Biomedical subjects
Publications and source records attributed to Y Fujiwara.
Three new carotenoids with a 3-methoxy-5-keto-5,6-seco-4,6-cyclo-beta end group (1-3) have been isolated from the seeds of Pittosporum tobira. Their structures were elucidated by detailed analyses of nuclear magnetic resonance and UV data.
The genes of tetraheme cytochrome c3 and hexadecaheme high-molecular-weight cytochrome c from Desulfovibrio vulgaris could be overexpressed as holoproteins in Shewanella oneidensis TSP-C using pUC-type vectors of E. coli. Surprisingly, S. oneidensis was transformed directly by pUC-type vectors through electroporation. The yields of the recombinant proteins in this expression system were much higher than the previously reported ones.
A novel method for the determination of residual solvents in pharmaceuticals by thermal desorption (TD)-GC/MS has been established. A programmed temperature pyrolyzer (double shot pyrolyzer) is applied for the TD. This method does not require any sample pretreatment and allows very small amounts of the sample. Directly desorbed solvents from intact pharmaceuticals (ca. 1 mg) in the desorption cup (5 mm x 3.8 mm i.d.) were cryofocused at the head of a capillary column prior to a GC/MS analysis. The desorption temperature was set at a point about 20 degrees C higher than the melting point of each sample individually, and held for 3 min. The analytical results using 7 different pharmaceuticals were in agreement with those obtained by direct injection (DI) of the solution, followed by USP XXIII. This proposed TD-GC/MS method was demonstrated to be very useful for the identification and quantification of residual solvents. Furthermore, this method was simple, allowed rapid analysis and gave good repeatability.
We describe a case of vinorelbine tartrate (VNR) associated acute respiratory failure. A 65-year-old man with non-small cell lung cancer developed acute respiratory failure 50 minutes after his first infusion with VNR in combination with mitomycin-C. The patient was treated with furosemide, dopamine and high-dose methylprednisolone, and recovered with no discernible sequelae. Although clinical trials have shown that respiratory symptoms associated with VNR treatment have only rarely been observed and the putative mechanism remains to be elucidated, patients receiving VNR should be monitored carefully, particularly in the first few hours after intravenous administration.
A reduction of dehydroerythorbic acid (DERA) to erythorbic acid (ERA) in vitamin C-deficient guinea pigs was evaluated and compared with that of dehydroascorbic acid (DASA). Thirty-six guinea pigs were fed with vitamin C-deficient diets for 18 days. On day 19, the guinea pigs were divided into four groups for the administration of 100 mg of DERA, ERA, ascorbic acid (ASA), or DASA every day. After 12 days of oral administration, the concentration of DERA, ERA, ASA, and DASA in the liver, adrenal, spleen, kidney, and plasma of guinea pigs was determined by HPLC. A recovery from scurvy was measured in terms of weight gain and serum alkaline phosphatase activity. All four groups showed similar recovery, indicating that the oral administration of relatively high concentrations of DERA reversed the effects of scurvy in vitamin C-deficient guinea pigs. In spite of DERA or DASA administration, ERA or ASA was mainly detected in the tissues. The reduction ratios of DEAR and DASA were similar (approximately 80%) in all tissues except spleen. These results suggest that both DASA and DERA are taken up and reduced to ASA or ERA in vivo.
To determine the limits of the duration of in vivo transferred foreign gene expression, we conducted electroporation (EP), a powerful non-viral means of gene transfer for living animals, into skeletal muscle of rats and mice with a luciferase, GFP or erythropoietin (EPO)-encoding reporter plasmid. The luciferase reporter plasmid was used for optimization of EP conditions, while GFP and EPO plasmids were used for monitoring the duration of gene expression. In the rat, increased hematocrit levels were maintained for at least 9 weeks with approximately a 3-fold increase in plasma EPO protein concentration at 4 weeks post-transfection. In the mouse, the GFP plasmid transfer confirmed that the reporter gene expression lasted as long as 3 months post-transfection. By introducing the EPO gene in vivo in the mouse, increased hematocrit levels revealed that duration of reporter gene expression was at least 14.5 months after in vivo gene EP into skeletal muscle. These results implicate an excellent potential of in vivo gene EP, applicable to both experimental and therapeutic purposes.
In order to detect a hematogenous spread of tumor cells in patients with hepatocellular carcinoma, reverse transcription-polymerase chain reaction assay has been used. In this study, we quantified alpha-fetoprotein (AFP) messenger RNA by real-time PCR approach using LightCyclertrade mark technique. AFP messenger RNA in the blood from 23 hepatocellular carcinoma patients undergoing hepatic resection, 31 healthy volunteers, 10 patients with liver cirrhosis and 5 patients underwent hepatectomy except for hepatocellular carcinoma was quantitated. In the real-time PCR, fluorescence was undetectable in any of the controls. On the contrary, fluorescent signals were detected in 10 out of 39 blood specimens collected from 23 HCC patients. AFP-positive status was significantly associated with the existence of multiple intrahepatic nodules. Out of 8 cases with AFP-positive status, intra- and/or extra-hepatic recurrence has been observed in 3 cases. The quantities of AFP messenger RNA in these 3 cases were relatively high among 8 cases with AFP-positive status. AFP messenger RNA was detectable by newly developed real-time PCR approach with LightCycler and it is suggested that this approach could be applicable in detection of small amounts of tumor cells in the blood of HCC patients.
H. pylori plays important roles in the pathogenesis of active gastritis, peptic ulcer, and gastric cancer. Culture of H. pylori from suspicious materials is one of gold standard methods for the diagnosis of its infection. H. pylori has two types of morphological manifestation, spiral (mobile) and coccoid (immobile) forms. Morphological conversion from spiral to coccoid form and its reversion may be important role in its pathophysiology including bacterial properties, pathogenicity, and etiology. However, the mechanism and significance of such transformation of H. pylori have remained unknown. In this chapter, we introduce the conventional and new culture methods of H. pylori and review the current knowledge regarding both two types of morphological manifestation and pathophysiological significance of such transformation.
A 12-year-old girl with atrial septal defect combined with pulmonary hypertension and 90% stenosis of the left main coronary artery caused by dilated pulmonary artery was scheduled for atrial septal closure and coronary artery bypass graft under general anesthesia. During the echocardiographic examination to evaluate the anatomical relationship between the pulmonary artery and left main coronary trunk, bradycardia and a depression of ST-segment on electrocardiogram appeared suddenly when the operator compressed the pulmonary artery with a probe of echocardiography from the operative field. The circulatory collapse and ischemic change on electrocardiogram might have been caused by a further reduction of blood flow to the left main coronary trunk narrowed originally by dilated pulmonary artery. Although various etiologies, such as atherosclerosis, syphilis, and congenital abnormalities are widely known to cause stenosis of the left main coronary trunk, external compression by dilated pulmonary artery has not been widely known. Malignant arrhythmias from coronary artery compression with subsequent ischemia could contribute to an incidence of sudden death. Coronary angiography and magnetic resonance imaging are useful for the preoperative evaluation. Careful management is needed to protect such a patient from ischemic event in the perioperative period.
OBJECTIVES: The incidence and severity of coronary artery disease were studied in patients with acute aortic dissection (AAD), and compared with coronary artery disease in patients with abdominal aortic aneurysm (AAA) or arteriosclerosis obliterans (ASO). METHODS: A total of 71 patients(42 males, 29 females, mean age 61.4 +/- 10.0 years) with AAD, undergoing coronary angiography between 1988 and 1999, were studied including 38 patients with open communication type and 33 patients with thrombosed type. According to the Stanford classification, 18 patients were type A and 53 patients were type B. Patients with AAD following Marfan syndrome or chest trauma were excluded from the study. Selective coronary angiography was performed in age- and sex-matched patients with AAA(n = 57; 42 males, 15 females, mean age 63.9 +/- 4.6 years) or ASO (n = 95; 66 males, 29 females, mean age 62.4 +/- 9.4 years). Coronary artery disease was defined as > or = 75% stenosis (left main trunk lesion of > or = 50% stenosis) by multidirectional imaging. RESULTS: Significant coronary artery disease was demonstrated in 14 patients with AAD (19.7%), 25 patients with AAA (43.9%), and 49 patients with ASO (51.5%). The incidence of coronary artery disease was significantly lower in the AAD group than in the other two groups (p < 0.05). One-vessel disease was present in approximately 70% of the patients with AAD and coronary artery disease. In contrast, multivessel disease was observed in approximately 50% of patients with AAA and ASO. Classification of the patients with AAD according to the blood flow in the false lumen showed coronary artery disease was more highly associated with the thrombosed type [10 (30.3%) of 33 patients] than the open communication type [4 (10.5%) of 38 patients]. Multivariate logistic regression analysis of the patients with AAD showed coronary artery disease was associated with a high serum total cholesterol level (p = 0.025) and the thrombosed type (p = 0.043). CONCLUSIONS: The incidence of coronary artery disease was significantly lower among patients with AAD than among age- and sex-matched patients with AAA or ASO. Coronary artery disease developed in 30% of the patients with the thrombosed type of AAD, although the prognosis seemed to be good.
To provide a detailed assessment of micrometastases of colorectal cancer by anatomical mapping of regional lymph nodes (LNs), we analyzed 237 LNs from 11 patients with colorectal cancer by reverse transcription-PCR (RT-PCR) using carcinoembryonic antigen and cytokeratin 20 as genetic markers. All dissected LNs were mapped anatomically and subjected to detection assays for micrometastases. Immunohistochemical analysis was also performed using anti-pancytokeratin antibody AE1/AE3 to confirm the existence of occult cancer cells. By histological analysis, 20 of 237 LNs contained metastatic cells, and they were all positive by both immunohistochemistry and RT-PCR. Of the 217 histologically negative LNs, 14 (6.5%) harbored micrometastases by immunohistochemistry, and 57 (26.2%) were positive for at least one of the two genetic markers. Lymphatic mappings of all patients showed that micrometastases were distributed not only at the pericolic LNs but often at distant LNS: Clinical follow-up study showed that two patients developed recurrence within 1 year after surgery, and both of them had RT-PCR-positive micrometastases in not less than 70% of LNs examined. Moreover, both patients had frequent micrometastases at distant LNs, i.e., those around the root or along the inferior mesenteric artery, when compared with patients with no recurrence. Our findings suggest that genetic diagnosis using the RT-PCR method may be clinically useful along with conventional pathological diagnosis, especially when micrometastases spread to distant LNS:
Surgery is the main therapy for malignancies of the gastrointestinal tract. Lymph node metastasis is one of the major factors in predicting patients' clinical course and choosing appropriate adjuvant therapy after surgery. The concept of micrometastasis to regional lymph nodes emerged over 10 years ago, but its significance has been controversial. To clarify the relevance of micrometastasis of gastrointestinal tract cancers, we have established RT-PCT based-diagnostic methods using multi-markers such as CEA, CK20, and Mage 3. Prospective studies have shown that not a few micrometastasis-positive patients with carcinoma of the colon, stomach, and esophagus suffered disease recurrence, even though they did not show histologically positive lymph node metastasis. They were initially diagnosed as node-negative, and thus predicted to be disease free. A retrospective study of 62 patients with stage II node-negative colorectal cancer showed that 5-year overall survival was 78.2% among micrometastasis-positive patients, against 95.3% micrometastasis-negative patients. Moreover, there was a marked difference in 5-year disease-free survival, with 61.4% versus 88.4%, respectively. These data warrant further prospective study with a large population since RT-PCR based detection systems for micrometastasis appear to have the potential to improve conventional diagnosis and therapy for colorectal cancer.
PURPOSE: Service dogs have received much attention as effective choices for promoting participation in society of disabled persons. The purpose of this study was to obtain the fundamental data to prepare guidelines regarding service dogs use in community life, by making clear the problems in terms of public health and community welfare through a survey of attitudes towards service dogs in community residents. METHODS: Study 1 was conducted as follows. The subjects were 423 community residents between their teens and seventies who participated in health and welfare lectures in 4 metropolitan areas. A questionnaire survey was conducted just after watching a promotion video about service dogs, including several items on aspects not accept, were anxious about or were unpleasant in the case of community life of a disabled person and a service dog. Study 2 was as follows. A questionnaire survey was conducted inside a supermarket in the central part of Tokyo. One group of subjects were 103 shopping customers who witnessed a shopping scene featuring a disabled person with her service dog, and the other subjects were 84 customers who were not witnessed to the scene. The same survey was simultaneously performed for impression about the shopping scene. RESULTS: Study 1 showed that 31.8% of all the respondents replied that they had some knowledge of service dogs before watching the video. A total of 20.4% (32.5% of the elderly) replied that there were places where service dogs should not enter. Concretely speaking, these included restaurants for 7.3-22.5% and medical institutions for 8.5-12.5% of the respondents. A third of all the respondents (51.3% of older persons) replied they could not endure some actions by service dogs, concretely "carrying food in the mouth" was highly pointed out by 27.3%. Zoonosis (15.3-19.0%), hair scattering (9.5-21.3%), injuries (4.4-7.4%) were also pointed out as main sources of anxiety or uncomfortable feelings. Such negative opinions were especially apparent among those aged 60 years and older. Also, 44.1% knew some disabled person. Cross tabulation showed those who had experience of taking care of a dog were significant less anxious about "vague anxiety or discomfort," and "zoonosis" than those who had not (P < 0.01). On the other hand, most of both witness (79.6%), and non-witness groups (81.0%) replied that it was heartwarming that the service dog assisted with shopping. None expressed discomfort about the presence of "service dog" inside the supermarket. In terms of carrying a commodity in the mouth, 92.2% and 89.1% in both groups replied that they don't mind, especially if safety was guaranteed for public health, and 91.3% and 95.2% in both groups replied that they had no other problems. Moreover, none of them would not entering stores. Significant differences were seldom in replies between the witness and non-witness groups. CONCLUSION: Although the respondents were extremely affirmative regarding the image of service dogs, it became clear that anxiety about public health aspects such as "zoonosis" or "utilization of medical institutions" was persisting. On the whole, negative opinions were more apparent in the elderly.
We investigated the effect of sofalcone, a synthetic flavonoid derivative of sophoradin, on tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta production in human monocytes stimulated by Helicobacter pylori water extract. H. pylori water extract significantly stimulated TNF-alpha and IL-1 beta production by monocytes while incubation with sofalcone (10 micrograms/ml and 50 micrograms/ml) significantly inhibited this increase in TNF-alpha and IL-1 beta production. These results suggest that sofalcone could be used to improve H. pylori-associated gastric mucosal inflammation through inhibition of proinflammatory cytokine production.
Peroxisome proliferator-activated receptor gamma (PPAR-gamma), a member of the nuclear hormone receptor superfamily, is involved in suppression of growth of several types of tumors such as liposarcoma, breast cancer, prostate cancer, and colon cancer, possibly through induction of cell cycle arrest and/or apoptosis. In this study, we demonstrated expression of PPAR-gamma mRNA and protein in human esophageal carcinoma cells. Expression of PPAR-gamma protein was higher in an adenocarcinoma cell line (TE-7 cells) than in a squamous cell carcinoma cell line (TE-1 cells). PPAR-gamma ligands such as 15-deoxy-Delta12,14-prostaglandin J2 and troglitazone significantly inhibited the growth of TE-7 cells but had less or no effect on growth of TE-1 cells. 15d-PGJ2 and troglitazone induced apoptosis in TE-7 cells but not in TE-1 cells. Troglitazone caused G1 cell cycle arrest and reduced ornithine decarboxylase activity (ODC) in TE-7 cells but not in TE-1 cells. Inhibition by PPAR-gamma ligands of growth of esophageal adenocarcinoma cells may thus be due to induction of apoptosis, G1 cell cycle arrest and reduction of ODC activity.
It is not known how Shiga toxins (Stxs), which are major virulence factors of enterohemorrhagic Escherichia coli, can affect the host immune system. We investigated the effect of Stx2 on murine thymic cells in vivo and in vitro. After intraperitoneal administration of Stx2, the body weight of mice (BW) and the organ index of thymocytes (OIT) gradually decreased from day 1 to day 4. Apoptosis of thymocytes, assessed by TUNEL staining, and DNA fragmentation assay, was marked on day 3 and day 4. Decrease in BW and OIT due to Stx2 was antagonized by the simultaneous administration of murine anti-Stx2 IgG antibody with Stx2. In vitro administration of Stx2 also induced apoptosis of cultured thymocytes on day 3 and day 4 in a dose-dependent fashion. These results showed that Stx2 directly caused apoptosis of thymocytes in vivo and in vitro. Our findings imply that StA2 may be intimately related to pathogenesis of E. coli O157:H7 infection, by causing apoptosis of thymus.
This longitudinal study examined numbers of homebound and bed bound individuals over time, and risk factors in a rural community elderly populations. A total of 731 older persons who needed no assistance of another person in traveling from their homes participated in the baseline survey in 1992 and were assessed annually for their ability to travel for subsequent six-years. We defined operationally a homebound status as the necessity for assistance of another person to be able to leave home, but without being bedbound. During the follow-up period, 86 homebound (23.7 per 1,000 person-years) and 32 bedbound cases (8.4 per 1,000 person-years) were noted, the former being 2.8 times more likely to occur. Of the newly bound cases 26% returned to a state capable of leaving home by themselves one year later, and maintained this independent living level even two years later. Of the other cases who remained as homebound or bedbound one year later, only one (1/31) had returned to independent living after two years; most of them became bedbound at home, hospital admitted or deceased. Stepwise multiple logistic regression analysis showed that independent risk factors for the homebound include older age, male gender, slower walking speed, history of hospital admission in the previous year, lower masticatory ability, and higher level of serum beta 2-microglobin. These results indicate that health promotion programs for prevention of becoming homebound among community elderly people should target more intensively assistance to maintaining walking and masticatory ability. In addition, public health professionals should pay more attention to elderly people shortly after hospital discharge.
Subcellular localization is important for proteins to function. For the prediction of subcellular localizations, we have developed a method, SortPred, using the amino acid composition and order. The composition represents the global features, e.g., the amino acid composition in the full or partial sequences, while the order represents the local features, e.g., the amino acid sequence order. The former was represented by neural networks and the latter was represented by a hidden Markov model. This method predicted the signal peptides (SP), the mitochondrial targeting peptides (mTP), the chloroplast transit peptides (cTP), and the nuclear or cytosolic sequences (other) comparing together the previous methods, this method achieved slightly higher prediction accuracy, 86% for plant and 91% for non-plant. We analyzed the trained neural networks and hidden Markov models and found out that these models well represent the biological features of the sequences.