Genetic and phenotypic profile of 112 patients with X-linked Charcot-Marie-Tooth disease type 1.

BACKGROUND AND PURPOSE: X-linked Charcot-Marie-Tooth disease type 1 (CMTX1), caused by mutations in gap junction protein beta 1 (GJB1), is characterized by various central nervous system symptoms and gender differences of clinical severity. The aim of this study was to identify the frequency and mutation spectrum of CMTX1 patients in Japan and to demonstrate their phenotypic diversities. METHODS: Using three high-throughput sequencing systems, targeted gene panel sequencing on 1483 unrelated index patients with suspected Charcot-Marie-Tooth (CMT) disease was performed. The peripheral and central nervous system involvements of all patients with GJB1 variants were assessed retrospectively and a detailed gender comparison was conducted with the CMT examination score. RESULTS: Twenty-three novel and 36 described GJB1 variants were identified from 88 pedigrees, in which 34 female and 78 male patients were enrolled. Mean age at onset of the male patients was much younger than the females, 21.56 ± 17.63 years vs. 35.53 ± 23.72 years (P = 0.007). Male patients presented with more severe phenotypes in every examination item, but statistical differences were observed only in motor dysfunctions of the lower extremities and vibration sensation. No significant sensory difference was identified between genders, either clinically or electrophysiologically. Central nervous system dysfunctions were found in 15 patients from 12 pedigrees. Therein, six patients developed stroke-like phenotypes, with dysarthria as the leading symptom. CONCLUSIONS: A relatively lower frequency of CMTX1 (5.9%) was demonstrated and a broad mutation spectrum of GJB1 was described. Detailed clinical differences between genders and various central nervous system symptoms were also illustrated, even in the same pedigree.

WNK1/HSN2 founder mutation in patients with hereditary sensory and autonomic neuropathy: A Japanese cohort study.

The clinical and genetic spectrum of hereditary sensory and autonomic neuropathy (HSAN) is still unknown in Japan. We collected a broad cohort of 33 unrelated patients with predominant sensory and/or autonomic dysfunctions, who were referred to our genetic laboratory. A gene panel sequencing targeting 18 HSAN-related genes was performed using a next-generation sequencing system. A recurrent frame shift mutation in the WNK1/HSN2 gene, c.3237_3238insT (p.Asp1080*), was detected in 5 patients. This mutation was homozygous in 4 cases and of a compound heterozygous genotype in 1 case. Geographic and haplotype analysis of all 5 patients suggested a founder event. In addition, a novel heterozygous nonsense variant, c.2615C>G (p.Ser872*), was identified. All the 5 patients presented with severe sensory and autonomic dysfunctions at birth or during adolescence. In 2 patients, an uncommon phenotype of acute pathological pain presented at ~50 years of age. Here, we present the first founder mutation of WNK1/HSN2, in addition to French Canadian, which accounts for ~15.2% of Japanese patients with HSAN in our cohort. We have also reviewed all previously described mutations in WNK1/HSN2 and reconciled their nomenclature strategy on the basis of the current longest transcript.

Clinical and mutational spectrum of Japanese patients with Charcot-Marie-Tooth disease caused by GDAP1 variants.

BACKGROUND: Mutations in GDAP1 are responsible for heterogeneous clinical and electrophysiological phenotypes of Charcot-Marie-Tooth disease (CMT), with autosomal dominant or recessive inheritance pattern. The aim of this study is to identify the clinical and mutational spectrum of CMT patients with GDAP1 variants in Japan. MATERIALS AND METHODS: From April 2007 to October 2014, using three state-of-art technologies, we conducted gene panel sequencing in a cohort of 1,030 patients with inherited peripheral neuropathies (IPNs), and 398 mutation-negative cases were further analyzed with whole-exome sequencing. RESULTS: We identified GDAP1 variants from 10 patients clinically diagnosed with CMT. The most frequent recessive variant in our cohort (5/10), c.740C>T (p.A247V), was verified to be associated with a founder event. We also detected three novel likely pathogenic variants: c.928C>T (p.R310W) and c.546delA (p.E183Kfs*23) in Case 2 and c.376G>A (p.E126K) in Case 8. Nerve conduction study or sural nerve biopsy of all 10 patients indicated axonal type peripheral neuropathy. CONCLUSION: We identified GDAP1 variants in approximately 1% of our cohort with IPNs, and established a founder mutation in half of these patients. Our study originally described the mutational spectrum and clinical features of GDAP1-related CMT patients in Japan.

Clinical and mutational spectrum of Charcot-Marie-Tooth disease type 2Z caused by MORC2 variants in Japan.

BACKGROUND AND PURPOSE: The microrchidia family CW-type zinc finger 2 gene (MORC2) was newly identified as a causative gene of Charcot-Marie-Tooth disease (CMT) type 2Z in 2016. We aimed to describe the clinical and mutational spectrum of patients with CMT harboring MORC2 mutations in Japan. METHODS: We analyzed samples from 781 unrelated patients clinically diagnosed with CMT using deoxyribonucleic acid microarray or targeted resequencing by next-generation sequencing, and samples from 434 mutation-negative patients were subjected to whole-exome sequencing. We extracted MORC2 variants from these whole-exome sequencing data and classified them according to American College of Medical Genetics standards and guidelines. RESULTS: We identified MORC2 variants in 13 patients. As the second most common causative gene of CMT type 2 after MFN2, MORC2 variants were detected in 2.7% of patients with CMT type 2. The mean age of onset was 10.3 ± 8.7 years, and the inheritance pattern was mostly sporadic (11/13 patients, 84.6%). The clinical phenotype was typically length-dependent polyneuropathy, and electrophysiological studies revealed sensory-dominant axonal neuropathy. Mental retardation was identified in 4/13 patients (30.8%). p.Arg190Trp, as a mutational hotspot, was observed in eight unrelated families. We also identified two novel probably pathogenic variants, p.Cys345Tyr and p.Ala369Val, and one novel uncertain significance variant, p.Tyr332Cys. CONCLUSIONS: Our study is the largest report of patients harboring MORC2 variants. We revealed a clinical and mutational spectrum of Japanese patients with MORC2 variants. More attention should be paid to cognitive impairment, and the responsible mechanism requires further research for elucidation.

Overexpression of CXCR4 on circulating B cells in patients with active systemic lupus erythematosus.

OBJECTIVES: To evaluate the roles of circulating B cells in the pathogenic process of systemic lupus erythematosus (SLE) by measuring the expression of chemokines and their receptors. METHODS: Peripheral-blood mononuclear cells were obtained from 17 active, 21 inactive SLE patients, and 13 healthy controls. The expression of CXCR4, CXCR5, and CCR7 on CD19+ B cells was determined by flow cytometry, serum concentration of CXCL12 was measured by enzyme-linked immunosorbent assay, and the chemotactic responsiveness of B cells toward CXCL12 was evaluated. B or plasma cells expressing CXCR4 in renal biopsy specimens were detected using immnofluorescent staining. RESULTS: Flow cytometric analysis revealed that expression level of CXCR4 on circulating B cells was significantly higher in patients with active disease than in those with inactive disease or controls. Serum CXCL12 concentration was not different between these groups. In addition, the migratory ability of B cells toward CXCL12 was enhanced in active SLE patients. Finally, CXCR4-expressing B cells were more frequently observed in the renal biopsy specimens of lupus nephritis. CONCLUSIONS: Up-regulated CXCR4 expression on circulating B cells in active SLE may enhance their chemotactic response toward CXCL12, which may promote infiltration of these cells into inflamed renal tissue and contribute to the development of SLE.

Insulinoma may mask the existence of Type 1 diabetes.

BACKGROUND: Insulinoma is a tumour of insulin-producing cells of the pancreas and is known to be one of the causes of hypoglycaemia. Usually, appropriate removal of the insulinoma results in normalization of blood glucose levels. However, we found novel cases of insulinoma, in which hyperglycaemia developed soon after resection of the insulinoma. CASE REPORT: We encountered two patients with repeated hypoglycaemia caused by insulinoma. Following removal of the insulinoma, unanticipated hyperglycaemia was observed in both patients. Thereafter, their blood tests revealed low levels of serum C-peptide and high titres of anti-glutamic acid decarboxylase antibody, indicating concomitant Type 1 diabetes. Indeed, histological examination of the resected specimen revealed that one patient showed insulitis in non-tumorous pancreatic tissue in which ß-cells had already disappeared. Moreover, inflammatory cells infiltrated the insulinoma, as if it were insulitis of Type 1 diabetes, suggesting the existence of anti-islet autoimmunity. CONCLUSION: These are first cases of insulinoma associated with underlying Type 1 diabetes. Physicians should be aware of the possibility that insulinoma may mask Type 1 diabetes, and measurement of anti-islet autoantibodies may be helpful to find underlying Type 1 diabetes, such as in these cases. It is pathologically interesting that the immune cell infiltration into insulinoma may be suggestive of anti-islet autoimmunity.

Posttranslational Modifications and Plant-Environment Interaction.

Posttranslational modifications (PTMs) of proteins such as phosphorylation and ubiquitination are crucial for controlling protein stability, localization, and conformation. Genetic information encoded in DNA is transcribed, translated, and increases its complexity by multiple PTMs. Conformational change introduced by PTMs affects interacting partners of each proteins and their downstream signaling; therefore, PTMs are the major level of modulations of total outcome of living cells. Plants are living in harsh environment that requires unremitting physiological modulation to survive, and the plant response to various environment stresses is regulated by PTMs of proteins. This review deals with the novel knowledge of PTM-focused proteomic studies on various life conditions. PTMs are focused that mediate plant-environment interaction such as stress perception, protein homeostasis, control of energy shift, and defense by immune system. Integration of diverse signals on a protein via multiple PTMs is discussed as well, considering current situation where signal integration became an emerging area approached by systems biology into account.

Augmentation of human leukemic cell invasion by activation of a small GTP-binding protein Rho.

OBJECTIVE: The functions of a small GTP-binding protein, Rho, in human leukemic cell invasion was investigated in vivo and in vitro. MATERIALS AND METHODS: Human leukemic KM3 and Reh cells (derived from B-cell-type common acute lymphoid leukemias) were inoculated into severe combined immundeficiency (SCID) mice. Alteration of invasion in SCID mice inoculated with KM3 cells that were introduced with the expression vector for Rho Val14 (Rho V14), an activated mutant form of Rho, was observed. RESULTS: SCID mice inoculated with KM3 and Reh cells developed paraplegia 21 days after inoculation. All died by day 26-27. The leukemic cells were localized to bone marrow and around the spinal cord, with no infiltration into peripheral blood, spleen, liver, thymus, or lymph nodes. SCID mice inoculated with Rho V14-transfected KM3 cells showed a 5-day reduction in the time to paraplegia and death compared with SCID mice inoculated with hygromycin-resistance gene-transfected KM3 (hyg(r)) cells. In addition, the mice inoculated with Rho V14 cells showed leukemic cell infiltration, not only into bone marrow and around the spinal cord but also into peripheral blood, liver, and spleen. There were no in vitro or in vivo differences in growth rates of Rho V14 and hyg(r) cells. However, the Rho V14 cells showed markedly increased cell adhesion compared to the hyg(r) cells. CONCLUSION: Results suggest that Rho activation accelerates human leukemic cell invasion via augmentation of cell adhesion.

Augmentation of megakaryocytopoiesis within the hematopoietic microenvironment of human granulocyte colony-stimulating factor transgenic mice.

OBJECTIVE: Megakaryocytopoiesis was dramatically augmented in human granulocyte colony-stimulating factor transgenic mice (G-Tg) compared to littermates. We examined the characteristics of megakaryocytes and megakaryocyte progenitor cells in these mice. MATERIALS AND METHODS: The numbers of colony-forming unit megakaryocytes (CFU-MK) and megakaryocytes in hematopoietic organs were counted. The megakaryocytes of G-Tg were examined ultrastructurally, and bone marrow transplantation studies using congenic G-Tg (Ly5.2) and C57BL/6 (Ly5.1) were performed. The number of day-14 colony-forming unit spleen (CFU-S) that contained megakaryocytes in [Ly5.1 > G-Tg] and [G-Tg > Ly5.1] mice also was counted. RESULTS: The number of CFU-MK increased markedly in the spleen, bone marrow, and peripheral blood. The number of megakaryocytes in the spleen and bone marrow also were increased in G-Tg mice. Ultrastructural analyses revealed that megakaryocytes in G-Tg mice were immature. Bone marrow transplantation studies of [Ly5.1 > G-Tg] mice resulted in a significantly increased number of megakaryocytes compared to [G-Tg > Ly5.1] mice. The number of day-14 CFU-S that contained megakaryocytes was increased markedly in [Ly5.1 > G-Tg] mice compared to [G-Tg > Ly5.1] mice. In vitro differentiation of megakaryocytes in [Ly5.1 > G-Tg] mice was induced by interleukin-11 and thrombopoietin. CONCLUSION: The results showed that the hematopoietic marrow microenvironment of G-Tg is important in augmenting megakaryocytopoiesis. [Ly5.1 > G-Tg] mice are potentially useful as a source of murine megakaryocytes and their progenitors.

The neuroprotective effect of a novel calmodulin antagonist, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate, in transient forebrain ischemia.

A novel calmodulin (CaM) antagonist DY-9760e, (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate), with an apparent neuroprotective effect in vivo, potently inhibits CaM-dependent nitric oxide synthase in situ. In the present study, we determined whether DY-9760e inhibits nitric oxide (NO) production and protein nitration by peroxynitrite (ONOO(-)) formation in the hippocampal CA1 region of gerbils after transient forebrain ischemia. In freely moving gerbils, NO production after 10-minute forebrain ischemia was monitored consecutively with in vivo brain microdialysis. Pretreatment with DY-9760e (50 mg/kg i.p.) significantly decreased the increased levels of NO(x)(-) (NO metabolites, NO(2)(-) plus NO(3)(-)) immediately after, 24 h after cerebral ischemia-reperfusion to the control levels of sham-operated animals. Western blot and immunohistochemical analyses using an anti-nitrotyrosine antibody as a marker of ONOO(-) formation indicated a marked increase in nitrotyrosine immunoreactivity in the pyramidal neurons of the CA1 region 2 h after reperfusion, and DY-9760e significantly inhibited increased nitrotyrosine immunoreactivity. Coincident with the inhibition of the NO production and protein tyrosine nitration, pretreatment with DY-9760e rescued the delayed neuronal death in the hippocampal CA1 region. These results suggest that the inhibitory effects of DY-9760e on the NO-ONOO(-) pathway partly account for its neuroprotective effects in cerebral ischemia.

A case of generalized Hailey-Hailey disease with fatal liver injury.

We report a case of a 59-year-old man with a severe generalized form of Hailey-Hailey disease that was complicated by fatal liver injury. Erosive lesions were first noted in the axillary and perianal regions at 15 year of age, and Hailey-Hailey disease was diagnosed based on the clinical features and histologic findings in skin biopsy specimens. The patient was treated with at first topical steroids and later a low dose of a corticosteroid, but the skin lesions gradually became generalized. At 45 years of age liver dysfunction was detected after azathioprine and vinblastine treatment for the generalized skin lesions. The liver injury gradually progressed and finally the patient died. The gene responsible for Hailey-Hailey disease was recently identified as ATP2C1, and it encodes a Ca(2+)-transport ATPase with broad expression, including in skin and liver. This finding suggests that mutation of the ATP2C1 gene may give rise to an extracutaneous phenotype, such as the liver dysfunction observed in severe cases, including our own. Further accumulation of cases is necessary to determine whether this is true.

Evaluation of the central nervous function in resuscitated comatose patients by multilevel evoked potentials.

Multilevel evoked potentials were examined in 17 patients who became comatose after cardiac arrest and resuscitation. In 4 patients, the P1 through N3 components of the somatosensory evoked cerebral potential (SECP) were present altogether within 100 ms after the ischemic insults. They all subsequently regained consciousness, though three of them developed intelligence and motor disturbances to some extent. In 11 patients who regained consciousness, or remained in a vegetative state, the evoked potentials which reflect brainstem functions, such as the auditory evoked brainstem potential, the R1 wave of the orbicularis oculi reflex and the slow positive wave of the somatosensory evoked brainstem potential, were recognized. The somatosensory evoked spinal potential and spinal monosynaptic reflex showed normal appearances in the state of vegetation and even after the determination of brain death. The measures of SECP could be useful in predicting restoration of consciousness.

Cardiopulmonary-cerebral resuscitation by using cardiopulmonary bypass through the femoral vein and artery in dogs.

Twenty-seven dogs, divided into three groups, were subjected to a normothermic ventricular fibrillation (VF) cardiac arrest of 15 min and resuscitated by using cardiopulmonary bypass through the femoral veins and artery (F-F bypass). Group I (n = 15): Cardiac beating did not return in any dogs during an initial 3-min conventional cardiopulmonary resuscitation, but it returned 5.2 +/- 3.8 min (mean +/- S.D.) after the successive initiation of the F-F bypass in all dogs, except in one with bypass trouble. Intermittent burst waves appeared on the electroencephalogram and continuous waves returned, 90.0 +/- 24.7 min and 130.7 +/- 28.1 min after the start of resuscitation, respectively. Values of blood glucose, lactate and potassium 5-15 min after the F-F bypass were significantly higher than those before induction of VF, while those of blood pH, base excess, hemoglobin, hematocrit, platelet and serum protein decreased significantly. Group II (n = 7): Both local cerebral (CBF) and myocardial blood flow (MCBF) returned to the pre-arrest level soon after the initiation of the F-F bypass, even though spontaneous cardiac beating was not yet restored. Closed or open chest cardiac massage could not produce as much blood flow as the F-F bypass did. In the early stage of restoration of spontaneous circulation, temporary interruption of the bypass led to a decrease in both local CBF and MCBF. Group III (n = 5): Spontaneous circulation was restored in all five dogs 5.2 +/- 1.1 min after the institution of the F-F bypass, which was continued for 164 +/- 30 min under mild hypothermia. After intensive care for a subsequent 6-36 h, the animals barked, moved their forelegs and could drink water. The mean neurological deficit score (normal: 0, brain death: 500) was 100.6. However, macroscopic examination of the brain in two dogs with prominent recovery revealed atrophy of the central gyrus and microscopic examination revealed injuries of the vulnerable neurons of the brain.

Function of 90-kDa heat shock protein in cellular differentiation of human embryonal carcinoma cells.

Heat shock proteins (HSPs) have been recognized as molecules that maintain cellular homeostasis during changes in the environment. Here we report that HSP90 functions not only in stress responses but also in certain aspects of cellular differentiation. We found that HSP90 showed remarkably high expression in undifferentiated human embryonal carcinoma (EC) cells, which were subsequently dramatically down-regulated during in vitro cellular differentiation, following retinoic acid (RA) treatment, at the protein level. Surprisingly, heat shock treatment also triggered the down-regulation of HSP90 within 48 h at the protein level. Furthermore, the heat treatment induced cellular differentiation into neural cells. This down-regulation of HSP90 by heat treatment was shifted to an up-regulation pattern after cellular differentiation in response to RA treatment. In order to clarify the functions of HSP90 in cellular differentiation, we conducted various experiments, including overexpression of HSP90 via gene transfer. We showed that the RA-induced differentiation of EC cells into a neural cell lineage was inhibited by overexpression of the HSP90alpha or -beta isoform via the gene transfer method. On the other hand, the overexpression of HSP90beta alone impaired cellular differentiation into trophoectoderm. These results show that down-regulation of HSP90 is a physiologically critical event in the differentiation of human EC cells and that specific HSP90 isoforms may be involved in differentiation into specific cell lineages.

Pineal germinomas with granulomatous inflammation. Report of two cases and review of the literature.

The authors report on two cases of pineal germinomas with granulomatous inflammation (granulomatous germinomas). Macroscopically, both tumors were relatively hard and grayish in color. Histological examination revealed a germinoma with multinucleated giant cells and Schaumann bodies in one case, and a germinoma with the background of acellular fibrillated matrix in the other. On immunohistochemical analysis, the granulomatous germinomas were shown to contain many macrophages, T- and B-lymphocytes, and glial fibrillary acid protein-positive cells infiltrating the specimens, compared to nongranulomatous germinomas. Analysis of Masson's trichrome staining tests showed that large areas of the granulomatous germinomas were occupied by a collagenous component; this was not the case in cases of nongranulomatous germinomas. Analysis of monoclonal anti-human Ki-67 results showed that the granulomatous germinomas had a lower score than nongranulomatous germinomas (p < 0.05, unpaired t-test), indicating that germinomas with granulomatous inflammation may have a better prognosis.

Radiation-induced cerebrovasculopathy of the distal middle cerebral artery and distal posterior cerebral artery--case report.

A 15-year-old girl underwent partial removal of a pituitary adenoma followed by local irradiation of the brain with a total of 70 Gy through two lateral opposing ports. Twenty years later, she experienced frequent transient ischemic attacks with left sensory disturbance. Cerebral angiography revealed stenoses of the right distal middle cerebral artery (MCA) and the right distal posterior cerebral artery without net-like vessels. There was a severe decrease of vasoreactivity in the right hemisphere. Right superficial temporal artery (STA)-MCA anastomosis was performed. Her neurological deficits were resolved and perfusion reserve capacity had markedly improved 6 months later. We recommend STA-MCA anastomosis in such cases.

[Emergency cardiopulmonary bypass (ECPB) for cardiopulmonary-cerebral resuscitation; (2). Physiologic changes].

After normothermic ventricular fibrillation (Vf) cardiac arrest of 15 min, 15 female mongrel dogs received conventional cardiopulmonary resuscitation (CPR) for 3 min and then cardiopulmonary bypass through the femoral artery and veins (F-F bypass). Cardiac beat did not return in any dogs during the initial 3-min CPR. Spontaneous circulation was restored by defibrillation 5.2 +/- 3.8 (mean +/- SD) min after the initiation of the F-F bypass in all dogs except in one with bypass trouble. Life-threatening ventricular dysrhythmias appeared in 8 of the 14 dogs (57%). Eleven of the 14 dogs (79%) were successfully weaned from the F-F bypass. Spontaneous respiration and circulation remained stable for the subsequent 1-h observation period in 8 dog (57%). The values of cardiac output measured just before weaning from the bypass and 30 to 60 min after weaning were significantly lower than those before inducing Vf (n = 5). On the electroencephalogram, intermittent burst waves reappeared 90.0 +/- 24.7 min after the initiation of resuscitation and EEG showed continuous waves 130.7 +/- 28.1 min (n = 7) after the initiation of resuscitation. The values of blood glucose, lactate and potassium 5 to 15 min after the initiation of F-F bypass were significantly higher than those before induction of Vf, while the values of hemoglobin, hematocrit, platelet and serum protein decreased significantly (n = 14).

[Studies of emergency cardiopulmonary bypass (ECPB) for cardiopulmonary-cerebral resuscitation--(3). Neurological outcome and pathological changes of the brain].

Neurological outcome and pathological changes of the brain were studied in 5 female mongrel dogs, which were subjected to normothermic ventricular fibrillation (Vf) cardiac arrest of 15 min and resuscitated by using cardiopulmonary bypass through the femoral artery and veins (F-F bypass). Spontaneous circulation was restored by one or two defibrillating countershocks in all 5 dogs 5.2 +/- 1.1 (mean +/- SD) min after initiation of the F-F bypass. The F-F partial bypass was continued for 164 +/- 30 min under mild hypothermia. After weaning from the bypass, intensive care including controlled ventilation was carried out for the subsequent 6 to 36 h. Intermittent slow waves appeared on the electroencephalogram 62.8 +/- 11.6 min after initiation of the F-F bypass resuscitation and continuous waves at 145.6 +/- 27.5 min. Soon after extubation, the animals barked, moved the forelegs and could drink water. Neurological deficit scores (normal: 0, brain death: 500) improved to become below 100 except in 1 dog. However, macroscopic examination of the brain in 2 dogs with prominent recovery disclosed atrophy of the central gyrus and microscopic examination revealed typical ischemic injuries of the vulnerable neurons at the cerebellum, hippocampus and cerebral cortex in the frontal lobe.

[Studies of emergency cardiopulmonary bypass (ECPB) for cardiopulmonary-cerebral resuscitation; (1) Introduction of a portable-percutaneous ECPB system].

The authors have developed an ECPB system, which can be applied quickly, safely and easily under an emergency condition requiring cardiac massage and artificial ventilation. Fundamentally, the ECPB system consists of 3 parts; a portable ECPB apparatus, a pair of percutaneous cannulae and a short circuit connecting an oxygenator with the cannulae. The ECPB apparatus is assembled with commercially available components (i.e., a centrifugal pump, a battery pack, a temperature controller, a compact membrane oxygenator with a heat exchanger, etc) and they are placed on a mobile cart. The circuit is primed with 300 ml of lactated Ringer solution. The priming can be done within 15 minutes via a reservoir. It is also possible to keep the primed circuit to be ready for emergency use at least for a week. The cannulae are placed intravascularly through the femoral artery and vein by using the Seldinger's percutaneous method. In an emergency situation, the arterial and venous cannulations are carried out separately on the both inguinal regions to save time. The tip of the venous cannula is adjusted to be placed near the right atrium under fluoroscopy. Initiation of ECPB via the femoro-femoral V-A cannulae assures instant and stable supply of oxygenated blood to all of the vital organs. At the present time, nothing is more important than a quick supply of oxygenated blood to the brain to ameliorate the post-ischemic brain damage.

[Dermal anesthesia: comparison of the analgesic effects of 2% and 10% lidocaine gel patch].

The analgesic effects of aqueous gel containing 2% or 10% lidocaine with 3% glycyrrhetinic acid mono 3-0 hemiphthalate sodium as an absorption promoter were compared in two volunteer groups of 12 persons each. A round sponge (25 mm in diameter and 1mm in thickness) filled with approximately 0.3g of either gel was applied on the volar surface of the forearm and kept covered with an adhesive plastic film (Tegaderm) for two hours. The analgesic effect was assessed every 30 min by pin-prick method at five places under the coverage for two hours, and after the gel was wiped away. The result from each place was scored 0 (no pain) or 1 (needle pain). The mean pain scores at 1 hr and 1.5 hr in the 10% group were 1.0 and 0.7, and significantly lower than 2.2 and 1.3 of the 2% group (P less than 0.05). Two hour application of the gel, five volunteers in the 2% group and eight volunteers in the 10% group produced a pain score under 1.0. In these subjects, a 26 gauge needle was stuck into the skin for further pain analysis. Four of the 5 subjects in the 2% group and 7 of the 8 subjects in the 10% group did not complain of any pain. Transient local redness under the coverage was observed in 3 subjects in each group. They were all known to be sensitive to alcoholic beverages. No other side effects were found. The plasma concentration of lidocaine was lower than 0.01 microgram.ml-1 at all times.