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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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Male Wistar rats weighing 230-300 g were used to characterize the participation of adrenergic and cholinergic receptors of the lateral hypothalamic area (LHA) in the control of renal water excretion. Since stimulation of adrenergic or cholinergic receptors has no effect on glomerular filtration rate, the antidiuresis and significant delay in urinary flow observed after lateral hypothalamic stimulation with carbachol (CCh) (0.036 +/- 0.005 to 0.019 +/- 0.003 microliters min-1 100 g body weight-1) and noradrenaline (Nad) (0.024 +/- 0.005 to 0.025 +/- 0.004 microliters min-1 100 g body weight-1) are secondary to an increase in distal tubular fluid reabsorption (DFR). Data are reported as means +/- SEM for ten rats in each group. Tubular water handling measured by lithium clearance demonstrated that LHA simulation with CCh (2.8 nmol in 1 microliter) and Nad (30.0 nmol in 1 microliter) leads to a significant reduction in proximal water reabsorption (CCh, 93.3 +/- 2.6 to 85.4 +/- 1.4%; Nad, 92.7 +/- 0.9 to 88.6 +/- 1.3%), with a simultaneous and significant increase in fluid reabsorption along the post-proximal nephron segments when compared to control (CNa) (CCh, 6.7 +/- 0.7 to 14.5 +/- 1.1%; Nad, 8.2 +/- 0.8 to 11.4 +/- 1.6%). These effects are blocked by muscarinic (atropine, 5 nmol in 1 microliter) and alpha-1 adrenoceptors (prazosin, 4 nmol in 1 microliter) antagonists. The results indicate the effective participation of the post-proximal nephron in the antidiuresis occurring after cholinergic and adrenergic LHA stimulation.

Studies were undertaken to characterize the participation of specific alpha-1,alpha-2 and beta adrenoceptors of the lateral hypothalamic area (LHA) in the urinary excretion of sodium and potassium. Alpha-1 and alpha-2 LHA receptors were shown to participate in the regulation of renal sodium and potassium excretion. The effects of noradrenaline microinjection (30 nmol in 1 microliter) into the LHA on urinary sodium excretion (UNaV) are blocked by previous injection of the alpha-1 antagonist prazosin (4 nmol in 1 microliter) from 3.22 +/- 0.25 to 0.59 +/- 0.04 microEq min-1 100 g body weight-1. Pre-injection of yohimbine, an alpha-2 antagonist (4 nmol in 1 microliter), synergistically potentiated the action of noradrenaline on UNaV (3.22 +/- 0.25 to 4.02 +/- 0.27 microEq min-1 100 g body weight-1) and on urinary potassium excretion (UKV) (0.70 +/- 0.08 to 1.15 +/- 0.12 microEq min-1 100 g body weight-1). The beta-adrenergic blockers metoprolol (100 nmol in 1 microliter) and propranolol (100 nmol in 1 microliter) had no synergistic or antagonistic action on the sodium excretion fraction, suggesting that neither of these receptors is present in LHA. Our results indicate that natriuresis occurs even in the absence of changes in glomerular filtration rate and demonstrate an inhibitory natriuretic effect of an alpha-1 blocker (prazosin) injected into the LHA before adrenaline, while an alpha-2 antagonist (yohimbine) yielded a potentiating effect.

Cholinergic stimulation of the lateral hypothalamic area with carbachol (1 microgram in 1 microliter) markedly inhibited gastric acid secretion in the anesthetized rat. Inhibition was blocked by prior micro-injection of atropine (4 micrograms/microliters) into the same brain area and was accompanied by an increased sodium content in the stomach. Muscarinic receptor mediated cholinergic inhibitory influence of the hypothalamus on gastric acid secretion is suggested by these results.

In the past, simian virus 40 (SV40) has been used as a cloning vehicle to clone foreign genes by substituting portions of the viral genome vital for viral replication. Propagation of these defective viruses required a helper virus and the recombinant viruses obtained could be grown only as a mixture. In this study, we describe a novel nondefective SV40 vector to clone small RNA polymerase III genes. Two small RNA polymerase III genes, an amber suppressor human serine tRNA gene and the adenovirus (Ad) VAI RNA gene, were cloned in the intron region of the large-T antigen gene of SV40 after deleting DNA sequences coding for the small-t polypeptide. The recombinant viruses grew to wild type levels and showed no growth defects. When CV-1p cells were infected with these viruses, the cloned RNA polymerase III genes were expressed at high levels at late times. Interestingly, large amounts VAI RNA in CV-1p cells infected with SV40-VA recombinant virus, did not enhance translation of viral mRNAs significantly but did lead to a 3 to 4 fold increase in the steady state levels of large-T mRNA suggesting a novel function for VAI RNA in SV40 infected monkey cells. Furthermore, VAI mutants which fail to function in Ad infected human cells also failed to enhance the levels of large-T mRNAs in monkey cells infected with SV40. The simple SV40 vector described here may be useful to study the structure and function of small RNA polymerase III genes in the context of a eucaryotic chromosome. In addition, the nondefective recombinant SV40 which expresses the suppressor tRNA gene at high levels may provide a useful helper system to propagate animal viruses with amber mutations in essential genes.

Different doses of anionic and cationic albumin (CA) were administered intravenously to 29 normal unanesthetized female rats. Administration of 20 and 30 mg of CA produced an increase in urinary excretion of endogenous albumin. Urinary CA levels in the urine samples collected during the first 60 min after injection of 30 mg of CA were higher than anionic albumin levels. A marked increase in the number of endocytic vacuoles, large numbers of images suggesting fusion of vacuoles with lysosomes, extrusion of cell elements into the tubular lumen and tubule rupture were observed in the animals injected with CA. These results show that CA may produce changes in proteinuria and in renal structure.

Using helical strips of isolated rabbit aortas it was studied the influence of a brief exposure to NH4Cl as well as of the subsequent washout phase. These changes did not affect the resting aortic tension but modified the responsiveness of the strip precontracted by norepinephrine (NE): in the presence of NH4Cl there occurred relaxation, and when ammonia was washed out the strip overcontracted transiently. All this was observed in the absence of external pH (pHe) changes. It is postulated that intracellular (IC) perturbations caused by ammonia led to IC alkalosis which was followed by an IC acidosis when the NH4Cl was removed from the preparation. If this assumption proves to be correct, the IC acid-base status--the intracellular pH (pHi)--could be regarded as a modulator of NE-induced contractility of vascular smooth muscle.

Urinary excretion of four plasma proteins having molecular weights between 44,100 and 90,000 daltons was studied by two-dimensional immunoelectrophoresis in normal individuals and in patients with different kinds of renal pathology. The proteins studied were: alpha 1-acid glycoprotein, 44,100 molecular weight (MW) and pH 2.7 isoelectric point (IP); alpha 1-antitrypsin, 54,000 MW and 4.0 IP; albumin, 69,000 MW and 4.9 IP; and transferrin, 90,000 MW and 5.6 IP. The proteins were measured in urine with an oligospecific serum produced by the immunization of rabbits with the 4S fraction obtained from normal human plasma by gel filtration on Sephadex G-200. The increase in urinary excretion of these proteins observed both among glomerulopathic and tubulopathic patients did not correlate with MW. Mean renal albumin excretion was 2.9 mg/24 h among normal individuals, 87.38 mg/24 h among patients with tubulopathy, and 3,228 mg/24 among patients with glomerulopathy. Among patients with glomerulopathy, there was a direct correlation between the increased excretion of these proteins and their IP, except for alpha 1-acid glycoprotein.