No description available.

The effect of acids on the growth, survival and detection of Salmonella enteritidis PT4 is particularly important in view of the number of outbreaks in which mayonnaise has been implicated as a vehicle. Capacitance measurement was compared with colony counting procedures for the enumeration and determination of sub-lethal injury of Salmonella enteritidis during storage under varied conditions of pH, acidulant and temperature. Capacitance monitoring was shown to offer an improved technique for the measurement of sub-lethal injury in cell populations. Higher levels of sub-lethal injury were detected by the extension of capacitance detection time than were indicated by differential colony counts on selective and non-selective media. The extension of detection time noted with sub-lethally injured cell populations was shown to be due to an extended lag phase when cells were placed in the capacitance growth medium and not the result of delayed detection of the growth of a small, uninjured sub-population. Plots of percentage survival and extension of detection time in survivors gave similar curves for acetic and lactic acid. These acids showed both greater lethality and greater ability to inflict sub-lethal injury than the stronger citric or hydrochloric acid. Sub-lethal injury and lethality were not simply related, as little sub-lethal injury was observed with the stronger acids even under conditions that were ultimately highly lethal. The results indicate that weak organic acids cause more reversible damage to cellular sites prior to death: an observation that has implications for choice of resuscitation procedures when examining acidified foods. Injured cells were found to contain lower levels of ATP than healthy unstressed cells. Inhibition with chloramphenicol did not appear to increase injury and total protein patterns for injured and uninjured cells were similar, suggesting that protection afforded by the synthesis of stress proteins is not a significant factor in this case. The recovery of sub-lethally injured cells in various pre-enrichment and selective enrichment media using capacitance detection times and colony counts on selective and non selective media was determined. Buffered peptone water appeared more effective in recovering injured Salmonella compared to Lactose broth. Additionally, selenite cystine was shown to recover cells faster than the other two selective broths tested; the Muller-Kauffmann Tetrathionate broth and the Rappaport Vassiliadis enrichment broth. In this study, different injury conditions were applied; these included acid stress, heating and freezing. According to the lag phases of injured cell populations, short pre-enrichment is not recommended in the present study.

No description available.

Neointimal proliferation is a key process underlying many cardiovascular diseases such as atherosclerosis and angioplasty-induced restenosis. Vascular smooth muscle cells (SMC) are significant contributors to the development and stability of the neointimal lesion. This is due, in part, to their capacity to be phenotypically modulated, facilitating SMC proliferation in response to mechanical injury, their subsequent migration, and deposition of extracellular matrix. The aim of this thesis was to characterize the function of two genes identified in our laboratory to be upregulated shortly after mechanical injury of vascular SMC and their exposure to fibroblast growth factor (FGF)-2, an injury-induced cytokine. The first is activation transcription factor (ATF) 4, which is upregulated by FGF-2 and mechanical injury in vascular SMC in vitro, and by balloon-injury in the artery wall. The induction of ATF4 by FGF-2 was shown to be mediated through the PI3K pathway, and preceded by phoshorylation of eIF2alpha, a known upstream effector of ATF4 activation. Knock-down of ATF4 expression inhibited balloon-injury induced neointimal hyperplasia, suggesting that ATF4 is a key player in the SMC response to injury. Furthermore, microarray analysis identified several genes whose transcription in response to FGF-2 may be regulated by ATF4. In particular, this work demonstrates that ATF4 is necessary for VEGF-A upregulation in SMC in response to FGF-2 and mechanical injury in vitro and in the artery wall following balloon-injury. The second is a translation factor, YrdC203. Using confocal fluorescence microscopy, YrdC203 was found to localize partially to the ER, and with RPL12, a component of the 60S ribosomal subunit. Immunoprecipitation studies demonstrate that YrdC203 also interacts with an initiation factor, eIF5B. Mutation of an initiation factor’s signature on the exterior of YrdC203 perturbed its interaction with RPL12 and eIF5B, and inhibited the increase in protein synthesis observed with overexpression of YrdC203. This implicates YrdC203 as a translation factor responsible for ensuring protein synthesis in vascular SMC in response to injury. The present work provides evidence for new molecular mechanisms, transcriptional and translational, regulating the response of vascular SMC to injury. This would provide leads for future therapeutic targets.

Neointimal proliferation is a key process underlying many cardiovascular diseases such as atherosclerosis and angioplasty-induced restenosis. Vascular smooth muscle cells (SMC) are significant contributors to the development and stability of the neointimal lesion. This is due, in part, to their capacity to be phenotypically modulated, facilitating SMC proliferation in response to mechanical injury, their subsequent migration, and deposition of extracellular matrix. The aim of this thesis was to characterize the function of two genes identified in our laboratory to be upregulated shortly after mechanical injury of vascular SMC and their exposure to fibroblast growth factor (FGF)-2, an injury-induced cytokine. The first is activation transcription factor (ATF) 4, which is upregulated by FGF-2 and mechanical injury in vascular SMC in vitro, and by balloon-injury in the artery wall. The induction of ATF4 by FGF-2 was shown to be mediated through the PI3K pathway, and preceded by phoshorylation of eIF2alpha, a known upstream effector of ATF4 activation. Knock-down of ATF4 expression inhibited balloon-injury induced neointimal hyperplasia, suggesting that ATF4 is a key player in the SMC response to injury. Furthermore, microarray analysis identified several genes whose transcription in response to FGF-2 may be regulated by ATF4. In particular, this work demonstrates that ATF4 is necessary for VEGF-A upregulation in SMC in response to FGF-2 and mechanical injury in vitro and in the artery wall following balloon-injury. The second is a translation factor, YrdC203. Using confocal fluorescence microscopy, YrdC203 was found to localize partially to the ER, and with RPL12, a component of the 60S ribosomal subunit. Immunoprecipitation studies demonstrate that YrdC203 also interacts with an initiation factor, eIF5B. Mutation of an initiation factor’s signature on the exterior of YrdC203 perturbed its interaction with RPL12 and eIF5B, and inhibited the increase in protein synthesis observed with overexpression of YrdC203. This implicates YrdC203 as a translation factor responsible for ensuring protein synthesis in vascular SMC in response to injury. The present work provides evidence for new molecular mechanisms, transcriptional and translational, regulating the response of vascular SMC to injury. This would provide leads for future therapeutic targets.

Objective: We proposed that women who develop preeclampsia have a low ratio of 'protective' toxicity preventing activity (TxPA) to 'toxic' very low density lipoproteins (VLDL) late in pregnancy. Having confirmed this hypothesis, we then tested whether this low ratio would manifest itself early in the pregnancy of women who develop preeclampsia. Methods: Serially collected plasma from women who developed preeclampsia and from matched controls was assayed blind for TxPA, triglycerides, cholesterol, high-density lipoproteins, albumin, and nonesterified fatty acids (NEFA). Main Outcome Measures: Plasma concentrations of lipids, NEFA, and proteins which bind NEFA (TxPA and albumin) were measured in normal and preeclamptic women. These parameters were formulated prior to data collection because of the low albumin/triglyceride' ratios and the elevated NEFA levels reported to occur in preeclampsia. Results: In late pregnancy, TxPA was lower (1.82 ± 0.63 vs. 2.30 ± 0.40 g/dL, P = 0.008) and VLDL higher (292 ± 130 vs. 206 ± 60 mg/dL, P = 0.013) in preeclamptics than in controls. Discrimination analysis (TxPA and triglyceride), correctly classified 95% of the preeclamptics and 79% of the controls in late pregnancy. The ratio of TxPA to non-TxPA and triglyceride correctly classified 92% of the preeclamptics and 85% of the controls in early pregnancy. Conclusions: The ratio of TxPA to VLDL accurately distinguishes preeclamptic from normal pregnant women, suggesting that both these factors are involved in the development of preeclampsia.

Isolated adult rat myocytes were subjected to 180 min of metabolic inhibition or incubated in ischaemic pellets, in the presence and absence of 10 μm okadaic acid (OA) or calyculin A (CL-A). Contracture and viability was determined by light microscopic analysis of trypan blue-stained preparations and ATP levels by HPLC. Osmotic fragility was assessed by brief hypotonic swelling of cells in 170 or 85 mOsm media prior to determination of viability. Neither drug significantly affected the relatively rapid rates of contracture of myocytes during metabolic inhibition, and both afforded significant protection from development of trypan blue permeability and osmotic fragility. Both OA and CL-A significantly accelerated the rates of contracture and ATP depletion of myocytes during ischaemic incubations. Despite an enhanced rate of ATP depletion, which would be expected to accelerate development of injury, neither drug accelerated development of loss of viability or development of osmotic fragility as measured by 170 mOsm swelling. Mathematical compensation for different rates of ATP depletion confirmed that a protective effect of the drugs, during ischaemic incubation, was masked by their enhancement of the rate of injury, following swelling at 170 mOsm. When the effects of CL-A on ischaemic cells were examined at 85 mOsm, a more stringent test for osmotic fragility, protection was found without compensation for differing rates of ATP depletion. A dose/response curve for CL-A showed some effect at 100 nm and a nearly full effect during metabolic inhibition at 1 μm concentrations. It is concluded that protein phosphatase inhibitors reduce the rates of development of osmotic fragility of metabolically inhibited cells and reduces the rate of injury relative to the rate of ATP depletion of ischaemic cardiomyocytes. Phosphorylation mechanisms may be important to development of irreversible myocardial cell injury.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19599号 / 医博第4106号 / 新制||医||1014(附属図書館) / 32635 / 京都大学大学院医学研究科医学専攻 / (主査)教授 柳田 素子, 教授 妹尾 浩, 教授 浅野 雅秀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM