Variation in the α 5 nicotinic acetylcholine receptor subunit gene predicts cigarette smoking intensity as a function of nicotine content.

A single-nucleotide polymorphism (SNP) in the α5 nicotinic acetylcholine receptor subunit gene, rs16969968, has been repeatedly associated with both smoking and respiratory health phenotypes. However, there remains considerable debate as to whether associations with lung cancer are mediated through effects on smoking behavior. Preclinical studies suggest that α5 receptor subunit expression and function may have a direct role in nicotine titration during self administration. The present study investigated the association of CHRNA5 polymorphisms and smoking topography in 66 smokers asked to smoke four nicotine-containing (nicotine yield=0.60 mg) and four placebo (nicotine yield <0.05 mg) cigarettes, during separate experimental sessions. Genotype at rs16969968 predicted nicotine titration, with homozygotes for the major allele (G:G) displaying significantly reduced puff volume in response to nicotine, whereas minor allele carriers (A:G or A:A) produced equivalent puff volumes for placebo and nicotine cigarettes. The present results suggest that puff volume may be a more powerful objective phenotype of smoking behavior than self-reported cigarettes per day and nicotine dependence. Further, these results suggest that the association between rs16969968 and lung cancer may be mediated by the quantity of smoke inhaled.

The nuclear envelope in the plant cell cycle: structure, function and regulation.

BACKGROUND: Higher plants are, like animals, organisms in which successful completion of the cell cycle requires the breakdown and reformation of the nuclear envelope in a highly controlled manner. Interestingly, however, while the structures and processes appear similar, there are remarkable differences in protein composition and function between plants and animals. SCOPE: Recent characterization of integral and associated components of the plant nuclear envelope has been instrumental in understanding its functions and behaviour. It is clear that protein interactions at the nuclear envelope are central to many processes in interphase and dividing cells and that the nuclear envelope has a key role in structural and regulatory events. CONCLUSION: Dissecting the mechanisms of nuclear envelope breakdown and reformation in plants is necessary before a better understanding of the functions of nuclear envelope components during the cell cycle can be gained.

Helping students become the medical teachers of the future--the Doctors as Teachers and Educators (DATE) Programme of Barts and the London School of Medicine and Dentistry, London.

CONTEXT: In the United Kingdom (UK), learning about teaching is an integral part of the General Medical Council's recommendations for the undergraduate medical curriculum. Yet often, implementing this aspect of learning presents a challenge to curriculum organisers in terms of content, timing and student interest. PROGRAMME OBJECTIVES AND STRUCTURE: The Doctors as Teachers and Educators (DATE) programme was set up at Barts and the London School of Medicine and Dentistry specifically to meet the requirements for development in teaching. Although largely practical, the two-day programme offers an introduction to educational theory and the teaching requirements for junior doctors in training. The methods used are lectures and group work within plenary sessions, followed by small group micro-teaching sessions. The DATE programme has now been undertaken by over 900 graduates. EVALUATION METHODS: We evaluated the Date programme by means of end-of-course questionnaires completed by two cohorts of students during the 2007/8 academic year and through the use of Nominal Group Technique in 2008/9. In line with the goals of the evaluation, the data on students' views were analysed to elicit self-reported learning and develop the programme. RESULTS: Response rates of the two cohorts to the surveys were high (80% and 98%). Nearly 100% of the students reported through the survey that they had gained confidence in teaching. In the nominal groups, students indicated that they had gained insight into educational principles like student-centredness and gained an appreciation for the nature of educational evidence and scholarship. They challenged the curriculum organisers to achieve an appropriate balance between theory and practice. CONCLUSIONS: A programme about teaching at the undergraduate medical level can be well-received by students; the DATE model could be transferred to other international contexts.

Cannabinoid receptor 1 (CNR1) gene variant moderates neural index of cognitive disruption during nicotine withdrawal.

Nicotine withdrawal-related disruption of cognitive control may contribute to the reinforcement of tobacco use. Identification of gene variants that predict this withdrawal phenotype may lead to tailored pharmacotherapy for smoking cessation. Variation on the cannabinoid receptor 1 gene (CNR1) has been related to nicotine dependence, and CNR1 antagonists may increase attention and memory functioning. We targeted CNR1 variants as moderators of a validated neural marker of nicotine withdrawal-related cognitive disruption. CNR1 polymorphisms comprising the 'TAG' haplotype (rs806379, rs1535255 and rs2023239) were tested independently, as no participants in this sample possessed this haplotype. Nicotine withdrawal-related cognitive disruption was indexed as increased resting electroencephalogram (EEG) alpha-1 power density across 17 electrodes. Seventy-three Caucasian Non-Hispanic smokers (≥15 cigarettes per day) visited the laboratory on two occasions following overnight smoking/nicotine deprivation. Either two nicotine or two placebo cigarettes were smoked prior to collecting EEG data at each session. Analyses showed that rs806379 moderated the effects of nicotine deprivation increasing slow wave EEG (P = 0.004). Smokers homozygous for the major allele exhibited greater nicotine withdrawal-related cognitive disruption. The current findings suggest potential efficacy of cannabinoid receptor antagonism as a pharmacotherapy approach for smoking cessation among individuals who exhibit greater nicotine withdrawal-related cognitive disruption.

Expression of nitric oxide synthase isoforms in bone and bone cell cultures.

Recent work has shown that nitric oxide (NO) acts as an important mediator of the effects of proinflammatory cytokines and mechanical strain in bone. Although several bone-derived cells have been shown to produce NO in vitro, less is known about the isoforms of NO synthase (NOS), which are expressed in bone or their cellular distribution. Here we investigated the expression, cellular localization, and regulation of NOS mRNA and protein in cultured bone-derived cells and in bone tissue sections. We failed to detect inducible NOS (iNOS) protein in normal bone using immunohistochemical techniques, even though low levels of iNOS mRNA were detected by sensitive reverse transcribed polymerase chain reaction (RT-PCR) assays in RNA extracted from whole bone samples. Cytokine stimulation of bone-derived cells and bone explant cultures caused dramatic induction of iNOS mRNA and protein in osteoblasts and bone marrow macrophages, but no evidence of iNOS expression was seen in osteoclasts by immunohistochemistry or in situ hybridization. Endothelial NOS (ecNOS) mRNA was also detected by RT-PCR in whole bone, and immunohistochemical studies showed widespread ecNOS expression in bone marrow cells and trabecular lining cells in vivo. Related studies in vitro confirmed that ecNOS was expressed in cultured osteoblasts, stromal cells, and osteoclasts. Neuronal NOS mRNA was detected by RT-PCR in whole bone, but we were unable to detect nNOS protein in bone cells in vivo or in studies of cultured bone-derived cells in vitro. In summary, our data show that mRNAs for all three NOS isoforms are expressed in bone and provide evidence for differential expression and regulation of the enzymes in different cell types. These findings confirm the likely importance of the L-arginine-NO pathway as a physiological mediator of bone cell function and demonstrate that it may be possible to exert differential effects on osteoblast and osteoclast activity in vivo by differential targeting of constitutive and inducible NOS isoforms by selective NOS inhibitors.

PM-type calcium pumps are associated with higher plant cell intracellular membranes.

The supposition that all eukaryotic cell types contain a plasma membrane (PM)-type Ca pump (i.e. a Ca pump which is directly-stimulated by calmodulin and located exclusively at the PM) has been questioned by recent data from higher plant cells. These studies suggest the presence of Ca pumps directly stimulated by calmodulin associated with an intracellular membrane (probably the endoplasmic reticulum, ER) in a variety of monocotelydonous and dicotelydonous species. Thus plants have a 'PM-type' Ca pump at an intracellular membrane. The evidence for this includes studies on isolated membranes, purification and functional reconstitution and phosphorylated intermediate formation. Plant cells also contain a homologue of the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) Ca pump, probably located at the ER. The implications of these new data for our appreciation of the structure, function and location of eukaryotic Ca pumps are discussed, together with recent data from the use of inhibitors specific to mammalian ER/SR Ca pumps.

Blockade of neonatal activation of the pituitary-testicular axis with continuous administration of a gonadotropin-releasing hormone agonist in male rhesus monkeys.

The objective of this study was to determine the effects of continuous GnRH agonist (Ag) treatment on neonatal activation of the pituitary-testicular axis in male rhesus monkeys. Five infants were treated continuously with Ag(10 micrograms/day; Wy-40972) for 112 days using osmotic minipumps beginning at 10-13 days of age. Two of five age-matched control animals were implanted sc with Silastic implants of comparable size to the minipumps; three did not receive sham implants. Ag treatment caused a fall in serum LH (bioassay) values to undetectable levels (much less than 0.1 micrograms/ml) within 3 weeks, where they remained throughout Ag treatment. Mean serum testosterone (T) levels fell from pretreatment values of 1.52 +/- 0.45 to 0.38 +/- 0.09 (+/- SE) ng/ml after 3 weeks of Ag treatment. The level of T never exceeded 0.60 ng/ml throughout the subsequent course of Ag treatment. In contrast, serum LH and T were elevated to levels that approached adult values during the first 2 postnatal months in control infants with or without sham implants. Both LH and T then gradually declined, and by 4 months of age, T levels were indistinguishable from those in Ag-treated animals. Control infants had an increase in serum LH from 0.56 +/- 0.10 to 2.67 +/- 0.49 micrograms/ml within 60 min of administration of 5 micrograms GnRH/kg BW at 60 days of age. Serum T values rose from 2.35 +/- 1.00 to 9.48 +/- 3.15 ng/ml during the same period. Seven weeks of Ag treatment abolished the LH and T responses to GnRH. Thirty days after the termination of Ag treatment (approximately 150 days of age), Ag-treated and control infants had comparable serum LH and T responses to GnRH, although the responses were reduced relative to the responses in controls at 60 days of age. These results suggest that continuous administration of Ag desensitizes the pituitary of the male infant rhesus monkey to GnRH and blocks neonatal activation of the pituitary-testicular axis.

Expression and characterization of PP7, a novel plant protein Ser/Thr phosphatase distantly related to RdgC/PPEF and PP5.

We have recently identified an Arabidopsis thaliana cDNA encoding a putative protein Ser/Thr phosphatase PP7, not closely related to any protein phosphatases in animals or fungi. Here, we describe the characterization of PP7 expressed in a bacterial system. The recombinant protein was inactive unless the longest insert in its catalytic domain was cleaved, suggesting that this insert is an autoinhibitory region. PP7 was resistant to okadaic acid, calyculin and fumonisin B1, and was stimulated by Mn2+ or Fe2+, while Ni2+ and Zn2+ were inhibitory. Polylysine stimulated PP7 activity towards p-nitrophenylphosphate but inhibited activity towards the most efficient protein substrate, myelin basic protein. A tentative model of the control of PP7 activity is proposed.

A calcium pump at the higher plant nuclear envelope?

Evidence for a Ca2+-pump at the nuclear envelope (NE) in plant cells has been obtained using confocal and electron microscope immunocytochemistry and antibodies raised to a plant homologue of the mammalian SERCA pump. This is the first evidence suggesting an NE Ca2+-pump in plants. In addition to being localised with the NE in interphase, the antigen was localised to membrane derived from the NE and associated ER during mitosis, correlating with known Ca2+-pools. The work suggests that a SERCA pump is present at the NE of plant as well as animal cells.

Babesiosis (Babesia bovis) stability in unstable environments.

Enzootic stability (herd immunity) in bovine babesiosis occurs when the rate of transmission (inoculation rate) of Babesia spp by the tick vector is sufficient to immunize a majority of susceptible calves before the loss of calfhood resistance. The effect of three tick (Boophilus microplus) control strategies (none, threshold, and strategic) on enzootic stability and the likelihood of babesiosis (Babesia bovis) outbreaks was studied using a spreadsheet age-class computer simulation model. The model was driven by weekly bovine tick counts from Brazil and Uruguay. The Eldorado do Sul, RS, Brazil bovine population (30 degrees 05' South latitude) was found to be in a naturally occurring state of enzootic stability, corresponding to an inoculation rate exceeding 0.005 throughout the year. Threshold dipping strategies should not increase the risk of babesiosis in cattle so managed. Strategic dipping resulted in an extended period of enzootic instability lasting 30 weeks, which requires protection of the herd through immunization. Because of the more prolonged low winter temperature conditions, the Tacuarembó, Uruguay bovine population (31 degrees 40' South latitude) was found to be in a naturally occurring state of enzootic instability, characterized by a 28 week period in which the inoculation rate was below 0.005. Strategic dipping should lead to eradication of the babesial parasite from tick and bovine populations, but would not result in eradication of the tick vector. This could lead to subsequent outbreaks if Babesia carrier animals were to be introduced into the herd. In both populations, strategic tick control could be accompanied by concurrent babesiosis vaccination.

Surface hydrophobicity and dispersal of Pseudomonas aeruginosa from biofilms.

A novel method of cell culture was employed to control the growth-rate of bacterial biofilms [1]. Cell-surface hydrophobicity increased progressively with growth rate for planktonic, chemostatgrown Pseudomonas aeruginosa and also for cells, resuspended from the biofilms. Dependence of surface hydrophobicity upon growth rate was greater for the planktonic cells. Newly-formed daughter cells, shed from the biofilms, were in all cases more hydrophilic than their adherent counterparts and demonstrated only slight growth rate dependency for this property.

Visual loss as a result of primary optic nerve neuropathy after intranasal corticosteroid injection.

A 25-year-old woman experienced acute visual loss after injection of methylprednisolone suspension into the concha nasalis inferioris. Results of ophthalmoscopic examinations were normal throughout the observation period, a finding not reported previously in cases of visual loss after head or neck injections. An afferent pupillary defect, which diminished as visual acuity improved, implicated otpic nerve damage, as did the visual field defect which also improved slightly. This is, to our knowledge, the first case report of acute visual loss caused by optic nerve neuropathy after injection to the head or neck.

Relative vulnerability of the brain and spinal cord to ischemia.

In this experiment CER and SER were monitored as blood flow was progressively lowered by lowering the systemic arterial pressure below the lower limits of autoregulation (bleeding). Blood flow in the brain and dorsal column of the spinal cord was monitored and recorded with the hydrogen clearance method. Long tract neural conduction in the spinal cord appeared quite refractory to the effects of ischemia and disappeared only after 8--18 min of essentially absolute ischemia. The CER was more sensitive to the effects of ischemia, disappearing first in one animal and returning later in all of the animals. The SER returned in all animals after re-infusion of the blood and re-establishment of the blood flow even after a 13--23 min period of absolute ischemia and a 5 min period of electrical silence.

The effect of sciatic nerve stimulation on spinal cord blood flow.

Focal spinal cord blood flow was measured in the left dorsal column during left sciatic nerve stimulation, and compared to blood flow in the same area during sciatic nerve inactivity. Blood flow was found to be significantly increased during stimulation. It is suggested that this increased blood flow is a reflection of increased metabolic demands of the neurons and synaptic systems within the internuncial neural pools, activated by stimulation of all components of the sciatic nerve, rather than a reflection of increased non-synaptic axonal conduction in the dorsal column.

Reduction of experimental intracranial hypertension by lidocaine.

Previous animal studies in our laboratory revealed that acute cerebral ischemia induced by air embolism is accompanied by a sharp and pronounced rise in both systemic blood pressure and intracranial pressure. To investigate possible approaches to the treatment of severe intracranial hypertension, we produced cerebral air embolism in anesthetized, ventilated cats by the infusion of air into the vertebral artery. Untreated animals consistently experienced severe acute intracranial hypertension. Pretreating the animals with lidocaine greatly attenuated the intracranial hypertension caused by air embolism. Lidocaine given after the injury was also effective in reducing intracranial pressure. Our results suggest that lidocaine may be a useful agent for reducing intracranial hypertension associated with ischemic or traumatic injuries to the brain.

Effects of progressive hypoxia on long tract neural conduction in the spinal cord.

In this experiment, the vulnerability of long tract neural conduction in the spinal cord to progressive hypoxia was studied. The physiological integrity of nonsynaptic spinal cord conduction was monitored with the spinal evoked response (SER). Focal spinal cord blood flow was measured with the hydrogen clearance method. Progressive hypoxia was created by progressively increasing the amount of nitrogen in the inspired gas mixture. The SER was seen to fail only after extended periods of severe hypoxia. Multisynaptic cerebral condition monitored by the cerebral evoked response (CER) seemed more sensitive in the three animals in which both SER and CER were recorded. Spinal cord blood flow (SCBF) was not affected by progressive hypoxia until the PO2 was below 40 torr, and then the SCBF rose dramatically with further progression of the hypoxia.

The effects of ischemia on long-tract neural conduction in the spinal cord.

In this experiment, the effects of ischemia on neural conduction in the monkey spinal cord were studied. In six monkeys generalized ischemia of the spinal cord was created by bleeding the animals to a hypotensive level below the lower limits of autoregulation in the spinal cord. The progressive development of spinal cord ischemia was documented by blood-flow measurement using the hydrogen clearance method. Physiological integrity of the spinal cord was monitored and recorded by the spinal evoked response. The spinal evoked response did not disappear until at least 10 minutes of profound ischemia. At levels of ischemia 20% to 25% of normal blood flow, the spinal evoked response was unchanged. It is concluded that long-tract neural conduction in the spinal cord is relatively resistant to the effects of ischemia.

Experimental acute balloon compression of the spinal cord. Factors affecting disappearance and return of the spinal evoked response.

Acute balloon compression of the thoracic spinal cord for 15, 7, 5, 3, and 1 minute in monkeys caused immediate disappearance of the spinal evoked response and complete focal ischemia of the compressed segment in all animals. Only the animals in the 1-minute group, however, demonstrated return of the evoked response. These data, coupled with data from previous experiments of slow balloon compression of the spinal cord and spinal cord ischemia, suggest that the major pathological substrate for neural dysfunction after balloon compression of the spinal cord, be it acute or slow, is physical injury of the neural membrane, irrespective of blood flow changes. These findings also suggest that the ability of that membrane to recover is related to rapidity and length of time of compression. Focal changes in blood flow do not appear to be significant in this mechanism.

Cardiac arrhythmias accompanying acute compression of the spinal cord.

This study was undertaken to determine the cardiovascular response to compression of the spinal cord and to determine the autonomic mechanisms involved. The electrocardiogram and arterial blood pressure were recorded in anesthetized monkeys during inflation of a balloon catheter in the epidural space of the mid-thoracic region. Acute spinal cord compression resulted in a wide variety of severe cardiac arrhythmias and acute hypertension. The arrhythmias were found to result from hyperactivity of both the sympathetic and parasympathetic divisions of the autonomic nervous system.

The effect of alpha adrenergic blockade on spinal cord autoregulation in the monkey.

Spinal cord blood flow (SCBF) was measured over a wide range of artificially varied mean systemic arterial blood pressures (MAP) in a group of monkeys with alpha adrenergic receptors blocked by the intravenous administration of phenoxybenzamine (Dibenzyline). The SCBF was found to vary linearly with changes in MAP. Autoregulation appeared to have been abolished. These data are cited as evidence for a dominant role of the sympathetic nervous system in control of the spinal circulation.