Genetic variants of human T-lymphotrophic virus type II in American Indian groups

The human T-lymphotropic virus type II (HTLV-II) is found in many New World Indian groups in North and South America and may have entered the New World from Asia with the earliest migration of ancestral Amerindians over 15,000 years ago. To characterize the phylogenetic relationships of HTLV-II strains infecting geographically diverse Indian populations, we used polymerase chain reaction to amplify HTLV-II sequences from lymphocytes of seropositive Amerindians from Brazil (Kraho, Kayapo, and Kaxuyana), Panama (Guaymi), and the United States (the Navajo and Pueblo tribes of the southwestern states and the Seminoles of Florida). Sequence analysis of a 780-base pair fragment (located between the env gene and the second exons of tax/rex) revealed that Amerindian viruses clustered in the same two genetic subtypes (IIa and IIb) previously identified for viruses from intravenous drug users. Most infected North and Central American Indians had subtype IIb, while HTLV-II infected members of three remote Amazonian tribes clustered as a distinct group within subtype IIa. These findings suggest that the ancestral Amerindians migrating to the New World brought at least two genetic subtypes, IIa and IIb. Because HTLV-II strains from Amazonian Indians form a distinct group within subtype HTLV-IIa, these Brazilian tribes are unlikely to be the source of IIa viruses in North American drug users. Finally, the near identity of viral sequences from geographically diverse populations indicate that HTLV-II is a very ancient virus of man.     

There's more to the picture than meets the eye: young children's difficulty understanding biased interpretation

5 experiments investigated children's understanding that expectations based on prior experience may influence a person's interpretation of ambiguous visual information. In Experiment 1, 4- and 5-year-olds were asked to infer a puppet's interpretation of a small, ambiguous portion of a line drawing after the puppet had been led to have an erroneous expectation about the drawing's identity. Children of both ages failed to ascribe to the puppet an interpretation consistent with the puppet's expectation. Instead, children attributed complete knowledge of the drawing to the puppet. In Experiment 2, the task was modified to reduce memory demands, but 4- and 5-year-olds continued to overlook the puppet's prior expectations when asked to infer the puppet's interpretation of an ambiguous scene. 6-year-olds responded correctly. In Experiment 3, 4- and 5-year-olds correctly reported that an observer who saw a restricted view would not know what was in the drawing, but children did not realize that the observer's interpretation might be mistaken. Experiments 4 and 5 explored the possibility that children's errors reflect difficulty inhibiting their own knowledge when responding. The results are taken as evidence that understanding of interpretation begins at approximately age 6 years.     

Preservation of myocyte contractile function after hypothermic, hyperkalemic cardioplegic arrest with 2, 3-butanedione monoxime

One proposed contributory mechanism for depressed ventricular performance after hypothermic, hyperkalemic cardioplegic arrest is a reduction in myocyte contractile function caused by alterations in intracellular calcium homeostasis. Because 2,3-butanedione monoxime decreases intracellular calcium transients, this study tested the hypothesis that 2,3-butanedione monoxime supplementation of the hyperkalemic cardioplegic solution could preserve isolated myocyte contractile function after hypothermic, hyperkalemic cardioplegic arrest. Myocytes were isolated from the left ventricles of six pigs. Magnitude and velocity of myocyte shortening were measured after 2 hours of incubation under normothermic conditions (37 degrees C, standard medium), hypothermic, hyperkalemic cardioplegic arrest (4 degrees C in Ringer's solution with 20 mEq potassium chloride and 20 mmol/L 2,3-butanedione monoxime). Because beta-adrenergic agonists are commonly employed after cardioplegic arrest, myocyte contractile function was examined in the presence of the beta-agonist isoproterenol (25 nmol/L). Hypothermic, hyperkalemic cardioplegic arrest and rewarming reduced the velocity (32%) and percentage of myocyte shortening (27%, p < 0.05). Supplementation with 2,3 butanedione monoxime normalized myocyte contractile function after hypothermic, hyperkalemic cardioplegic arrest. Although beta-adrenergic stimulation significantly increased myocyte contractile function under normothermic conditions and after hypothermic, hyperkalemic cardioplegic arrest, contractile function of myocytes exposed to beta-agonist after hypothermic, hyperkalemic cardioplegic arrest remained significantly reduced relative to the normothermic control group. Supplementation with 2,3-butanedione monoxime restored beta-adrenergic responsiveness of myocytes after hypothermic, hyperkalemic cardioplegic arrest. Thus, supplementation of a hyperkalemic cardioplegic solution with 2,3-butanedione monoxime had direct and beneficial effects on myocyte contractile function and beta-adrenergic responsiveness after cardioplegic arrest. A potential mechanism for the effects of 2,3-butanedione monoxime includes modulation of intracellular calcium transients or alterations in sensitivity to calcium. Supplementation with 2,3-butanedione monoxime may have clinical utility in improving myocardial contractile function after hypothermic, hyperkalemic cardioplegic arrest.     

Retroviral display of antibody fragments; interdomain spacing strongly influences vector infectivity

Five different single-chain antibody fragments (scFv) against human cell-surface antigens were displayed on murine ecotropic retroviral vectors by fusing them to the Moloney SU envelope glycoprotein. The spacing between the scFv and the SU glycoprotein was varied by fusing the scFv to residue +7 or to residue +1 of Moloney SU and by inserting linker sequences of different lengths between the domains. All of the chimeric envelopes were efficiently incorporated into vector particles and could bind to human cells through their displayed antibody fragments, but did not infect them. The spacing between the scFvs and the SU glycoproteins had no significant effect on the efficiency of envelope expression or viral incorporation and did not affect the binding properties of the chimeric envelopes, nor did it influence the efficiency of targeted gene delivery to human cells by scFv-displaying vectors. However, on murine fibroblasts the infectivity of vectors incorporating the chimeric envelopes was strongly influenced by the length of the interdomain spacer. The titers were very low when the single-chain antibodies were fused through a tripeptide linker to SU residue +7 and were greatly enhanced (up to 10(5)-fold) when they were fused to SU residue +1 through a heptapeptide linker. These results point to the importance of steric interactions between the domains of chimeric envelope glycoproteins and may have implications for retroviral vector design for human gene therapy.     

Decreased release of glutathione into the systemic circulation of patients with HIV infection

The human DDX6 gene (alias RCK) at chromosome 11 band q23 was identified through the study of the breakpoint of t(11;14)(q23;q32) translocation in a B-cell lymphoma cell line, RC-K8. DDX6 encodes a DEAD box protein/RNA helicase. Positive mouse genomic and cDNA recombinant clones were obtained by screening mouse B-cell genomic and cDNA libraries with a human DDX6 cDNA probe. The deduced amino acid sequence of an open reading frame from a cDNA clone revealed a protein with 92.5% identity to human ddx6/p54. All positive mouse genomic recombinant clones, and cDNA clones containing mouse Ddx6 (previous gene symbol: Rck), were localized by fluorescent in situ hybridization to band B of mouse Chromosome 9, a region showing conserved linkage homology to human chromosome 11 band q23. Mouse Ddx6 was localized to the region between Ncam and D9Mit45 by molecular linkage analysis. A 7.5-kb mRNA and a 54-kDa protein were identified as mouse Ddx6 gene products which are similar in size to products of the human DDX6 gene, as shown by Northern and Western blot analyses.     

Transfected glucocorticoid receptor and certain GR fragments evoke cell death in malignant lymphoid, not myeloid cell lines

We compared the data from four growth hormone (GH) immunoassays for analyzing 24-h GH profiles in four apparently normal subjects and four obese subjects (508 serum samples). The detection limit was 0.02 microgram/L for one immunochemiluminometric assay (ICMA), 0.1 microgram/L for two IRMAs, and 0.4 microgram/L for one RIA. All GH pulses with a peak ICMA value > 1 microgram/L were detected by each of the other methods. Overall, the correlation coefficient between the values obtained with all four assays exceeded 0.90. However, for GH concentrations < or = 0.25 microgram/L, acceptable concordance (r2 > or = 0.80) was reached only between the ICMA and one IRMA; between the ICMA and the RIA, concordance was acceptable only for GH concentrations > or = 10 micrograms/L. In the normal subjects, the percentage of undetectable values was 0% with the ICMA but 29% with one of the IRMAs; in obese subjects, the corresponding values were 12% and 38%.     

Some pharmacologic and toxicologic studies on Rhazya stricta decne in rats, mice and rabbits

1. This work examines some in vivo and in vitro pharmacologic and toxicologic effects of extracts of Rhazya stricta, a medicinal plant in the United Arab Emirates. 2. R. stricta extracts at doses of 0.1-10 mg reduced the mean arterial blood pressure (MBP) of anesthetized rats in a dose-dependent manner. The depressor effect was partially sensitive to atropine (5 microM). Although the MBP was reduced by 50% by both doses of extracts, the normal electrocardiogram pattern and the heart rate remained unaltered. 3. Acute treatment of rats with the lyophilized extract at doses of 4 g/kg produced a significant rise in insulin concentration. In streptozotocin-diabetic rats loaded orally with glucose (1 g/kg), R. stricta at doses of 8 g/kg produced significant decreases in plasma glucose concentration at 0.5 and 1 h after treatment. 4. Chronic treatment of rats and mice for 28 days with the lyophilized extract of R. stricta did not affect the plasma glucose or insulin concentration or any of the hematological or biochemical indices measured. 5. The extracts of R. stricta (0.5-4 g/kg) dose-dependently decreased the gastrointestinal transit time in mice by 4-50%. 6. The butanolic extract of R. stricta (1 and 2 g/kg) significantly reduced the carrageenan-induced increase in raw paw edema 3 and 4 h after the extract administration. 7. The rectal temperatures of normothermic and pyrexic rats were reduced significantly 0.5 and 1 h after administration of butanolic R. stricta at doses of 1 and 2 g/kg. 8. The butanolic extract of R. stricta at doses of 1 and 2 g/kg significantly increased the reaction time on the hot plate 30 and 60 min after administration to rats. 9. At concentration < 0.05 mg/ml (bath concentration), lyophilized water and butanol extracts of R. stricta potentiated the twitch responses induced by indirect electrical stimulation in the rat phrenic nerve diaphragm preparation. The responses were inhibited by concentrations > 0.05 mg/ml. Neostigmine (2 x 10(-4)M) did not alter these effects of the extracts. 10. R. stricta extracts dose-dependently decreased the force of contraction and heart rate of the isolated rabbit heart. Atropine (1 x 10(-5)M) had no effect on the inhibitory activity of these extracts. The lyophilized water extract (> 10 mg) and butanol extract (> 5 mg) produced irreversible inhibition and disturbances in the force of contraction and heart rate.     

Insulin and insulin-like growth factor-I signal transduction requires p21ras

We have investigated the role of cellular p21ras protein in insulin and insulin-like growth factor-I (IGF-I) signaling pathways. Insulin stimulation increased Ras-GTP formation in Rat-1 fibroblasts overexpressing normal human insulin receptors (HIRc-B), far greater than in parental Rat-1 fibroblasts, indicating that competent insulin receptors mediate this response. Cellular microinjection of a dominant-negative mutant p21ras protein (N17 ras) or anti-p21ras monoclonal antibody (Y13-259) into HIRc-B cells reduced insulin- and IGF-I-stimulated DNA synthesis by 75-90%. Insulin-induced c-fos protein expression was also inhibited by 74%. Microinjection of oncogenic p21ras (T-24 ras) into HIRc-B cells activated the mitogenic pathway, and coinjection of N17 ras and T-24 ras showed that oncogenic p21ras rescued the cells from the N17 ras blockade. This later finding indicates that T-24 ras acts downstream of N17 ras. In conclusion, 1) microinjection of a dominant interferring ras mutant into quiescent cells abrogated subsequent insulin and IGF-I mitogenic signaling; 2) oncogenic ras protein rescued cells from the N17 ras blockade, indicating that T24 ras action is downstream of the site of N17 inhibition; and 3) p21ras is an intermediate signaling molecule in the insulin/IGF-I signal transduction pathway and is required for gene expression and DNA synthesis.     

Serotonin and serotonin receptors in the central auditory system

Single channel currents were activated by GABA (0.5 to 5 microM) in cell-attached and inside-out patches from cells in the dentate gyrus of rat hippocampal slices. The currents reversed at the chloride equilibrium potential and were blocked by bicuculline (100 microM). Several different kinds of channel were seen: high conductance and low conductance, rectifying and "nonrectifying." Channels had multiple conductance states. The open probability (Po) of channels was greater at depolarized than at hyperpolarized potentials and the relationship between Po and potential could be fitted with a Boltzmann equation with equivalent valency (z) of 1. The combination of outward rectification and potential-dependent open probability gave very little chloride current at hyperpolarized potentials but steeply increasing current with depolarization, useful properties for a tonic inhibitory mechanism.