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.

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.

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.

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.

Effect of NH4Cl on the contractility of isolated vascular smooth muscle.

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.

Effects of injection of selective adrenergic receptor antagonists into the lateral hypothalamic area on renal function.

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.

Inhibition of gastric acid secretion by cholinergic stimulation of the lateral hypothalamic area.

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.

Crossed-immunoelectrophoresis analysis of the urinary excretion of alpha 1-acid glycoprotein, alpha 1-antitrypsin, albumin, and transferrin in normal subjects and in patients with renal disease.

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.

Renal water handling evaluated by lithium clearance following adrenergic and cholinergic stimulation of the lateral hypothalamic area.

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.

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.

[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.

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.

Inhibition of the permeability response to vasopressin and oxytocin in the toad bladder: Effects of bradykinin, kallidin, eledoisin, and physalaemin.

It has been shown by means of Bentley'sin vitro preparation of the isolated urinary bladder of the toad,Bufo marinus paracnemis Lutz, that bradykinin reversibly inhibited the increase brought about by vasopressin on the permeability to water of the toad bladder. The increased hydro-osmotic response of the bladder to oxytocin was also inhibited by the kinin. The effect on water permeability was observed when bradykinin was added either to the serosal Ringer's solution or to the mucosal solution. The addition of bradykinin alone did not alter the basal osmotic water transfer across the bladder. In this context, bradykinin acted as a competitive antagonist of vasopressin (and oxytocin). Although lacking intrinsic activity, bradykinin exhibited affinity for receptor sites that are also common to the neurohypophysial hormones, causing a parallel shift of the log-dose/response curve for vasopressin without changing the maximal responses. The effects of other kinins (namely kallidin, eledoisin and physalaemin) on the toad bladder were also tested. Each of these drugs alone did not change the basal water flux across the bladder wall. Like bradykinin, these peptides inhibited the increase in water permeability evoked by vasopressin and oxytocin in the bladder. In view of the importance of neurohypophysial hormones and their target tissues to the osmotic homeostasis of amphibians, and the observation of antagonism between the kinins and the pituitary hormones coupled to the abundance of kinins in the amphibian organism, particularly in the skin and urinary bladder, teleological reasoning predicts a physiological role for the kinins, possibly functioning to dampen excesses and oscillations in membrane permeability that could occur in face of a constant and variable secretion of neurohypophysial hormone, thus adding to the homeostatic response of the amphibian organism.

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.

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.

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.

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.

Characteristics of H+ current transients induced by adverse H+ gradient pulses in toad bladder.

Acidification in the toad bladder occurs as a result of electrogenic H+ secretion (JH). When a pH gradient is applied in a stepwise fashion in the absence of exogenous CO2, JH decreases linearly with the mucosal (M) solution pH and is null when pHm is approximately 4.5. When pHm is returned to initial values (7.4) in a stepwise fashion, JH increases linearly with pHm. However, on this return, higher values of JH are initially obtained. To investigate this hysteresis, hemibladders mounted in chambers were used to measure the change in the H+ current before and after acid pulses were applied to the mucosal solution. In the absence of exogenous CO2, the application of graded acid pulses to mucosa for 1, 2, 4, and 8 min resulted in a graded decrease in JH. The restoration of pHm to 7.4 was followed by an immediate transient overshoot of reversed short-circuit current (Irsc), which was related to the time of exposure and the magnitude of the acid pulse. The longer the acid pulse or the larger the pulse, the greater the Irsc overshoot. The addition of protonophores, dinitrophenol, or salicylate, into the mucosal solution enhanced this overshoot. Similar Irsc overshoots could be obtained with the application of pulses of adverse electrical gradients. Introduction of exogenous CO2 into the system (3%) completely inhibited the overshoot in JH after an acid pulse. In conclusion, when pHm is decreased JH is reduced and the cell pH presumably decreases because of continued exit of alkali at the serosal side of the cell and entry of H+ from the mucosal solution. The decrease in cell pH then triggers the pump to produce a sharp overshoot in JH when pHm returns to 7.4.