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Early, low-risk International Prognostic Scoring System (IPSS) myelodysplastic syndrome (MDS) is a heterogeneous disorder where the molecular and cellular hematopoietic defects are poorly understood. To gain insight into this condition, we analyzed gene expression profiles of marrow CD34+ progenitor cells from normal-karyotype, low-blast-count MDS patients, age-matched controls, and patients with non-MDS anemia. Given the heterogeneity of early MDS, a surprisingly consistent finding was decreased expression of B-cell lineage–affiliated genes in MDS patients compared with healthy controls and 3 of 5 samples with non-MDS anemia. Both patients with non-MDS anemia with reduced B-cell gene expression were on chemotherapy. In 25 of 27 of the original samples and 9 further MDS samples, Taqman real-time polymerase chain reaction (PCR) confirmed these data. Flow cytometry on unfractionated marrow from independent samples also demonstrated reduced B-cell progenitors in MDS patients compared with healthy controls. These novel findings suggest a common perturbation in early MDS hematopoiesis. They also provide the rationale for a larger study to evaluate the diagnostic utility of reduced B-cell progenitor number as a diagnostic biomarker of early low-risk MDS, which can pose a diagnostic challenge.

ATP-sensitive potassium (KATP) channels link membrane excitability to metabolism. They are regulated by intracellular nucleotides and by other factors including membrane phospholipids, protein kinases and phosphatases. KATP channels comprise octamers of four Kir6 pore-forming subunits associated with four sulphonylurea receptor subunits. The exact subunit composition differs between the tissues in which the channels are expressed, which include pancreas, cardiac, smooth and skeletal muscle and brain. KATP channels are targets for antidiabetic sulphonylurea blockers, and for channel opening drugs that are used as antianginals and antihypertensives. This review focuses on non-pancreatic KATP channels. In vascular smooth muscle, KATP channels are extensively regulated by signalling pathways and cause vasodilation, contributing both to resting blood flow and vasodilator-induced increases in flow. Similarly, KATP channel activation relaxes smooth muscle of the bladder, gastrointestinal tract and airways. In cardiac muscle, sarcolemmal KATP channels open to protect cells under stress conditions such as ischaemia or exercise, and appear central to the protection induced by ischaemic preconditioning (IPC). Mitochondrial KATP channels are also strongly implicated in IPC, but clarification of their exact role awaits information on their molecular structure. Skeletal muscle KATP channels play roles in fatigue and recovery, K efflux, and glucose uptake, while neuronal channels may provide ischaemic protection and underlie the glucose-responsiveness of hypothalamic neurones. Current therapeutic considerations include the use of KATP openers to protect cardiac muscle, attempts to develop openers selective for airway or bladder, and the question of whether block of extra-pancreatic KATP channels may cause adverse cardiovascular side-effects of sulphonylureas.

Presents tips for master of business administration (MBA) graduates on starting their first job as a seasonal associate or as a full-time employee. Advice on travelling with the boss; Communication laws an MBA graduate should always consider; Rules for client meetings.

A novel protocol for the generation of sulfur ylides is described. The overall process involves thermal decarboxylation of a carboxymethylsulfonium betaine to give a sulfur ylide that, in the presence of an aldehyde, affords the corresponding terminal oxirane. Yields were found to correlate with the electron deficiency of the aryl aldehyde. In situ generation of betaine in the presence of an aldehyde successfully afforded the desired oxirane in moderate yield, thus demonstrating the feasibility of a catalytic process.

1We have investigated the possible role of A‐kinase anchoring proteins (AKAPs) in protein kinase A (PKA) signalling to ATP‐sensitive K+(KATP) channels of rat isolated mesenteric arterial smooth muscle cells using whole‐cell patch clamp and peptides that inhibit PKA‐AKAP binding.2Intracellular Ht31 peptide (20 μm), which inhibits the PKA‐AKAP interaction, blocked KATPcurrent activation by either dibutyryl cAMP or calcitonin gene‐related peptide. Ht31‐proline (20 μm), which does not inhibit PKA binding to AKAP, did not block KATPcurrent activation.3Ht31 reduced KATPcurrent activated by pinacidil and also prevented its inhibition by Rp‐cAMPS, effects consistent with Ht31 blocking steady‐state KATPchannel activation by PKA. However, Ht31 did not prevent KATPcurrent activation by the catalytic subunit of PKA.4An antibody to the RII subunit of PKA showed localization of PKA near to the cell membrane. Our results provide evidence that both steady‐state and receptor‐driven activation of KATPchannels by PKA involve the localization of PKA by an AKAP.

The KATPchannel opener diazoxide has been proposed to protect cardiac muscle against ischaemia by opening mitochondrial KATPchannels to depolarize the mitochondrial membrane potential, ΔΨm. We have used the fluorescent dye TMRE to measure ΔΨmin adult rat freshly isolated cardiac myocytes exposed to diazoxide and metabolic inhibition.Diazoxide, at concentrations that are highly cardioprotective (100 or 200 μM), caused no detectable increase in TMRE fluorescence (n=27 cells). However, subsequent application of the protonophore FCCP, which should collapse ΔΨm, led to large increases in TMRE fluorescence (>300%).Metabolic inhibition (MI: 2 mMNaCN+1 mMiodoacetic acid (IAA) led to an immediate partial depolarization of ΔΨm, followed after a few minutes delay by complete depolarization which was correlated with rigor contracture. Removal of metabolic inhibition led to abrupt mitochondrial repolarization followed in many cells by hypercontracture, indicated by cell rounding and loss of striated appearance.Prior application of diazoxide (100 μM) reduced the number of cells that hypercontracted after metabolic inhibition from 63.7±4.7% to 24.2±1.8% (P<0.0001). 5‐hydroxydeanoate (100 μM) reduced the protection of diazoxide (46.8±2.7% cells hypercontracted, P<0.0001 vsdiazoxide alone).Diazoxide caused no detectable change in flavoprotein autofluorescence (n=26 cells).Our results suggest that mitochondrial depolarization and flavoprotein oxidation are not inevitable consequences of diazoxide application in intact cardiac myocytes, and that they are also not essential components of the mechanism by which it causes protection.

The authors have previously presented a methodology to guide the development of virtual learning environments (VLEs) for use by people with learning disabilities (Brown, Neale, Cobb, & Reynolds 1999). This paper presents the results of two sets of studies and shows how these studies enhance this methodology. The first set of studies are user-centered designs for VLE to teach horticultural-related employment, leisure, travel, and public presentation skills to young people with learning disabilities. These projects have allowed us for the first time to distill a set of design guidelines for the production of VLEs for use by people with a learning disability. The second set of studies aims to determine the tutoring strategies employed by support workers, teachers, and care providers when supporting people with learning disabilities using VLEs. These strategies will be embedded in the VLEs to further scaffold the learning process of our target users and allow us to expand the guidance given within the implementation component of the development methodology. These enhancements of the existing methodology are clearly developed within the paper and illustrated diagrammatically.

The neuronal adaptor protein X11α participates in the formation of multiprotein complexes and intracellular trafficking. It contains a series of discrete protein-protein interaction domains including two contiguous C-terminal PDZ domains. We used the yeast two-hybrid system to screen for proteins that interact with the PDZ domains of human X11α, and we isolated a clone encoding domains II and III of the copper chaperone for Cu,Zn-superoxide dismutase-1 (CCS). The X11α/CCS interaction was confirmed in coimmunoprecipitation studies plus glutathioneS-transferase fusion protein pull-down assays and was shown to be mediated via PDZ2 of X11α and a sequence within the carboxyl terminus of domain III of CCS. CCS delivers the copper cofactor to the antioxidant superoxide dismutase-1 (SOD1) enzyme and is required for its activity. Overexpression of X11α inhibited SOD1 activity in transfected Chinese hamster ovary cells which suggests that X11α binding to CCS is inhibitory to SOD1 activation. X11α also interacts with another copper-binding protein found in neurons, the Alzheimer's disease amyloid precursor protein. Thus, X11α may participate in copper homeostasis within neurons.

Selective protein kinase C (PKC) activators and inhibitors were used to investigate the involvement of specific PKC isoforms in the modulation of voltage‐sensitive Ca2+channels (VSCCs) in bovine adrenal chromaffin cells. Exposure to the phorbol ester phorbol‐12,13‐dibutyrate (PDBu) inhibited the Ca2+currents elicited by depolarizing voltage steps. This inhibition was occluded by the PKC‐specific inhibitor Ro 31‐8220 but remained unaffected by Gö 6976, a selective inhibitor of conventional PKC isoforms. PDBu treatment caused the translocation of PKC‐α and ‐ϵ isoforms from cytosol to membranes. PKC‐ι and ‐ζ showed no signs of translocation. It is concluded that VSCCs are specifically inhibited by the activation of PKC‐ϵ in chromaffin cells. This may be relevant to the action of phospholipase‐linked receptors involved in the control of Ca2+influx, both in catecholaminergic cells and other cell types.

OBJECTIVES

Selective protein kinase C (PKC) activators and inhibitors were used to investigate the involvement of specific PKC isoforms in the modulation of voltage-sensitive Ca 2+channels (VSCCs) in bovine adrenal chromaffin cells. Exposure to the phorbol ester phorbol-12,13-dibutyrate (PDBu) inhibited the Ca 2+currents elicited by depolarizing voltage steps. This inhibition was occluded by the PKC-specific inhibitor Ro 31-8220 but remained unaffected by Gö 6976, a selective inhibitor of conventional PKC isoforms. PDBu treatment caused the translocation of PKC-α and -ϵ isoforms from cytosol to membranes. PKC-ι and -ζ showed no signs of translocation. It is concluded that VSCCs are specifically inhibited by the activation of PKC-ϵ in chromaffin cells. This may be relevant to the action of phospholipase-linked receptors involved in the control of Ca 2+influx, both in catecholaminergic cells and other cell types.

1We used whole‐cell patch clamp to investigate steady‐state activation of ATP‐sensitive K+channels (KATP) of rat arterial smooth muscle by protein kinase A (PKA) and the pathway by which angiotensin II (Ang II) inhibits these channels.2Rp‐cAMPS, an inhibitor of PKA, did not affect KATPcurrents activated by pinacidil when the intracellular solution contained 0.1 mM ATP. However, when ATP was increased to 1.0 mM, inhibition of PKA reduced KATPcurrent, while the phosphatase inhibitor calyculin A caused a small increase in current.3Ang II (100 nM) inhibited KATPcurrent activated by the K+channel opener pinacidil. The degree of inhibition was greater with 1.0 mM than with 0.1 mM intracellular ATP. The effect of Ang II was abolished by the AT1receptor antagonist losartan.4The inhibition of KATPcurrents by Ang II was abolished by a combination of PKA inhibitor peptide 5‐24 (5 μM) and PKC inhibitor peptide 19‐27 (100 μM), while either alone caused only partial block of the effect.5In the presence of PKA inhibitor peptide, the inhibitory effect of Ang II was unaffected by the PKC inhibitor Gö 6976, which is selective for Ca2+‐dependent isoforms of PKC, but was abolished by a selective peptide inhibitor of the translocation of the ε isoform of PKC.6Our results indicate that KATPchannels are activated by steady‐state phosphorylation by PKA at normal intracellular ATP levels, and that Ang II inhibits the channels both through activation of PKCε and inhibition of PKA.

1We have used patch clamp to record large‐conductance Ca2+‐activated K+(BKCa) currents from a human embryonic kidney cell line (HEK293) expressing wild‐type and mutant hSloα channels.2When we mutated F380 in the S6 region, thought to contribute to the intracellular vestibule of the pore, to isoleucine (F380I), very little channel activity was recorded. In contrast, mutation to tyrosine (F380Y) resulted in significant voltage‐dependent currents.3The unitary conductances of F380I, F380Y and wild‐type channels were 92 ± 6 pS (n= 3), 166 ± 5 pS (n= 3) and 294 ± 5 pS (n= 5), respectively.4Both mutant and wild‐type hSloα channels were sensitive to 100 nM iberiotoxin.5The F380Y mutant produced channels that were active at negative membrane potentials, even in the absence of Ca2+.6We conclude that this conserved residue within BKCachannels may line the conduction pathway and forms a key element of the gating mechanism.

The Fe65s and XIIs are two families of adaptor proteins that bind to the Alzheimer's disease amyloid precursor protein (APP). Although both the XIIs and Fe65s bind to similar regions of APP, they have opposing effects on Aβ production and hence may represent novel therapeutic targets. However, there is no evidence that the Fe65s and XIIs are present within the same cell type or cell compartment and are thus capable of competing for binding to APP. Here we show that in neurones and transfected cells, APP, Fe65 and X11β show overlapping subcellular distributions. Furthermore, we demonstrate that Fe65 and X11β compete for binding to APP.

X11α and X11β are two neuronal adaptor proteins that interact with the Alzheimer's disease amyloid precursor protein (APP). X11α and X11β stabilise APP and inhibit production of proteolytic APP fragments including the Aβ peptide that is deposited in the brains of Alzheimer's disease patients. The mechanisms by which X11α and X11β modulate APP processing are not clear but one possibility is that they influence the activity of the secretases that cleave APP to give rise to Aβ. Presenilin-1 is required for γ-secretase activity and here we demonstrate that both X11α and X11β interact with presenilin-1. X11/presenilin-1 binding is via two X11 PDZ domains and sequences within the carboxy-terminus of presenilin-1. We also demonstrate that both X11α and X11β mediate the formation of complexes between APP and presenilin-1. These results suggest that the X11 regulation of APP processing is controlled, at least in part, via their interactions with APP and presenilin-1.

This paper examines the value of particle‐size counting and turbidimeters at a groundwater supply works in various modes, from the early commissioning of a full‐scale microfiltration plant through to process optimisation and monitoring. The relative strengths and weaknesses of particle‐size counters and turbidimeters are discussed in terms of their application.

Pneumomediastinum may result from a variety of causes that may be either intrathoracic (eg, narrowed or plugged airway, straining against a closed glottis, blunt chest trauma, alveolar rupture) or extrathoracic (eg, sinus fracture, iatrogenic manipulation in dental extraction, perforation of a hollow viscus [corrected]. The radiographic signs of pneumomediastinum depend on the depiction of normal anatomic structures that are outlined by the air as it leaves the mediastinum. These signs include the thymic sail sign, "ring around the artery" sign, tubular artery sign, double bronchial wall sign, continuous diaphragm sign, and extrapleural sign. In distal esophageal rupture, air may migrate from the mediastinum into the pulmonary ligament. Pneumomediastinum may be difficult to differentiate from medial pneumothorax and pneumopericardium. Occasionally, normal anatomic structures (eg, major fissure, anterior junction line) may simulate air within the mediastinum. Iatrogenic entities that may simulate pneumomediastinum include helium in the balloon of an intraaortic assist device. In addition, pneumomediastinum may be simulated by the Mach band effect, which manifests as a region of lucency adjacent to structures with convex borders. The absence of an opaque line, which is typically seen in pneumomediastinum, can aid in differentiation. Computed tomographic (CT) digital radiography and conventional CT can also be helpful in establishing or confirming the diagnosis.

Objectives. The purpose of this study was to apply Weiner s (1986) attributional model of helping to the care of clients presenting with challenging behaviour. A number of predictions were tested: (a) that aggressive, destructive, and selfinjurious behaviours would differentially affect carers ratings of attributional dimensions; (b) that carers propensity to help would be mediated by positive affect rather than optimism; (c) that optimism would be reduced by a perceived stable cause, such as client s level of dependency.

‘Immunonutrition’ is inarguably one of the more essential components of critical care. Basic aims are to modulate the immune response with naturally occurring nutrients so as to limit tissue injury, reduce infection rates and morbidity, so ultimately improving survival. While some controversy surrounds the optimal enteral formula, enrichment with glutamine or the combination of arginine, purine nucleotides and omega-3 fatty acids has been the most widely administered in clinical practice. A number of clinical trials and two meta-analyses suggest that these forms of immunonutrition are of benefit in specific groups of patients. In the future, the optimal combination of immunonutrients for different patient groups and diseases will lead to further advances in patient care.

1We have used the patch‐clamp technique in combination with fluorimetric recording to study the mechanisms that regulate intracellular Ca2+, [Ca2+]i, following depolarization in cells isolated from the rat femoral artery.2Depolarization to 0 mV from a holding potential of −70 mV increased [Ca2+]i. Little Ca2+release from sarcoplasmic reticulum, SR, was detected during depolarization since application of 30 μM ryanodine, a Ca2+‐release inhibitor, had no significant effect on total Ca2+buffering power.3Upon repolarization to −70 mV, 7 out of 13 cells showed three phases of Ca2+removal; an initial rapid first phase, a slow second phase, and a faster third phase. Six cells, in which Ca2+recovered quickly, lacked the third phase. The third phase was also absent in cells treated with a SR Ca2+‐pump inhibitor, cyclopiazonic acid.4The peak first‐phase Ca2+removal rate observed upon repolarization to −70 mV was significantly reduced in cells treated with a mitochondrial Ca2+uptake inhibitor, carbonyl cyanide m‐chlorophenylhydrazone. However, an ATP‐synthase inhibitor, oligomycin B, had no significant effect.5The Ca2+removal rate was little affected by clamping the cell at +120 mV rather than −70 mV, suggesting that Ca2+removal processes are largely voltage independent. Also, little inward current was associated with Ca2+clearance, indicating that Ca2+removal does not involve an electrogenic process.6Our results suggest that Ca2+‐induced Ca2+release contributes little to the elevation of Ca2+in these cells. The SR Ca2+pump may contribute to Ca2+removal over a low [Ca2+]irange in cells where [Ca2+]iremains high for long enough, while mitochondrial Ca2+uptake may be important when [Ca2+]iis high.

The molecular forms of gonadotropin-releasing hormone (GnRH) were examined in the bonytongue fishes (Osteoglossomorpha), one of the most ancient living teleost groups. These fish represent a phylogenetic link between the early ray-finned fishes and the modern teleosts. Five representative species from four of six bonytongue families were examined for GnRH using high-performance liquid chromatography and radioimmunoassay techniques with antisera raised against salmon (s), chicken-II (c-II), and mammalian (m) forms of GnRH. Salmon GnRH and cGnRH-II were identified in four of the species (arawana, elephantnose, false featherfin, Asiatic featherfin) whereas in the butterfly fish, mGnRH and cGnRH-II were identified. Our data suggest that teleosts such as eels and butterfly fish, which have mGnRH like that of even earlier ray-finned fishes, may have evolved before fish with sGnRH. We also suggest that sGnRH first appeared in the Osteoglossomorpha. The phylogenetic relationship of the eels (Anguillidae), butterfly fish (Pantodontidae), and bonytongue fish among other teleosts needs to be reexamined using additional characteristics.

The aim of the current study was to characterize which cannabinoid receptors, if any, are present on rat carotid artery smooth muscle. Additionally, the effects of cannabinoids on carotid artery tone, on cyclic AMP accumulation and on forskolin‐induced relaxation were examined in the same tissue.Stimulation of carotid arteries with forskolin (10 μM) significantly increased cyclic AMP accumulation, an effect that was inhibited in a concentration‐dependent manner by the cannabinoid receptor agonist, methanandamide.Similar inhibition was seen with the CB1agonist HU‐210 but this inhibition was not mimicked by the CB2agonist, WIN 55,2212‐2.The inhibitory effect of methanandamide on cyclic AMP accumulation was prevented by incubation of the arteries with pertussis toxin and was significantly reduced by LY320135, a selective CB1antagonist, but not by SR 144528, a CB2‐selective antagonist.Methanandamide failed to relax carotid arteries pre‐contracted with phenylephrine, but inhibited forskolin‐induced relaxation of these arteries. This functional inhibition of relaxation by methanandamide was inhibited by CB1‐selective (LY320135 and SR 141716A), but not a CB2‐selective antagonist (SR 144528).These data demonstrate the presence of functional G protein‐linked cannabinoid receptors of the CB1subtype in the rat carotid artery, but show that these receptors inhibit cyclic AMP accumulation rather than cause relaxation.

We have investigated the inhibition of inwardly rectifying potassium channels by the α‐adrenergic agonist/antagonist chloroethylclonidine (CEC). We used two preparations; two‐electrode voltage‐clamp of rat isolated flexor digitorum brevis muscle and whole‐cell patch‐clamp of cell lines transfected with Kir2.1 (IRK1).In skeletal muscle and at a membrane potential of −50 mV, chloroethylclonidine (CEC), an agonist at α2‐adrenergic receptors and an antagonist at α1x‐receptors, was found to inhibit the inward rectifier current with a Kiof 30 μM.The inhibition of skeletal muscle inward rectifier current by CEC was not mimicked by clonidine, adrenaline or noradrenaline and was not sensitive to high concentrations of α1‐(prazosin) or α2‐(rauwolscine) antagonists.The degree of current inhibition by CEC was found to vary with the membrane potential (approximately 70% block at −50 mV c.f. ∼10% block at −190 mV). The kinetics of this voltage dependence were further investigated using recombinant inward rectifier K+channels (Kir2.1) expressed in the MEL cell line. Using a two pulse protocol, we calculated the time constant for block to be ∼8 s at 0 mV, and the rate of unblock was described by the relationship τ=exp((Vm+149)/22) s.This block was effective when CEC was applied to either the inside or the outside of patch clamped cells, but ineffective when a polyamine binding site (aspartate 172) was mutated to asparagine.The data suggest that the clonidine‐like imidazoline compound, CEC, inhibits inward rectifier K+channels independently of α‐receptors by directly blocking the channel pore, possibly at an intracellular polyamine binding site.

The functional and electrophysiological effects of IMID‐4F (2‐[N‐(2,6‐dichlorophenyl)‐N‐(4‐flurorobenzyl)amino]imidazoline), a fluoro‐benzyl derivative of clonidine, on vascular KATPchannels were investigated. In pig coronary artery, IMID‐4F inhibited the vasorelaxation response to the KATPchannel opener levcromakalim with a pKBvalue of approximately 7.1. IMID‐4F (30 μM) did not affect the vasorelaxation response to sodium nitroprusside (SNP).In rat mesenteric artery smooth muscle cells IMID‐4F (1–10 μM) caused a concentration‐dependent depolarization of membrane potential. IMID‐4F (10 μM) abolished the hyperpolarizing effects of levcromakalim (10 μM).In patch clamp experiments using rat mesenteric artery smooth muscle cells, KATPchannel currents induced by levcromakalim (10 μM) were inhibited by IMID‐4F (0.3–3 μM) in a concentration‐dependent manner. The calculated IC50for IMID‐4F inhibiting KATPchannel current was approximately 0.8 μM.Radioligand binding studies using bovine aortic smooth muscle cell membranes showed that IMID‐4F (30 μM) did not displace binding to the KATPchannel opener [3H]‐P1075. However, both levcromakalim (10 μM) and glibenclamide (10 μM) caused significant displacement of [3H]‐P1075.These studies show that the imidazoline compound IMID‐4F is one of the most potent antagonists of arterial KATPchannels identified. Vasorelaxation, hyperpolarization and K+currents through KATPchannels were all inhibited by IMID‐4F at micromolar concentrations. Radioligand binding studies indicate that IMID‐4F does not bind to the same site as levcromakalim or as glibenclamide. Considering other evidence, it is likely that IMID‐4F acts by interacting directly with the pore of the KIRchannel, rather than through the sulphonylurea subunit of the KATPchannel complex.

We used whole‐cell patch clamp to investigate the currents activated by nicorandil in smooth muscle cells isolated from rat small mesenteric arteries, and studied the relaxant effect of nicorandil using myography.Nicorandil (300 μM) activated currents with near‐linear current‐voltage relationships and reversal potentials near to the equilibrium potential for K+.The nicorandil‐activated current was blocked by glibenclamide (10 μM), but unaffected by iberiotoxin (100 nM) and the guanylyl cyclase inhibitor LY 83583 (1 μM). During current activation by nicorandil, openings of channels with a unitary conductance of 31 pS were detected.One hundred μMnicorandil had no effect on currents through Ca2+channels recorded in response to depolarizing voltage steps using 10 mMBa2+as a charge carrier. A small reduction in current amplitude was seen in 300 μMnicorandil, though this was not statistically significant.In arterial rings contracted with 20 mMK+Krebs solution containing 200 nMBAYK 8644, nicorandil produced a concentration‐dependent relaxation with mean pD2=4.77±0.06. Glibenclamide (10 μM) shifted the curve to the right (pD2=4.32±0.05), as did 60 mMK+. LY 83583 caused a dose‐dependent inhibition of the relaxant effect of nicorandil, while LY 83583 and glibenclamide together produced greater inhibition than either alone.Metabolic inhibition with carbonyl cyanide m‐chlorophenyl hydrazone (30 nM), or by reduction of extracellular glucose to 0.5 mM, increased the potency of nicorandil.We conclude that nicorandil activates KATPchannels in these vessels and also acts through guanylyl cyclase to cause vasorelaxation, and that the potency of nicorandil is increased during metabolic inhibition.

Southern blotting was performed with cDNA probes for the human von Willebrand factor (vWF) gene on six patients with severe type III von Willebrand's disease (vWD). A partial deletion in the 3' end of the vWF gene was demonstrated in one individual whose parents were related and who had an alloantibody inhibitor to vWF. A resulting novel 2.0-kilobase (kb) EcoRI fragment was used for carrier detection within the patient's family, and seven carriers of this recessive trait were identified. Of the six tested, five had normal or only slightly reduced levels of vWF antigen, but with generally higher levels of factor VIII. The sixth carrier had moderately severe vWD and it is proposed that this patient is heterozygous for the defective vWF gene and a second recessive vWF defect. The novel 2.0-kb EcoRI restriction fragment was cloned and sequenced, and compared with that of the corresponding normal 4.2-kb EcoRI fragment that includes exons 41 and 42 of the vWF gene. A deletion of 2,320 base pairs (bp) which included exon 42, was identified and a novel 182-bp insert was found between the breakpoints. This insert was detected by polymerase chain reaction amplification both in the patient's DNA and in his carrier relatives.

We have previously demonstrated within intron 40 of the von Willebrand factor (vWF) gene a region of ATCT repeats that was shown to vary in length between two different DNA clones from unrelated individuals. The polymerase chain reaction (PCR) was used to examine the variability in length of this variable number tandem repeat (VNTR) in 53 normal individuals, using primers to DNA sequence flanking the repeat region. Overall, eight different length allelic bands were seen. These were individually sequenced and shown to contain from 6 to 14 ATCT repeats (a nine-repeat band was not seen). Seventy-five percent of individuals were shown to be heterozygous for this vWF.VNTR, and family studies showed Mendelian inheritance with allelic frequencies from 1% (vWF.VNTR [8] and vWF.VNTR [14]) to 39% (vWF.VNTR [7]). In the family of a patient with type III severe von Willebrand disease (vWD), vWF.VNTR results mirrored the phenotypic data and results with previously reported intragenic vWF restriction fragment length polymorphisms (RFLP). The patient was shown to be a compound heterozygote. In a family with a child with severe type III vWD, prenatal diagnosis by vWF.VNTR analysis on DNA obtained by chorionic villus sampling at 10 weeks gestation during a subsequent pregnancy indicated a severely affected fetus. This diagnosis was confirmed by fetal blood sampling at 18 weeks.

We have shown previously that the Rhesus (Rh) polypeptides are the commonest targets for pathogenic anti-red blood cell (RBC) autoantibodies in patients with autoimmune hemolytic anemia (AIHA). The aim of the current work was to determine whether activated T cells from such patients also mount recall responses to epitopes on these proteins. Two panels of overlapping 15-mer peptides, corresponding to the sequences of the 30-kD Rh proteins associated with expression of the D and Cc/Ee blood group antigens, were synthesized and screened for the ability to stimulate the in vitro proliferation of mononuclear cells from the peripheral blood or spleen of nine AIHA cases. Culture conditions were chosen that favor recall proliferation by previously activated T cells, rather than primary responses. In seven of the patients, including all four cases with autoantibody to the Rh proteins, two or more peptides elicited proliferation, but cells from eight of nine patients with other anemias and seven of nine healthy donors failed to respond to the panels. Multiple peptides were also stimulatory in two positive control donors who had been alloimmunized with Rh D-positive RBCs. Six different profiles of peptides elicited responses in the AIHA patients, and this variation may reflect the different HLA types in the group. Stimulatory peptides were identified throughout domains shared between, or specific to, each of the related 30-kD Rh proteins, but T cells that responded to nonconserved regions did not cross-react with the alternative sequences. Anti-major histocompatibility complex class II antibodies blocked the responses and depletion experiments confirmed that the proliferating mononuclear cells were T cells. Notably, splenic T cells that proliferated against multiple Rh peptides also responded to intact RBCs. We propose that pathogenic autoantibody production in many cases of AIHA is driven by the activation of T-helper cells specific for previously cryptic epitopes on the Rh proteins.

A patient with superior vena cava syndrome secondary to a small cell carcinoma of the lung was recently encountered. Diuretic therapy, radiotherapy, and chemotherapy failed to provide relief of symptoms. Consequently, we constructed a bypass graft with a long segment of the left saphenous vein. The vein was drawn through a subcutaneous tunnel over the abdomen and chest to be attached to the left external jugular vein. The resultant decompression of the superior vena cava provided almost immediate relief of symptoms. This simple bypass procedure allows decompression of the superior vena cava with minimal risk of morbidity or death and provides excellent palliation of a most disabling condition.

AbstractAllowing employees to work at home for at least part of the week is a management concept said to be rapidly gaining popularity. There is evidence that employees working at home are more productive, satisfied and committed, and that homeworking allows organisations to reduce office overheads, recruit from a wider pool, and retain valued workers. This paper examines the spread of homeworking and its telecommunications-based variant teleworking in Australia. A survey of human resource managers in Western Australia shows that, while 28 percent of large organisations have homeworkers, they are generally few in number and permitted only on an informal basis. Factors inhibiting use of homeworking include a lack of awareness and support amongst senior and middle managers, concerns over issues such as security and occupational health, and unsupportive organisational cultures. It is concluded that, contrary to media reporting, home-working and teleworking are not likely to become widespread in Australia in the near future.

1We used patch clamp to study whole‐cell K+currents activated by calcitonin gene‐related peptide (CGRP) in smooth muscle cells freshly dissociated from pig coronary arteries.2CGRP (50 nm) activated an inward current at −60 mV in symmetrical 140 mmK+that was blocked by glibenclamide (10 μm), an inhibitor of ATP‐sensitive potassium (KATP) channels. CGRP‐induced currents were larger in cells dialysed with 0.1 mmATP than with 3.0 mmATP.3Forskolin (10 μm) activated a glibenclamide‐sensitive current, as did intracellular dialysis with cAMP (100 μm). The catalytic subunit of cAMP‐dependent protein kinase (protein kinase A, PKA), added to the pipette solution, activated equivalent currents in five out of twelve cells.4CGRP‐induced currents were reduced by the PKA inhibitors adenosine 3′,5′‐cyclic monophosphorothioate, RP‐isomer, triethylammonium salt (Rp‐cAMPS; 100 μm) and N‐[2‐((p‐bromocinnamyl)amino)ethyl]‐5‐isoquinolinesulphonamide dihydrochloride (H‐89; 1 μm), and abolished by inclusion of a PKA inhibitor peptide in the pipette solution.5The β‐adrenergic agonist isoprenaline (10 μm) also activated a glibenclamide‐sensitive K+current.6CGRP‐induced currents were unaffected by the inhibitor of cGMP‐dependent protein kinase (PKG) KT5823 (1 μm). Sodium nitroprusside (10 μm) did not activate a glibenclamide‐sensitive current in cells held at −60 mV, but did activate an outward current at +60 mV that was abolished by KT5823, or by 100 nmiberiotoxin (an inhibitor of BKCachannels).7Our findings suggest that CGRP activates coronary KATPchannels through a pathway that involves adenylyl cyclase and PKA, but not PKG.

In eighty-seven hypertensive patients in general practice, treated with either methyldopa or the combination of methyldopa and a diuretic, oxprenolol 80 mg was added to the regime with subsequent reduction in methyldopa dosage. In the two groups of patients studied, those whose blood pressure was ‘well controlled’ and another group with a higher blood pressure, the control of blood pressure levels was improved and the incidence of reported tiredness decreased.

1. We have used single‐channel recording to investigate the block by extracellular tetraethylammonium ions (TEA+) of delayed rectifier potassium channels of frog skeletal sarcolemma. 2. TEA+ blocks by reducing the apparent amplitude of unitary currents, without detectable increase in open‐level current variance. 3. The block by TEA+ appeared to be 1:1, the fractional current being halved at 5.8 mM and ‐3 mV. The dissociation constant for the block was voltage dependent, increasing e‐fold for a 138 mV depolarization. 4. Activation of delayed rectifier potassium currents is not altered by TEA+. 5. Open times, which in the presence of TEA+ represents bursts of open and blocked events, are not increased by TEA+, indicating that blocked channels are able to close normally.

1. Unitary currents were measured through delayed rectifier potassium channels of frog skeletal muscle, under conditions where either potassium or rubidium ions carried current. 2. Unitary currents were reduced in amplitude when Rb+ was the charge carrier, indicating that Rb+ permeated the channel less readily than did K+. On the other hand permeability ratios (PRb/PK) measured from the change in reversal potential upon ionic substitution were 0.92 for the external and 0.67 for the internal mouth of the channel. 3. Ensemble‐averaged currents activated under depolarization along a similarly S‐shaped time course whether K+ or Rb+ carried current, though slightly more slowly in Rb+. However, under repolarization to a negative level, tail currents were prolonged about tenfold in Rb+. 4. The duration of channel opening was substantially prolonged in Rb+. The distribution of open times was fitted by a single exponential whether K+ or Rb+ was the charge carrier, indicating a single open state. But the mean open time, averaged over all voltages investigated, was 2.65 times greater in Rb+. 5. The prolongation in Rb+ of tail currents under repolarization was associated with increases in the number of openings per burst and in the number of bursts during each tail. 6. The implications of these results for channel gating are discussed. It is argued that an early step in channel activation is more voltage dependent than later steps.

1. Patch‐clamp methods were used to study the action of tetraethylammonium ions (TEA+) and other quaternary ammonium ions on adenosine‐5'‐triphosphate (ATP)‐sensitive K+ channels in sarcolemmal vesicles from frog skeletal muscle. The blocking ions were applied either to the external or the internal surface of the membrane patch. 2. External TEA+ caused a very fast block, so that the amplitude of single‐channel currents was reduced. Open‐channel variance was decreased. The block was 1:1, with a dissociation constant (Kd) of 6‐7 mM. We could detect no voltage dependence of Kd. 3. External TEA+ prolonged open times in a manner consistent with the channel being unable to close when blocked by TEA+. 4. TEA+ also blocked when applied to the internal side of the membrane. This block showed two components with different kinetics and different affinities. The slow block chopped up openings into much briefer events and had a Kd of about 1.4 mM at ‐3 mV. The fast block reduced the amplitude of unitary currents and was of lower affinity, with Kd around 26 mM. 5. The slow block by internal TEA+ was markedly voltage dependent, the Kd decreasing e‐fold for a 37 mV depolarization. Both the association and dissociation rates were dependent on voltage. In contrast, the fast block by internal TEA+ appeared virtually independent of voltage. 6. The effects of internally applied tetramethylammonium (TMA+) and tetrapentylammonium (TPA+) ions were also investigated. Internal TMA+ produced a flickery block while the block by internal TPA+ was similar to that caused by TEA+, although TPA+ was about 10 times more effective. 7. Our results suggest that the channel has three binding sites for TEA+, one of which is accessible from the outside of the membrane. Only one of the internal sites is located so as to experience a substantial fraction of the membrane voltage field.

1. We have used patch‐clamp methods to study the effects of pH at the cytoplasmic surface of the membrane on ATP‐dependent K+ channels (KATP channels) in patches excised from frog (Rana temporaria) skeletal muscle, and to study the kinetics of ATP binding. 2. In the absence of ATP, a reduction in pH led to a slight decrease in single‐channel current amplitude, an increase in the number of very brief closings, an increase in the apparent mean open time, and an increase in burst duration. After correction for missed closings, the change in mean open time was slight. Despite these changes in detailed kinetics, the channel open‐state probability, Popen, changed little with changes in pH in the absence of ATP. 3. In the presence of ATP, a decrease in internal pH (pHi) reduced the degree of channel inhibition by ATP, shifting the curve relating Popen and [ATP] to higher concentrations of ATP without altering its steepness. The ATP concentration for half‐inhibition of channel activity (Ki) was 17 microM at pH 7.2 and 260 microM at pH 6.3. 4. The effect of pH could be modelled by assuming that one or two protons bind to the channel and prevent ATP binding to exert its effect of causing channel closure. The predicted dissociation constants for ATP and H+ respectively were 5.4 and 0.11 microM. 5. The rate constants for binding and unbinding of ATP were estimated from the dependence of the mean open time on [ATP] and from the Ki. The apparent rate constants for ATP binding were 0.6 and 0.04 mM‐1 ms‐1 at pH 7.2 and 6.3 respectively, while the rate constant for unbinding was 0.01 ms‐1. In terms of our model the calculated true rate constant for ATP binding was 1.85 mM‐1 ms‐1. ATP binding also led to a reduction in burst duration. 6. The effect of pH described here differs from findings in cardiac muscle and pancreatic B‐cells. The results are discussed in relation to the possible function of KATP channels in skeletal muscle during exercise.