| 171. |
- Zhang, Hong, 1957-
(författare)
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Alloxan Toxicity to macrophages and Insulinoma Cells
- 1995
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Alloxan induces damage and death of pancreatic islet B-cells in severalexperimental animal models, thus causing insulin-dependent diabetes mellitus (IDDM or type I diabetes). This unique cytotoxicity of alloxan has been studied for more than fifty years. The mechanisms behind the cytotoxicity of alloxan have, however, never been fully understood, although an increasing number of authors now suggest formation of reactive oxygen species, targeting the plasma membrane, mitochondria and DNA. In the present study, we have investigated: (i) the production of superoxide and hydrogen peroxide during reactions between alloxan and reducing agents such as cysteine, reduced glutathione, and ascorbic acid; (ii) the cytotoxic effects of alloxan in the absence of reducing agents, on model systems of cultured macrophages and insulinoma cells; (iii) the cytotoxicity of alloxan together with the reducing agents on these cultured cells; (iv) the cytotoxicity of hydrogen peroxide, used at concentrations similar to those formed during the reactions of alloxan with reducing agents; (v) the influence of iron and the iron-chelator, desferrioxamine, on the alloxan-induced cytotoxicity; and (vi) the influence of starvation-induced autophagocytosis on the sensitivity of cells to hydrogen peroxide-induced oxidative stress. Cell viability was estimated by a delayed trypan blue dye exclusion test and plasma membrane permeability by a modified microfluorometric combined fluorescein diacetate-propidium iodide staining technique. Lysosomal membrane stability was microfluorometrically assayed by acridine orange and neutral red relocalization techniques. The intracellular amounts of iron, reduced glutathione, antioxidant enzymes, and ATP were biochemically and cytochemically studied under a variety of conditions. The results showed that: (i) superoxide artion radicals and hydrogen peroxide were produced by reactions between alloxan and several reducing agents (e.g. cysteine, reduced glutathione and ascorbic acid). Hydrogen peroxide readily diffused through cellular membranes into the lysosomes if it was not previously degraded by the cellular antioxidative defence systems. Hydroxyl radicals might be produced by intralysosornal Fenton reactions, if reactive iron was present, resulting in lysosomal membrane damage followed by a leakage of lysosomal lytic enzymes with ensuing cell degeneration and eventually cell death. (ii) If iron was adsorbed to plasma membranes, extracellularly produced superoxide anion radicals and hydrogen peroxide might cause the plasma membrane damage due to Fenton reactions. (iii) Preincubation with desferrioxamine, or the presence of catalase inhibited the cytotoxicity induced by alloxan and reducing agents. (iv) The antioxidative defence activity of insulinoma cells was low. (v) Starvation in PBS enhanced the sensitivity of both macrophages and insulinoma cells to oxidative stress induced by hydrogen peroxide mediated through increased activity of autophagocytotosis. Thus, the amount of intralysosomal reactive iron consequently resulted from the degradation of various iron-containing metallo-proteins. We conclude that the exposure of cells to alloxan together with a reducing agent created cellular oxidative stress through extracellular formation of superoxide anion radicals and hydrogen peroxide. The latter compound easily penetrated plasma and lysosomal membranes, reaching the lysosomal interior. If enough reactive iron was present within lysosomes and the hydrogen peroxide was not degraded by catalase or glutathione peroxidase before entering the acidic vacuolar apparatus hydroxyl radicals could be produced via intralysosomal Fenton reactions.The hydroxyl radicals, in turn, would attack and damage the lysosomalmembranes, causing a leakage of lysosomal enzymes to the cytosol and eventually leading to cell death. The sensitivity of cells to alloxan-induced cytotoxicity in the presence of reducing agents was therefore a function of (i) the rate of hydrogen peroxide production, (ii) the cellular antioxidative defence systems, (iii) the lysosomal amount of reactive iron, and (iv) the capacity of autophagocytosis.
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| 172. |
- Zhuang, Shi-Mei, 1965-
(författare)
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Molecular genetic alterations in chemically-induced lymphomas
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Lymphoma is one of the most common malignancies in humans. Its incidence has increased rapidly in the past 30 years. However, the molecular mechanisms underlying the development of lymphomas are largely unknown.Environmental carcinogens play an important role in tumorigenesis. 1,3-butadiene (BD) and 2~,3-dideoxycytidine (ddC) are two carcinogens to which humans are exposed. Cancer bioassays in mice have revealed that both BD and ddC induce high frequencies of lymphomas. The present study provides a genetic dissection of these chemically-induced lymphomas, with a focus on identification of potential turner suppressor loci and genetic alterations in genes involved in the pRb, p53 and Ras/Raf pathways. These pathways are important in the control of cell proliferation.Approximately 87% of BD-induced and 75% of ddC-induced lymphomas show allelic losses or mutations in genes examined. Similar frequencies for inactivation of the p53 pathway were observed in BD- and ddC-induced tumors, whereas disruption of the pRb pathway is more common in ddC-induced lymphomas. On the other hand, BD-induced tumors display more frequent activation of the Ras/Raf pathway. These data indicate the genotoxicity of both ddC and BD, and also confirm the carcinogenicity of these chemicals at a molecular level.This study also reveals that different genetic alterations occur in distinct stages of the development of BD-induced lymphomas. Ras mutations were detected in tumors derived from mice exposed to BD for only 26 weeks or at a rather low concentration (20 ppm), suggesting that ras mutations may occur early in tumor formation. In contrast, all six tumors with aberrations of p53 occurred in the high dose (625 ppm), continuous long-term exposure group, and these tumors appear to have a more aggressive phenotype, indicating that inactivation of p53 may be a late event, associated with progression of BD-induced lymphomas. Furthermore, two or more genetic alterations were found in 67% of tumors from the 625 ppm dose group and in only 46% of lymphomas derived from mice exposed to s312 ppm of BD. In addition, more than five genetic aberrations occurred only in the 625 ppm dose group. These results support the contention that there is a dose-dependent increase of genetic alterations in BD-induced tumors.The mutational pattern resulting from carcinogen-exposure has been observed in both human and animal turners. In the present study, the specific K-ras codon 13 CGC mutation and allelic loss of the Rafl locus on chromosome 6 were detected only in BD-induced lymphomas, while frequent allelic loss of the telomeric region of chromosome 2 was observed only in ddC-induced tumors, suggesting an agent-specific effect.The genome-wide screen of allelic losses revealed that multiple potential tumor suppressor genes contribute to the development of BD- and ddC-induced lymphomas. Moreover, most of the identified regions with frequent allelic losses carry unknown tumor suppressor genes, whose isolation and identification are of great interest for further investigation.
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| 173. |
- Ziedén, Bo
(författare)
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LDL Oxidation Susceptibility and Its Relation To Antioxidants and Fatty Acids : A Clinical and Experimental Study
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Atherosclerosis is an arterial disease characterized by lipid deposits in the artery wall. These lipid deposits partly consist of oxidatively modified low density lipoprotein (LDL). Several lines of evidence indicate a role of oxidized LDL in atherogenesis. The susceptibility of human circulating LDL towards oxidation was therefore considered to be of interest.The LDL oxidation susceptibility was followed spectrophotometrically after addition of copper and was expressed as LDL lag time. We also investigated the plasma concentrations of oxidized cholesterol, lipophilic antioxidative vitamins and fatty acids to get a better understanding of the mechanisms governing LDL oxidation.We compared 50-54 year old men from Vilnius, Lithuania who have a four fold higher coronary heart disease (CHD) mortality compared to men from Link6ping, Sweden. Plasma or LDL cholesterol, blood pressure and smoking could not account for this difference. We found a significantly shorter Jag time in Vilnius men, higher serum concentrations of 7-ß hydroxycholesterol, lower concentrations of y-tocopherol, ß-carotene and lycopene and also higher amount of easily oxidized highly unsaturated fatty acids, such as eicosapentaenioc acid ( EPA). Important determinants of the LDL lag time were found to be EPA (negative), a-tocopherol (positive), but also LDL cholesterol and plasma triglyceride concentrations were associated with a shorter LDL lag time.The effect of ω-3 fatty acid compared to corn oil supplementation was investigated in patients with severe hypertriglyceridernia. Plasma triglyceride concentrations decreased by 48% in the ω-3 group. In both groups the LDL lag time shortened after 12 weeks of supplementation.In vitro addition of the antihypertensive drug captopril to LDL was investigated. Captopril prolonged the LDL lag time, decreased other oxidation products such as thiobarbituric acid reactive substances (TBARS) and lipid peroxides in a dose dependent manner.These studies show that the LDL oxidation susceptibility differs between two groups of healthy men from two populations with a marked difference in CHD mortality. Supplementation with ω-3 fatty acids shortened the LDL lag time. LDL oxidation susceptibility is related to low serum concentrations of lipophilic antioxidative vitamins and high content of long chain highly unsaturated fatty acids.
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| 174. |
- Öberg, Ulrika
(författare)
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Functional assessment system of lower-extremity dysfunction
- 1996
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Functional Assessment System (FAS) is a new instrument for evaluation of lower-extremity dysfunction. It consists of 20 variables, representing major lower--extremity functions related to daily life activities. The variables are divided into five groups: hip impairment, knee impairment, physical disability, social disability, and paiu variables. The grouping agrees fairly well with the WHO classification of impairment, disabilities and handicaps. Every variable is given a disability score on a five-point scale. The scores are plotted into a diagram, giving an individual functional profile. The profile can be used to document present functional status, for goal-setting, and for follow-up after treatment. It can also be used to design individual training programs.The aim of this thesis was:• to present the new Functional Assessment System (FAS)• to examine the metric properties of the FAS, and• to apply the FAS in some clinical situations.The FAS was applied on a series of patients with osteoarthritis of the hip or knee. Content validity was tested with factor analysis. The obtained factor structure agreed very well with the preliminary grouping of the variables. Concurrent validity was tested by comparison with measurements from the AIMS (Arthritis Impact Measurement Scales), the Rosser-Kind index (for evaluation of quality of life), and a radiographic scale for grading of osteoarthritis. There was a good agreement between the measurements of the FAS and the physical variables of the AIMS. There was also a good agreement between the FAS and the results obtained by the Rosser-Kind index. There was a low agreement between the FAS and the psychosocial variables of the AIMS. There was also a low correlation between the radiographic grading of osteoarthritis and the functional status recorded with the FAS. Inter-tester reliability was evaluated as correlation between the measurements performed by two independent physiotherapists. There was an almost perfect agreement between the two testers. The FAS was also tested for discriminatory power. It showed a good ability to differentiate between healthy subjects and patients with osteoarthritis. It also had a good power to discriminate between different degrees of the disease. The disability group of variables had better discriminstory power than the impairment group of variables. Most variables had a high specificity, whereas high sensitivity was mainly found in the disability group of variables and pain. The FAS was used to measure outcome after arthroplasty. A baseline functional status was recorded before surgery, and a goal profile was made. Six months later there was a striking improvement, especially in the disability variables and pain, and there was a high degree of goal achievement of most patients in most variables. Patients admitted for arthroplasty were examined for age-related diffirences in founctional status, Despite the fact that age was not included in the criteria for operation, old people had significantly lower functional status. This finding may indicate a hidden age criterion for referral of patients for arthroplasty. The age-related changes were mainly found in the disability group of variables. These findings may speak in favor of early surgery of patients with osteoarthritis.To summarize: The FAS had very good metric properties, such as validity, intertester reliability, discriminatory power, sensitivity and specificity. It was also a useful instrument in a clinical setting to record functional status, to set an individual treatment goal, and as an instrument for follow-up and outcome measurements. It was also sensitive in detecting age-related differences in patients admitted for arthroplasty. It can be used to communicate functional status to other health professionals, and to establish realistic goals for the patient.
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| 175. |
- Öhman, K. Peter
(författare)
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The kallikrein-kinin system in primary hypertension : dynamics of circulating components of the kallikrein-kinin system in relation to the renin-angiotensin-aldosterone system
- 1997
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Kinins are potent vasodilatory and blood pressure lowering peptides, with additional effects on renal handling of electrolytes and water. The kallikrein-kinin system consists of two separate entities, one confined to the circulation and the other to both tissues and the circulation. The role of the plasma system and the circulating part of the tissue system in circulatory homeostasis and electrolyte and metabolism is unclear, as well as the pathogenetic involvement of these systems in primary hypertension.The aims of the studies were to investigate the possible participation of the plasma kallikrein~ kinin system and the circulating components of the tissue kallikrein-kinin system in the regulation of blood pressure, electrolyte and water balance; possible differences between normotensive and primary hypertensive subjects with respect to these systems; relations between the kallikrein-kinin systems and the renin angiotensin-aldosterone system; evidence of regulated release of prorenin and the possible participation of the kallikrein-kinin systems in the conversion of prorenin to renin.Twentyseven patients with primary hypertension and 16 normotensive control subjects were given furosemide 80 mg p.a. in the first study. In the following 3 studies 15 hypertensive and 15 normotensive subjects participated. Graded i.v. infusion of angiotensin 11 during 3h, i.v. infusion of 2000ml 0.9% sodium chloride during 4h, and fludrocortisone 0.2 mg o.d. p.a. during 7 days were administered in separate protocol.1. Plasma prekallikrein levels did not differ between normotensive and hypertensive subjects. Furosemide increased plasma prekallikrein, angiotensin II had no effect and sodium chloride infusion and fludrocortisone administration reduced plasma prekallikrein. 2. Tissue kallikrein in plasma did not change during these experiments. 3. There were minute differences between normotensive and hypertensive subjects in the activation of both the plasma and the tissue kallikrein-systems and in their relation to blood pressure levels and renal sodium and water handling. 4. Alterations of prorenin levels in plasma evoked by changes in blood pressure, angiotensin II levels, electrolyte and water balance were parallel with those of renin and without any signs of separate specific regulatory mechanisms. 5. There were no signs that the plasma or circulating tissue kallikrein-kinin system participated in the conversion of prorenin to renin. 6. There were no clear functional relations between endogenous mineralocorticoids and circulating tissue kalikrein or plasma prekallikrein.In conclusion: The data demonstrate alterations in the activity of the plasma kallikrein-kinin system, related to electrolyte and volume balance; differences between normotensive and hypertensive subjects, the pathophysiological relevance of which remains to be elucidated; minor differences in the reactivity of circulating tissue kallikrein in hypertensive subjects, but no obvious participation of the circulating components of the tissue system. Hypertensives have higer renin/prorenin ratio there are no signs of specific regulation of prorenin release or participation of circulating kallikreins in the conversion of prorenin to renin. Some of these differences may have relevance to the abnormal blood pressure levels in patients with primary hypertension.
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