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A man in his early 60 s who worked at a waste disposal plant had fallen into the refuse pit and was immediately taken to the emergency department for treatment. After 8 days without recovering consciousness, the man died. Antemortem contrast-enhanced computed tomography at the emergency department indicated Stanford type B/DeBakey type IIIb aortic dissection. The autopsy showed a sharp and transverse intimal tear 0.6 cm in length in the aortic isthmus and fractures in the 5th-6th thoracic vertebrae. No structural abnormalities in arterial walls were noted on histopathological examination. The traumatic aortic dissection induced by falling is rare, compared with vehicle crash. Although the verification process was challenging, the cause of death was ultimately concluded as traumatic aortic dissection due to falling into the refuse pit. The following observations were cited as evidence: (1) the location and feature of the intimal tear, (2) the positional relationship between the impact site and the entry tear, and (3) the circumstance of clash impact onto the "cushion" of accumulated waste in the refuse pit. Inquiries into the cause of death, such as those made in this report, are required to provide detailed information on the circumstances of the accident, postmortem examinations, and careful consideration.

Thiamylal is an ultrashort-acting barbiturate used for intravenous administration or general anesthesia induction. However, some cases of poisoning and suicide with thiamylal administration have been reported. Additionally, there are few reports on its analysis in the organs and adipose tissue, which requires purification by column chromatography and evaporation. A rapid and sensitive method was developed for quantifying thiamylal and its metabolite, secobarbital, in the adipose tissue, serum, and liver using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Samples were prepared using modified QuEChERS extraction. For adipose tissue samples, an acetonitrile-hexane partitioning step was added to the extraction. This method was applied to investigate a suspected self-poisoning autopsy case. The quantitation accuracy for thiamylal added to porcine pericardial fat (0.18 µg/g), human serum (0.015 µg/mL), and porcine liver (0.18 µg/g) was 103%, 113%, and 95.3%, respectively. The quantitation limits calculated for porcine pericardial fat, human serum, and porcine liver at a signal-to-noise ratio of 10 were 0.06 µg/g, 0.005 µg/mL, and 0.06 µg/g, respectively. In addition, the thiamylal and secobarbital levels in the forensic autopsy case were 140 and 1.5 µg/g, respectively, in myocardial fat; 3.5-4.9 and 0.12-0.20 µg/mL, respectively, in serum; and 6.2-42 and 0.58-1.1 µg/g, respectively, in liver tissue. Thiamylal is especially distributed in the adipose tissue. The thiamylal-to-fat ratio may help estimate the time from administration to death. The developed modified QuEChERS extraction method with acetonitrile-hexane partitioning is suitable for analyzing hydrophobic compounds, such as thiamylal, in the adipose tissue.

CD31, a transmembrane protein expressed on endothelial and hematopoietic cells, plays important roles in leukocyte trafficking, mechanotransduction, angiogenesis, vascular permeability, and regulation of cellular responsiveness. CD31 immunoreactivity is employed as a sensitive and specific endothelial marker in diagnostic pathology. In this study, CD31 expression in myocardial tissues from deceased patients with ischemic heart disease and a mouse model of acute myocardial infarction were examined by immunohistochemical staining. We examined 24 neutral formalin-fixed, paraffin-embedded myocardial tissue samples obtained within 48 h postmortem from the autopsies of patients who were diagnosed with ischemic heart disease. CD31 expression was observed in vascular endothelial and endocardial cells. In necrotic myocardium, diffusion of CD31 antigen was observed. Elevated CD31 expression was observed around myocardial cells undergoing remodeling, suggesting that endothelial proliferation occurred at these sites. In contrast, fibrotic myocardial foci did not show upregulated CD31 expression. The same CD31 expression characteristics as those observed in the human samples were observed in the mouse model. CD31 immunostaining as an endothelial and microvasculature marker may be a useful complement to conventional staining techniques currently used in the diagnosis of ischemic heart disease, and may allow the timing and process of myocardial remodeling to be analyzed in detail.

The deceased was a 44-year-old male who was treated for a suspected Ebstein's anomaly observed using transthoracic echocardiogram. He was found dead in his bed at home. Autopsy revealed that the septal tricuspid leaflet was intact; however, a large anterior tricuspid leaflet cleft and right atrioventricular cavity dilation were observed. Pathological examination revealed a normal tricuspid valve, except for the presence of a cleft with local fibrosis of the left ventricle papillary muscle and hemosiderin-containing macrophages at both lungs. There were no other abnormalities that may have led to death. It was concluded that he died a cardiac death based on the right heart overload associated with the anterior tricuspid leaflet cleft. This case indicates the possibility that the anterior tricuspid leaflet cleft can cause death and also highlights the necessity of a detailed autopsy to accurately diagnose the cause of death.

The plant species Gloriosa superba and Colchicum autumnale produce extremely poisonous colchicine as a major toxic metabolite. Almost all previous studies on colchicine poisoning have focused on drug analysis and clinical and pathological aspects. In this study, we developed a rapid, highly sensitive method to identify G. superba and C. autumnale. This method, which can distinguish between G. superba and C. autumnale using even minute amounts of plant material, is based on duplex real-time PCR in combination with melting curve analysis. To discriminate between the two genera of colchicine-containing plants, we designed new primer pairs targeting the region of the ycf15 gene, which is present in C. autumnale but not G. superba. By producing PCR amplicons with easily distinguishable melting temperatures, we were able to rapidly and accurately distinguish G. superba from C. autumnale. The new primer pairs generated no PCR amplicons from commercially available human DNA or various plant DNAs except for G. superba and C. autumnale. Sensitivity testing indicated that this assay can accurately detect less than 0.031 ng of DNA. Using our method in conjunction with colchicine drug analysis, we successfully identified G. superba in the stomach contents of a suicide victim who ingested massive quantities of a colchicine-containing plant. According to these results, duplex real-time PCR analysis is very appropriate for testing forensic samples, such as stomach contents harboring a variety of vegetables, and enables discrimination between G. superba and C. autumnale in forensic and emergency medical fields.

An allergic anaphylactic reaction is a potentially fatal cascade consisting of an initial sensitization, antigen exposure, crosslinking of immunoglobulin E (IgE) specific for the antigen, activation of mast cells, and release of bioactive substances. Postmortem diagnosis of anaphylaxis is challenging because of the limited availability of antemortem history and minimal macroscopic evidence at autopsy. The elevated activity of a neutral proteinase, such as tryptase, can be a surrogate marker for the activation and degranulation of mast cells. However, it does not directly indicate the involvement of antigen-specific IgE, which has an important role in IgE-mediated allergic anaphylaxis. In the present study, we examined blood from a case of death following infusion of the antibiotic ceftriaxone (CTRX), with a control case for comparison. The aim was to detect IgE specific for CTRX. A pull-down assay with N-hydroxysuccinimide-activated sepharose identified IgE specific for CTRX only in the serum obtained from the case of CTRX exposure, and not in the control case. The specificity of IgE was confirmed by adsorption to an excess of CTRX, which resulted in the signal for IgE disappearing in the pull-down assay. This antigen-specific IgE is a key molecule in the IgE-mediated allergic anaphylaxis and seldom investigated in postmortem examinations. Its detection can provide support for the postmortem diagnosis of allergic anaphylaxis, especially when combined with an antemortem history of allergen exposure and elevated neutral proteinase levels in serum.

We report an autopsy case of hemopericardium caused by rupture of a ventricular aneurysm associated with acute myocarditis in an infant boy aged 2years and 10months. Three days before his death, the patient developed fever. On the day of death, he described an urge to defecate and attempted to do so in an upright position. While straining to defecate without success for a prolonged period, he stopped breathing and collapsed. On autopsy, his heart weighed 91.7g and cardiac tamponade was evident, the pericardial cavity being filled with 140mL of blood that had come from a 1.5-cm-long rupture in a 2.7×1.5cm ventricular aneurysm in the posterior left ventricular wall. Patchy grayish-white discoloration was noted in the myocardium. Histologically, CD3-positive T lymphocytic infiltration accompanied by pronounced macrophage infiltration was observed in the myocardium. Hemorrhagic necrosis was detected in the area of the ventricular aneurysm. Staining for matrix metalloproteinase (MMP) expression revealed abundant MMP-2, MMP-7, and MMP-9. Polymerase chain reaction to detect viruses failed to identify any specific causative viruses in the myocardium. In this case of lymphocytic (viral) and histiocytic myocarditis with pronounced macrophage infiltration and upregulation of MMP expression, myocardial remodeling and associated wall weakening had resulted in formation and rupture of an aneurysm.

In forensic toxicology studies, drug concentrations must be estimated by the analytical data of formalin-fixed tissues if fresh or frozen tissue specimens are not available. We wished to investigate the stability and time-course of metabolism/degradation of drugs in formalin-fixed tissues using porcine liver homogenates (PLHs) instead of human tissue. Ten psychotropic drugs (amitriptyline, brotizolam, diazepam, diphenhydramine, estazolam, etizolam, levomepromazine, paroxetine, quetiapine and triazolam) were added to PLHs. After the PLHs had been fixed with neutral buffered formalin at room temperature, the concentrations of the drugs in the PLHs were determined by liquid chromatography-tandem mass spectrometry after 3 days, 1 week, 2 weeks, 4 weeks, 2 months, 4 months and 6 months. After 6 months, the residual ratio of amitriptyline, diphenhydramine and quetiapine was 80 %-95 %; that of diazepam, paroxetine and triazolam was 10 %-45 %; and that of brotizolam, etizolam and levomepromazine was 1 %-5 %. Estazolam was not detected from the first day of formalin fixation. These data suggest that the concentrations of drugs in PLHs measured after formalin fixation decreased to varying degrees compared with their initial concentrations. These time-dependent changes in drug concentration were due to degradation during preservation in formalin solution and metabolism by hepatic microsomal enzymes.

A Japanese man in his 30s who had congenital cerebral palsy was found unresponsive in bed. His death was confirmed after resuscitation attempts. He had a history of occasional falling (despite the use of walking sticks and a wheelchair) owing to a slowly progressive gait disturbance, and had a medical examination without full neurological re-examination. Autopsy revealed gangliocytoma in the medulla oblongata, which was diagnosed as the cause of death. Although gangliocytoma is a well-differentiated benign tumor, the almost total replacement of the medulla oblongata by the tumor cells was assumed to result in ataxia via the olivocerebellar tract and secondary cerebellar atrophy, followed by central hypoventilation and death of the patient. The symptoms caused by gangliocytoma may be overlooked owing to long-standing cerebral palsy.

A man in his thirties was found dead in bed. He had undergone repair of a ventricular septal defect in his infancy and had a 2-month history of antemortem chest pain. On autopsy, a ruptured saccular aneurysm of the ascending aorta was identified, and the right thoracic cavity was found to contain coagulated blood. The oval ostium of the aneurysm was smoothly endothelialized and a black suture was found near its edge. Histological analysis revealed a defect of the intimal and medial layers in the wall of the aneurysm and hypertensive changes in several organs. The cause of death was presumed to be the rupture of a pseudoaneurysm at the aortic cannulation site after a long postoperative period. In the present case, preexisting hypertension was suspected as the cause of the formation and rupture of the pseudoaneurysm. In case of unexpected death, it is important to examine the past medical history even after a long postoperative period.

Chain collapse and secondary structure formation are frequently observed during the early stages of protein folding. Is the chain collapse brought about by interactions between secondary structure units or is it due to polymer behavior in a poor solvent (coil-globule transition)? To answer this question, we measured small-angle X-ray scattering for a series of β-lactoglobulin mutants under conditions in which they assume a partially folded state analogous to the folding intermediates. Mutants that were designed to disrupt the secondary structure units showed the gyration radii similar to that of the wild type protein, indicating that chain collapse is due to coil-globule transitions. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 651–658, 2014.

A 21-day-old Japanese male infant became inactive and then collapsed unexpectedly. On autopsy, there was no connection between the ascending and descending thoracic aorta. The site of interruption was the aortic isthmus. The heart showed hypertrophy and exhibited intracardiac malformations, including subaortic valve stenosis resulting from posterior deviation of the ventricular outlet septum and ventricular septal defect. The cause of death was diagnosed as prolonged physiological closure of the ductus arteriosus complicated by interrupted aortic arch and followed by assumed ductal shock.

Coronary artery aneurysm is a fairly uncommon clinical entity, which is defined by a characteristic dilatation that exceeds 1.5 times the width of normal adjacent coronary artery segments. In the present report, we describe a case of rupture of a massive coronary artery aneurysm. A man in his 40 s was found dead in his bed. The pericardial cavity contained 270 mL of blood with 428.2 g of coagulation. Two true aneurysms of the right coronary artery were identified. A proximal aneurysm, adjacent to the right auricle, had ruptured on the right. A distal unruptured aneurysm was identified 5.1 cm distal to the proximal ruptured aneurysm. Atherosclerosis of the coronary arteries and aorta was severe. The heart weighed 799.1 g and showed concentric ventricular hypertrophy, myocardial thinning, and patchy fibrosis. Histological analysis showed that both aneurysms were purely atherosclerotic true aneurysms without considerable inflammation. The cause of death was determined as cardiac tamponade due to rupture of a giant coronary atherosclerotic aneurysm.

Environmental factors such as outside temperature at the time of death are very important for forensic diagnoses and police investigations. In particular, death in a cold environment is associated with factors of forensic interest, including hypothermia, drowning in cold water, or postmortem body movement by a suspect. Hypothermia raises a special problem because of the difficulty of evaluation during autopsy. We describe here a unique method of estimating antemortem environmental temperature, involving the immunohistochemical analysis of HSP70 expression patterns in glomerular podocytes. Using this method, we found that HSP70 was present in glomerular podocytes at autopsy and that HSP70 was highly expressed, mainly in the nucleus of podocytes, in deaths associated with exposure to cold. Interestingly, this expression pattern was specific to death in a cold environment, including hypothermia and drowning in cold water. Analysis of the pattern of HSP70 expression in glomeruli may therefore be very useful in forensic diagnosis, for determining whether the antemortem environmental temperature was low. Moreover, immunohistochemical and real-time PCR assays of the molecular mechanism of HSP70 and HSF1 expression in glomeruli following exposure to cold indicated that HSP70 was rapidly translocated to the nucleus of podocytes following exposure to cold, but without new protein synthesis.

We proposed a pedestrian movement support system using visible-light communication (VLC) with light-emitting diodes (LEDs) for the people of weak eyesight and the elderly. It provides the user safety information in the neighborhood or the route information, and receives a request from the user and traffic information by the LED light. We conducted visibility evaluation experiments with visually impaired people to satisfy the quality of experience for flickerfree. We created three-types of hands-free receiver (neck, arm, and white cane) of the navigation system to inform danger condition and/or obstacles such as steps and signals. The prototype was evaluated by visually impaired users, clarifying the effectiveness of the system.

This chapter analyzes the role of women’s self-help networks in anti-caste discrimination movements in Nepal. Focusing on the women of the Khadgi caste, who have historically been engaged in slaughtering, processing, and trading livestock (their caste-ordained role) and suffered from discrimination as “low caste,” I will describe shifts in strategies in their networking process during anti-caste discrimination movements of the 1950s, 1990s, and 2000s.

Equine β-lactoglobulin is a 162-residue β-sheet protein. A partially folded form of equine β-lactoglobulin contains a β-hairpin and an α-helix. The β-hairpin converts into non-native α-helices at temperatures0°C. CHIBL, a truncated equine β-lactoglobulin (residues 88-142), contains the low-temperature α-helical structure even at room temperature, indicating that the interactions responsible for the stability of the β-hairpin reside in non-CHIBL residues. For the study reported herein, we characterized two truncated mutants and their leucine103 → proline103 variants to identify residues that stabilize the β-hairpin. The dependence of their circular dichroism spectra on chloride ion concentration and temperature revealed that the ability to transition from the non-native α-helices to the β-hairpin depends on the polypeptide chain length and improves as the chain length increases despite the apparent absence of any ordered structure in the extended sequences.

Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells.

Background and Aims: Enteroscopic observation has clearly demonstrated that non-steroidal anti-inflammatory drugs/low-dose aspirin (usually enteric-coated) induces hemorrhagic lesions, including ulcers and bleeding, in the small intestine of patients at a high incidence. Such intestinal lesions induced by NSAIDs have been confirmed in animal experiments. With aspirin, however, it has long been believed that it is difficult to induce any damage in the intestinal mucosa of laboratory animals. Therefore, we established a new method of inducing intestinal hemorrhagic lesions in rats by injecting aspirin into the proximal duodenum. Methods: Under ether anesthesia, aspirin (50–200 mg/body), suspended in 2% methylcellulose (with or without 0.1 N HCl), was injected into the proximal duodenum of normally fed or 20-h non-fed rats (male Sprague–Dawley, 9 weeks old). At 1 h after treatment, the animals were killed with ether and the entire small intestine was removed for histological examination. In some experiments, 1% Evans blue was injected (i.v.) into the rats 1 h after aspirin treatment to visualize the lesions. An image analyzer determined the total area of the intestinal lesions. Oral proton pump inhibitors and histamine H2-receptor blockers were given 1 h before aspirin injection. 16,16-dimethyl prostaglandin E2 (dmPGE2) was given s.c. 30 min before aspirin injection. Results: Aspirin alone clearly induced severe lesions (including bleeding and ulcers) mainly in the jejunum at 100% incidence. Total score of lesions per rat obtained by histological examination was similar to the damaged area quantified with the dye method. Dose-related induction of lesions by aspirin was confirmed both by the histological and dye methods. The irritable effect of aspirin suspended in 0.1 N HCl solution was the same as that of aspirin alone; 0.1 N HCl alone induced only minor lesions in the intestine. Both proton pump inhibitors and histamine H2-receptor blockers, at doses that inhibit gastric acid secretion, had no or little effect on aspirin-induced intestinal lesions. Pretreatment with dmPGE2 (3, 10, 30 µg/kg) showed significant prevention of both aspirin- and HCl/aspirin-induced intestinal lesions. Conclusion: This new aspirin lesion model will be useful for screening defensive drugs against aspirin-induced intestinal lesions and to elucidate the underlying mechanism.

Cadmium (Cd) is a serious toxic metal, which is classified as a possible human carcinogen. We assessed the effects of Cd on the expression levels of homeobox genes, which are associated with carcinogenesis. Among 6 homeobox genes examined in this study, only HOXB8 exhibited increased mRNA expression in COS-7 cells treated with 10 microM CdCl(2). Semiquantitative reverse transcription-polymerase chain reaction analysis revealed that the HOXB8 mRNA level was increased by a maximum of 5.4-fold after 6h of Cd exposure. The levels of HOXA7, A9, C4, C9 and C10 mRNAs decreased from 0.1 to 0.3-fold. Silencing of HOXB8 mRNA expression using a siRNA increased HOXC9 and C10 mRNA expression levels by 6.6- and 1.9-fold, respectively. These results suggest that HOXB8 upregulation is associated with suppression of HOXC9 and C10, and that decreased expression of HOXC9 and C10 after Cd exposure is partly due to HOXB8 induction. In conclusion, Cd disrupts the HOX network. Comprehensive analyses of all the HOX gene expression levels in the presence of Cd may afford clues toward understanding Cd-induced carcinogenesis and teratogenesis.

The chloride-ion concentration dependence of the molecular dimension in the acid-denatured state of equine β-lactoglobulin (ELG) was investigated by small-angle X-ray scattering. In the presence of chloride ion, ELG has a globular and compact conformation (the A state). The molecular dimension of ELG increases little with decreasing chloride-ion concentration. A remarkable dependence was observed for a mutant protein in which both Cys66 and Cys160 were replaced with Ala (C66A/C160A). In the presence of chloride ion, C66A/C160A has a globular and compact conformation, like the wild type. In the absence of chloride ion, however, the molecular dimension and shape was close to that in the urea-unfolded state. Previously, we have shown that the helix content in the acid-denatured state increases with decreasing chloride-ion concentration [Yamada et al. (2006). Proteins Struct. Funct. Bioinf. 63, 595–602]. These results suggest that the secondary structure in the A state is mainly determined by non-local interactions. When they are absent in an expanded conformation, the local interactions become predominant and the amount of non-native α-helix increases.

Islet transplantation is a prospective treatment for restoring normoglycemia in patients with type 1 diabetes. Islet isolation from pancreases by decomposition with proteolytic enzymes is necessary for transplantation. Two collagenases, collagenase class I (ColG) and collagenase class II (ColH), from Clostridium histolyticum have been used for islet isolation. Neutral proteases have been added to the collagenases for human islet isolation. A neutral protease from C. histolyticum (NP) and thermolysin from Bacillus thermoproteolyicus has been used for the purpose. Thermolysin is an extensively studied enzyme, but NP is not well known. We therefore cloned the gene encoding NP and constructed a Bacillus subtilis overexpression strain. The expressed enzyme was purified, and its substrate specificity was examined. We observed that the substrate specificity of NP was higher than that of thermolysin, and that the protein digestion activities of NP, as determined by colorimetric methods, were lower than those of thermolysin. It seems that decomposition using NP does not negatively affect islets during islet preparation from pancreases. Furthermore, we designed a novel substrate that allows the measurement of NP activity specifically in the enzyme mixture for islet preparation and the culture broth of C. histolyticum. The activity of NP can also be monitored during islet isolation. We hope the purified enzyme and this specific substrate contribute to the optimization of islet isolation from pancreases and that it leads to the success of islet transplantation and the improvement of the quality of life (QOL) for diabetic patients.

Background The pathogenesis of ischemia–reperfusion involves generation of reactive oxygen and resulting lipid peroxidation. However, investigation that ischemia–reperfusion following tourniquet release enhances lipid peroxidation is insufficient. Methods Tourniquet was applied to a unilateral hind limb of mice for 3 h followed by 5-, 15-, 30- and 60-min release. To examine superoxide production immunohistochemically in ischemia–reperfusion muscles, a primary antibody directed to 4-hydroxy-nonenal (HNE) was used. Furthermore, we analyzed 7α- and 7β-hydroperoxycholest-5-en-3β-ol, 7α- and 7β-hydroxycholesterol, and 7-ketocholesterol by HPLC in the gastrocnemius muscles, kidneys, liver, heart and lungs of mice after 1-h reperfusion. Results Increased HNE immunoreactivitiy was observed in the tourniquet-applied side of gastrocnemius muscles of hind limb particularly after 5-min reperfusion. All the oxysterols were significantly higher in the gastrocnemius muscles of the tourniquet-applied side than of the contralateral muscles. Oxysterols were elevated in the kidneys and the liver. Together with the presence of high blood urea nitrogen, these data indicate that the kidney is vulnerable to ischemia–reperfusion. Conclusions The enhanced oxidative stress due to ischemia–reperfusion appears to increase HNE in muscle and oxysterols by peroxidation not only in the gastrocnemius muscles but also in the kidneys and liver.

β-lactoglobulin forms a non-native α-helix during an early stage of folding. To address the role of the non-native structure in the folding process, we designed several mutants of equine β-lactoglobulin with reduced helical propensity in the non-native helix region. One of them, A123T, showed a similar structure to that of the wild-type protein; its folding kinetics was investigated by stopped-flow circular dichroism (CD) and fluorescence. Although A123T showed a reduced burst-phase CD intensity, its folding rate was similar to that of the wild-type protein, which indicated that the formation of the non-native helix does not accelerate or decelerate the folding reaction.

Sudden death while bathing is a major issue in elderly Japanese individuals. Despite its growing incidence, the exact processes underlying sudden death while bathing remain unknown. This study was designed to establish reliable diagnostic markers for drowning while bathing, as well as to assess the pathological conditions associated with sudden death while bathing, by analyzing the immunohistochemical intensity and localization of surfactant protein-A (SP-A), heat shock protein 70 (HSP70) and c-Fos protein in the lungs and central nervous system, including the cerebral cortex and hippocampus. We found that a distinct pattern of immunohistochemical expression of SP-A, HSP70 and c-Fos in the lung can be utilized to diagnose drowning. In most of these individuals, c-Fos protein was absent from the central nervous system, providing insight into patterns of ischemia in the central nervous system, or a loss in consciousness while bathing. We also found that rapid hypothermia or a cold stimulus increased c-Fos expression in the hippocampus (CA4). Furthermore, our results suggest that HSP70 expression in the hippocampus (CA4) can be used to estimate the length of time between the initial occurrence of stress and death.

Oxidative stress contributes to the pathogenesis of alcoholic liver disease. The purpose of this study is to estimate the amount of oxidative stress that is present when healthy humans consume moderate amounts of ethanol. Blood was collected from healthy volunteers before, 1 h, and 3 h after drinking 400 ml of Japanese rice wine at the rate of 100 ml per 5 min. The aldehyde dehydrogenase 2 genotype and the concentrations of blood ethanol, total lipid hydroperoxides (LOOH), and cholesterol hydroperoxides were determined. The plasma LOOH was found to have significantly increased 1 h after drinking. Cholesterol hydroperoxides were not detected in plasma, either before or after drinking. There was no relationship between the LOOH and the ethanol concentration. We showed that one-shot of moderate ethanol consumption temporarily increases the plasma LOOH in healthy volunteers but excessive plasma LOOH compounds were eliminated within a short time.

Chain collapse and secondary structure formation are frequently observed during the early stages of protein folding. Is the chain collapse brought about by interactions between secondary structure units or is it due to polymer behavior in a poor solvent (coil-globule transition)? To answer this question, we measured small-angle X-ray scattering for a series of β-lactoglobulin mutants under conditions in which they assume a partially folded state analogous to the folding intermediates. Mutants that were designed to disrupt the secondary structure units showed the gyration radii similar to that of the wild type protein, indicating that chain collapse is due to coil-globule transitions.

The repair of large cartilage defects with hyaline cartilage continues to be a challenging clinical issue. We recently reported that the forced expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) can induce chondrogenic cells from mouse dermal fibroblast culture without going through a pluripotent state. We here generated induced chondrogenic (iChon) cells from human dermal fibroblast (HDF) culture with the same factors. We developed a chondrocyte-specific COL11A2 promoter/enhancer lentiviral reporter vector to select iChon cells. The human iChon cells expressed marker genes for chondrocytes but not fibroblasts, and were derived from non-chondrogenic COL11A2-negative cells. The human iChon cells formed cartilage but not tumors in nude mice. This approach could lead to the preparation of cartilage directly from skin in human, without going through pluripotent stem cells., ヒトの皮膚細胞から軟骨様細胞へ直接変換に成功. 京都大学プレスリリース. 2013-10-14.

Equine β-lactoglobulin (ELG) assumes non-native helices during refolding and in partially folded states. Previously, circular dichroism (CD) combined with site-directed mutagenesis identified helical regions in the acid- and cold-denatured states of ELG. It is also known that a fragment of ELG, CHIBL (residues 88-142), has a structure similar to that of the cold-denatured state. For the study reported herein, the structure of a shorter fragment, CHIBLΔF (residues 97-142), was investigated by CD and nuclear magnetic resonance spectroscopy. The secondary chemical shifts clearly showed that non-native α-helices are present in two different regions, residues 98-107 and 114-135, and are connected by a native disulfide bond. The CD spectra of two peptides that correspond to the helical regions are characterized by weak helical signatures, and the sum of their CD spectra is nearly the same as the spectrum of disulfide-reduced CHIBLΔF. Therefore, the non-native helices are stabilized by the disulfide, and non-native helix formation may occur only during the refolding of the disulfide-intact protein. Supporting this conclusion is the observation that tear lipocalin, a homologue of ELG that lacks the disulfide, does not form non-native helices during folding.

Enteroscopic observation has clearly demonstrated that non-steroidal anti-inflammatory drugs/low-dose aspirin (usually enteric-coated) induces hemorrhagic lesions, including ulcers and bleeding, in the small intestine of patients at a high incidence. Such intestinal lesions induced by NSAIDs have been confirmed in animal experiments. With aspirin, however, it has long been believed that it is difficult to induce any damage in the intestinal mucosa of laboratory animals. Therefore, we established a new method of inducing intestinal hemorrhagic lesions in rats by injecting aspirin into the proximal duodenum.Under ether anesthesia, aspirin (50-200 mg/body), suspended in 2% methylcellulose (with or without 0.1 N HCl), was injected into the proximal duodenum of normally fed or 20-h non-fed rats (male Sprague-Dawley, 9 weeks old). At 1 h after treatment, the animals were killed with ether and the entire small intestine was removed for histological examination. In some experiments, 1% Evans blue was injected (i.v.) into the rats 1 h after aspirin treatment to visualize the lesions. An image analyzer determined the total area of the intestinal lesions. Oral proton pump inhibitors and histamine H(2)-receptor blockers were given 1 h before aspirin injection. 16,16-dimethyl prostaglandin E(2) (dmPGE(2)) was given s.c. 30 min before aspirin injection.Aspirin alone clearly induced severe lesions (including bleeding and ulcers) mainly in the jejunum at 100% incidence. Total score of lesions per rat obtained by histological examination was similar to the damaged area quantified with the dye method. Dose-related induction of lesions by aspirin was confirmed both by the histological and dye methods. The irritable effect of aspirin suspended in 0.1 N HCl solution was the same as that of aspirin alone; 0.1 N HCl alone induced only minor lesions in the intestine. Both proton pump inhibitors and histamine H(2)-receptor blockers, at doses that inhibit gastric acid secretion, had no or little effect on aspirin-induced intestinal lesions. Pretreatment with dmPGE(2) (3, 10, 30 microg/kg) showed significant prevention of both aspirin- and HCl/aspirin-induced intestinal lesions.This new aspirin lesion model will be useful for screening defensive drugs against aspirin-induced intestinal lesions and to elucidate the underlying mechanism.

A 12-year-old severely disabled woman child had been suffering from the refractive respiratory infection due to gastroesophageal reflux (GER) in years. However two transnasal catheters inserted to control GER, one was for feeding to the jejunum and the other was for decompression of the stomach, they were not effective against respiratory infection. Then, to resolve the problems, a button-shaped double lumen transgastric jejunal catheter was inserted into her jejunum via PEG in two-stage. After the procedure, the refractive respiratory infection due to GER could be successfully controlled. Additionally, by using the button-shaped catheter, any position came to be acceptable in daily life, for example in rehabilitation, sleeping and so on. Her ADL (activity of daily life) was well preserved.

The secondary structure in the molten globule state (an equilibrium analogue of a burst-phase folding intermediate) of equine beta-lactoglobulin was investigated by changes in the circular dichroic spectrum induced by a series of site-directed proline substitutions. The results challenge the structural picture obtained from previous hydrogen/deuterium exchange experiments. A stable non-native alpha-helix was found to exist in the region corresponding to the eighth strand (H strand) in the native structure, where the backbone amide protons are the most strongly protected from exchange. Therefore, the backbone topology in the folding core is significantly different from that in the native structure. This indicates that the burst-phase folding intermediate of beta-lactoglobulin is a trapped species because of misfolded backbone topology.

Equine beta-lactoglobulin forms a compact intermediate at an acidic pH (A state). It also forms an expanded and helical conformation at low temperatures (C state). The structure of a single disulfide mutant C66A/C160A is similar to the A state in the presence of salts, while it is similar to the C state at low anion concentrations. We have investigated the temperature-dependent change in the secondary structure using circular dichroism and proline scanning mutagenesis. At low anion concentrations, the helical content increased linearly as temperature decreased. In the presence of salts, the A state was cooperatively transformed into the C state at low temperatures. This suggests the importance of hydrophobic interactions for stabilizing the A state. Peptides encompassing native-like and non-native alpha-helices were synthesized to investigate the interactions responsible for helix formation in the A and C states. These did not form stable helices, indicating that not only the helices in the A state but also the helices in the C state are stabilized by long-range interactions. A longer fragment, CHIBL, which encompasses the structured region in the A and C states, showed a helical structure. Proline-substituted mutants of CHIBL showed CD spectral changes similar to the corresponding mutants of the full-length protein in the C state. Therefore, CHIBL has a structure similar to the corresponding region of the full-length protein in the C state. This result indicates that interactions responsible for helix formation in the C state reside in the sequence of CHIBL, and that the sequences outside CHIBL are essential for secondary structure formation in the A state.

Ethanol causes extensive damage to the intestinal tract from the oropharynx to the rectum. The jejunum has also been shown to be particularly vulnerable to the deleterious effects of ethanol. We hypothesized that (I) the pathogenesis of acute alcohol-mediated injury in the small intestine involves generation of reactive oxygen species, and consequentially, enhanced lipid peroxidation; (II) the pathogenic changes due to alcohol can be ameliorated with daidzein pretreatment. To test these hypotheses male Wistar rats (n=24) were divided into four groups as follows (pretreatment followed by treatment): [A] carrier+saline (control); [B] daidzein+saline; [C] carrier+ethanol; [D] daidzein+ethanol. Daidzein (100 mg/kg) or carrier (Intralipid) pretreatment was twice administered as a single dose, whereas ethanol (75 mmol/kg) or saline (0.15 mol/l NaCl) treatment was administered once only. At 24 h after ethanol or saline was administered, rats were sacrificed. The analytes 7alpha-and 7beta-hydroperoxycholest-5-en-3beta-ol (7alpha-OOH and 7beta-OOH), 7alpha-and 7beta-hydroxycholesterol (7alpha-OH and 7beta-OH), and 7-ketocholesterol (7-keto) in jejunum were analyzed by HPLC. The data showed that daidzein per se did not affect levels of cholesterol hydroperoxides nor oxysterols. However, there were significant increases in 7alpha- and 7beta-OOHs, 7alpha- and 7beta-OHs, and 7-keto after ethanol dosage compared to controls. Daidzein ameliorated these effects, i.e., values in the daidzein+ethanol group were similar to those in the carrier+saline (control) group. This is the first report showing that (1) cholesterol-derived markers of oxidative stress are increased in the rat jejunum in response to ethanol, indicative of metabolic damage; (2) daidzein pretreatment has protective effects against ethanol-induced injury.

A disulfide bond between cysteine 66 and cysteine 160 of equine β-lactoglobulin was removed by substituting cysteine residues with alanine. This disulfide bond is conserved across the lipocalin family. The conformation and stability of the disulfide-deleted mutant protein was investigated by circular dichroism. The mutant protein assumes a native-like structure under physiological conditions and assumes a helix-rich molten globule structure at acid pH or at moderate concentrations of urea as the wild-type protein does. The urea-induced unfolding experiment shows that the stability of the native conformation was reduced but that of the molten globule intermediate is not significantly changed at pH 4 by removal of the disulfide bond. On the other hand, the molten globule at acid pH was destabilized by removal of the disulfide bond. This difference in the stabilizing effect of the disulfide bond was interpreted by the effect of the disulfide in keeping the molecule compact against the electrostatic repulsion at acid pH. In contrast to the wild-type protein, the circular dichroism spectrum in the molten globule state at acid pH depends on anion concentration, suggesting that the expansion of the molecule through electrostatic repulsion induces α-helices as observed in the cold denatured state of the wild-type protein. Proteins 2006. © 2006 Wiley-Liss, Inc.

An improved technique for the analysis of phosphatidylcholine (PC) and lyso-phosphatidylcholine (lyso-PC) oxidation products was developed using quadrupole time of flight (Q-TOF) mass spectrometry with electrospray ionization. We separated these products using an HPLC C(8) column with a gradient of methanol and 10mM aqueous ammonium acetate. Monohydroxides, oxo derivatives, and trihydroxides of palmitoyl-linoleoyl (C16:0/C18:2) PC, stearoyl-linoleoyl (C18:0/C18:2) PC, and oleoyl-linoleoyl (C18:1/C18:2) PC were detected mainly as MH(+) and [M+Na](+) ions in the heart of the intact rat. Using standard synthetic PC-OH (C16:0/C18:2-OH), the lipid extract component was identified as (C16:0/C18:2-OH) PC based on the product ions of ESI-MS-MS and, the PC-OH concentration was quantitated. Four oxidatively modified 1-lyso-phosphatidylcholines (lyso-PCs) were also detected. This is the first report showing the presence of monohydroxides, oxo derivatives, and trihydroxides of (C16:0/C18:2)PC, (C18:0/C18:2)PC, and (C18:1/C18:2) PC in the rat heart.

A case of sexual asphyxial death by hanging is presented. A 35-year-old male, found dead lying on his face in a bed of a truck cab, had hanged himself from a window frame using a leather belt. He was completely naked. There were pornographic and sadomasochistic magazines beneath his face, opened to pages depicted nude photographs of a woman. Autopsy findings revealed a ligature mark on the neck and petechial hemorrhages in the conjunctivae, but there were no hemorrhages in the neck muscles or fractures of the hyoid bone or the thyroid cartilage. The alcohol levels in the blood and urine were 0.78 and 0.45 mg/ml, respectively. The circumstances suggested that his death was accidental, and due to asphyxia by hanging performed to enhance sexual gratification during masturbation. Sexual asphyxia is reviewed and discussed.

An accidental atypical hanging with the collar of a sweater is reported. A 67-year-old man was found dead in the sitting position with the collar of his sweater hanging off the brake handle of a motorcycle. Autopsy findings revealed a ligature mark on the surface of the neck; hemorrhages in the sternohyoid muscles; submucosal hemorrhages in the left greater horn of the hyoid bone; a large degree of submucosal petechial hemorrhages in the larynx, oral mucosa, and palpebral conjunctivae; and dark-red liquid blood with little clotting in the heart cavities. The alcohol levels in the blood and urine were 2.84 mg/ml and 3.52 mg/ml, respectively. It was concluded that the man had died from hanging by the sweater, and it was speculated that when he became inebriated, he might have lost consciousness and then fell with the collar of his sweater hanging from the brake handle of the motorcycle.