Femtosecond charge and molecular dynamics of I-containing organic molecules induced by intense X-ray free-electron laser pulses.

We studied the electronic and nuclear dynamics of I-containing organic molecules induced by intense hard X-ray pulses at the XFEL facility SACLA in Japan. The interaction with the intense XFEL pulse causes absorption of multiple X-ray photons by the iodine atom, which results in the creation of many electronic vacancies (positive charges) via the sequential electronic relaxation in the iodine, followed by intramolecular charge redistribution. In a previous study we investigated the subsequent fragmentation by Coulomb explosion of the simplest I-substituted hydrocarbon, iodomethane (CH3I). We carried out three-dimensional momentum correlation measurements of the atomic ions created via Coulomb explosion of the molecule and found that a classical Coulomb explosion model including charge evolution (CCE-CE model), which accounts for the concerted dynamics of nuclear motion and charge creation/charge redistribution, reproduces well the observed momentum correlation maps of fragment ions emitted after XFEL irradiation. Then we extended the study to 5-iodouracil (C4H3IN2O2, 5-IU), which is a more complex molecule of biological relevance, and confirmed that, in both CH3I and 5-IU, the charge build-up takes about 10 fs, while the charge is redistributed among atoms within only a few fs. We also adopted a self-consistent charge density-functional based tight-binding (SCC-DFTB) method to treat the fragmentations of highly charged 5-IU ions created by XFEL pulses. Our SCC-DFTB modeling reproduces well the experimental and CCE-CE results. We have also investigated the influence of the nuclear dynamics on the charge redistribution (charge transfer) using nonadiabatic quantum-mechanical molecular dynamics (NAQMD) simulation. The time scale of the charge transfer from the iodine atomic site to the uracil ring induced by nuclear motion turned out to be only ∼5 fs, indicating that, besides the molecular Auger decay in which molecular orbitals delocalized over the iodine site and the uracil ring are involved, the nuclear dynamics also play a role for ultrafast charge redistribution. The present study illustrates that the CCE-CE model as well as the SCC-DFTB method can be used for reconstructing the positions of atoms in motion, in combination with the momentum correlation measurement of the atomic ions created via XFEL-induced Coulomb explosion of molecules.

Sinistral portal hypertension after pancreaticoduodenectomy with splenic vein ligation.

BACKGROUND: Splenic vein ligation may result in sinistral (left-sided) portal hypertension and gastrointestinal haemorrhage. The aim of this study was to analyse the pathogenesis of sinistral portal hypertension following splenic vein ligation in pancreaticoduodenectomy. METHODS: Patients who underwent pancreaticoduodenectomy for pancreatic cancer between January 2005 and December 2012 were included in this retrospective study. The venous flow pattern from the spleen and splenic hypertrophy were examined after surgery. RESULTS: Of 103 patients who underwent pancreaticoduodenectomy with portal vein resection, 43 had splenic vein ligation. There were two predominant venous flow patterns from the spleen. In the varicose route (27 patients), flow from the spleen passed to colonic varices and/or other varicose veins. In the non-varicose route, flow from the spleen passed through a splenocolonic collateral (14 patients) or a spontaneous splenorenal shunt (2 patients). The varicose route was associated with significantly greater splenic hypertrophy than the non-varicose route (median splenic hypertrophy ratio 1·52 versus 0·94; P < 0·001). All patients with the varicose route had colonic varices, and none had a right colic marginal vein at the hepatic flexure. CONCLUSION: Pancreaticoduodenectomy with splenic vein ligation may lead to sinistral portal hypertension. To avoid the development of varices, it is important to preserve the right colic marginal vein. Reconstruction of the splenic vein should be considered if the right colic marginal vein is divided.

Emerin deficiency at the nuclear membrane in patients with Emery-Dreifuss muscular dystrophy.

Mutations in the STA gene at the Xq28 locus have been found in patients with X-linked Emery-Dreifuss muscular dystrophy (EDMD). This gene encodes a hitherto unknown protein named 'emerin'. To elucidate the subcellular localization of emerin, we raised two antisera against synthetic peptide fragments predicted from emerin cDNA. Using both antisera, we found positive nuclear membrane staining in skeletal, cardiac and smooth muscles in the normal controls and in patients with neuromuscular diseases other than EDMD. In contrast, a deficiency in immunofluorescent staining of skeletal and cardiac muscle from EDMD patients was observed. A 34 kD protein is immunoreactive with the antisera--the protein is equivalent to that predicted for emerin. Together, our findings suggest the specific deficiency of emerin in the nuclear membrane of muscle cells in patients with EDMD.

Abnormal functional and morphological regulation of the gastric mucosa in histamine H2 receptor-deficient mice.

To clarify the physiological roles of histamine H2 receptor (H2R), we have generated histamine H2R-deficient mice by gene targeting. Homozygous mutant mice were viable and fertile without apparent abnormalities and, unexpectedly, showed normal basal gastric pH. However, the H2R-deficient mice exhibited a marked hypertrophy with enlarged folds in gastric mucosa and an elevated serum gastrin level. Immunohistochemical analysis revealed increased numbers of parietal and enterochromaffin-like (ECL) cells. Despite this hypertrophy, parietal cells in mutant mice were significantly smaller than in wild-type mice and contained enlarged secretory canaliculi with a lower density of microvilli and few typical tubulovesicles in the narrow cytoplasm. Induction of gastric acid secretion by histamine or gastrin was completely abolished in the mutant mice, but carbachol still induced acid secretion. The present study clearly demonstrates that H2R-mediated signal(s) are required for cellular homeostasis of the gastric mucosa and normally formed secretory membranes in parietal cells. Moreover, impaired acid secretion due to the absence of H2R could be overcome by the signals from cholinergic receptors.

Risk Factors for and Management of Postpancreatectomy Hepatic Steatosis.

BACKGROUND: Relatively little is known about the risk factors and treatments for postpancreatectomy hepatic steatosis. METHODS: The records of patients who underwent pancreaticoduodenectomy or total pancreatectomy between 2005 and 2010 and were followed up by periodic imaging were reviewed retrospectively. Risk factors and treatment for postpancreatectomy hepatic steatosis were analyzed. RESULTS: A total of 253 patients were included in the analysis, including 137 males and 116 females, of median (5, 95 percentile) age 67 (47, 81) years. Of these 253 patients, 75 (29.6%) developed postpancreatectomy hepatic steatosis. Multivariable logistic regression analysis showed that female gender ( p = 0.005; odds ratio: 2.387; 95% confidence interval: 1.293-4.386), body mass index > 22.5 kg/m2 ( p = 0.007; odds ratio: 2.330; 95% confidence interval: 1.261-4.307), operative duration > 540 min ( p = 0.018; odds ratio: 2.286; 95% confidence interval: 1.153-4.533), and delayed gastric emptying ( p < 0.001; odds ratio: 4.598; 95% confidence interval: 1.979-10.678) were independent risk factors associated with postpancreatectomy hepatic steatosis. Treatment consisted of maintenance- or high-dose digestive enzyme replacement therapy. Of patients without obvious tumor recurrence after 6 months, 12 of 15 treated with high dose and only 6 of 35 treated with maintenance-dose digestive enzyme replacement therapy showed improvements in postpancreatectomy hepatic steatosis ( p = 0.006). CONCLUSION: Female gender, obesity, longer operative time, and occurrence of delayed gastric emptying are risk factors for postpancreatectomy hepatic steatosis. High-dose digestive enzyme replacement therapy may improve postpancreatectomy hepatic steatosis.

[Intralobar pulmonary sequestration with high level of serum CEA; report of a case].

We reported a case of intralobar pulmonary sequestration with a high level of the serum CEA. A 53-year-old woman whose chief complaint was cough was admitted to our hospital. Enhanced chest computed tomography (CT) revealed the mass in the left lower lung, lymph-nodes swelling, and the aberrant artery. Magnetic resonance angiography (MRA) conformed the aberrant artery from the descending aorta. The level of serum CEA elevated at 9.6 ng/ml. Left lower lobectomy was performed. A diagnosis of intralobar pulmonary sequestration (Pryce type II) was established in this case. Histopathologically, the peribronchial epithelial cells in pulmonary sequestration showed weak positive for anti-CEA monoclonal antibody. Postoperative course was uneventful and the serum CEA level was 3.5 ng/ml in the normal range at the postoperative 17th day.

Immunoblot analysis of dystrophin-related protein (DRP).

Polyclonal antibodies against the carboxy-terminal portion of dystrophin-related protein (DRP), the putative autosomal gene product which shares sequence homology with dystrophin, show the clear expression of DRP in mouse fetal muscle and in cultured human muscle cells, but not in mature mouse or human muscle. DRP has the same molecular mass as X-linked dystrophin and is recovered from the membrane fraction, but is associated with membranes more loosely than dystrophin.

[Mucoepidermoid carcinoma with high level of serous carcinoembryonic antigen; report of a case].

We reported a case of mucoepidermoid carcinoma with a high level of the serum CEA. A 38-year-old woman was admitted because of abnormal chest shadow. Bronchoscopy revealed polypoid tumor occluding the lumen of right B3 bronchus. Bronchoscopic biopsy suggested a diagnosis of tubular adenocarcinoma. Chest computed tomography (CT) confirmed the mass in the right upper lung field and the swelling of right bronchial lymph node. The CEA level of serum elevated at 12.4 ng/ml. A right upper and middle lobectomy with mediastinal lymph nodes dissection was performed on August 26, 2003. Histopathologically, the polypoid tumor was a low grade mucoepidermoid carcinoma with partially extrabronchial extension. However, no lymph nodes metastasis were noted. The cytoplasms of about 45% of tumor cells showed positive for anti-CEA monoclonal antibody. Pathological stage was IB (T2N0M0). Seventeen months has passed with no evidence of recurrence and the CEA level of serum was in the normal range.

[Pulmonary pleomorphic carcinoma].

Pleomorphic carcinoma is a rare primary pulmonary malignancy. We report 2 surgical cases of pulmonary pleomorphic carcinoma. The first case was a 71-year-old male. Chest computed tomography (CT) showed a rapidly growing tumor with irregular density. Transbronchial lung biopsy revealed the tumor to be malignant. Left lower lobectomy was performed. Pathological diagnosis was pleomorphic carcinoma (pT2N2M0, stage IIIA). He died 8 months after surgery due to brain metastasis and mediastinal lymph node metastasis. The second case was a 74-year-old male who complained of bloody sputum. Chest CT showed a tumor with cavity in the right middle lobe. Brushing cytology under bronchofiberscopy revealed atypical cell. Right middle lobectomy and partial resection of the right lower lobe were performed. Pathological diagnosis was also pleomorphic carcinoma (pT2N0M0, stage IB). He has no findings of recurrence nor metastasis 15 months after the operation.

Immunohistochemical analysis of perforin and granzyme A in inflammatory myopathies.

Perforin (PF) and granzyme A (GA) are candidates suspected of being cytolytic proteins of the granules of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. We analysed PF and GA in muscles from patients with inflammatory myopathies. Five cases of polymyositis (PM), two cases of inclusion body myositis (IBM), and five cases of dermatomyositis (DM) were studied immunohistochemically using anti-PF and GA antibodies raised against each synthetic peptide of human PF and mouse GA, together with a panel of monoclonal antibodies reactive for lymphocyte subsets. In PM and IBM, PF positive cells were colocalized with GA positive cells and occasionally invaded into the non-necrotic muscle fibres. The percentage of PF positive cells among the endomysial CD8 positive cell population was 9.9% (PM) and 12.5% (IBM), and the majority of the endomysial CD8 positive cells were alpha/beta T cells. In contrast, in DM, both PF and GA positive cells were very few in all cases. Only few inflammatory cells were CD16+ or CD57+ NK cells among these diseases. Our results suggest that PF and GA are secreted mainly from alpha/beta T cells, and may play a key role in muscle fibre damage in at least some PM and IBM, but not in DM.

A severe muscular dystrophy patient with an internally deleted very short (110 kD) dystrophin: presence of the binding site for dystrophin-associated glycoprotein (DAG) may not be enough for physiological function of dystrophin.

We report a 4-yr and 5-month-old boy with severe clinical features of an early-onset Duchenne muscular dystrophy, who had a very short (110 kDa) dystrophin at the sarcolemma. The patient had a large deletion (exons 2-44) of the dystrophin gene which was predicted to cause a reading frame shift. Sequence analysis of dystrophin mRNA in muscle revealed an alternatively spliced gene product from exons 1 to 51 that caused restoration of the reading frame, in addition to an mRNA corresponding to the DNA deletion. A consistent result was obtained by immunocytochemical analysis of muscle; i.e. positive staining for dystrophin at the sarcolemma using antibodies against the C-terminus, cysteine-rich region and last three of 24 repeat units of the central rod-domain, but not for the remaining antibodies for dystrophin that recognize the N-terminal and proximal rod-domains. Immunostaining for dystrophin-associated glycoproteins (DAGs: 43 and 50 K) and merosin were preserved. Utrophin staining was positive but fainter than other DMD muscles. These results suggest that an extremely short dystrophin lacking the entire actin-binding site in the N-terminus cannot function properly even if the protein possesses the putative DAG-binding cysteine-rich and the C-terminal domains, and still has an ability to associate with sarcolemmal membrane.

Biologically active oligodeoxyribonucleotides. 5. 5'-End-substituted d(TGGGAG) possesses anti-human immunodeficiency virus type 1 activity by forming a G-quadruplex structure.

A series of hexadeoxyribonucleotides (6-mers), d(TGGGAG), substituted with a variety of aromatic groups at the 5'-end were synthesized and tested for anti-human immunodeficiency virus type 1 (HIV-1) activity. While unmodified d(TGGGAG) (31) had no anti-HIV-1 activity, compound 23 with a 3,4-di(benzyloxy)benzyl (DBB) group at the 5'-end potently inhibited the HIV-1IIIB-induced cytopathicity of MT-4 cells in vitro (IC50 = 0.37 microM) without cytotoxicity up to 40 microM. A thermal denaturation study on the 5'-end-substituted 6-mers by means of the circular dichroism (CD) spectra demonstrated that the aromatic substituent attached at the 5'-end of the 6-mer strongly enhanced the formation of a parallel helical structure consisting of four strands (quadruplex). On the contrary, compound 36, in which one of the guanosines of 23 was replaced by a thymidine, did not form a quadruplex, thus exhibiting no anti-HIV-1 activity. Moreover, both compound 15, with a tert-butyldiphenylsilyl group solely at its 3'-end, and compound 21, with a relatively small substituent, a benzyl group, at the 5'-end, formed quadruplexes but had no anti-HIV-1 activity. These findings led us to the conclusion that both the quadruplex structure and the aromatic substituent with adequate size at the 5'-end are crucial for the interaction of the 5'-end-substituted 6-mers with the V3 loop as well as the CD4 binding site on viral gp120, resulting in anti-HIV-1 activity.

Protection of hu-PBL-SCID/beige mice from HIV-1 infection by a 6-mer modified oligonucleotide, R-95288.

We analyzed the anti-HIV-1 activity of an oligonucleotide derivative, R-95288, in severe combined immunodeficient (SCID/beige) mice transplanted with normal human peripheral blood leukocytes (PBLs), designated hu-PBL-SCID/beige mice. The human chimeric mice were inoculated with HIV-1(CC1) 3 weeks after the transplantation and sacrificed 2 weeks later. Virus infection was determined by coculture of splenocytes with fresh human PBLs and also by detection of HIV- specific DNA sequences using the polymerase chain reaction. No evidence of infection was observed in mice treated with R-95288 (100 mg/kg/day) using intraperitoneal delivery by osmotic minipumps starting 1 day before virus challenge. In contrast, virus infection was observed in over 80% of the saline-treated control mice. In addition, partial inhibition of HIV-1 infection was obtained in mice treated subcutaneously with R-95288 (100 mg/kg/day). Toxicity towards the engrafted human cells was not observed by flow cytometric analysis. Moreover, R-95288 failed to inhibit lymphocyte proliferation (CC50 > 400 microg/ml), while 90% inhibition of HIV-1 replication was achieved at 3.1 microg/ml in vitro. These results suggest the ability of R-95288 to protect the human chimeric mice against HIV-1 infection.

Preparation of protein components exhibiting myosin light chain kinase activities from bovine aorta: discrepancies between its enzyme activity and actomyosin activating effect.

1) Two protein components, 155 and 130 kDa in their electrophoretic molecular weights, respectively, were isolated in a homogeneous state from bovine aorta; they showed both the superprecipitation-inducing effect on desensitized natural actomyosin and the myosin light chain kinase (MLCK) action on gizzard myosin. 2) The superprecipitating activity of the 155 kDa component was 5 time higher than that of the 130 kDa component on the basis of equivalent MLCK activity. 3) The same procedure was applied to bovine stomach, giving rise to a 155 kDa component in a homogeneous state as in the case of aorta, but the 130 kDa component thus prepared was contaminated by higher molecular weight components. 4) If compared on the basis of equivalent MLCK activity, bovine stomach 155 kDa component showed more than 10 times higher superprecipitating activity than the fraction that contained the 130 kDa component as the main constituent. 5) The discrepancy between the superprecipitating activity and MLCK activity mentioned above was discussed in relation to the Ca2+ regulation mechanism in smooth muscle contraction. The possibility that the 130 kDa component might be a proteolytic product of the 155 kDa component was also discussed.

Antibody against the C-terminal portion of dystrophin crossreacts with the 400 kDa protein in the pia mater of dystrophin-deficient mdx mouse brain.

The mdx mouse is an animal model for X-linked Duchenne muscular dystrophy. A polyclonal antibody against a synthetic peptide IV equivalent to the C-terminal portion (amino acids 3495-3544) of dystrophin crossreacted with a 400 kDa protein in the brain and the spinal cord of mdx mouse, as well as in the control B10 mouse. However, the protein did not crossreact with the polyclonal antibody raised against the N-terminal portion of dystrophin peptide I (amino acids 215-264). Immunofluorescent micrography revealed that the outside of the small arteries and the pia mater of the brain strongly reacted with the anti-peptide IV antibody. These results strongly suggest the presence of a crossreactive protein other than dystrophin, possibly a dystrophin-related autosomal gene product, in the pia mater.

Association between blood pressure and insulin resistance in obese females during weight loss and weight rebound phenomenon.

The purpose of this study was to investigate the effect of weight loss on blood pressure and its related variables in moderately obese Japanese females, including an investigation of the rebound phenomenon. Study I examined the effects of weight loss on blood pressure in 138 moderately obese, nondiabetic females (BMI 29.3+/-0.3 kg/M2; age, 46.3+/-0.8 years) during a 3-month therapeutic dietary and exercise program. Study II investigated the effect of weight rebound on blood pressure over an additional 21 months of exercise in 48 subjects from Study I subjects. After 3 months, the BMI significantly decreased to 27.9+/-0.3 kg/m2. Abdominal total fat, visceral fat (V), and subcutaneous fat (S) also decreased significantly. In addition, the summation of insulin (sigmaIRI), plasma glucose (sigmaPG) and HOMA during 75 g oral glucose tolerance test also all significantly decreased. Significant decreases in both the SBP and DBP were observed after the 3 month weight reduction program. Multiple regression analysis revealed that the reduction in SBP was significantly and positively associated with the reduction in log sigmaIRI and the reduction in log 24h-urinary norepinephrine excretion at the end of Study I. The DBP showed a significantly positive association with the log sigmaIRI. With regard to the weight rebound phenomenon, Study II showed that the SBP, DBP and sigmaIRI all increased significantly, and a positive correlation was observed between the changes in the SBP and those in the log sigmaIRI. However, no such correlation was observed regarding the abdominal total fat and visceral fat during both periods. These results suggest that weight loss therefore caused the BP to decrease due to both an improvement in hyperinsulinemia and a decrease in the adrenergic activity which may be involved in the urinary catecholamine. As a result, hyperinsulinemia is thus considered to play an important role in the pathogenesis of blood pressure due to obesity not only during weight loss, but also during the weight rebound phenomenon.

Abnormal localization of laminin subunits in muscular dystrophies.

To address potential involvement of muscle basal lamina and membrane cytoskeleton proteins in the etiology of non-dystrophinopathy muscular dystrophies, we examined the immunostaining intensity and distribution of laminin subunits (A, B1, B2 and M), type IV collagen, dystrophin and spectrin in skeletal muscle biopsies from 64 myopathic patients (17 Fukuyama congenital muscular dystrophy: FCMD, 13 congenital muscular dystrophy unrelated to FCMD: other CMD, 16 Duchenne muscular dystrophy: DMD, and 18 other neuromuscular diseases. In FCMD muscle, we found a significant reduction of laminin M (merosin; a striated muscle specific basal lamina-associated protein) with approximately 26% of levels seen in controls by quantitative immunofluorescence. Other CMD and DMD muscles showed less dramatic reductions (78%, 80%, respectively). The localization of laminin M was also abnormal in FCMD muscle. Laminin B1 and B2 showed abnormalities similar to those observed with laminin M, but were less marked. Laminin A was only detected in rare regenerating fibers in control biopsies, whereas it was seen around most muscle fibers in FCMD patients, and in dystrophin deficient muscle fibers from DMD patients and its carrier. Staining intensity of type IV collagen in FCMD muscle was not significantly different from the other diseases. These findings may implicate a primary or central role for the basal lamina in FCMD muscle.

[Dystrophin-related protein in diaphragm, limb and myoblast transferred muscles of mdx mouse].

Expression of the dystrophin-related protein (DRP or Utrophin) was examined with Western blot and immunohistochemical methods in diaphragm, limb and also in myoblast transferred muscles of the mdx mouse. Although we have hypothesized that progressive fibrosis in the diaphragm of the mdx mouse has been due to a smaller amount of DRP expression compared with limb muscles, we could not find any difference in the amount of DRP or in the DRP localization pattern between the two muscle sites. In limb muscles treated with myoblast transfer, dystrophin-positive muscle fibers had no DRP on their surface membrane, although dystrophin-negative muscle fibers were DRP-positive. These findings suggest that excessive expression of DRP is suppressed in the normalized muscle fiber with dystrophin. It also appears that the histological differences seen in the different muscles of the mdx mouse are not due to the amount of DRP present.

[Chronological change of EEG findings in a case of pyridoxine dependency seizures].

A patient of pyridoxine dependent seizures was reported. He was born at 34 weeks' gestation and weighted 2,760 g. Apgar scores were 6 and 9 at 1 and 5 minutes, respectively. He showed the first seizure 2 hours after his birth. Phenobarbital, phenytoin, sodium valproate, diazepam and clonazepam were not effective. Pyridoxal phosphate (50 mg) was given intravenously, resulting in suppression of convulsions. However, muscle tonus was severely depressed. In EEG, a discontinuous pattern was found in quiet and indeterminate sleep on the 2nd day of life. At 5th week multifocal spikes were found, and the discontinuous pattern persisted. Ictal discharges at 13th week showed generalized, continuous, irregular and high voltage slow waves with multifocal spikes. At 27th week of life, high voltage slow waves disappeared and multifocal spike discharges decreased. At 2 years and 10 months of age, the patient was suffering from athetotic cerebral palsy and severe mental retardation. Pyridoxal phosphate at the doses of 35-40 mg/kg/day had been administered. Irritability sometimes occurred and additional 50 mg of pyridoxal phosphate controlled this irritability effectively.

[Chronic hemorrhagic pyothorax treated with preoperative internal thoracic and intercostal arterial embolization and perioperative non-invasive positive pressure ventilation].

We reported successful surgery for chronic hemorrhagic empyema with severe right heart insufficiency. The preoperative embolization of right internal thoracic artery and intercostals arteries was effective for the control of intraoperative bleeding. Non-invasive positive pressure ventilation (NIPPV) was useful for the perioperative respiratory management. A 62-year-old female with a history of right pneumonectomy and thoracoplasty for pulmonary tuberculosis was admitted because of dyspnea on effort on Dec 5th 2002. Her right heart insufficiency was worsened gradually. On May 20th 2003, we performed the transcatheter embolization of right internal thoracic and intercostals arteries for the control of intraoperative bleeding. The next day, the curettage and fenestration was performed for intraoperative cardiac dysfunction. The intraoperative bleeding was 1,596 ml and operative time was 2 hours 24 minutes. Due to CO2 narcosis, the ventilator under the intratracheal tube was needed for respiratory management in the postoperative course. The switching of the respiratory management with NIPPV from the intratracheal tube during 8 days, her respiratory and general conditions had been improved gradually. Because of methicillin-resistant Staphylococcus aureus (MRSA) infection of thoracic cavity, the radical thoracoplasty following the latissimus dorsi muscules flap and the omentopexy was performed. The operative course was uneventful and she needed overnight NIPPV without O2 inhalation and was discharged.