Physical mapping of HIV reverse transcriptase to the 5' end of RNA primers.

Enzymatic analysis of RNA cleavage products has suggested that human immunodeficiency virus (HIV) reverse transcriptase (RT) binds to the 5' end of RNAs that are recessed on a longer DNA template (RNA primers) yet binds to the 3' end of DNA primers. One concern is that RT molecules bound at the 3' end of RNA would not be easily detected because RT may not catalyze substantial RNA extension or cleavage when bound to the 3' end. We used physical mapping to show that RT binds preferentially to the 5' end of RNA primers. An HIV-RT that lacked RNase H activity (HIV-RT(E478Q)) was incubated with the RNA-DNA hybrid followed by the addition of Escherichia coli RNase H. RT protected a approximately 23-base region at the 5' end of the RNA and 4 additional bases on the DNA strand. This footprint correlated well with the crystal structure of HIV-RT. No protection of the RNA 3' end was observed, although when dNTPs were included, low levels of extension occurred, indicating that RT can bind this end. Wild-type HIV-RT cleaved the RNA and then extended a small portion of the cleaved fragments, suggesting that very small RNAs may be bound similar to DNA primers.

Genetic and pharmacological disruption of neurokinin 1 receptor function decreases anxiety-related behaviors and increases serotonergic function.

Alterations in serotonin (5-hydroxytriptamine, 5-HT), norepinephrine, and gamma-aminobutyric acid have been linked to the pathophysiology of anxiety and depression, and medications that modulate these neurotransmitters are widely used to treat mood disorders. Recently, the neuropeptide substance P (SP) and its receptor, the neurokinin 1 receptor (NK1R), have been proposed as possible targets for new antidepressant and anxiolytic therapies. However, animal and human studies have so far failed to provide a clear consensus on the role of SP in the modulation of emotional states. Here we show that both genetic disruption and acute pharmacological blockade of the NK1R in mice result in a marked reduction of anxiety and stress-related responses. These behavioral changes are paralleled by an increase in the firing rate of 5-HT neurons in the dorsal raphe nucleus, a major source of serotonergic input to the forebrain. NK1R disruption also results in a selective desensitization of 5-HT1A inhibitory autoreceptors, which resembles the effect of sustained antidepressant treatment. Together these results indicate that the SP system powerfully modulates anxiety and suggest that this effect is at least in part mediated by changes in the 5-HT system.

Perioperative management of the left ventricular assist device recipient.

An understanding of the unique preoperative, intraoperative, and postoperative considerations of left ventricular assist device implantation is essential for the successful anesthetic management of these challenging cases. This article discusses the different stages of anesthetic care of the left ventricular assist device recipient, including preoperative assessment, lining, induction, separation from cardiopulmonary bypass, and the postbypass period.

The development of the nociceptive responses in neurokinin-1 receptor knockout mice.

An important yet unanswered question is how neonates respond to painful stimuli, given the immaturity of their neural pathways. We examined the development of the neurokinin system using a novel approach, examining changes of this system by observing the pain responses of mice lacking the NK1 receptor at different stages of development We show that the NK1 receptor is not involved in nociception to heat, mechanical or chemical stimuli, at 3 days. In contrast, the NK1 receptor is involved in nociceptive responses to high intensity heat and mechanical stimuli, and mediates the second phase of the formalin response in 21-day-old mice. This indicates that nociception in neonates does not require the NK1 receptor and that the functional maturation of the NK1 receptor allows diversity in both the type of stimuli that activate the pain system and the types of responses elicited by nociceptive stimuli.

Molecules that specify modality: mechanisms of nociception.

Damage-sensing neurons express a unique cohort of genes encoding channels and receptors that play a role in nociception. However, there are redundant pathways involved in, for example, inflammatory pain and many channels and receptors have additional physiologic roles unrelated to nociception. Thus, it is impossible to ascribe a particular sensory modality to the expressions of a single molecular entity. Despite this, the relatively selective expression of a number of channels and receptors in nociceptive neurons provide potentially interesting analgesic drug targets that can be examined through the generation of knockout mice.

Habitability of planets around red dwarf stars.

Recent models indicate that relatively moderate climates could exist on Earth-sized planets in synchronous rotation around red dwarf stars. Investigation of the global water cycle, availability of photosynthetically active radiation in red dwarf sunlight, and the biological implications of stellar flares, which can be frequent for red dwarfs, suggests that higher plant habitability of red dwarf planets may be possible.

Mechanism of extracellular Ca2+ receptor-stimulated hormone release from sheep thyroid parafollicular cells.

1. Expression of receptors to extracellular calcium enables parafollicular cells of the thyroid gland (PF cells) to release calcitonin (CT) and serotonin (5-HT) in response to increased external Ca2+. Recently, a calcium-sensing receptor (CaR), similar to the G protein-coupled receptor for external Ca2+ cloned from parathyroid gland, was shown to be expressed in PF cells. Using a highly purified preparation of sheep PF cells, we have examined the electrical and biochemical processes coupling CaR activation to hormone release. 2. Whole-cell recordings in the permeabilized-patch configuration show that elevated extracellular Ca2+ concentration ([Ca2+]0) depolarizes these cells and induces oscillations in membrane potential. In voltage clamp, high [Ca2+]0 activates a cation conductance that underlies the depolarization. This conductance is cation selective, with a reversal potential near -25 mV indicating poor ion selectivity. 3. The CaR expressed in these cells is activated by other multivalent cations with a rank order potency of Gd3+ > Ba2+ > Ca2+ > > Mg2+. The insensitivity of these cells to high external Mg2+ contrasts with the reported sensitivity of the cloned CaR from parathyroid. 4. Elevation of [Ca2+]0 also stimulates increases in intracellular Ca2+ concentration ([Ca2+]i) and this effect is largely inhibited by the Ca2+ channel blocker nimodipine, indicating that L-type voltage-gated Ca2+ channels contribute to the response to elevated [Ca2+]0. 5. Elevated [Ca2+]0 induces an inward current under conditions where the only permeant external cation is Ca2+, indicating that influx via the cation conductance is another source of the increases in [Ca2+]i. 6. Extracellular Ca2+ stimulates 5-HT release with an EC50 of 1.5 mM. Nimodipine blocks 90% of the Ca2+0-induced 5-HT release, while other inhibitors of voltage-gated calcium channels had no effect. These data support an important role for L-type Ca2+ channels in CaR-induced hormone secretion. Although earlier studies indicate that high [Ca2+]0 induces release of Ca2+ from intracellular stores, thapsigargin-induced depletion of these stores did not affect secretion from these cells, indicating that Ca2+ influx is necessary and sufficient for the Ca2+0-induced 5-HT secretion. 7. Inhibition of protein kinase C (PKC) using chelerythrine, staurosporine, or calphostin C inhibited Ca2+0-induced 5-HT release by 50% while phorobol ester-induced 5-HT secretion was completely inhibited. Thus, PKC is an important component of the pathway linking CaR activation to hormone release. However, another as yet unknown second messenger also contributes to this pathway. 8. We tested the contribution of two different phospholipases to the CaR responses to determine the source of the PKC activator diacylglycerol (DAG). Selective inhibition of phosphatidylinositol-specific phospholipase C (PI-PLC) with U73122 had no effect on the response to elevated [Ca2+]0. However, pretreatment with D609, a selective inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), inhibited Ca(2+)-induced 5-HT release to 50% of control indicating that phosphatidylcholine is a likely source of DAG in the response of PF cells to elevated [Ca2+]0.

GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis.

We have found that, during the early stages of cortical neurogenesis, both GABA and glutamate depolarize cells in the ventricular zone of rat embryonic neocortex. In the ventricular zone, glutamate acts on AMPA/kainate receptors, while GABA acts on GABAA receptors. GABA induces an inward current at resting membrane potentials, presumably owing to a high intracellular Cl- concentration maintained by furosemide-sensitive Cl- transport. GABA and glutamate also produce increases in intracellular Ca2+ in ventricular zone cells, in part through activation of voltage-gated Ca2+ channels. Furthermore, GABA and glutamate decrease the number of embryonic cortical cells synthesizing DNA. Depolarization with K+ similarly decreases DNA synthesis, suggesting that the neurotransmitters act via membrane depolarization. Applied alone, GABAA and AMPA/kainate receptor antagonists increase DNA synthesis, indicating that endogenously released amino acids influence neocortical progenitors in the cell cycle. These results demonstrate a novel role for amino acid neurotransmitters in regulating neocortical neurogenesis.

Nicotine enhancement of fast excitatory synaptic transmission in CNS by presynaptic receptors.

The behavioral and cognitive effects of nicotine suggest that nicotinic acetylcholine receptors (nAChRs) participate in central nervous system (CNS) function. Although nAChR subunit messenger RNA (mRNA) and nicotine binding sites are common in the brain, there is little evidence for synapses mediated by nAChRs in the CNS. To test whether, CNS nAChRs might modify rather than mediate transmission, the regulation of excitatory synaptic transmission by these receptors was examined. Nanomolar concentrations of nicotine enhanced both glutamatergic and cholinergic synaptic transmission by activation of presynaptic nAChRs that increased presynaptic [Ca2]i. Pharmacological and subunit deletion experiments reveal that these presynaptic nAChRs include the alpha 7 subunit. These findings reveal that CNS nAChRs enhance fast excitatory transmission, providing a likely mechanism for the complex behavioral effects of nicotine.

Serotonin receptors. Genetic insights into serotonin function.

Targeted disruption of the genes for the 5-HT1B and 5-HT2C serotonin receptors and monoamine oxidase A have confirmed pharmacological experiments and revealed unexpected behavioral roles for serotonin.

Functional expression of the tachykinin NK1 receptor by floor plate cells in the embryonic rat spinal cord and brainstem.

1. The floor plate is a ventral mid-line structure that plays a pivotal role in the organization of the developing vertebrate central nervous system. Previous studies have demonstrated that the floor plate may provide signals that induce neuronal differentiation and guide axons; however, it is not known whether the floor plate can itself respond to signals that derive from surrounding tissue. 2. The peptide substance P is one of the first transmitters to be expressed in the developing spinal cord. To determine whether the floor plate may respond to substance P we have examined the expression of the principal substance P receptor (the tachykinin NK1 receptor) by floor plate cells of the rat embryonic spinal cord using immunocytochemistry, in situ hybridization and fura-2 calcium imaging. 3. Immunocytochemistry demonstrated selective expression of the NK1 receptor by cells at the ventral mid-line of the spinal cord. Double immunofluorescence labelling with the specific floor plate marker FP3 indicated that NK1 receptor expression is confined to cells in the lateral region of the floor plate. 4. In order to confirm the specificity of the NK1 receptor immunoreactivity we performed in situ hybridization histochemistry using antisense cRNA probes directed against the NK1 receptor. In situ hybridization demonstrated selective expression of NK1 receptor mRNA by floor plate cells. 5. The ontogeny of NK1 receptor protein and mRNA expression in the floor plate was defined. NK1 receptor expression occurred in a rostrocaudal progression that begins at embryonic day 10-11 (E10-E11) and is complete by E12-E14. The restriction of NK1 receptor expression to the lateral part of the floor plate was conserved throughout embryonic development. 6. NK1 receptor signalling was assessed by monitoring substance P-evoked changes in the intracellular concentration of calcium ions ([Ca2+]i) of acutely dissociated cells from the floor plate region. Application of substance P (5 nM) elevated [Ca2+]i in 10% of cells examined. 7. Selective neurokinin agonists were used to identify the receptor subtype involved in the substance P-evoked elevation of [Ca2+]i. Acetyl-[Arg6,Sar9,Met(O2)11]-substance P(6-11) (5 nM) and [Sar9,Met(O2)11]-substance P (5 nM), two highly selective NK1 receptor agonists, both elevated [Ca2+]i in floor plate cells that responded to substance P. [beta-Ala8]-neurokinin A(4-10) (50 nM) and senktide (50 nM), selective agonists respectively of NK2 and NK3 receptors, had no effect on [Ca2+]i.

Calcium entry through a subpopulation of AMPA receptors desensitized neighbouring NMDA receptors in rat dorsal horn neurons.

1. A Ca(2+)-dependent interaction between non-NMDA and NMDA receptors was studied in embryonic rat dorsal horn neurons grown in tissue culture using perforated-patch recording. Specifically, non-NMDA receptors were found to induce reversible inhibition of NMDA receptors in a manner dependent on the presence of extracellular Ca2+. 2. Non-NMDA receptor-induced inhibition of NMDA receptors was mediated by the elevation of intracellular Ca2+ concentration produced by Ca2+ entry through a subpopulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) non-NMDA receptors. Furthermore, Ca2+ entry through the AMPA channels alone is sufficient for desensitization of NMDA channels to occur. 3. Imaging of neuritic sites of Ca2+ revealed that Ca(2+)-permeable AMPA channels are often co-localized with NMDA channels on dorsal horn neurons, indicating that the Ca(2+)-mediated interaction between receptors may occur within small dendritic domains. 4. The ability of Ca(2+)-permeable AMPA channels to inhibit adjacent NMDA channels may contribute to the postsynaptic integration of excitatory input.

Substance P elevates intracellular calcium in both neurons and glial cells from the dorsal horn of the spinal cord.

1. We used microfluorimetric measurement of [Ca2+]i to identify substance P-sensitive cells acutely isolated from the dorsal horn of neonatal rats. We then used morphological, physiological, and immunocytochemical criteria to delineate two distinct populations of substance P-sensitive dorsal horn cells. 2. One population of cells with small-diameter cell bodies and many fine processes responds to substance P by releasing Ca2+ from internal stores. Many of these cells express the O4 surface antigen, and are thus likely to be glial cells, probably from the oligodendrocyte lineage. None of the cells with glial attributes respond to N-methyl-D-aspartate (NMDA), providing further evidence that they are nonneuronal. 3. In a second population of dorsal horn cells, substance P elevates [Ca2+]i by promoting Ca2+ entry. This class of cells is morphologically distinct from substance P-sensitive glial cells in that it exhibits large-diameter cell bodies, has smooth tapering processes, and is sensitive to NMDA. This second class of cells is therefore likely to consist of neurons. 4. Consistent with the identification of different mechanisms of Ca2+ elevation in the two cell types, the kinetics of the substance P-evoked release of Ca2+ in glial cells is very different than the kinetics of the Ca(2+)-entry response evoked in neurons. The glial cell response had a rapid average rate of rise (mean = 260 +/- 105 nM/s) and relatively brief duration (mean = 7.6 +/- 2.2 s) whereas the neuronal response had a much slower rate of rise (mean = 10 +/- 9 nM/s) with a much longer duration.(ABSTRACT TRUNCATED AT 250 WORDS)

An exceptional case of lethal disulfiram-alcohol reaction.

A case of fatal disulfiram-alcohol reaction due to ingestion of ethanol and antabuse is presented. Unusual autopsy findings and toxicological results are described. This case represents a report of corrosion lesion in the lower oesophagus and stomach due to a violent chemical reaction in situ and also the highest blood concentration of acetaldehyde (41 mg/l) ever recorded.

Measurement of 'free' gold in patients receiving disodium aurothiomalate and the association of high free to total gold levels with toxicity.

Serum from patients with rheumatoid arthritis (RA) receiving disodium aurothiomalate was analysed for total gold by atomic absorption spectrometry and for unbound (free) gold by the same method after ultrafiltration by an inert membrane. It was shown that it is possible to obtain reliable free gold concentrations by this method. Good correlations were shown between total and 'free' gold and between total and protein bound gold (PBG) for 54 patients with RA who were stabilised on gold therapy. Significant correlation was also shown between the same parameters for a second group of 15 patients starting gold therapy who were bled at weekly intervals for nine weeks immediately before medication. A single correlation with regression for all patients studied again showed good correlation between total and free gold and between total and PBG. Of the 189 paired values plotted, 182 fell within 2SD of the regression lines for the two plots. Of the seven patients with results outside 2SD of the regression line, six presented with side effects during the study.

Intragastric pH measurement using a novel disposable sensor.

An unique disposable pH sensor molded into the end of a nasogastric tube was tested in twelve healthy human volunteers. A Spearman's rank correlation coefficient (rs) of 0.90 was observed for the sensor and an indwelling miniature glass membrane electrode. The sensor did not correlate as well with aspirated stomach fluid (rs = 0.68). No sensor calibration was necessary and the sensors measured +/- 0.1 pH in laboratory pH buffers before and after the clinical study. Both bare and shielded disposable sensors closely agreed with a shielded miniature glass electrode.

Patterns of urinary excretion of gold in patients with rheumatoid arthritis undergoing chrysotherapy.

Thirty patients receiving gold therapy for rheumatoid arthritis (RA) were studied for the urinary excretion of gold. Statistical analysis of all the urine specimens passed over a period of four days by each patient showed that a definite rhythm of gold excretion exists for each patient which is possibly related to water excretion but not to creatinine excretion. The study indicates possible reasons for the inability of earlier workers to relate gold excretion to the general body gold status of patients and suggests that as the study of 24 hour excretions of gold may be an insensitive marker of gold excretion, closer examination of individual patient rhythms of gold excretion could possibly provide a more useful method of analysis.

Patterns of gold levels in urine, serum, and saliva in patients with rheumatoid arthritis undergoing chrysotherapy.

Twenty patients undergoing treatment with aurothiomalate for rheumatoid arthritis (RA) were studied for the presence of gold in all urine specimens passed over four days and for gold in the serum of blood drawn by venous section at 10.00, 16.00, and 22.00 hours on a single day of the study. Specimens of saliva collected at the same times as the blood specimens were also analysed for (total) gold content. Eighteen patients showed rhythmic urinary gold excretion. Variations were observed in the serum levels for total, free, and protein bound gold at different times of the day and night together with similar variations in the salivary total gold levels. It was established that a possible relation exists between urinary gold, serum gold, and salivary gold such that at times of higher urinary gold excretion the serum gold levels (total, free, and protein bound) and the total salivary gold levels were decreased. Conversely, at times of lower urinary gold excretion serum and salivary gold levels were increased.