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ObjectiveWe examined the correlation between the dosing regimen of oral prednisolone (PSL) and the achievement of minimal manifestation status or better on PSL ≤5 mg/day lasting >6 months (the treatment target) in patients with generalised myasthenia gravis (MG).MethodsWe classified 590 patients with generalised MG into high-dose (n=237), intermediate-dose (n=187) and low-dose (n=166) groups based on the oral PSL dosing regimen, and compared the clinical characteristics, previous treatments other than PSL and prognosis between three groups. The effect of oral PSL dosing regimen on the achievement of the treatment target was followed for 3 years of treatment.ResultsTo achieve the treatment target, ORs for low-dose versus high-dose regimen were 10.4 (PConclusionA low-dose PSL regimen with early combination of other treatment options may ensure earlier achievement of the treatment target in generalised MG.

Objectives To clarify the social disadvantages associated with myasthenia gravis (MG) and examine associations with its disease and treatment. Design Cross-sectional study. Setting and participants We evaluated 917 consecutive cases of established MG seen at 13 neurological centres in Japan over a short duration. Outcome measures All patients completed a questionnaire on social disadvantages resulting from MG and its treatment and a 15-item MG-specific quality of life scale at study entry. Clinical severity at the worst condition was graded according to the MG Foundation of America classification, and that at the current condition was determined according to the quantitative MG score and MG composite. Maximum dose and duration of dose ≥20 mg/day of oral prednisolone during the disease course were obtained from the patients9 medical records. Achievement of the treatment target (minimal manifestation status with prednisolone at ≤5 mg/day) was determined at 1, 2 and 4 years after starting treatment and at study entry. Results We found that 27.2% of the patients had experienced unemployment, 4.1% had been unwillingly transferred and 35.9% had experienced a decrease in income, 47.1% of whom reported that the decrease was ≥50% of their previous total income. In addition, 49.0% of the patients reported feeling reduced social positivity. Factors promoting social disadvantages were severity of illness, dose and duration of prednisolone, long-term treatment, and a depressive state and change in appearance after treatment with oral steroids. Early achievement of the treatment target was a major inhibiting factor. Conclusions Patients with MG often experience unemployment, unwilling job transfers and a decrease in income. In addition, many patients report feeling reduced social positivity. To inhibit the social disadvantages associated with MG and its treatment, greater focus needs to be placed on helping patients with MG resume a normal lifestyle as soon as possible by achieving the treatment target.

Background We have previously reported using two-step cluster analysis to classify myasthenia gravis (MG) patients into the following five subtypes: ocular MG; thymoma-associated MG; MG with thymic hyperplasia; anti-acetylcholine receptor antibody (AChR-Ab)-negative MG; and AChR-Ab-positive MG without thymic abnormalities. The objectives of the present study were to examine the reproducibility of this five-subtype classification using a new data set of MG patients and to identify additional characteristics of these subtypes, particularly in regard to response to treatment. Methods A total of 923 consecutive MG patients underwent two-step cluster analysis for the classification of subtypes. The variables used for classification were sex, age of onset, disease duration, presence of thymoma or thymic hyperplasia, positivity for AChR-Ab or anti–muscle-specific tyrosine kinase antibody, positivity for other concurrent autoantibodies, and disease condition at worst and current. The period from the start of treatment until the achievement of minimal manifestation status (early-stage response) was determined and then compared between subtypes using Kaplan-Meier analysis and the log-rank test. In addition, between subtypes, the rate of the number of patients who maintained minimal manifestations during the study period/that of patients who only achieved the status once (stability of improved status) was compared. Results As a result of two-step cluster analysis, 923 MG patients were classified into five subtypes as follows: ocular MG (AChR-Ab-positivity, 77%; histogram of onset age, skewed to older age); thymoma-associated MG (100%; normal distribution); MG with thymic hyperplasia (89%; skewed to younger age); AChR-Ab-negative MG (0%; normal distribution); and AChR-Ab-positive MG without thymic abnormalities (100%, skewed to older age). Furthermore, patients classified as ocular MG showed the best early-stage response to treatment and stability of improved status, followed by those classified as thymoma-associated MG and AChR-Ab-positive MG without thymic abnormalities; by contrast, those classified as AChR-Ab-negative MG showed the worst early-stage response to treatment and stability of improved status. Conclusions Differences were seen between the five subtypes in demographic characteristics, clinical severity, and therapeutic response. Our five-subtype classification approach would be beneficial not only to elucidate disease subtypes, but also to plan treatment strategies for individual MG patients.

Introduction In this study we sought to clarify the effects of early fast-acting treatment (EFT) strategies on the time course for achieving the treatment target in generalized myasthenia gravis (MG). Methods This retrospective study of 923 consecutive MG patients analyzed 688 generalized MG patients who had received immunotherapy during the disease course. The time to first achieve minimal manifestations (MM) or better while receiving prednisolone at ≤5 mg/day for ≥6 months (MM-or-better-5mg) up to 120 months after starting immunotherapy was compared between EFT and non-EFT patients. Results Achievement of MM-or-better-5mg was more frequent and earlier in the EFT group (P = 0.0004, Wilcoxon test; P = 0.0001, log-rank test). Multivariate Cox regression analysis calculated a hazard ratio of 1.98 (P < 0.0001) for utilization of EFT. Dosing regimens of oral steroids in EFT produced no differences in the time course. Conclusions EFT strategies are advantageous for early achievement of MM-or-better-5mg. Muscle Nerve 55: 794–801, 2017

From a screen of 36 plant-associated strains of Burkholderia spp., we identified 24 strains that suppressed leaf and pseudobulb necrosis of orchid caused by B. gladioli. To gain insights into the mechanisms of disease suppression, we generated a draft genome sequence from one suppressive strain, TC3.4.2R3. The genome is an estimated 7.67 megabases in size, with three replicons, two chromosomes, and the plasmid pC3. Using a combination of multilocus sequence analysis and phylogenomics, we identified TC3.4.2R3 as B. seminalis, a species within the Burkholderia cepacia complex that includes opportunistic human pathogens and environmental strains. We generated and screened a library of 3,840 transposon mutants of strain TC3.4.2R3 on orchid leaves to identify genes contributing to plant disease suppression. Twelve mutants deficient in suppression of leaf necrosis were selected and the transposon insertions were mapped to eight loci. One gene is in a wcb cluster that is related to synthesis of extracellular polysaccharide, a key determinant in bacterial-host interactions in other systems, and the other seven are highly conserved among Burkholderia spp. The fundamental information developed in this study will serve as a resource for future research aiming to identify mechanisms contributing to biological control.

When foot-and-mouth-disease (FMD) was identified in Miyazaki prefecture in March 2000, Japan conducted an intensive serological and clinical survey in the areas surrounding the index herd. As a result of the survey during the 21 days of the movement-restriction period, two infected herds were detected and destroyed; there were no other cases in the months that followed. To evaluate the survey used for screening the disease-control area and surveillance area, we estimated the herd-level sensitivity of the survey (HSe) through a spreadsheet model using Monte-Carlo methods. The Reed–Frost model was incorporated to simulate the spread of FMD within an infected herd. In the simulations, 4, 8 and 12 effective-contact scenarios during the 5-day period were examined. The estimated HSes of serological tests (HSeE) were 71.0, 75.3 and 76.3% under the 4, 8 and 12 contact scenarios, respectively. The sensitivity analysis showed that increasing the number of contacts beyond 12 did not improve HSeE, but increasing the number of sampled animals and delaying the dates of sampling did raise HSeEs. Small herd size in the outbreak area (>80% of herds have

Isothermal adsorption and desorption processes of In/Si(1 1 1) were studied by reflection high energy electron diffraction (RHEED) and fluorescent X-ray spectroscopy. In the isothermal adsorption process at room temperature, a sticking probability of In on Si(1 1 1) is unity even in a multilayer region, and the 7×7 structure is replaced by 1×1 during adsorption of In up to 2 monolayers (ML). In the isothermal desorption process at temperatures between 360 and 460 °C, superstructure of In/Si(1 1 1) changes to 1×1, 4×1, 31 × 31 , 3 × 3 , 7×7, in turn. Structure change and In desorption from each phase go together and all desorption rates are zeroth order. Activation energies of In desorption from 1×1, 4×1, 31 × 31 and 3 × 3 phases are estimated to 0.48, 1.5, 1.6 and 1.9 eV, respectively. From RHEED and isothermal desorption results, saturation coverage of 4×1 and 31 × 31 phases are estimated to be 0.96±0.15 and 0.56±0.06 ML, respectively.

Ullrich disease is a form of congenital muscular dystrophy characterized clinically by generalized muscle weakness, contractures of the proximal joints, and hyperflexibility of the distal joints from birth or early infancy. Recently, mutations of the collagen VI gene have been associated with Ullrich disease. The authors report on a boy with Ullrich disease who has complete deficiency of collagen VI and harbors compound heterozygous mutations in the collagen VI alpha 2 gene. Absence of microfibrils on EM, together with normal collagen fibrils and basal lamina, suggests that loss of a link between interstitium and basal lamina may be a new molecular pathomechanism of muscular dystrophy.

Structures of the initial stage of Fe silicide grown on Si(1 1 1) by reactive deposition epitaxy method are studied by reflection high-energy electron diffraction and scanning tunneling microscopy. At the beginning, triangular two-dimensional Fe silicide islands of a 2×2 structure appear. During iron deposition, they grow and part of them stick to each other, while their flatness and periodic structure remain unchanged. At multilayer coverage, a diffraction pattern due to three-dimensional islands appears. This is considered to be a new Fe silicide phase as a precursor of β-FeSi 2 .

Photoluminescence (PL) from fullerene (C 60 and C 70 )-doped polymers such as poly(methyl methacrylate) (PMMA), polystyrene (PS), poly(methyl phenyl silane) (PMPS) and poly(phenyl silsesquioxane) (PPSQ) increases gradually under laser irradiation in air (but not in vacuum and in nitrogen) and eventually becomes visible to the naked eye. Concomitantly, the PL peak is broadened and, in most cases, blue-shifted. No such PL increases are observed for pure C 60 films made by vacuum vapor deposition and pure polymer films. Among the polymers used, fullerene-doped PMMA has the greatest PL increase after several hours of laser irradiation and fullerene-doped PMPS has the highest rate of PL increase at the initial stage of the laser irradiation. To gain an insight into the mechanism of the PL increase, laser-irradiated fullerene-doped PMMA samples were analyzed by UV-Vis spectrophotometer, FT-IR, mass spectrometry, GPC and NMR. The results show that the PL increase can be attributed to C 60 O n -polymer (or C 70 O n -polymer) and oxidized fullerene-polymer adducts formed by some laser-induced photochemical reactions among fullerenes, oxygen and polymers.

The kinetics of ethylene hydrogenation on a Ni(100) surface were investigated using a reactor cell directly attached to a UHV chamber under a total pressure of ca. 100 Pa and a temperature range of 270–320 K. An activation energy of 10 kJ/mol and kinetic orders with respect to hydrogen and ethylene partial pressure of 1.3 and −0.3 were obtained, respectively. The rate of ethane formation gradually decreased during the course of the reaction even in the presence of excess hydrogen. RAIR spectra taken at 295 K using IR cell showed vinyl species in the presence of hydrogen and ethylene mixture. Preadsorption of ethylene at 295 K, forming ethylene decomposed fragments, decreased the reaction rate, but half of the hydrogenation rate on the clean surface still remained upon saturated ethylene preadsorption. It indicates that the surface is not covered completely by ethylene decomposed fragments. Then, the rate of ethane production in the very beginning of the reaction was measured under vacuum by introducing pulses of hydrogen and ethylene mixture containing ca. 10 15 ethylene molecules to the hydrogen preadsorbed Ni surface kept at 240–310 K. The reaction probability calculated from the response caused by the first pulse was 10 times larger than that obtained for the initial reaction rate in the reactor cell. The activation energy of 25 kJ/mol from ethylene hydrogenation in the pulse experiment coincides with that of 24 kJ/mol reported on Pt(111) corresponding to the hydrogenation of weakly adsorbed ethylene.

Thin carbon films have been prepared by ArF excimer laser ablation of hard fullerene‐based carbon in vacuum. The target material was obtained from pressure–temperature treatment of C60 fullerene. Micro‐Raman, electron energy loss, and electron diffraction measurements of deposited films reveal distinct evidence of a small microcrystalline diamond fraction. This appears to be the observation of crystalline diamond produced by laser ablation of a nondiamond target.

Thin carbon films have been prepared by ArF excimer laser ablation of hard fullerene-based carbon in vacuum. The target material was obtained from a pressure - temperature treatment of fullerene. Micro-Raman, electron energy loss and electron diffraction measurements of deposited films reveal distinct evidence of microcrystalline diamond. Appearance of the diamond fraction in the films can be related to a peculiar structure of the target substance.

The interaction of hydrogen atoms with the Si(100)(2 × 1)-K surface has been studied by using thermal desorption and electron energy loss spectroscopy. We have found that hydrogen adsorption induces the formation of potassium hydride islands at 90 K for even one-monolayer coverage of K, indicative of restructuring of the K-covered surface induced by hydrogen adsorption. The proposed reaction mechanism is as follows: incoming H atoms react with surface SiK species to generate surface SiH species and K atoms with the K atoms subsequently reacting with incoming H atoms to form potassium hydride. We discuss the relevance of the present results to the reaction of the metal-modified Si surface with hydrogen atoms.

The interaction of K with Si(100)(2 × 1)-H has been studied by using thermal desorption and electron energy loss spectroscopy. We have found that above 360 K potassium adsorption induces concomitant H2 desorption. This mechanism has been ascribed to a two step process consisting of the reaction of an incoming K atom with surface SiH species to form a surface complex, followed by the decomposition of this complex to give rise to the H2 desorption. We have found that the isolated reaction intermediate, whose decomposition leads to the H2 desorption around 450 K, is formed by the deposition of a small amount of K at 90 K. For the deposition of a large amount of K, the reaction intermediates form two types of potassium hydrides upon annealing and give rise to sharp H2 desorption peaks at 360 and 380 K. Analysis of the K uptake curves above 360 K in terms of the Kisliuk model shows that K adsorption occurs via a precursor-mediated process, which is similar to that on clean Si(100)(2 × 1) but with a much low probability (< 110) for the precursor to chemisorb. We suggest that the low probability for chemisorption is due to the activation barrier, estimated to be ∼ 3 kcal/mol, for the precursor K atoms to react with surface SiH species.

Developmental block of early embryos is due to the lack of some components in the culture medium and/or caused by inappropriate environmental conditions for embryonic development. It was shown in our experiments that the development of mouse and bovine embryos under the influence of oviducts in vitro can provide us with a model to analyze the oviductal factors that promote embryonic development. In the first experiment, it was shown that mouse zygotes developed to the blastocyst stage when a coculture system with ampulla was applied, and the duration of embryo coculture significantly affected the further development of embryos. In the second experiment, it was shown that the proportion of blastocysts of cleaved ova obtained after coculture with mouse ampulla was significantly higher than that obtained after coculture with a granulosa cell monolayer or culture without cells.

The role of nitric oxide (NO) in neurotransmitter release was studied using bovine adrenal medullary chromaffin cells. L-Arginine and sodium nitroprusside (SNP) slightly increased the intracellular free calcium concentration ([Ca2+]i), and the effects of the agents were dependent on the presence of the extracellular Ca2+ ([Ca2+]o), but were not blocked by verapamil (30 microM) or diltiazem (30 microM). SNP enhanced the acetylcholine (ACh)-induced rise in [Ca2+]i in the presence but not in the absence of [Ca2+]o. The effects of L-arginine but not those of SNP were inhibited by N omega-nitro-L-arginine (L-NNA). L-NNA significantly reduced the ACh-induced rise in [Ca2+]i and catecholamine (CA) release, and the reduction was restored by L-arginine but not by D-arginine. These results suggest a possible involvement of NO in ACh-induced [Ca2+]i rise and CA release in bovine adrenal chromaffin cells.

The growth of ice clusters on the Si(100)(2 × 1)-H surface has been investigated mainly by the use of high-resolution electron energy loss spectroscopy and thermal desorption spectroscopy. At 90 K, H2O molecules are adsorbed on the (2 × 1)-H surface to form ice clusters by the hydrogen-bonding interaction. Four H2O peaks are observed at 165, 185, 215 and 270 K in the thermal desorption spectra for the ice-covered surface. The peaks at 165 and 185 K correspond to the ice clusters and the peaks at 215 and 270 K to the strongly-bound H2O species which play a role as the nucleation centers of the ice clusters desorbing as H2O at 185 and 165 K, respectively.

Dysferlinopathy is caused by mutations of the dysferlin gene (DYSF) on chromosome 2p13. This gave rise to a spectrum of neuromuscular disorders including: limb-girdle (LGMD2B) and distal muscular dystrophies (Miyoshi myopathy: MM). Aim: to study the clinical, pathological and genetic characteristics of dysferlinopathy patients in Egypt. Subjects & Methods: Patients were selected from those with progressive muscular dystrophy referred to Muscle and Nerve Research Laboratory, Cairo, Egypt. We studied 77 patients with dystrophic muscle biopsy. Patients had neurological assessment, family pedigree study, Serum Creatine Kinase, ECHO cardiography, electromyography and Dystrophin gene testing. A battery of histochemical tests, immunohistochemistry using both fluorescent and automated methods against a panel of antibodies were done. Mini-multiplex Western Blotting was also done. Genetic study of Dysferlin gene was done for some patients. We found 40 patients with limb-girdle muscular dystrophy, 12 with dysferlinopathy, 6 with calpainopathy, 6 with sarcoglycanopathy and 16 with non-specified limb-girdle muscular dystrophy. Dysferlinopathy patients showed 3 patients with proximal type, 3 patients with Miyoshi myopathy and 6 patients with anterior compartment myopathy. Weakness was asymmetrical in 5 patients and symmetrical in 7 patients. Some muscles were uniformly affected in most patients: gluteus maximus, hamstrings, gastrocnemius, tibialis anterior and deltoids. Pathological study showed dystrophic changes of all biopsies, mitochondrial changes in 2 biopsies and inflammatory changes in 3 biopsies. Dysferlin gene analysis was done for only 6 patients of 4 families, showed homozygous mutations in two families, one heterozygous mutation in one family, and no mutation (only polymorphisms) in one family. Dysferlinopathy is a common condition in Egypt. Many clinical, pathological and genetic characteristics are found and help in its diagnosis.

The bundle structure and the sliding of single-walled carbon nanotubes (SWNTs) have been observed by frictional-force microscopy. The diameter of the nanotube and the nearest distance between any two nanotubes were estimated to be 1.4 and 0.3 nm, respectively. The frictional force required to move SWNTs on a KCl(001) surface and its energy dissipation were estimated to be 11 nN and 0.75×10−16 J, respectively.

Isolation of cDNA clones encoding the beta-chain of the human T cell surface glycoprotein CD8 revealed the presence of five distinct forms of cDNA resulting from alternative splicing. In the process of analysis of the gene organization, we found that there exist two recently duplicated genes for CD8 beta. These genes, designated CD8 beta 1 and beta 2, consist of nine and seven exons, respectively. The organization of CD8 beta 1 and beta 2 genes is almost identical except in their 3'-ends. There are nine nucleotide differences between the coding regions of the CD8 beta 1 and beta 2 genes in spite of the extremely high similarity of these genes which extends over the entire genes including introns. Pulse field gel analysis demonstrated that CD8 beta 1 and beta 2 genes are located more than 1.5 Mb apart. It was found that the CD8 beta 1 gene is approximately 25 kb upstream from the CD8 alpha gene in the same transcriptional orientation on chromosome 2. Although both CD8 beta 1 and beta 2 genes appear functional from the nucleotide sequence, the five distinct forms of CD8 beta cDNAs and corresponding mRNAs found in thymus, PBL, and leukemic cell line HPB-ALL are all derived by alternative splicing from CD8 beta 1 transcripts.

The interaction of the K-covered Si(100) (2\ifmmode\times\else\texttimes\fi{}1) surface with oxygen has been investigated by the use of high-resolution electron-energy-loss spectroscopy and thermal-desorption spectroscopy. It was found that the mechanism of enhanced oxidation of the K-modified Si(100) (2\ifmmode\times\else\texttimes\fi{}1) surface is dependent on the fractional K coverage ${\mathrm{\ensuremath{\Theta}}}_{\mathrm{K}}$. At low coverage (${\mathrm{\ensuremath{\Theta}}}_{\mathrm{K}}$\ensuremath{\le}1), K atoms reduce the work function of the Si surface, thus increasing the initial sticking probability of oxygen; the oxygen atoms are bonded to Si and potassium oxides are not formed. However, at high coverage (${\mathrm{\ensuremath{\Theta}}}_{\mathrm{K}}$g1), K adatoms are bonded to oxygen and potassium oxides (${\mathrm{K}}_{2}$${\mathrm{O}}_{2}$, ${\mathrm{KO}}_{2}$) are formed; the promoting mechanism is related to the thermal decomposition of these oxides which play the role of an ``oxygen reservoir.''

A unique sib pair afflicted by limb girdle muscular dystrophy type 2A (LGMD2A) is described showing a slowly progressive autosomal recessive type of muscular dystrophy with onset in the third and fourth decades. The patients had early asymmetric muscle involvement characterized by prominent biceps brachii atrophy with sparing of the knee extensors. Additional findings included elevation of serum creatine kinase level, myopathic EMG changes and dystrophic type of pathology on muscle biopsy. Asymmetrical wasting of muscles in the extremities exhibited uniform and highly selective CT imaging patterns. RNA and DNA analyses confirmed novel compound heterozygous mutations (R147X/L212F) in the human CAPN3 gene.

To clarify the role of disintegrin-like and metalloproteinase with thrombospondin type I motifs-1 (ADAMTS-1) in ovarian function, we examined abnormalities in ovulatory processes, folliculogenesis and the vascular system of ADAMTS-1 null ovaries. First, when immature female mice were treated with pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG), the number of ovulated oocytes was markedly decreased in ADAMTS-1 null mice in comparision to ADAMTS-1 (+/−) controls. The proportion of anovulated follicles to total mature follicles was significantly higher in ADAMTS-1 null females when compared with controls. The numbers of growing follicles at each stage were counted. The number of follicles at type 5b (late preantral) and later stages was markedly reduced in ADAMTS-1 null mice, irrespective of gonadotropin treatment (no gonadotropins, PMSG alone or PMSG/hCG). These data demonstrate that impairment of ovarian function to ovulate oocytes in ADAMTS-1 null mice occurs at two different levels: in the development of growing follicles and ovulatory processes. Furthermore, ADAMTS-1 null ovaries included a number of unusual atretic follicles that showed no sign of oocyte degeneration but lost the surrounding granulosa cell layers and were considered to be derived from type 4 or 5a follicles. These results suggest that ADAMTS-1 is important for follicular development beyond the type 4 and/or 5a and for maintaining normal granulosa cell layers in follicles. Finally, the number of large blood vessels in the medullar zone was significantly decreased in ADAMTS-1 null mice ovaries, suggesting that ADAMTS-1 is also involved in the organization of the medullary vascular network.

In our laboratory, limb-girdle muscular dystrophy (LGMD) accounted for 20% of all patients with muscular dystrophy. To determine the incidence of various forms of LGMD phenotypes, we looked for mutations in the calpain 3 gene and, for deficiencies in dysferlin and sarcoglycan by immunohistochemical studies with specific antibodies on muscle biopsies from patients with probable autosomal recessive inheritance (LGMD2), which were mostly sporadic cases of LGMD. Fourteen of 276 (5%) patients examined had sarcoglycan complex deficiency (sarcoglycanopathy) and 21 of 80 (26%) had mutations in the calpain 3 gene. Although we have not performed gene analysis in all patients, 10 of 64 (15%) patients examined had no apparent immunoreactivity against the dysferlin antibody. Thus, approximately 46% of LGMD2 patients had the above 3 distinct disorders, but in 54% the causative defects remain unknown.

In the present study, the sequential expression and cellular localization of cyclin B1 was examined in two-cell mouse embryos to elucidate the mechanism of the two-cell block. One-cell embryos derived from in vitro fertilization were cultured with oviductal tissue (nonblocking condition) or without oviductal tissue (blocking condition) to establish the experimental conditions in which the embryos either overcome the two-cell block or do not. The amount of cyclin B1 gradually increased through the second cell cycle (through S to G2 phase). However, the difference was not observed between culture conditions. This showed that even embryos exhibiting the two-cell block normally synthesize cyclin B1 through the cell cycle. Cyclin B1 in embryos cultured under nonblocking condition accumulates in the nucleus during the transition from the G2 to the M phase, whereas that in embryos cultured in blocking condition localizes in the cytoplasm throughout the cell cycle. These data indicate that two-cell embryos cultured in blocking condition are able to normally synthesize cyclin B1 but have defects in nuclear accumulation of the protein. However, when two-cell blocked embryos were treated with okadaic acid, an activator of Cdc2 kinase, part of cyclin B1 in the embryos translocated into the nucleus. Moreover, treatment with butyrolactone I, a specific inhibitor of Cdc2 kinase, inhibits nuclear translocation of cyclin B1 in those embryos. These results suggest that Cdc2 kinase regulates the nuclear accumulation of cyclin B1 in mouse two-cell embryos.

Dendritic cells (DC) with the potential to induce anti-tumour immunity represent one of the promising candidates for cancer vaccines. Efficiency of ex vivo DC generation depends on culture conditions, especially protein components in the plasma or serum used. Using human serum albumin (HSA), we devised a constant and reproducible culture method for DC generation from peripheral blood CD14+ cells. The number of DC obtained with 2% HSA-supplemented cultures containing granulocyte-macrophage colony-stimulating factor and interleukin 4 were consistently higher than in cultures with various concentrations of autologous plasma or serum. The concentrations and time points tested for plasma or serum considerably affected the number of DC recovered. DC prepared with HSA acquired the ability to uptake dextran, and expressed high levels of major histocompatibility (MHC) and co-stimulatory molecules similar to DC cultured with autologous plasma or serum. Although DC cultured with autologous plasma or serum consisted of CD1a+ and CD1a- populations, DC differentiated in the presence of HSA expressed CD1a. DC obtained with HSA primed and induced immunogenic peptide-specific cytotoxic T lymphocytes against a tumour rejection antigen, HER2. These findings suggest that our method for preparation of DC with HSA should prove valuable in DC generation for immunotherapy.

Between June 1992 and May 2000, transsternal extended thymectomy was performed for 70 patients with myasthenia gravis in our hospital. We were able to evaluate 64 of them in terms of prediction of the need for postoperative mechanical ventilation using the score systems reported by Leventhal et al., Kimura et al. and the criteria of Adachi et al.. For these systems, the rates of agreement between predictions and results were 85.9%, 82.8%, and 64.1%, respectively. The two former systems had some false negative cases (i.e., they predicted that ventilation would not be needed when in fact it was), but the last one gave no false negatives. We recommend Adachi's criteria for clinical safety. In our cases the patients whose value of %VC multiplied by FEV1.0% was less than 7,000 (Adachi's criterion is less in 8,300), especially, needed careful management with regard to respiratory crisis.

A 45-year-old housewife had proximal dominant limb muscle weakness from around 25 years of age. Her parents were cousins. None of family members was affected. Progressive muscle weakness and atrophy were prominent at the posterior compartments of legs and trunk. Serum CK was moderately elevated. Muscle pathology revealed variation in fiber size, moderate increase in numbers of internal nuclei and abundant lobulated fibers. On immunostaining using by monoclonal antibody against human calpain 3 (NCL-CALP-2 C4; Novocastra) to the biopsied muscle, calpain 3 was completely absent in the sarcoplasm, while granular debris and in part positive striation were noted in control muscle. By Western blot analysis, a band corresponding to 94 kDa of calpain 3 was not detected. A genetic analysis of calpain 3 revealed homozygous C-565-G mutation (Leu189Val). From the present study. Western blot analysis and immunostaining by using calpain 3 antibody were suggested to be useful to diagnose LGMD2A in LGMD patients.

The effect of the oviductal environment on gene expression in 2-cell mouse embryos was examined with mRNA differential display. Embryos used for experiments were cultured in modified Whitten medium with or without oviductal tissue until late 2-cell stage. The results of sequencing indicated that the genes for ATP synthase (ATPase 6), S:-adenosylmethionine decarboxylase (S:-AMDC) and nuclear autoantigenic sperm protein (NASP) were differentially expressed in embryos cultured in the oviductal environment (nonblocking culture condition). The ATPase 6 gene is encoded by mitochondrial DNA and is essential for the production of ATP. This indicates that the expression of ATP synthesis-related genes at the 2-cell stage may be required to maintain normal development in vitro. S:-Adenosylmethionine decarboxylase decarboxylates adenosylmethionine, which is a substrate of DNA methylation. The expression of S:-AMDC may be responsible for the low level of methylation of preimplantation development. As NASP is a histone-binding protein that is thought to be testis and sperm specific, its function in embryos remains unclear. On the other hand, the Tcl1 gene and a novel gene, the c-1 gene, were strongly expressed in embryos cultured without oviductal tissue (blocking culture condition). The expression patterns of these genes are quite similar. However, the detailed functions of these genes in embryos remain to be determined.

A new and simple method for achieving Color Electro-Holographic Display systems was developed and tested with very promising results. In this method, holography techniques utilizing a single white light source, instead of the traditional three color laser lights, and three liquid crystal display panels (LCD) were proposed. A metal halide lamp with good specifications in each wavelength of the color components was adopted as the light source. Each of the three color light components required for the reconstruction of an image was extracted by color separation using cold mirrors. The separated color images were then reconstructed by the hologram formed on the LCD panels which is then combined together through the use of a prism to form a single 3D image. Moreover the image position is adjusted with focal adjustment method. Compared with usual reconstruction methods using three color lasers, this new approach using the single white light source seems to have better color clarity.

Peripheral blood monocytes are common precursor cells of dendritic cells (DCs) and macrophages. We have searched for factors with the potential to regulate the differentiation of monocytes to DCs and macrophages. When CD14+ monocytes are cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL) 4, the CD14+CD1a- population, which consists of macrophages, was found in the serum-containing cultures but not in the serum-free cultures. Addition of IL-6 receptor-neutralizing monoclonal antibody (mAb) or gp130-neutralizing mAb to the serum-containing cultures resulted in a decreased population of CD14+CD1a- cells. An increase in the CD14+CD1a- population with reduction in CD14-CD1a+ DCs was observed with the addition of IL-6 to cultures, whereas IL-11, leukaemia inhibitory factor, oncostatin M or macrophage colony-stimulating factor did not affect the differentiation of monocytes in the presence of GM-CSF plus IL-4. This effect of IL-6 was blocked by tumour necrosis factor alpha (TNF-alpha), lipopolysaccharide (LPS), IL-1beta, CD40 ligand (CD40L) and transforming growth factor beta1 (TGF-beta1). Among these factors, TNF-alpha was most potent in interfering with the action of IL-6. These results suggest that IL-6 inhibits the differentiation of monocytes to DCs by promoting their differentiation toward macrophages, which is modulated by factors such as TNF-alpha, LPS, IL-1beta, CD40L and TGF-beta1.

We examined 159 patients with thrombotic thrombocytopenic purpura and hemolytic uremic syndrome in Japan. The subjects were divided in three groups; 90 patients with thrombotic thrombocytopenic purpura, 51 patients with verotoxin-induced hemolytic uremic syndrome, and 18 patients with drug-induced hemolytic uremic syndrome. Eighty-two percent of the patients with thrombotic thrombocytopenic purpura had associated neurological disorders and 78% of drug-induced hemolytic uremic syndrome associated with pulmonary edema. Renal insufficiency was noted in the 69% cases with both hemolytic uremic syndrome groups. Seventeen patients with thrombotic thrombocytopenic purpura had systemic lupus erythematosus and 6 were pregnant. Autoantibody were positive in 53% of thrombotic thrombocytopenic purpura. Seventy-seven percent of patients with thrombotic thrombocytopenic purpura received plasma exchange at 4,000 mL/day three times a week, 71% antithrombotic agents, and 78% steroid administration, respectively. However, 27% of the patients with hemolytic uremic syndrome were treated by hemodialysis in addition to antithrombotic agents. When drug-induced hemolytic uremic syndrome was diagnosed, the drug was immediately discontinued and the patients were treated with antiplatelet agents. Seventy-four percent of the patients with thrombotic thrombocytopenic purpura were alive at 26 weeks compared with 95% of those with hemolytic uremic syndrome. As thrombotic thrombocytopenic purpura had a high mortality rate in Japan, we should carry out early diagnosis and early treatment.

The objective of this study is to develop underwater-feasible and high corrosion-proof surface treatment technology for an extended life span of offshore structures. Cavity formation is a prerequisite to underwater plasma spraying using a fluid-stabilized local dry wet method. It was examined that the effects of the slit angle of a cavity protection tube, the jet velocity of water curtain and the clearance between the protection tube end and the plate surface (below described as the clearance ) , etc. on the cavity formation. By using pure Ti wire as a spraying material, plasma-sprayed coating was formed underwater. As a result, when the clearance was 2mm, the protection tube having the slit angle of 75°, formed broader cavity than that having the angle of 45°, however the clearance of 6mm brought the opposite result. A cavity which has enough dimensions of spraying distance and diameter for the purpose of underwater spraying was formed under the conditions of the clearance of 2-6mm. In the macroscopic appearance, neither cracks nor peelings were observed in the underwater sprayed coating. X-ray analysis revealed that the underwater sprayed coating obtained was consisted mostly of Ti phase. Key words : thermal spraying, underwater, protection tube, water curtain, cavity formation

We studied the growth of hematopoietic progenitors at different progressive stages of differentiation and focused especially on changes in cell-cycling. Hematopoietic progenitors from 5-fluorouracil (5-FU)-treated mice were separated into three groups on the basis of differentiation, Stages I, II, and III, and have studied their cell-cycling. Primary marrow cells collected from 5-FU-treated mice were categorized as Stage I progenitors. Stages II and III progenitors are early and late progenies of Stage I progenitors, respectively. The rate of growth of hematopoietic progenitors supported by interleukin-3 (IL-3) and steel factor (SF) was estimated by sequential analysis of colony formation and studying replating efficiency of individual colonies. The time required for hematopoietic progenitors to go through the cell-cycle shortened as their stage of differentiation progressed. Similar results were obtained with other growth factor combinations. The analysis of DNA content of cells suggests that shortening of cell-cycling is mainly due to a decrease in the time of G1 phase of the cell-cycle. Our results demonstrate that in early hematopoiesis, the cell-cycling of hematopoietic progenitors accelerates as they differentiate.

The nuclear localization signal of the major structural protein, Vp1, of simian virus 40 was further defined by mutagenesis. The targeting activity was examined in cells microinjected with SV-Vp1 variant viral DNAs bearing either an initiation codon mutation of the agnoprotein or mutations in the Vp1 coding sequence or microinjected with pSG5-Vp1 and pSG5-Vp1 mutant DNAs in which Vp1 or mutant Vp1 is expressed from simian virus 40 early promoter. The Vp1 nuclear localization signal functioned autonomously without agno-protein once the Vp1 protein was synthesized in the cytoplasm. The targeting activity was localized to the amino-terminal 19 residues. While replacement of cysteine 10 with glycine, alanine, or serine did not affect the activity, replacement of arginine 6 with glycine caused the cytoplasmic phenotype. When multiple mutations were introduced among residue 5, 6, 7, 16, 17, or 19, the targeting activity was found to reside in two clusters of basic residues, a cluster of lysine 5, arginine 6, and lysine 7 and a cluster of lysine 16, lysine 17, and lysine 19. The clusters are independently important for nuclear localization activity.

We examined red cell fragmentation syndrome (RCFS) induced by mitomycin C (MMC) (13 patients), by thrombotic thrombocytopenic purpura (TTP) (17 patients), and by disseminated intravascular coagulation (DIC) (15 patients). Plasma cytokine levels were increased in the TTP and DIC patients, but not in those whose RCFS was induced by MMC, suggesting that the activation of the immune system plays an important role in the pathogenesis of RCFS due to TTP and DIC but did not in RCFS due to MMC. Plasma thrombomodulin, tissue type plasminogen activator, and plasminogen activator inhibitor-I levels were increased in all RCFS patients, suggesting that RCFS, whether MMC induced, or due to TTP or DIC, might be associated with vascular endothelial cell injury. In TTP, von Willebrand factor (vWF) antigen and high molecular weight vWF multimer levels were reduced, possibly as a result of microthrombus consumption. The hemostatic data in this study showed that the TTP patients were in a hypercoagulable state without hyperfibrinolysis, and that DIC patients were in both a hypercoagulable and a hyperfibrinolytic state, whereas hemostatic abnormalities were slight in patients with MMC induced RCFS. These findings suggest that vascular endothelial cell injuries might be associated with RCFS, and that those injuries in MMC-induced RCFS might not be related to microthrombi or an activated immune system.

Vesicular amine transport is crucial for activity of monoaminergic neurons since translocation of cytosolic amine to storage vesicles is required for both exocytotic release and reuptake of the neurotransmitter. We developed a convenient assay system for vesicular amine transport based on permeabilizing the plasma membrane of pheochromocytoma PC12 cells with digitonin at concentrations of 100 to 150 microM. Serotonin (5HT) is a better substrate than either epinephrine or norepinephrine in this assay system. In the presence of 2 mM ATP, 5HT uptake by the permeabilized cells increased linearly for at least 30 min at 25 degrees C, whereas without addition of exogenous ATP, 5HT uptake reached an apparent plateau after 20 min incubation. Reserpine (500 nM) completely blocked the ATP-dependent 5HT uptake in this system whereas nomifensine (10 microM) had no effect, indicating specificity for vesicular transport. Treatment of intact PC12 cells with AMP dose-dependently decreased 5HT uptake with an EC50 of 20-30 microM as measured after cell permeabilization. Treatment of the intact PC12 cells with forskolin and phorbol 12-myristate 13-acetate prior to permeabilization also down-regulated vesicular 5HT transport, whereas the addition of either of these agents into the reaction mixture for the amine uptake by already permeabilized cells did not alter the vesicular uptake activity. Thus, the system can be applied to studies on regulation of the vesicular amine transport by physiological signaling molecules, intracellular messengers, and drugs.

For a successful study on strength evaluation of ceramic-metal joints with interface crack based on interfacial fracture mechanics, there is a study by Yuuki et al. [1] on Si3N4/Mo joints. In this study, they assumed that a metal was elastic, since Mo had a high yield stress. However, in the actual ceramic-metal joints, the soft metal layer such as copper and aluminum is generally inserted between a ceramics and a joint metal, in order to decrease a thermal residual stress by plastic deformation of soft metal. In this problem, it is necessary to evaluate the strength considering the plastic deformation of the soft metal layer and the joint metal. But the strength evaluation method of this problem have not been found, since the interfacial fracture mechanics considering plasticity has not been established yet.

The mechanism of the regulation of Ca++ influx via Na(+)-Ca++ exchange in response to Na+ deprivation was studied in bovine adrenal medullary cells. Protein kinase inhibitors staurosporine and (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl2,3,9,10-te trahydro-8, 11-epoxy-1H,8H, 11H-2,7b, 11a-triazadibenzo[a,g]cycloocta[c,d,e]trinden- 1-one depressed Na+ deprivation-induced 45Ca++ uptake and catecholamine secretion in a concentration-dependent manner. However, 1 mM dibutyryl cyclic AMP and 1 microM forskolin, an activator of adenylate cyclase, had little effect on Na+ deprivation-induced 45Ca++ uptake and catecholamine secretion. Dibutyryl cyclic GMP (1 mM) and muscarine (30 microM), which increased intracellular cyclic GMP level via stimulation of muscarinic receptors, had also little effect on the responses. Although the phorbol esters 12-O-tetradecanoyl-phorbol-13-acetate and phorbol 12,13-dibutyrate, activators of protein kinase C, enhanced Na+ deprivation-induced catecholamine secretion, these compounds failed to affect Na+ deprivation-induced 45Ca++ uptake. On the other hand, a variety of calmodulin antagonists such as calmidazolium, trifluoperazine, pimozide and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide inhibited Na+ deprivation-induced 45Ca++ uptake and catecholamine secretion in a concentration-dependent manner. Furthermore, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipera zin e, which is known as an inhibitor of Ca++/calmodulin-dependent protein kinase II, also reduced Na+ deprivation-induced 45Ca++ uptake and catecholamine secretion. Chelation of intracellular Ca++ with Quin-2 acetoxymethyl ester resulted in a decrease in Na+ deprivation-induced 45Ca++ uptake. However, these compounds that inhibited the Na+ deprivation-induced responses in the cells did not cause solely nonspecific and direct inhibition on Na(+)-Ca++ exchanger. These pharmacological observations suggest that Ca++/calmodulin-dependent protein kinase is involved in the regulation of Na(+)-Ca++ exchange in bovine adrenal medullary cells.