Novel Brugada syndrome-causing mutation in ion-conducting pore of cardiac Na+ channel does not affect ion selectivity properties

AIM: Brugada syndrome is an inherited cardiac disease with an increased risk of sudden cardiac death. Thus far Brugada syndrome has been linked only to mutations in SCN5A, the gene encoding the alpha-subunit of cardiac Na+ channel. In this study, a novel SCN5A gene mutation (D1714G) is reported, which has been found in a 57-year-old male patient. Since the mutation is located in a segment of the ion-conducting pore of the cardiac Na+ channel, which putatively determines ion selectivity, it may affect ion selectivity properties. METHODS: HEK-293 cells were transfected with wild-type (WT) or D1714G alpha-subunit and beta-subunit cDNA. Whole-cell configuration of the patch-clamp technique was used to study biophysical properties at room temperature (21 degrees C) and physiological temperature (36 degrees C). This study represents the first measurements of human Na+ channel kinetics at 36 degrees C. Ion selectivity, current density, and gating properties of WT and D1714G channel were studied. RESULTS: D1714G channel yielded nearly 80% reduction of Na+ current density at 21 and 36 degrees C. At both temperatures, no significant changes were observed in V(1/2) values and slope factors for voltage-dependent activation and inactivation. At 36 degrees C, but not at 21 degrees C, D1714G channel exhibited more slow inactivation compared with WT channel. Ion selectivity properties were not affected by the mutation at both temperatures, as assessed by either current or permeability ratio. CONCLUSION: This study shows no changes in ion selectivity properties of D1714G channel. However, the profoundly decreased current density associated with the D1714G mutation may explain the Brugada syndrome phenotype in our patient.     

Novel Brugada syndrome‐causing mutation in ion‐conducting pore of cardiac Na+ channel does not affect ion selectivity properties

Aim: Brugada syndrome is an inherited cardiac disease with an increased risk of sudden cardiac death. Thus far Brugada syndrome has been linked only to mutations in SCN5A, the gene encoding the α‐subunit of cardiac Na⁺ channel. In this study, a novel SCN5A gene mutation (D1714G) is reported, which has been found in a 57‐year‐old male patient. Since the mutation is located in a segment of the ion‐conducting pore of the cardiac Na⁺ channel, which putatively determines ion selectivity, it may affect ion selectivity properties. Methods: HEK‐293 cells were transfected with wild‐type (WT) or D1714G α‐subunit and β‐subunit cDNA. Whole‐cell configuration of the patch‐clamp technique was used to study biophysical properties at room temperature (21 °C) and physiological temperature (36 °C). This study represents the first measurements of human Na⁺ channel kinetics at 36 °C. Ion selectivity, current density, and gating properties of WT and D1714G channel were studied. Results: D1714G channel yielded nearly 80% reduction of Na⁺ current density at 21 and 36 °C. At both temperatures, no significant changes were observed in V_1/2 values and slope factors for voltage‐dependent activation and inactivation. At 36 °C, but not at 21 °C, D1714G channel exhibited more slow inactivation compared with WT channel. Ion selectivity properties were not affected by the mutation at both temperatures, as assessed by either current or permeability ratio. Conclusion: This study shows no changes in ion selectivity properties of D1714G channel. However, the profoundly decreased current density associated with the D1714G mutation may explain the Brugada syndrome phenotype in our patient.     

Improvement of Schwann cell attachment and proliferation on modified hyaluronic acid strands by polylysine

Hyaluronic acid (HyA) has the intrinsic ability to promote cell proliferation and reduce scar formation. However, the clinical use of HyA has so far been limited because of its water solubility and nonadhesive characteristics. Increasing interest in HyA as a clinically useful biomaterial has prompted our study of altering HyA's physical properties to render it a potential component of nerve grafts. In this study, strands of HyA were cross-linked by glutaraldehyde (Glut), coated with polylysine, and then inoculated with Schwann cells (SCs). Results in vivo and in vitro demonstrated that cross-linked HyA strands were water insoluble and thus less biodegradable. Poly-D-lysine-resurfaced strands showed significant SC attachment of 350-400 cells/mm(2), compared to uncoated controls (0-10 cells/mm(2), p < 0.01). Fibroblast control groups showed an attachment of 40-100 cells/mm(2) on coated strands. Immunostaining for proliferating cells showed SCs as and fibroblasts as +. Cells neither adhered to nor proliferated on the modified HyA strands that were not resurfaced. The results suggest that polylysine promotes SC attachment and proliferation to glutaraldehyde-cross-linked HyA strands, the product being a three-dimensional composite with low solubility that may have potential application in nerve grafts.     

Modification of Si(100) surface by the grafting of poly(ethylene glycol) for reduction in protein adsorption and platelet adhesion

The modification of argon plasma-pretreated single-crystal Si(100) wafer surfaces via the UV-induced graft polymerization of poly(ethylene glycol) methacrylate (PEGMA) macromonomer (molecular weight approximately 340) for biomaterials applications was explored. The modified Si(100) surfaces were characterized by X-ray photoelectron spectroscopy and atomic force microscopy. Surface peroxide concentrations resulting from the argon plasma treatment and subsequent atmospheric exposure were determined by a coupling reaction with diphenylpicrylhydrazyl. The results suggested that a short plasma treatment time of 10 s and brief air exposure were sufficient for generating an optimum amount of peroxides and hydroperoxides for the subsequent UV-induced graft polymerization. The graft concentration of the PEGMA polymer increased with increasing PEGMA macromonomer concentration for the graft polymerization and with increasing UV graft polymerization time. The PEGMA graft-polymerized silicon surface with a high poly(ethylene glycol) graft concentration was very effective in preventing protein adsorption and platelet adhesion. The grafted PEGMA polymer layer on the Si(100) surface exhibited fairly good stability during storage in a buffer solution.     

Projections of the bed nucleus of the stria terminalis to the mesencephalon,pons, and medulla oblongata in the cat

Summary Injections of HRP in the nucleus raphe magnus and adjoining medial reticular formation in the cat resulted in many labeled neurons in the lateral part of the bed nucleus of the stria terminalis (BNST) but not in the medial part of this nucleus. HRP injections in the nucleus raphe pallidus and in the C2 segment of the spinal cord did not result in labeled neurons in the BNST. Injections of ³H-leucine in the BNST resulted in many labeled fibers in the brain stem. Labeled fiber bundles descended by way of the medial forebrain bundle and the central tegmental field to the lateral tegmental field of pons and medulla. Dense BNST projections could be observed to the substantia nigra pars compacta, the ventral tegmental area, the nucleus of the posterior commissure, the PAG (except its dorsolateral part), the cuneiform nucleus, the nucleus raphe dorsalis, the locus coeruleus, the nucleus subcoeruleus, the medial and lateral parabrachial nuclei, the lateral tegmental field of caudal pons and medulla and the nucleus raphe magnus and adjoining medial reticular formation. Furthermore many labeled fibers were present in the solitary nucleus, and in especially the peripheral parts of the dorsal vagal nucleus. Finally some fibers could be traced in the marginal layer of the rostral part of the caudal spinal trigeminal nucleus. These projections appear to be virtually identical to the ones derived from the medial part of the central nucleus of the amygdala (Hopkins and Holstege 1978). The possibility that the BNST and the medial and central amygdaloid nuclei must be considered as one anatomical entity is discussed.Abbreviations AA anterior amygdaloid nucleus - AC anterior commissure - ACN nucleus of the anterior commissure - ACO cortical amygdaloid nucleus - AL lateral amygdaloid nucleus - AM medial amygdaloid nucleus - APN anterior paraventricular thalamic nucleus - AQ cerebral aqueduct - BC brachium conjunctivum - BIC brachium of the inferior colliculus - BL basolateral amygdaloid nucleus - BNSTL lateral part of the bed nucleus of the stria terminalis - BNSTM medial part of the bed nucleus of the stria terminalis - BP brachium pontis - CA central nucleus of the amygdala - Cd caudate nucleus - CI inferior colliculus - CL claustrum - CN cochlear nucleus - CP posterior commissure - CR corpus restiforme - CSN superior central nucleus - CTF central tegmental field - CU cuneate nucleus - D nucleus of Darkschewitsch - EC external cuneate nucleus - F fornix - G gracile nucleus - GP globus pallidus - HL lateral habenular nucleus - IC interstitial nucleus of Cajal - ICA internal capsule - IO inferior olive - IP interpeduncular nucleus - LC locus coeruleus - LGN lateral geniculate nucleus - LP lateral posterior complex - LRN lateral reticular nucleus - MGN medial geniculate nucleus - MLF medial longitudinal fascicle - NAdg dorsal group of nucleus ambiguus - NPC nucleus of the posterior commissure - nV trigeminal nerve - nVII facial nerve - OC optic chiasm - OR optic radiation - OT optic tract - P pyramidal tract - PAG periaqueductal grey - PC cerebral peduncle - PO posterior complex of the thalamus - POA preoptic area - prV principal trigeminal nucleus - PTA pretectal area - Pu putamen - PUL pulvinar nucleus - R red nucleus - RF reticular formation - RM nucleus raphe magnus - RP nucleus raphe pallidus - RST rubrospinal tract - S solitary nucleus - SC suprachiasmatic nucleus - SCN nucleus subcoeruleus - SI substantia innominata - SM stria medullaris - SN substantia nigra - SO superior olive - SOL solitary nucleus - SON supraoptic nucleus - spV spinal trigeminal nucleus - spVcd spinal trigeminal nucleus pars caudalis - ST stria terminalis - TRF retroflex tract - VC vestibular complex - VTA ventral tegmental area of Tsai - III oculomotor nucleus - Vm motor trigeminal nucleus - VI abducens nucleus - VII facial nucleus - Xd dorsal vagal nucleus - XII hypoglossal nucleus     

Audit of start of anticoagulation treatment in inpatients.

AIMS: To develop a method for evaluating the start of anticoagulation treatment in inpatients. METHODS: One hundred case notes were audited using a proforma based on local guidelines in accordance with British Society for Haematology recommendations. RESULTS: Confirmatory investigations were done in 93% and 79% of patients with symptomatic deep venous thrombosis and pulmonary embolism, respectively. Identification of patients' risk factors for anticoagulation by history taking and laboratory tests was often inadequate: baseline coagulation screen, platelet count, liver function and renal function tests were done in 52%, 95%, 70% and 87% of cases, respectively. There was a tendency to undertreat patients: 33% of the activated partial thromboplastin times (APTT) and 58% of the International Normalised Ratios (INR) were subtherapeutic. The heparin-warfarin crossover period was particularly problematic: 37% stopped heparin without an INR that day, or had an INR of less than 2. Microscopic haematuria was monitored occasionally. Of the 62 patients continuing anticoagulation, 72% were discharged with the final INR in the therapeutic range. At discharge, only 74% of patients had documented appointments for the anticoagulant Clinic, the period between discharge and appointment ranging from 0 to 12 days. Of the 25 cases with an appointment exceeding four days after discharge, only six (24%) had arrangements for an interim INR check. CONCLUSIONS: The experience allowed the proforma to become streamlined to a more practical, reliable, and valid tool for use elsewhere. Findings will be fed back to the hospital staff to promote practice improvements before closing the audit loop by re-evaluating practice. Further studies are in progress to identify barriers experienced by doctors in implementing the guidelines and problems in the process of referral to the anticoagulant clinic.     

Circulating immune complexes, complement and complement component levels in childhood Hodgkin's disease.

Serum levels of circulating immune complexes (CIC) assayed by the Raji cell radioimmunoassay, total haemolytic complement (TCH50), Clq and C3 were correlated with clinical stage, histological type, age, sex and treatment of eighty-six children with Hodgkin's disease over a period of 4 years. Most significant findings were the changes of levels of CIC, TCH50, Clq and C3 during disease activity and following treatment. Significant perturbations were also seen in association with relapse. Levels of C and CIC were significantly elevated (P less than 0.001) at the time of diagnosis prior to splenectomy and/or any treatment. In the group before treatment, 81 percent of CIC levels were above 16 micrograms/ml with a maximum value of 1120 micrograms/ml. During treatment 33 percent were still above normal with a maximum of 320 micrograms/ml. Within 1 year after cessation of treatment, 37 percent also remained above normal levels with a maximum of 240 micrograms/ml. At relapse prior to treatment, 63 percent were again elevated with a maximum of 1280 micrograms/ml. The most significant difference on TCH50 levels relates to treatment periods. Sera of patients with active disease who are previously untreated show elevation of TCH50 levels (P less than 0.001) (average 127 CH50 mu/ml. During and after treatment eht TCH50 levels drop to 96 and 102 CH50 mu/ml, as compared to normal control of 100 CH50 mu/ml. In sera of patients at the first, second or third relapse, the combined TCH50 levels are significantly different from controls and across treatment periods (P less than 0.005).     

Treg-Cell Control of a CXCL5-IL-17 Inflammatory Axis Promotes Hair-Follicle-Stem-Cell Differentiation During Skin-Barrier Repair

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Spontaneous wheel running attenuates cardiovascular responses to stress in rats

We investigated the effect of chronic, 10-week spontaneous wheel running (SWR) exercise on stress-induced cardiovascular responses in free-moving male rats, using a biotelemetry system. During cage-switch stress or immobilization stress, blood pressure and heart rate were significantly increased in both the SWR (P<0.001 for each stress) and control groups (P<0.001 for each stress). However the blood pressure response was attenuated significantly in the SWR group (P<0.001) during cage-switch stress, and the blood pressure and heart rate responses were attenuated significantly in the SWR group (P<0.0001 and 0.01, respectively) during immobilization stress. The plasma norepinephrine (NE) response induced by immobilization stress tended to be attenuated in the SWR group, but the groups showed no significant differences in the plasma NE and epinephrine (E) responses to both stresses. These results suggest that daily SWR in rats has beneficial effects in suppressing excessive blood pressure and heart rate responses induced by two different types of stress. The mechanisms responsible for the greater resistance to these stresses in the SWR rats should be investigated further.