Observation of Edge Magnetoplasmon Squeezing in a Quantum Hall Conductor.

Squeezing of the quadratures of the electromagnetic field has been extensively studied in optics and microwaves. However, previous works focused on the generation of squeezed states in a low impedance (Z_{0}≈50 Ω) environment. We report here on the demonstration of the squeezing of bosonic edge magnetoplasmon modes in a quantum Hall conductor whose characteristic impedance is set by the quantum of resistance (R_{K}≈25 kΩ), offering the possibility of an enhanced coupling to low-dimensional quantum conductors. By applying a combination of dc and ac drives to a quantum point contact, we demonstrate squeezing and observe a noise reduction 18% below the vacuum fluctuations. This level of squeezing can be improved by using more complex conductors, such as ac driven quantum dots or mesoscopic capacitors.

A novel triple secured technique for pancreatic reconstruction following pancreaticoduodenectomy for a soft pancreas.

BACKGROUND/AIMS: Soft pancreases are susceptible to developing pancreatic fistula following pancreaticoduodenectomy. To reduce the incidence of pancreatic fistula after pancreaticoduodenectomy in patients with a soft pancreas, we developed a triple secured technique. In this study, we describe the details of this technique and also report on the postoperative outcomes. METHODOLOGY: The triple secured technique employed an ultrasonic dissector for pancreatic transection with skeletonizing and ligating of the small pancreatic branch ducts, duct-invagination or duct-to-mucosa anastomosis for main pancreatic duct management, and, finally, four large stitches between the pancreatic stump parenchyma and the jejunal seromuscular layer to prevent minor pancreatic leakage. A total of 28 consecutive patients with a soft pancreas who underwent pancreaticoduodenectomy using our technique were included in this study. RESULTS: Postopetrative complications occurred in 16 patients. Grade B pancreatic fistula developed in 6 patients. However, no grade C pancreatic fistula occurred in this series. Neither any reoperation nor in-hospital mortality was observed in this series. CONCLUSIONS: Our triple secured technique after pancreaticoduodenectomy was feasible and safe, with an acceptable rate of grade B pancreatic fistula and no grade C pancreatic fistula for patients with a soft pancreas.

Short communication: Molecular typing of Prototheca zopfii from bovine mastitis in Japan.

Prototheca zopfii causes bovine mastitis, resulting in reduced milk production and the secretion of thin watery milk with white flakes. Prototheca zopfii has been biochemically and serologically divided into at least 2 genotypes, P. zopfii genotype 1 and P. zopfii genotype 2. The latter is known to be the main causative agent of bovine protothecal mastitis. Prototheca zopfii was later reclassified into 5 varieties: var. zopfii (genotypes 1 and 2), var. 1 (formerly Prototheca blaschkeae), var. 3 (formerly P. moriformis), and var. portoricensis. In this study, the 18S ribosomal DNA sequences of diverse clinical specimens from different areas in Japan were studied to clarify the pathogenicity of P. zopfii var. zopfii. The phylogenetic tree revealed that all genotype 2 isolates were grouped in a cluster of P. zopfii var. zopfii SAG 2021(T) (type strain genotype 2), and were independent from the cluster of the genotype 1 isolates. Thus, all isolates from bovine mastitis in Japan were identified as P. zopfii genotype 2. Therefore, P. zopfii var. zopfii genotype 2 is associated with bovine mastitis.

Long-term forskolin stimulation induces AMPK activation and thereby enhances tight junction formation in human placental trophoblast BeWo cells.

BeWo cells, derived from human choriocarcinoma, have been known to respond to forskolin or cAMP analogues by differentiating into multinucleated cells- like syncytiotrophoblasts on the surfaces of chorionic villi of the human placenta. In this study, we demonstrated that long-term treatment with forskolin enhances the tight junction (TJ) formation in human placental BeWo cells. Interestingly, AMPK activation and phosphorylation of acetyl-CoA carboxylase (ACC), a molecule downstream from AMPK, were induced by long-term incubation (>12h) with forskolin, despite not being induced by acute stimulation with forskolin. In addition, co-incubation with an AMPK inhibitor, compound C, as well as overexpression of an AMPK dominant negative mutant inhibited forskolin-induced TJ formation. Thus, although the molecular mechanism underlying AMPK activation via the forskolin stimulation is unclear, the TJ formation induced by forskolin is likely to be mediated by the AMPK pathway. Taking into consideration that TJs are present in the normal human placenta, this mechanism may be important for forming the placental barrier system between the fetal and maternal circulations.

Health-related QOL of elderly patients with pulmonary M. avium complex disease in a university hospital.

BACKGROUND: Little is known about the clinical characteristics and health-related quality of life (HQOL) of elderly patients with pulmonary Mycobacterium avium complex (pMAC) disease. OBJECTIVES: To evaluate HQOL using the 36-Item Short-Form Health Survey and St George's Respiratory Questionnaire (SGRQ) and to investigate the predictors of HQOL changes among elderly patients with pMAC disease. METHODS: This prospective cohort registry was conducted at Keio University Hospital, Tokyo, Japan, between May 2012 and July 2015 and included 84 patients with pMAC disease aged 75 years who had completed the HQOL questionnaire and 48 patients with pMAC disease who had been followed up and completed the HQOL questionnaire in cross-sectional and longitudinal analyses, respectively. RESULTS: In cross-sectional analyses, elderly patients with pMAC disease had significantly lower role-physical, general health, vitality, social functioning, role-emotional and role/social component scores than the general Japanese elderly population. Analysis of covariance revealed that patients with cavitary lesions had significantly worse physical functioning and SGRQ scores (P < 0.05). Longitudinal analysis showed that under-treatment, short duration of disease and positive sputum smear at baseline were predictors of worse HQOL at 12 months. CONCLUSIONS: Elderly patients with pMAC disease have reduced HQOL. Further large studies on HQOL are required to refine the use of this parameter in the treatment of these patients.

Mutation of the p53 tumour suppressor gene and overexpression of its protein in 62 Japanese non-Hodgkin's lymphomas.

To clarify whether p53 mutation could be involved in the pathogenesis of various subtypes of lymphoma, we investigated 62 Japanese cases of non-Hodgkin's lymphomas (NHLs) for p53 gene mutations and their relationship with the expression of p53 protein. Mutations in exons 5-9 of the p53 gene were screened for using the non-isotopic RNase cleavage assay (NIRCA) and confirmed by direct sequencing, followed by immunohistochemical analysis for p53 protein. Missense and/or nonsense mutations of p53 were detected in 3 (10.7%) of 28 diffuse large B-cell lymphomas (DLBLs) and 2 (15.4%) of 13 T-cell NHLs (15.4%). A single missense mutation at codon 157 (Val to Phe) in exon 5 and at codon 273 (Arg to Pro) in exon 8 was found respectively in 2 DLBLs and in one peripheral T-cell lymphoma (unspecified). In these 3 cases harbouring a missense mutation, overexpression of p53 protein was observed in more than 80% of tumour cells. Double transversion mutations comprising of a missense mutation at codon 167 (Gln to His) in exon 5 and a nonsense mutation at codon 183 (Ser to stop codon) in exon 5 were detected in one DLBL that had apparently transformed from follicular lymphoma and in one advanced adult T-cell lymphoma (ATL). In these two cases harbouring p53 nonsense mutation, no cells positive for p53 protein immunostaining were detected, as well as lymphomas without p53 mutation.

Inhibitory effect of NaN3 on the F0F1 ATPase of submitochondrial particles as related to nucleotide binding.

The inhibitory effects of NaN3 on the F0F1 ATPase of beef heart submitochondrial particles were investigated. It was shown that NaN3 inhibited the ATPase activity only in the presence of ATP or ADP and the inhibition proceeded slowly. Analysis of the time-course of the inhibition process lead to a conclusion that an ATP binding site which has an apparent Kd of 14.0 +/- 8.7 microM is responsible for the increase of NaN3 sensitivity. This value agreed well with the low Km of ATP hydrolysis characterized before (Muneyuki, E., and Hirata, H. (1988) FEBS Lett. 234, 455-458) and in the range of so-called bi-site catalysis. The same conclusion was derived as for isolated F1 ATPase. From similar analysis, the Kd of this site for ADP was deduced to be 1.34 +/- 0.45 microM, which also agreed with that reported by Pedersen (Pedersen, P.L. (1975) Biochem. Biophys. Res. Commun. 64, 610-616) and also in the same range as reported for the low Km of ATP synthesis by activated submitochondrial particles. These results suggest that hydrolysis through the low Km mode of ATPase reaction leads the enzyme NaN3 sensitive form and this reaction cycle corresponds to the low Km mode of ATP synthesis.

Redox regulation of cellular signalling.

Extracellular stimuli elicit a variety of responses, such as cell proliferation and differentiation, through the cellular signalling system. Binding of growth factors to the respective receptor leads to the activation of receptor tyrosine kinases, which in turn stimulate downstream signalling systems such as mitogen-activated protein (MAP) kinases, phospholipase Cgamma (PLCgamma) and phosphatidylinositol 3-kinase. These biochemical reactions finally reach the nucleus, resulting in gene expression mediated by the activation of several transcription factors. Recent studies have revealed that cellular signalling pathways are regulated by the intracellular redox state. Generation of reactive oxygen species (ROS), such as H2O2, leads to the activation of protein tyrosine kinases followed by the stimulation of downstream signalling systems including MAP kinase and PLCgamma. The activation of PLCgamma by oxidative radical stress elevates the cellular Ca2+ levels by flux from the intracellular Ca2+ pool and from the extracellular space. Such reactions in the upstream signalling cascade, in concert, result in the activation of several transcription factors. On the other hand, reductants generally suppress the upstream signalling cascade resulting in the suppression of transcription factors. However, it is well known that cysteine residues in a reduced state are essential for the activity of many transcription factors. In fact, in vitro, oxidation of NFkappaB results in its activation, whereas reductants promote its activity. Thus, cellular signalling pathways are generally subjected to dual redox regulation in which redox has opposite effects on upstream signalling systems and downstream transcription factors. Not only are the cellular signalling pathways subjected to redox regulation, but also the signalling systems regulate the cellular redox state. When cells are activated by extracellular stimuli, the cells produce ROS, which in turn stimulate other cellular signalling pathways, indicating that ROS act as second messengers. It is thus evident that there is cross talk between the cellular signalling system and the cellular redox state. Cell death and life also are subjected to such dual redox regulation and cross talk. Death signals induce apoptosis through the activation of caspases in the cells. Oxidative radical stress induces the activation of caspases, whereas the oxidation of caspases results in their inactivation. Furthermore, some cell-death signals induce the production of ROS in the cells, and the ROS produced in turn stimulate the cell-death machinery. All this evidence shows that the cell's fate is determined by cross talk between the cellular signalling pathways and the cellular redox state through a complicated regulation mechanism.

Clinical features of ovarian cancer in Japanese women with germ-line mutations of BRCA1.

We analyzed the clinical features of 25 ovarian cancer patients who were associated with germ-line mutations of BRCA1 from four site-specific ovarian cancer families and seven breast-ovarian cancer families in Japan. The average age at diagnosis was 51.1 years (range, 38-77 years). Histological examination revealed 24 serous cyst adenocarcinomas in 25 patients. In 23 patients with clear clinical records, 3 patients had stage I disease, 17 had stage III disease, and 3 had stage IV disease. Thirteen patients with stage III disease who were treated with cisplatin-containing chemotherapy following tumor reduction surgery showed more favorable outcomes in both the survival rate and disease-free intervals, compared with age- and treatment course-matched controls (5-year survival rate, 0.786 versus 0.303; median disease-free interval, 91.43 versus 40.92 months; P < 0.05 for both, by logarithmic rank test). Our statistical model for the inheritance of susceptibility to ovarian cancer was derived from the analysis of 26 patients and 19 healthy carriers of 12 families. The expected lifetime risk of ovarian cancer is about 80% for women with mutations of BRCA1. These results suggest that the clinical outcome of ovarian cancer with germ-line mutations of BRCA1 appears to be more favorable than that with sporadic cases and that the disease penetrance among pedigrees with germ-line mutations of the BRCA1 gene is substantially high.

Induction of canine pyometra by inoculation of Escherichia coli into the uterus and its relationship to reproductive features.

To characterize oestrus-related factors affecting the induction of and recovery from pyometra in bitches, 60 clinically healthy beagle bitches were used for induction of pyometra by inoculation of Escherichia coli into the uterus during oestrous and metoestrous stages. The animals were classified into the following six groups according to inoculation time: Days 1-10, 11-20, 21-30, 31-40, 41-50 and 51-60 after LH surge. The incidence of pyometra during the periods Days 11-20 and 21-30 after LH surge was 90.9% and 78.9% respectively, while that during Days 1-10 and 51-60 after LH surge was less than 20%, and the patterns of the incidence of pyometra and the serum progesterone levels were similar. There was no difference in the incidence of pyometra induced in bitches less than 5 years old compared to bitches over 6 years old. Oestrus in all of the bitches with pyometra induced by E. coli returned with or without PGF 2alpha treatment, unlike in bitches with spontaneous pyometra. The duration of the oestrous cycle in the non-treated and PGF 2alpha-treated groups was 231.4+/-55.2 days and 162.1+/-40.6 days (P < 0.001), respectively, and there was no difference in the rate of return of oestrus between the two groups. The conception rate in all of the bitches in which oestrus had returned was 81.8%. The above findings indicate that the period during which severe pyometra could be induced was limited to the early stage in metoestrus.

A novel insertion sequence (IS)-like element of the thermophilic bacterium PS3 promotes expression of the alanine carrier protein-encoding gene.

A novel insertion sequence (IS)-like element was found in the 5'-upstream region of the alanine carrier protein-encoding gene (acp) in the thermophilic bacterium PS3 chromosomal DNA. The sequence contained an open reading frame (ORF) encoding a polypeptide of 369 amino acids which revealed high similarity with ORFs from IS891 from the cyanobacterium Anabaena and IS1136 from Saccharopolyspora erythraea. The direction of transcription was the same as that of acp, and typical inverted and direct repeats characteristic of IS were found in both the 5' and 3' region of the ORF. Southern hybridization analysis of the chromosomal DNA revealed that multiple copies of the ORF sequence were contained in the PS3 genome. This element might well be a member of a new IS family including IS891 and IS1136, and we have designated this element IS1341. The analysis of acp expression in Escherichia coli cells indicated that IS1341 promotes the expression of acp.

N-acetylcysteine suppresses TNF-induced NF-kappaB activation through inhibition of IkappaB kinases.

Here, we used a reductant, N-acetyl-L-cysteine (NAC), to investigate the redox-sensitive step(s) in the signalling pathway from the tumor necrosis factor (TNF) receptor to nuclear factor kappaB (NF-kappaB). We found that NAC suppressed NF-kappaB activation triggered by TNF or by overexpression of either the TNF receptor-associated death domain protein, TNF receptor-associated factor 2, NF-kappaB-inducing kinase (NIK), or IkappaB kinases (IKKalpha and IKKbeta). NAC also suppressed the TNF-induced activation of IKKalpha and IKKbeta, phosphorylation and degradation of IkappaB, and nuclear translocation of NF-kappaB. Furthermore, NAC suppressed the activation of IKKalpha and IKKbeta triggered by the overexpression of NIK. These results indicate that IKKalpha and IKKbeta are subject to redox regulation in the cells, and that NAC inhibits NF-kappaB activation through the suppression of these kinases.

Cycloprodigiosin hydrocloride suppresses tumor necrosis factor (TNF) alpha-induced transcriptional activation by NF-kappaB.

Cycloprodigiosin hydrochloride (cPrG.HCl) obtained from a marine bacterium Pseudoalteromonas denitrificans induces apoptotic cell death in various cancerous cell lines. cPrG.HCl alone caused a little cytotoxicity in HeLa cells, but it enhanced the apoptotic process progressively when co-administered with tumor necrosis factor (TNF)alpha. Here we studied the effect of cPrG.HCl on TNFalpha-induced activation of the transcription factor nuclear factor kappaB (NF-kappaB). Luciferase gene reporter assays revealed that cPrG.HCl potently suppressed the TNFalpha- and the phorbol myristate acetate-induced activation of NF-kappaB. The suppression occurred in the presence of imidazole, indicating that it was not related to the intracellular acidification resulting from the intrinsic H(+)/Cl(-) symporter activity of cPrG.HCl. cPrG.HCl inhibited neither the TNFalpha-induced phosphorylation and degradation of inhibitor of nuclear factor-kappaB, nor the subsequent nuclear translocation and DNA binding of NF-kappaB. cPrG.HCl also suppressed NF-kappaB-enhanced gene expression induced by Rac1, Cdc42, MEKK1, inhibitor of nuclear factor-kappaalpha (IKKalpha), IKKbeta, and a subunit of NF-kappaB, p65. These results indicate that cPrG.HCl suppresses NF-kappaB-dependent gene expression through the inhibition of transcriptional activation.

Overexpression of the alanine carrier protein gene from thermophilic bacterium PS3 in Escherichia coli.

The alanine transporter (alanine carrier protein, ACP) gene of thermophilic bacterium PS3 was previously cloned and expressed in a functionally active form in Escherichia coli cells. To achieve controlled overproduction of the ACP protein, we designed a plasmid encoding a fusion protein comprising ACP joined to the carboxyl terminus of the maltose binding protein (MBP-ACP). Upon transduction of the plasmid into E. coli RM1 cells defective in alanine/glycine transport, the transport activity was expressed even before induction with 1-thio-beta-D-galacto-pyranoside (IPTG), and increased slightly on induction with IPTG at low concentrations. However, overexpression of the MBP-ACP gene, induced by higher concentrations of IPTG, resulted in death of the host cells. Hence we screened other host cells and found that the MBP-ACP fusion protein was produced in a large quantity in E. coli TB1 cells 3 h after IPTG induction. The MBP-ACP fusion protein was accumulated in cytoplasmic membranes in an amount reaching more than 20% of the total membrane protein. The affinity-purified MBP-ACP exhibited very low transport activity when reconstituted into proteoliposomes.

Partial purification and reconstitution of inositol 1,4,5-trisphosphate receptor/Ca2+ channel of bovine liver microsomes.

The binding of inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] to bovine liver microsomes was characterized. The Ins(1,4,5)P3 receptor of the microsomes was solubilized by 1% Triton X-100 and purified by sucrose density gradient, Heparin-Sepharose, DEAE-Toyopearl, ATP-Agarose, and Ins(1,4,5)P3-Sepharose column chromatographies. More than 1,000-fold enrichment of the Ins(1,4,5)P3-binding activity was achieved. Kd values of the binding activity were 2.8 nM in microsomes and 3.0 nM in the partially purified receptor, respectively, and the binding activity was optimal in the medium containing 100 mM KCl and at pH between 7.5 and 8.5. The presence of Ca2+ failed to inhibit the binding. Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PtdIns), and phosphatidylinositol-4-monophosphate [PtdIns(4)P] showed no effect on the Ins(1,4,5)P3 binding. However, soybean phospholipids asolectin and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] strongly inhibited the binding activity. PtdIns(4,5)P2 inhibited the activity competitively with a half-maximal inhibitory concentration of 30 micrograms/ml. The partially purified Ins(1,4,5)P3 receptor was reconstituted into proteoliposomes. Fluorescence measurements using Quin 2 indicated that Ins(1,4,5)P3 stimulated Ca2+ influx into the proteoliposomes. The EC50 of Ins(1,4,5)P3 on Ca2+ influx was 50 nM. This result strongly suggest that Ins(1,4,5)P3 binding protein of liver microsomes acts as a physiological Ins(1,4,5)P3 receptor/Ca2+ channel.

Expression in baculovirus vector system of the nucleocapsid protein gene of rinderpest virus.

The rinderpest (RV) nucleocapsid (NP) gene segment was inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) adjacent to the polyhedrin promoter. The expression of NP protein in Sf9 cells was confirmed by indirect immunofluorescence and by Western blotting analysis with monoclonal antibodies. Recombinant RV-NP protein was purified by ultracentrifugation on a sucrose density gradient, and used as an antigen for an enzyme linked immunosorbent assay to detect anti RV-NP antibody. Both IgM and IgG antibodies against RV-NP were detected in the sera of rabbits infected with the L strain of RV. The pattern of development of IgG anti RV-NP antibody closely correlated with that of virus neutralizing antibody. In rabbits inoculated with recombinant vaccinia virus expressing RV-H gene (RRV-H), anti RV-NP was not detected. The results indicated that the baculovirus vector system can be used for the preparation of the diagnostic antigen of rinderpest as well as to distinguish between natural infection and vaccination with RRV-H.

Nerve growth factor and forskolin prevent H2O2-induced apoptosis in PC12 cells by glutathione independent mechanism.

PC12 cells died by apoptosis at relatively low concentrations of H2O2, of which cytotoxicity was effectively suppressed by nerve growth factor (NGF), forskolin, and dbt-cAMP. Treatment with NGF or forskolin for 24 h increased the level of cellular antioxidant glutathione (GSH) by 1.6-2.0-fold. However, both NGF and forskolin protected cells against H2O2-stress even when cellular GSH was depleted by treatment with L-buthionine-(S,R)-sulfoximine (BSO). The GSH-independent protection effects of NGF and forskolin did not require new protein or RNA synthesis. Exogenous expression of an oncogenic ras suppressed apoptosis caused by H2O2 indicating that Ras protein also plays a role in suppressing apoptosis caused by oxidative radical stress.

Protective effects of serum thymic factor to Leptospira interrogans serovar Copenhageni infection in Mongolian gerbils.

The susceptibility to Leptospira interrogans serovar copenhageni in Mongolian gerbils treated with 10 micrograms of serum thymic factor (FTS) 1 day before infection was examined. Susceptibility of gerbils treated 5 times with 10 micrograms of FTS was also investigated. Mortality of FTS-treated gerbils was significantly lower than that of controls when small challenge doses were used. To analyse the FTS-induced resistance to leptospiral infection, natural killer (NK) cell activity and macrophage activity were studied. Macrophage activity was unaltered but NK cell activity was enhanced in FTS-treated gerbils, with or without leptospiral infection. Since no side-effects of FTS were observed, this compound should be considered for the treatment of leptospirosis.

Immunizing effect of vaccinia virus expressing the nucleoprotein of rinderpest virus on systemic rinderpest virus infection in rabbits.

A recombinant vaccinia virus (RVV) expressing the nucleoprotein (NP) of rinderpest virus (RPV) was examined in rabbits for the involvement of the NP protein in protection from the RPV infection. Despite their production of anti-NP antibody, the RVV-immunized rabbits succumbed to the RPV challenge, although there was a slight delay in the onset of disease after the low-dose challenge. On the other hand, the animals immunized with RVV expressing the hemagglutinin (H) protein of the RPV were completely protected. These results indicate that the NP protein might be not so effective as the H protein for the protection against viremic and systemic infection with RPV.

Analysis of c-myc DNA amplification in non-small cell lung carcinoma in comparison with small cell lung carcinoma using polymerase chain reaction.

Previous studies of c-mvc DNA amplification in lung cancer have focused primarily on analysis of small cell carcinoma or its tumor cell lines. There are few data about c-myc DNA amplification in histological types of lung cancer other than small cell carcinoma. Therefore the present study was conducted to investigate c-myc oncogene amplification in non-small cell lung carcinoma. We studied 46 lung tumor specimens for c-myc DNA amplification (15 adenocarcinomas, 15 squamous cell carcinomas, 6 large cell carcinomas, and 10 small cell carcinomas). Polymerase chain reaction, digoxigenin DNA labeling, and electrophoresis were utilized to investigate the c-myc copy number in the lung tumor specimens. The c-myc copy number of non-small cell carcinoma ranged from 1.5 to more than 20.0 in adenocarcinoma and squamous cell carcinoma, and from 6.0 to 12.0 in large cell carcinoma. That of small cell carcinoma ranged from 1.8 to 12.0. The c-myc copy number of non-small cell carcinoma was significantly higher than that of small cell carcinoma (Wilcoxon rank sum test, Z=2.06 P=0.040). However, the differences in c-myc copy number among these four histological types were not statistically significant. Amplification of c-myc (more than 4 copies) was observed not only in small cell carcinoma but also in nonsmall cell carcinoma at similarly high frequency (12/15 in adenocarcinoma and squamous cell carcinoma, 6/6 in large cell carcinoma, and 9/10 in small cell carcinoma).