliced mRNAs encoding the vif, vpr, env, nef, tat, and rev proteins were characterized. An evaluation of the coding potentials of these transcripts recognized both leaky scanning and reinitiation at downstream initiation codons as mechanisms that may operate during translation of many of the polycistronic messages. Two new splice acceptor sites, one at nt 6018 defining a new mRNA coding for the env and vpu proteins and another at nt 8671 defining a novel tat-env fusion transcript, were characterized. The latter transcript expressed a novel protein p17tev that was immunoprecipitated by both polyclonal tat antibodies and monoclonals directed towards the C-terminal region of gp41. The p17tev protein was able to transactivate transcription from the HIV-1 LTR in transient transfection assays. The use of multiple alternative splice donor and acceptor sites and the generation of novel proteins may confer evolutionary advantages on the viral mutants encoding them and influence the course of clinical disease.