Dynamic behaviour of the silica-water-bio electrical double layer in the presence of a divalent electrolyte.

Electronic devices are becoming increasingly used in chemical- and bio-sensing applications and therefore understanding the silica-electrolyte interface at the atomic scale is becoming increasingly important. For example, field-effect biosensors (BioFETs) operate by measuring perturbations in the electric field produced by the electrical double layer due to biomolecules binding on the surface. In this paper, explicit-solvent atomistic calculations of this electric field are presented and the structure and dynamics of the interface are investigated in different ionic strengths using molecular dynamics simulations. Novel results from simulation of the addition of DNA molecules and divalent ions are also presented, the latter of particular importance in both physiological solutions and biosensing experiments. The simulations demonstrated evidence of charge inversion, which is known to occur experimentally for divalent electrolyte systems. A strong interaction between ions and DNA phosphate groups was demonstrated in mixed electrolyte solutions, which are relevant to experimental observations of device sensitivity in the literature. The bound DNA resulted in local changes to the electric field at the surface; however, the spatial- and temporal-mean electric field showed no significant change. This result is explained by strong screening resulting from a combination of strongly polarised water and a compact layer of counterions around the DNA and silica surface. This work suggests that the saturation of the Stern layer is an important factor in determining BioFET response to increased salt concentration and provides novel insight into the interplay between ions and the EDL.

[Evaluation of a new compact prothrombin time-international normalized ratio measurement device in pediatric management].

BACKGROUND: In a pediatric setting, the need for lifetime oral anticoagulation is increasing because of currency of extracardiac total cavo-pulmonary connection (TCPC) and pediatric valve surgery. We evaluated a new compact device "CoaguChek XS" for measuring prothrombin time-internatinal normalized ratio (PT-INR). METHODS: The international normalized ratio (INR) values obtained from 71 patients (223 samples) by a CoaguChek XS were compared with those obtained by a laboratory-based coagulation analyzer. RESULTS: The values from the CoaguChek XS had a significant correlation with the laboratory based results. (r2 = 0.92, p < 0.01, regression line y = 1.05 x -0.02). CONCLUSION: The CoaguChek XS will be useful in pediatric management.

[Senning operation for transposition of great arteries in a premature baby].

A male baby was delivered by emergency cesarean section due to fetal distress at 30 weeks of gestational age with a birth weight of 813 g. By fetal echocardiography, the patient had been diagnosed with transposition of great arteries (type 1). Early two-staged arterial switch operation was planned after 34 gestational age avoiding intracranial hemorrhage under cardiopulmonary bypass. At 19 days of life, vegetation was revealed on the pulmonary valve by echocardiography, so he was diagnosed as infectious endocarditis. Cefotaxime and gamma-globulin were given intravenously for 4 weeks. While waiting for the increase in the body weight, desaturation from chronic respiratory distress syndrome was exacerbated. At 8 months old, urgent Senning operation was performed to improve desaturation. The patient was discharged at 20 post operative day. We conclude that Senning operation can be feasible operation in such a complicated case.

[Aortic valve replacement through right thoracotomy; report of a case].

A 78-year-old female was admitted to our hospital with a diagnosis of severe aortic valve regurgitation. She had had dyspnea on effort and syncope twice in 5 months. She had also suffered from right pneumonia 8 years before, and her respiratory function was severely constrictive. Chest X-ray showed her mediastinum significantly shifted toward the right side. Chest computed tomography (CT) revealed the main pulmonary artery, right atrium (RA) and right pulmonary veins also shifted toward the right. We planned right thoracotomy at 4th intercostals space to obtain a good surgical field. A cardiopulmonary bypass was established by RA appendage drainage and femoral artery perfusion. Aortic valve replacement(AVR) was performed successfully after aortic clamp. Though defibrillator pads were placed on her back and the anterior wall of the left chest during operation, no ventricular fibrillation occurred. AVR via right thoracotomy is considered to be a good option for such a mediastinum shifted case.

[Aortic root replacement for aortic dissection and aortic regurgitation due to aortitis syndrome].

Aortic aneurysms and aortic regurgitation (AR) with aortitis syndrome are occasionally reported in young women. We report a case of aortic dissection with severe AR in an 8-year-old girl. The patient underwent aortic root replacement with a composite graft. Pathological report revealed aortitis syndrome and steroid therapy was continued to suppress further inflammatory vascular reaction.

[Beating heart surgery for the patient of severe mitral regurgitation with a episode of ventricular fibrillation; report of a case].

A 77-year-old female was admitted to our hospital with a diagnosis of severe mitral regurgitation. Cardiopulmonary revival was done by an emergent resuscitation for the ventricular fibrillation before admission. She had mild anoxic brain damage and brain magnetic resonance imaging (MRI) revealed severe brain atrophy. Chest X-ray showed severe cardiomegaly and congestion. Beating heart mitral valve replacement was planned for the prevention of reperfusion injury. A cardiopulmonary bypass was established by bicaval drainage and aortic return. The prolapse of anterior leaflet was recognized through transeptal approach after aortic clamp. We selected continuous infusion of antegrade cardioplegia for intraoperative coronary perfusion. Mitral valve replacement was done successfully. During intraoperation and postoperation, ventricular fibrillation did not occur. On-pump beating mitral valve replacement is a good procedure to prevent perioperative ventricular arrhythmia especially such the case with a decompressed myocardial function and with a preoperative episode of lethal ventricular arrhythmia necessary for cardiopulmonary resuscitation.

Glycine substitution mutations in the type VII collagen gene (COL7A1) in dystrophic epidermolysis bullosa: implications for genetic counseling.

Dystrophic epidermolysis bullosa (DEB) is an inherited mechanobullous disorder characterized by fragility of the skin and mucous membranes. The anchoring fibril protein, type VII collagen, is encoded by COL7A1, which harbors mutations in this group of diseases. In this study, we report novel glycine substitution mutations in COL7A1 in two Japanese families with DEB. The mutation detection strategy consisted of PCR amplification of genomic DNA, followed by heteroduplex analysis and nucleotide sequencing of the PCR products demonstrating altered mobility. The first case is a patient with clinically severe recessive DEB. The proband was shown to have a homozygous glycine-to-valine substitution (G2671V) in exon 108. The clinically unaffected parents were heterozygous carriers of this mutation, indicating that this glycine substitution in one allele is "silent" when combined with a normal COL7A1 allele. Thus, this patient appeared to be affected with DEB inherited in an autosomal recessive pattern. The second case was a DEB patient with a heterozygous glycine-to-glutamic acid substitution (G2079E) in exon 75. The parents were clinically unaffected and neither had this mutation in their peripheral blood leukocyte DNA. Haplotype analyses suggested that this case arose as a de novo occurrence of autosomal dominant DEB. These cases illustrate the consequences of COL7A1 glycine substitution mutations underlying DEB in terms of the mode of inheritance and the phenotype, with profound implications for genetic counseling of individuals at risk for recurrence of DEB in subsequent offspring or future generations.

Purification of small polydisperse circular DNA of eukaryotic cells by use of ATP-dependent deoxyribonuclease.

Small polydisperse circular (spc) DNAs of mouse thymocytes were purified by a procedure involving nitrocellulose column chromatography and the treatment of ATP-dependent DNase, which acts only upon linear DNA molecules. Nitrocellulose column chromatography prior to the enzyme treatment was essential because digestion of linear DNA duplexes by the enzyme was inhibited by the presence of concomitant single-stranded DNAs. Mitochondrial DNAs were eliminated by linearization with XhoI and digestion with ATP-dependent DNase. The size distribution of the purified spc DNA molecules ranged from 0.2 micron to more than 28 micron, with a mean length of 5.4 micron. Circular molecules of more than 0.4 micron long (or 1.2 kb) were free from the contamination of linear DNA fragments and pure enough to be cloned into plasmids.

Amiloride at pH 7.0 inhibits the Na(+)-driven flagellar motors of Vibrio alginolyticus but allows cell growth.

Amiloride, a specific inhibitor for the Na(+)-driven flagellar motors of alkalophilic Bacillus, is known to inhibit secondarily the growth of alkalophiles. The motility of a marine Vibrio, V. alginolyticus, was almost completely inhibited by 2 mM amiloride either at pH 7.0 or 8.5. We found that this concentration of amiloride inhibited the cell growth completely at pH 8.5 but only slightly at pH 7.0. Kinetic analysis of the inhibition of motility by amiloride at pH 7.0 showed that the inhibition was competitive with Na+ in the medium. Thus, amiloride at pH 7.0 is really a specific and useful tool for the analysis of the Na(+)-driven flagellar motors of Vibrio.

Different cytokines are required for induction and maintenance of the Th2 type response in DBA/2 mice resistant to infection with Leishmania major.

Experimental cutaneous leishmaniasis is a useful model in studying the mechanism regulating immune responses between T helper type 1 (Th1) and Th2. Mice susceptible to Leishmania major infection such as BALB/c (H-2(d)) are associated with the induction of the disease-promoting Th2 response, while the resistant mice such as DBA/2 (H-2(d)) develop the protective Th1 response. To understand the induction mechanism of Th1 and Th2 responses, it is necessary to establish an immunization scheme by which the induction of each Th response can be easily and experimentally controlled. Adjuvants are known to enhance the immune responses through the combined effect of several factors: prolonged release of antigen, migration of cells, mitogenic effect and so forth. When the genetically resistant DBA/2 mice were immunized twice with soluble leishmanial antigen (SLA), emulsified in incomplete Freund's adjuvant (IFA) before L. major inoculation, these mice mounted a Th2 cell response and suffered from progressive infection. While IL-4 and IL-13 were upregulated early after the infection in both healer and non-healer groups of mice, IL-5 and IL-10 were upregulated only in non-healer mice. From these results, IL-5 and IL-10 appear to have an important role, at least in the early phases of the infection, rather than IL-4 and IL-13 in establishing the disease-promoting Th2 response in leishmaniasis. Further, IL-9 was found to be expressed in both BALB/c and DBA/2 mice immunized with IFA/SLA. This cytokine may support the establishment of a Th2 response in these mice. Therefore it is suggested that Th2 cytokines play different roles between priming and maintaining the Th2 immune response after the infection.

Expression and role of heat-shock protein 65 (HSP65) in macrophages during Trypanosoma cruzi infection: involvement of HSP65 in prevention of apoptosis of macrophages.

The 65-kDa heat-shock protein (HSP65) is thought to play a role in host defense against infections with various microbial pathogens and in autoimmune inflammatory disorders. We investigated the biological function and expression mechanism of HSP65 in macrophages of mice infected with Trypanosoma cruzi. BALB/c mice, which are susceptible to T. cruzi, showed high levels of parasitemia, and 80% of these mice died within 42 days after the infection, whereas resistant C57BL/6 or DBA/2 mice showed low levels of transient parasitemia and all survived. HSP65 expression was correlated with resistance to T. cruzi infection; HSP65 was more strongly expressed in macrophages of resistant C57BL/6 and DBA/2 mice than in macrophages of susceptible BALB/c mice. Immunodeficient BALB/c-nu/nu (nude) and C.B-17 scid/scid (SCID) mice were shown to be highly susceptible to this infection, and they did not express detectable levels of HSP65, suggesting that T cells play essential roles in the expression of HSP65 as well as in protective immunity against the infection. CD4(+) T cells, but not CD8(+) T cells or gammadelta T cells, were the cell population responsible for the induction of HSP65 expression in macrophages. Furthermore, depletion of asialo GM-1(+) NK cells made resistant C57BL/6 mice more susceptible to the infection, and HSP65 expression in their macrophages was abolished. Semiquantitative reverse transcription PCR analyses showed that both interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) mRNA levels in CD4(+) T cells became low when resistant C57BL/6 mice were depleted of NK cells, suggesting that NK cells contribute to functional differentiation of CD4(+) T cells and thereby affect the induction of HSP65 expression. To determine the function of HSP65, macrophages were treated in vitro with antisense oligonucleotide for HSP65 prior to inducing HSP65 with IFN-gamma plus TNF-alpha or T. cruzi infection. This treatment did not affect the production of nitric oxide following activation, but the treated macrophages became susceptible to apoptosis. These results indicate that HSP65 plays a role in preventing the apoptosis of macrophages and thereby contributes to host resistance against T. cruzi infection.

Clinical course of atopic dermatitis in Japanese patients.

We conducted a questionnaire study of 117 patients, who had first consulted the Department of Dermatology Kumamoto University Hospital 20 or 30 years ago, regarding the clinical course of atopic dermatitis (AD). Forty-one patients responded to the questionnaire. Seventeen patients (41.4%) had recovered and 24 patients (58.6%) still had AD. The AD had resolved in 12 of 13 (92.2%) with mild disease severity, four of 18 (22.2%) with moderate severity and one of ten (10%) with severe disease. The outcome was significantly better in the mild group than in the moderate or severe group (chi2=15.5, P=0.0008/chi2=14.8, P=0.00012, respectively). The resolution of the disease was not correlated with the sex of patient, age at onset, period of disease or family history.

Cloning and nucleotide sequences of the BanI restriction-modification genes in Bacillus aneurinolyticus.

The genes of the BanI restriction-modification system specific for GGPyPuCC were cloned from the chromosomal DNA of Bacillus aneurinolyticus IAM1077, and the coding regions were assigned on the nucleotide sequence on the basis of the N-terminal amino acid sequences and molecular weights of the enzymes. The restriction and modification genes coded for polypeptides with calculated molecular weights of 39,841 and 42,637, respectively. Both the enzymes were coded by the same DNA strand. The restriction gene was located upstream of the methylase gene, separated by 21 bp. The cloned genes were significantly expressed in E. coli cells, so that the respective enzymes could be purified to homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration indicated that the catalytically active form of the endonuclease was dimeric and that of the methylase was monomeric. Comparison of the amino acid sequences revealed no significant homology between the endonuclease and methylase, though both enzymes recognize the same target sequence. Sequence comparison with other related enzymes indicated that BanI methylase contains sequences common to cytosine-specific methylases.

Extracorporeal removal of anticancer drugs in hepatic artery infusion: the effect of direct hemoperfusion combined with venovenous bypass.

The effect of a new extracorporeal system combining direct hemoperfusion (DHP) with venovenous bypass was evaluated in the elimination of anticancer drugs in hepatic artery infusion. Adriamycin (3 mg/kg) and mitomycin C (1 mg/kg) were given to mongrel dogs through the hepatic artery with three different durations of 1, 10, and 20 minutes. Plasma drug levels were determined at the inlet and outlet of DHP and right external jugular vein (systemic level). Blood flow through DHP averaged 200 ml/min. In dogs without DHP (group I; n = 4), systemic levels of adriamycin and mitomycin C increased rapidly with 1-minute infusion, reaching the peak values of 6.61 +/- 2.44 (mean +/- SD) and 2.20 +/- 1.05 micrograms/ml, respectively. With DHP under single venous bypass (group II; n = 5), the peak values were reduced to 1.25 +/- 1.02 and 0.79 +/- 0.52 microgram/ml. Moreover, the peak levels were markedly reduced by DHP under hepatic venous isolation (group III; n = 6), the values being 0.41 +/- 0.15 and 0.13 +/- 0.07 microgram/ml with 1-minute infusion. The drug-removal rates were improved substantially in group III compared with group II. The longer the duration of infusion, the higher the removal rates tended to be in group III. These results indicate that effective elimination of anticancer drugs can be accomplished by this system during intraarterial chemotherapy of the liver.

Diaper area granuloma of the aged.

Nine women, aged 77 to 89 years, suffered from diaper dermatitis, and many granulomas were seen in the genital or gluteal region restricted to the lesion of dermatitis. Because these granulomas resembled granuloma gluteale infantum of Tappeiner and Pfleger, we propose to call this condition diaper area granuloma of the aged.

Intraarterial infusion of high-dose adriamycin for unresectable hepatocellular carcinoma using direct hemoperfusion under hepatic venous isolation.

A 67-year-old man with an extensive hepatocellular carcinoma (HCC) was treated successfully with intraarterial infusion of high-dose adriamycin (ADR), 150 mg/m2, five minutes continuous infusion using an extra-corporeal system consisting of direct hemoperfusion (DHP) under hepatic venous isolation (HVI). During drug infusion, hepatic effluent was isolated and adsorbed by the DHP for 30 mins. Plasma ADR levels in the radial artery reached a peak of 2.00 micrograms/ml at five mins after the initiation of drug infusion. Peak values at the inlet and outlet of the DHP were 19.71 micrograms/ml and 1.75 micrograms/ml, respectively, indicating substantial drug adsorption by the DHP. The estimated drug removal rate was 31.1%. This treatment led to a marked regression of tumors with tolerable systemic toxicities. Although the patient subsequently died 9 months after treatment of progression of disease, this treatment resulted in a remission of significant duration.

Cathepsin B-inhibitor promotes the development of Th1 type protective T cells in mice infected with Leishmania major.

BALB/c mice are genetically susceptible to infection with Leishmania major (L major). When such mice infected with L. major were treated with specific inhibitors of cathepsin B, a lysosomal cysteine protease that digests exogenous antigenic proteins, the mice acquired resistance against L. major infection. T cells from these mice produced large amounts of IFN-gamma and low amounts of IL-4 as compared with those of untreated BALB/c mice. In addition, the mice treated with cathepsin B inhibitor produced a high titer of IgG2a specific antibodies and only low titers of IgG1 and IgE antibodies. This type of response is in contrast with the high specific IgG1 or IgE antibody responses which are the usual antibody responses in BALB/c mice infected with L. major. These findings indicate that cathepsin B may be critically involved in processing antigens of L. major to promote exclusively the development of Th2 type CD 4+T cell responses.

Molecular cloning of a cystatin from parasitic intestinal nematode, Nippostrongylus brasiliensis.

A novel member of the cystatin family, nippocystatin (NbCys), was identified from excretory-secretory (ES)-products of a nematode Nippostrongylus brasiliensis, and the cDNA was cloned and sequenced. The mRNA of NbCys was confirmed to be expressed in both larvae and adults of the parasite. NbCys was translated as a proform with a single domain for secretion and was detected as a 14-kDa mature form in ES-products of the adult worm. Recombinant protein of NbCys profoundly inhibited the activity of cysteine proteases such as cathepsin L and B, but not that of cathepsin D, an aspartic protease. Furthermore, the ES-products had also been confirmed to inhibit cysteine proteases. Taken together, NbCys may play a role in evasion of N. brasiliensis from host defense systems, since cysteine proteases are known to participate in immune systems of infected hosts.

Complicating systemic amyloidosis in dystrophic epidermolysis bullosa, recessive type.

An autopsy case of dystrophic epidermolysis bullosa, recessive type, complicated by systemic secondary amyloidosis is described. The patient had developed multiple bullous lesions and erosions from birth, followed by repeated infection. At autopsy, chronic persistent inflammation was observed in the skin and in various visceral organs, accompanied by systemic amyloidosis. By the peroxidase-antiperoxidase (PAP) method, amyloid deposits stained positively for anti-AA-protein antiserum. In the present case, we concluded that the systemic amyloidosis was of the AA type, and developed secondarily to the chronic persistent inflammation in the prolonged course of dystrophic epidermolysis bullosa, recessive type.

Role of innate immune cells in protection against Toxoplasma gondii at inflamed site.

The intraperitoneal infection with Toxoplasma gondii (T. gondii) caused accumulation of gamma delta T, NK, NK1.1+T-like (NKT) cells at inflamed sites. To clarify the roles of these cells in protection against T. gondii at the inflamed sites, BALB/c mice were depleted of gamma delta T, NK, NK and NKT cells by treatment with antibody against TCR-gamma delta, asialoGM1 or Interleukin-2 receptor beta-chain (IL-2 R beta), respectively, prior to infection. Mice treated with anti-TCR-gamma delta monoclonal antibody (mAb) became more susceptible to infection, whereas mice treated with anti-IL-2R beta mAb acquired resistance. Treatment with anti-asialoGM1 Ab showed no effect. We previously reported that heat shock protein 65 (HSP65) in macrophages induced by gamma delta T cells plays an essential role in protective immunity against T. gondii infection, by preventing apoptotic death of infected macrophages. In the present study, we showed that treatment with anti-IL-2R beta mAb, but not with anti-asialoGM1 Ab, enhanced the HSP65 induction in macrophages, and inhibited Interleukin-4 (IL-4) expression in nonadherent peritoneal exudate cells. Furthermore, neutralization of endogenous IL-4 by anti-IL-4 mAb enhanced the HSP65 induction in macrophages. These findings suggest that NKT cells, but not NK cells, negatively regulate the protective immunity against T. gondii infection possibly by producing IL-4 and suppressing HSP65 induction.