Showing papers in "Molecular and Cellular Biochemistry in 2001"
TL;DR: The therapeutic role of Trigonella seed powder in type1 diabetes as exemplified in this study can be attributed to the change of glucose and lipid metabolising enzyme activities to normal values, thus stabilizing glucose homeostasis in the liver and kidney.
Abstract: Trigonella foenum graecum (fenugreek) seed powder has been suggested to have potential antidiabetic effects. The effect of oral administration of Trigonella whole seed powder (5% in the diet) for 21 days on glycolytic, gluconeogenic and NADPlinked lipogenic enzymes were studied in liver and kidney tissues of alloxan-induced diabetic Wistar rats. Diabetic rats were characterised by a 4fold higher blood glucose level and a 0.7fold lower body weight compared to normal controls. The activities of the glycolytic enzymes were significantly lower in the diabetic liver and higher in the diabetic kidney. The activities of gluconeogenic enzymes were higher in both liver and kidney during diabetes, however the activities of the lipogenic enzymes were decreased in both tissues during diabetes. Trigonella seed powder treatment to diabetic rats for 21 days brought down the elevated fasting blood glucose levels to control levels. The altered enzyme activities were significantly restored to control values in both the liver and kidney after Trigonella seed powder treatment. The therapeutic role of Trigonella seed powder in type1 diabetes as exemplified in this study can be attributed to the change of glucose and lipid metabolising enzyme activities to normal values, thus stabilizing glucose homeostasis in the liver and kidney. These biochemical effects exerted by Trigonella seeds make it a possible new therapeutic in type1 diabetes.
297 citations
TL;DR: The results from this study encourage examination of the efficacy of origanum oil in other forms of systemic and superficial fungal infections and exploration of its broad spectrum effect against other pathogenic manifestations including malignancy.
Abstract: The antimicrobial properties of volatile aromatic oils from medicinal as well as other edible plants has been recognized since antiquity. Origanum oil, which is used as a food flavoring agent, possesses a broad spectrum of in vitro antimicrobial activities attributed to the high content of phenolic derivatives such as carvacrol and thymol. In the present study, antifungal properties of origanum oil were examined both in vitro and in vivo. Using Candida albicans in broth cultures and a micro dilution method, comparative efficacy of origanum oil, carvacrol, nystatin and amphotericin B were examined in vitro. Origanum oil at 0.25 mg/ml was found to completely inhibit the growth of C. albicans in culture. Growth inhibitions of 75% and >50% were observed at 0.125 mg/ml and 0.0625 mg/ml level, respectively. In addition, both the germination and the mycelial growth of C. albicans were found to be inhibited by origanum oil and carvacrol in a dose-dependent manner. Furthermore, the therapeutic efficacy of origanum oil was examined in an experimental murine systemic candidiasis model. Groups of mice (n = 6) infected with C. albicans (5 × LD50) were fed varying amounts of origanum oil in a final vol. of 0.1 ml of olive oil (vehicle). The daily administration of 8.6 mg of origanum oil in 100 μl of olive oil/kg body weight for 30 days resulted in 80% survivability, with no renal burden of C. albicans as opposed to the group of mice fed olive oil alone, who died within 10 days. Similar results were obtained with carvacrol. However, mice fed origanum oil exhibited cosmetically better clinical appearance compared to those cured with carvacrol. The results from our study encourage examination of the efficacy of origanum oil in other forms of systemic and superficial fungal infections and exploration of its broad spectrum effect against other pathogenic manifestations including malignancy.
242 citations
TL;DR: Taking together, oxidative stress and oxidative tissue damage, and a cascade of cellular events including modulation of apoptotic regulatory gene p53 are involved in chromium (VI)‐induced toxicity and carcinogenesis.
Abstract: Chromium (VI) is a widely used industrial chemical, extensively used in paints, metal finishes, steel including stainless steel manufacturing, alloy cast irons, chrome, and wood treatment. On the contrary, chromium (III) salts such as chromium polynicotinate, chromium chloride and chromium picolinate, are used as micronutrients and nutritional supplements, and have been demonstrated to exhibit a significant number of health benefits in rodents and humans. However, the cause for the hexavalent chromium to induce cytotoxicity is not entirely understood. A series of in vitro and in vivo studies have demonstrated that chromium (VI) induces an oxidative stress through enhanced production of reactive oxygen species (ROS) leading to genomic DNA damage and oxidative deterioration of lipids and proteins. A cascade of cellular events occur following chromium (VI)‐induced oxidative stress including enhanced production of superoxide anion and hydroxyl radicals, increased lipid peroxidation and genomic DNA fragmentation, modulation of intracellular oxidized states, activation of protein kinase C, apoptotic cell death and altered gene expression. In this paper, we have demonstrated concentration‐ and time‐dependent effects of sodium dichromate (chromium (VI) or Cr (VI)) on enhanced production of superoxide anion and hydroxyl radicals, changes in intracellular oxidized states as determined by laser scanning confocal microscopy, DNA fragmentation and apoptotic cell death (by flow cytometry) in human peripheral blood mononuclear cells. These results were compared with the concentration-dependent effects of chromium (VI) on chronic myelogenous leukemic K562 cells and J774A.1 murine macrophage cells. Chromium (VI)‐induced enhanced production of ROS, as well as oxidative tissue and DNA damage were observed in these cells. More pronounced effect was observed on chronic myelogenous leukemic K562 cells and J774A.1 murine macrophage cells. Furthermore, we have assessed the effect of a single oral LD50 dose of chromium (VI) on female C57BL/6Ntac and p53‐deficient C57BL/6TSG p53 mice on enhanced production of superoxide anion, lipid peroxidation and DNA fragmentation in the hepatic and brain tissues. Chromium (VI)‐induced more pronounced oxidative damage in p53 deficient mice. This in vivo study highlighted that apoptotic regulatory protein p53 may play a major role in chromium (VI)‐induced oxidative stress and toxicity. Taken together, oxidative stress and oxidative tissue damage, and a cascade of cellular events including modulation of apoptotic regulatory gene p53 are involved in chromium (VI)‐induced toxicity and carcinogenesis.
224 citations
TL;DR: The primary aim of this review was to critically evaluate the literature that has examined the regulation of cellular total Cr content and identify two CreaT genes, which are primarily responsible for cellular Cr uptake.
Abstract: The cellular role of creatine (Cr) and Cr phosphate (CrP) has been studied extensively in neural, cardiac and skeletal muscle. Several studies have demonstrated that alterations in the cellular total Cr (Cr + CrP) concentration in these tissues can produce marked functional and/or structural change. The primary aim of this review was to critically evaluate the literature that has examined the regulation of cellular total Cr content. In particular, the review focuses on the regulation of the activity and gene expression of the Cr transporter (CreaT), which is primarily responsible for cellular Cr uptake. Two CreaT genes (CreaT1 and CreaT2) have been identified and their chromosomal location and DNA sequencing have been completed. From these data, putative structures of the CreaT proteins have been formulated. Transcription products of the CreaT2 gene are expressed exclusively in the testes, whereas CreaT1 transcripts are found in a variety of tissues. Recent research has measured the expression of the CreaT1 protein in several tissues including neural, cardiac and skeletal muscle. There is very little information available about the factors regulating CreaT gene expression. There is some evidence that suggests the intracellular Cr concentration may be involved in the regulatory process but there is much more to learn before this process is understood. The activity of the CreaT protein is controlled by many factors. These include substrate concentration, transmembrane Na+ gradients, cellular location, and various hormones. It is also likely that transporter activity is influenced by its phosphorylation state and by its interaction with other plasma membrane proteins. The extent of CreaT protein glycosylation may vary within cells, the functional significance of which remains unclear.
182 citations
TL;DR: These studies illustrate that in vivo EPR spectroscopy and magnetic resonance imaging can be very useful and powerful tools for studying paramagnetic metal ions in chemical and biochemical reactions occurring in intact animals.
Abstract: Chromium (VI) compounds are widely recognized as human carcinogens. Extensive studiesin vitroand in model systems indicate that the reactive intermediate, Cr (V), generated by cellular reduction of Cr (VI), is likely the candidate for the ultimate carcinogenic form of chromium compounds. Here we review our current understanding of the in vivo reduction of Cr (VI) and its related free radical generation. Our results demonstrate that Cr (V) is indeed generated from the reduction of Cr (VI)in vivoand that Cr (V) thus formed can mediate the generation of free radicals. Cr (V) and its related free radicals are very likely to be involved in the mechanism of Cr (Vl)-induced toxicity and carcinogenesis. These studies also illustrate thatin vivoEPR spectroscopy and magnetic resonance imaging can be very useful and powerful tools for studying paramagnetic metal ions in chemical and biochemical reactions occurring in intact animals. (Mol Cell Biochem 222: 41-47, 2001)
151 citations
TL;DR: Os may be of therapeutic and prophylactic value in the treatment of MI and was found to demonstrate maximum cardioprotective effect, which was confirmed by histopathological findings.
Abstract: Myocardial infarction (MI∥ was produced in rats with 85, 200 and 300 mg/kg of isoproterenol (ISO) administered subcutaneously (sc) twice at an interval of 24 h. Shift in antioxidant parameters, lactate dehydrogenase (LDH) together with morphological and histopathological changes were investigated. Two hundred mg/kg ISO dose was selected for the present study as this dose offered significant alteration in biochemical parameters along with moderate necrosis in heart. Effect of pre‐ and co‐treatment of hydroalcoholic extract of Ocimum sanctum (Os) at different doses (25, 50, 75, 100, 200 and 400 mg/kg) was investigated against ISO (200 mg/kg) induced myocardial infarction in rats. Modulation of various biochemical parameters and membrane integrity was studied. Os at the dose of 25, 50, 75 and 100 mg/kg reduced significantly glutathione (GSH), superoxide dismutase (SOD) and LDH levels. It also inhibited the lipid peroxidation as observed by the reduced thiobarbituric acid reactive substances (TBARS) levels. In the present study Os at the dose of 50 mg/kg was found to demonstrate maximum cardioprotective effect. Above results were further confirmed by histopathological findings. Thus from the present study it is concluded that Os may be of therapeutic and prophylactic value in the treatment of MI.
143 citations
TL;DR: The results show arsenic contaminated groundwater from tubule‐type wells of depths about 15–30 m was serious compared with open-type wells where depth is about 3–5 m, and the prevalence of arsenical dermatosis was highest in the over 40‐year‐old age group.
Abstract: An investigation was carried out on arsenic contamination of groundwater and prevalence of arsenical dermatosis in the Hetao plain of Inner Mongolia Autonomous Region, China. Based on the screening of water samples from 96 randomly selected wells in this Region, two areas (Wuyuan and Alashan) were chosen as highly contaminated areas because arsenic in the water samples was higher than 50 μg/l. Arsenic was measured using an arsenic silver diethyl dithiocarbamate method for 326 water samples from all the wells in these areas. The results show arsenic contaminated groundwater from tubule‐type wells of depths about 15–30 m was serious compared with open-type wells where depth is about 3–5 m. In the Wuyuan area, 96.2% of water samples from tubule-type wells contained arsenic above 50 μg/l and 69.3% in Alashan area; the highest value was 1354 μg/l and 1088 μg/l, respectively. In these two areas, a health survey was carried out for arsenical dermatosis. The results show the prevalence of arsenical dermatosis in the Wuyuan area was 44.8%, higher than 37.1% prevalence of arsenical dermatosis in the Alashan area. The prevalence of arsenical dermatosis was highest in the over 40‐year‐old age group. There was no sex difference in the prevalence. Further investigation is needed to clarify the actual situation of arsenic pollution of groundwater in Inner Mongolia, China in order to reduce the adverse health effect among residents exposed to arsenic.
141 citations
TL;DR: This study shows that the cardioprotection of arjunolic acid pre and post treatment could possibly be due to the protective effect against the damage caused by myocardial necrosis.
Abstract: Arjunolic acid, a new triterpene and a potent principle from the bark of Terminalia arjuna, has been shown to provide significant cardiac protection in isoproterenol induced myocardial necrosis in rats. To further explore the mechanism of action of arjunolic acid, antiplatelet activity, anticoagulant assays, electrocardiographic changes, serum marker enzymes, antioxidant status, lipid peroxide and myeloperoxidase (MPO) have been measured and the results are compared with a potent cardioprotective drug, acetyl salicylic acid (ASA). Administration of isoproterenol produces electrocardiographic changes such as decreased R amplitude and increased ST segment elevation and has resulted in an increase in serum marker enzyme levels as well as a decrease in enzymatic and nonenzymatic antioxidant levels. Arjunolic acid at an effective dosage of 15 mg/kg body wt. (pre and post treatment), when administered intraperitoneally (i.p.), effects a decrease in serum enzyme levels and the electrocardiographic changes get restored towards normalcy. Arjunolic acid treatment is also shown to prevent the decrease in the levels of superoxide dismutase, catalase, glutathione peroxidase, ceruloplasmin, alpha-tocopherol, reduced glutathione (GSH), ascorbic acid, lipid peroxide, MPO and the cardioprotection is confirmed by the histopathological studies. This study shows that the cardioprotection of arjunolic acid pre and post treatment could possibly be due to the protective effect against the damage caused by myocardial necrosis.
136 citations
TL;DR: CK2 may promote breast cancer through dysregulation of key pathways of transcriptional control in the mammary epithelium, and inhibition of CK2 has a potential role in the treatment of breast and other cancers.
Abstract: Breast cancer is a major cause of cancer death in women, and the genetic abnormalities leading to the common sporadic forms of the disease are still under active investigation. CK2 has been reported to be upregulated in human breast cancer, which these studies confirm; CK2 is also upregulated in rat carcinogen-induced breast tumors. Transgenic mice overexpressing CK2α in the mammary gland develop mammary hyperplasia, dysplasia, and eventually adenocarcinomas, demonstrating that dysregulated expression of CK2 can contribute to transformation of the mammary epithelium. These mammary tumors have evidence of activation of the Wnt and NFκB pathways and upregulation of c-Myc. CK2 is capable of phosphorylating the key signaling molecule in the Wnt pathway, the transcriptional cofactor β-catenin, and regulating its turnover. CK2 is known to phosphorylate IκB and thereby regulate basal NFκB levels; in the mammary cell lines and tumors, CK2 activity correlates with NFκB levels and inhibition of CK2 downregulates NFκB. Thus, CK2 may promote breast cancer through dysregulation of key pathways of transcriptional control in the mammary epithelium, and inhibition of CK2 has a potential role in the treatment of breast and other cancers.
126 citations
TL;DR: This overview provides a description of the essential points and technical improvements enable the distinction of active and latent TGF-β proteins in vivo and have started to reveal anomalies in the control of activation in relation to various pathological situations.
Abstract: The latency associated with the transforming growth factor-betas (TGF-betas) was discovered in 1984. Since the two publications on this subject in that year, there has been on average over sixty reports in which latency was the dominant theme for each of the past 10 years, proof enough of the interest in this field of growth factor research. As the mature 25 kD forms of the TGF-betas are required for them to exert their many, diverse biological effects, it was inevitable that an explanation of the structure and of the activation of the latent complexes be sought. This overview provides a description of these essential points. Now that it has been clearly shown that dysregulation of particular components of the TGF-β signalling pathway is implicated in many human diseases, the activation of the latent TGF-β complexes has taken on added importance. Technical improvements enable the distinction of active and latent TGF-β proteins in vivo and have started to reveal anomalies in the control of activation in relation to various pathological situations.
116 citations
TL;DR: The hypothesis that the stability of mRNA mediated by the 3′-untranslated region (3′-UTR) plays a role in changing gene expression in cardiovascular pathophysiology is presented and concepts will aid in better understanding cardiovascular diseases and in developing new therapies are discussed.
Abstract: Knowledge of transcription and translation has advanced our understanding of cardiac diseases. Here, we present the hypothesis that the stability of mRNA mediated by the 3′-untranslated region (3′-UTR) plays a role in changing gene expression in cardiovascular pathophysiology. Several proteins that bind to sequences in the 3′-UTR of mRNA of cardiovascular targets have been identified. The affected mRNAs include those encoding β-adrenergic receptors, angiotensin II receptors, endothelial and inducible nitric oxide synthases, cyclooxygenase, endothelial growth factor, tissue necrosis factor (TNF-α), globin, elastin, proteins involved in cell cycle regulation, oncogenes, cytokines and lymphokines. We discuss: (a) the types of 3′-UTR sequences involved in mRNA stability, (b) AUF1, HuR and other proteins that bind to these sequences to either stabilize or destabilize the target mRNAs, and (c) the potential role of the 3′-UTR mediated mRNA stability in heart failure, myocardial infarction and hypertension. We hope that these concepts will aid in better understanding cardiovascular diseases and in developing new therapies.
TL;DR: In vitro carnosine inhibited lipid peroxidation and oxidative modification of protein in muscle tissue prepared from rat hind limb homogenates exposed to in vitro Fenton reactant (Fe2+, H2O2)‐generated free radicals.
Abstract: Carnosine, a beta-alanyl-L-histidine dipeptide with antioxidant properties is present at high concentrations in skeletal muscle tissue. In this study, we report on the antioxidant activity of carnosine on muscle lipid and protein stability from both in vitro and in vivo experiments. Carnosine inhibited lipid peroxidation and oxidative modification of protein in muscle tissue prepared from rat hind limb homogenates exposed to in vitro Fenton reactant (Fe2+, H2O2)-generated free radicals. The minimum effective concentrations of carnosine for lipid and protein oxidation were 2.5 and 1 mM, respectively. Histidine and beta-alanine, active components of carnosine, showed no individual effect towards inhibiting either lipid or protein oxidation. Skeletal muscle of rats fed a histidine supplemented diet for 13 days exhibited a marked increase in carnosine content with a concomitant reduction in muscle lipid peroxidation and protein carbonyl content in skeletal muscle caused by subjecting rats to a Fe-nitrilotriacetate administration treatment. This significant in vitro result confirms the in vivo antioxidant activity of carnosine for both lipid and protein constituents of muscle under physiological conditions.
TL;DR: The results indicate that AII, ET-1 and TGF-β (released by cardiac and vascular cell types) act as autocrine/paracrine mediators of stretch-induced cardiomyocyte hypertrophy.
Abstract: Cardiac hypertrophy is a compensatory response of myocardial tissue upon increased mechanical load. Of the mechanical factors, stretch is rapidly followed by hypertrophic responses. We tried to elucidate the role of angiotensin II (AII), endothelin-1 (ET-1) and transforming growth factor-beta (TGF-beta) as autocrine/paracrine mediators of stretch-induced cardiomyocyte hypertrophy. We collected conditioned medium (CM) from stretched cardiomyocytes and from other stretched cardiac cells, such as cardiac fibroblasts, endothelial cells and vascular smooth muscle cells (VSMCs). These CMs were administered to stationary cardiomyocytes with or without an AII type 1 (AT1) receptor antagonist (losartan), an ET-1 type A (ET(A)) receptor antagonist (BQ610), or anti-TGF-beta antibodies. By measuring the mRNA levels of the proto-oncogene c-fos and the hypertrophy marker gene atrial natriuretic peptide (ANP), the molecular phenotype of the CM-treated stationary cardiomyocytes was characterized. Our results showed that c-fos and ANP expression in stationary cardiomyocytes was increased by All release from cardiomyocytes that had been stretched for 60 min. Stretched cardiomyocytes, cardiac fibroblasts and endothelial cells released ET-1 which led to increased c-fos and ANP expression in stationary cardiomyocytes. ET-1 released by stretched VSMCs, and TGF-beta released by stretched cardiac fibroblasts and endothelial cells, appeared to be paracrine mediators of ANP expression in stationary cardiomyocytes. These results indicate that AII, ET-1 and TGF-beta (released by cardiac and vascular cell types) act as autocrine/paracrine mediators of stretch-induced cardiomyocyte hypertrophy. Therefore, it is likely that in stretched myocardium the cardiomyocytes, cardiac fibroblasts, endothelial cells and VSMCs take part in intercellular interactions contributing to cardiomyocyte hypertrophy.
TL;DR: The findings that liver PPARγ2 expression is significantly elevated in the ob/ob model of diabetes and positively associated with an index of insulin resistance, suggests a possible compensatory response through which type II diabetic and obese organisms strive to maintain insulin sensitivity of the liver.
Abstract: Studies of the molecular basis of insulin resistance have focused on the peroxisome proliferator activated receptor gamma (PPARgamma, gamma1 and gamma2). The aim of this study was to determine whether the insulin resistance in liver of diabetic animals is associated with abnormal expression of these receptors. PPARgamma mRNA and protein expression levels were quantified in liver of 9-week-old male ob/ob mice as a model of diabetes and compared to age- and gender-matched wild type control animals of the same genetic background. Semi-quantitative reverse transcription-polymerase chain reaction, using 18S rRNA as an internal standard, indicated that PPARgamma2 mRNA was significantly upregulated in ob/ob liver vs. that in wild type mice. Western blotting revealed greater immunoreactivity of PPARgamma2 in liver from ob/ob mice relative to that in wild type mice. An index of insulin resistance (product of serum glucose and insulin concentration) was correlated with liver PPARgamma2 mRNA expression (r = 0.776; p < 0.001). The findings that liver PPARgamma2 expression is (1) significantly elevated in the ob/ob model of diabetes and (2) positively associated with an index of insulin resistance, suggests a possible compensatory response through which type II diabetic and obese organisms strive to maintain insulin sensitivity of the liver.
TL;DR: The apparent molecular mass of L-asparaginase by SDS-PAGE was found to be 33 kDa, whereas by its mobility on Sephacryl S-300 superfine column was around 200 k Da, indicating that the enzyme at the native stage acts as hexamer.
Abstract: L-asparaginase EC 3511 was purified to homogeneity from Thermus thermophilus The apparent molecular mass of L-asparaginase by SDS-PAGE was found to be 33 kDa, whereas by its mobility on Sephacryl S-300 superfine column was around 200 kDa, indicating that the enzyme at the native stage acts as hexamer The purified enzyme showed a single band on acrylamide gel electrophoresis with pI = 60 The optimum pH was 92 and the Km for L-asparagine was 28 mM It is a thermostable enzyme and it follows linear kinetics even at 77°C Chemical modification experiments implied the existence of histidyl, arginyl and a carboxylic residues located at or near active site while serine and mainly cysteine seems to be necessary for active form
TL;DR: It is concluded that the particularly effective antioxidant properties of EGCG noted in both chemical and biological biphasic systems were related to a unique hydrophilic and lipophilic balance which enabled effective free radical scavenging.
Abstract: The antioxidant activity of epigallocatechin gallate (EGCG) was studied in different in vitro model systems, which enabled evaluation of both chemical and physical factors involved in assessing the role of EGCG in oxidative reactions. EGCG suppressed the initiation rate and prolonged the lag phase duration of peroxyl radical-induced oxidation in a phospholipid liposome model to a greater extent (p < 0.01) compared to both Trolox and α-tocopherol. Effectiveness of these antioxidants to prolong the peroxyl radical-induced lag phase was inversely related to lipophilic character. EGCG also protected against both peroxyl radical and hydroxyl radical-induced supercoiled DNA nicking. The rate constant describing EGCG reaction against hydroxyl radical was 4.22 ± 0.07 × 1010 M–1·sec–1, which was comparable to those of Trolox and α-tocopherol, respectively. EGCG exhibited a synergistic effect with α-tocopherol in scavenging 1,1-diphenyl-2-picylhydrazyl (DPPH) radical, thus displaying a direct free radical scavenging capacity. In vitro Cu2+-induced-human LDL oxidation was accelerated in the presence of EGCG and attributed to the conversion of Cu2+ to Cu+. We conclude that the particularly effective antioxidant properties of EGCG noted in both chemical and biological biphasic systems were related to a unique hydrophilic and lipophilic balance which enabled effective free radical scavenging. The same chemical-physical properties of EGCG also enabled prooxidant activity, only when in contact with unbound transition metal ions in a multiphasic system.
TL;DR: This mini-review addresses the mechanisms of cell death induced by some carcinogenic metals, including arsenic (III), chromium (VI) and vanadium (V).
Abstract: Well-documented evidence suggests that environmental and occupational exposure of toxic metals or metal-containing compounds can cause a number of human diseases, including inflammation and cancer, through DNA damage, protein modifications, or lipid peroxidation. This mini-review addresses the mechanisms of cell death induced by some carcinogenic metals, including arsenic (III), chromium (VI) and vanadium (V). A possible contribution of reactive oxygen species to metal-induced cell death is also discussed.
TL;DR: This in vitro study demonstrated a lymphocyte proliferative response to both Co‐Cr‐Mo and Ti alloy metalloprotein degradation products, and was greatest when the metals were complexed with high molecular weight proteins, and with metal‐protein complexes formed from Co‐ Cr‐Mo alloy degradation.
Abstract: Metal toxicity from sources such as orthopaedic implants was investigated in terms of immune system hyper-reactivity to metal implant alloy degradation products. Lymphocyte response to serum protein complexed with metal from implant alloy degradation was investigated in thisin vitrostudy using primary human lymphocytes from healthy volunteers (n = 10). Cobalt chromium molybdenum alloy (Co-Cr-Mo, ASTM F-75) and titanium allay (Ti-6A1-4V, ASTM F-136) beads (70 um) were incubated in agitated human serum at 37 degrees Celsius to simulate naturally occurring metal implant alloy degradation processes. Particulate free serum samples, which were incubated with metal, were then separated into molecular weight based fractions. The amounts of soluble Cr and Ti within each serum fraction were measured and correlated with lymphocyte proliferation response to the individual serum fractions. Lymphocytes from each subject were cultured with 11 autologaus molecular weight based serum fractions either with or without added metal. Two molecular weight ranges of human serum proteins were associated with the binding of Cr and Ti from Co-Cr-Mo and Ti implant alloy degradation (at < 30 and 180-330 kDa). High molecular weight serum proteins 180 kDa) demonstrated greater lymphocyte reactivity when complexed with metal released from Co-Cr-Mo alloy and Ti alloy than with low (5-30 kDa) and midrange (30-77 kDa) serum proteins. When the amount of lymphocyte stimulation was normalized to both the moles of metal and the moles of protein within each fraction (Metal-Protein Complex Reactivity Index, MPCRI), Cr from Co-Cr-Mo alloy degradation demonstrated approximately 10 fold greater reactivity than Ti in the higher molecular weight serum proteins (~ 180-250 kDa). Thisin vitrostudy demonstrated a lymphocyte proliferative response to both Co-Cr-Mo and Ti alloy metalloprotein degradation products. This response was greatest when the metals were complexed with high molecular weight proteins, and with metal-protein complexes formed from Co-Cr-Mo alloy degradation. (Mol Cell Siochem222:127-136, 2001)
TL;DR: Significantly, the effects of the antisense ODN on the CK2 activity in the nuclear matrix were relatively small compared to the much stronger induction of apoptosis in cells, suggesting that modest down-regulation of CK2 can evoke a much greater apoptotic response in cancer cells.
Abstract: Protein kinase CK2 is one of the key cellular signals for cell survival, growth, and proliferation. It is has been observed to be elevated in various cancers that have been examined. Various observations suggest that moderate dysregulation of CK2 may profoundly influence the cell response. We have examined the effects of interfering with the CK2 signal in various cancer cell lines by employing antisense oligodeoxynucleotides (ODN) against the α and β subunits of CK2. Our results demonstrate that antisense CK2-α and antisense CK2-β ODNs markedly influence cell viability of these cancer cells in a dose and time-dependent manner. Antisense CK2-α was slightly more effective than antisense CK2-β in most of the cells tested. The efficacy of the antisense ODN seemed to vary with the cell type; however, in all cases potent induction of apoptosis was observed. Significantly, the effects of the antisense ODN on the CK2 activity in the nuclear matrix were relatively small compared to the much stronger induction of apoptosis in cells. This suggests that modest downregulation of CK2 can evoke a much greater apoptotic response in cancer cells. (Mol Cell Biochem 227: 167-174, 2001)
TL;DR: The potent inhibitory effect of low concentrations of GSPE on agonist-induced VCAM-1 expression suggests therapeutic potential of this extract in inflammatory conditions and other pathologies involving altered expression of VCAM -1.
Abstract: Altered expression of cell adhesion molecule expression has been implicated in a variety of chronic inflammatory conditions. Regulation of adhesion molecule expression by specific redox sensitive mechanisms has been reported. Grape seed proanthocyanidins have been reported to have potent antioxidant properties. We evaluated the effects of grape seed proanthocyanidin extract (GSPE) on the expression of TNFα-induced ICAM-1 and VCAM-1 expression in primary human umbilical vein endothelial cells (HUVEC). GSPE at low concentrations (1-5 μg/ml), down-regulated TNFα-induced VCAM-1 expression but not ICAM-1 expression in HUVEC. Such regulation of inducible VCAM-1 by GSPE was also observed at the mRNA expression level. A cell-cell co-culture assay was performed to verify whether the inhibitory effect of GSPE on the expression of VCAM-1 was also effective in down-regulating actual endothelial cell/leukocyte interaction. GSPE treatment significantly decreased TNFα-induced adherence of T-cells to HUVEC. Although several studies have postulated NF-κB as the molecular site where redox active substances act to regulate agonist-induced ICAM-1 and VCAM-1 gene expression, inhibition of inducible VCAM-1 gene expression by GSPE was not through a NF-κB-dependent pathway as detected by a NF-κB reporter assay. The potent inhibitory effect of low concentrations of GSPE on agonist-induced VCAM-1 expression suggests therapeutic potential of this extract in inflammatory conditions and other pathologies involving altered expression of VCAM-1.
TL;DR: This study indicates that activation of either A1 or A3 adenosine receptors in the rat can attenuate myocyte injury during hypoxia, and highly selective A1R and A3R agonists may have potential as cardioprotective agents against ischemia or heart surgery.
Abstract: Adenosine (ADO) is a well-known regulator of a variety of physiological functions in the heart. In stress conditions, like hypoxia or ischemia, the concentration of adenosine in the extracellular fluid rises dramatically, mainly through the breakdown of ATP. The degradation of adenosine in the ischemic myocytes induced damage in these cells, but it may simultaneously exert protective effects in the heart by activation of the adenosine receptors. The contribution of ADO to stimulation of protective effects was reported in human and animal hearts, but not in rat hearts. The aim of this study was to evaluate the role of adenosine A1 and A3 receptors (A1R and A3R), in protection of isolated cardiac myocytes of newborn rats from ischemic injury. The hypoxic conditions were simulated by exposure of cultured rat cardiomyocytes (4–5 days in vitro), to an atmosphere of a N2 (95%) and CO2 (5%) mixture, in glucose-free medium for 90 min. The cardiotoxic and cardioprotective effects of ADO ligands were measured by the release of lactate dehydrogenase (LDH) into the medium. Morphological investigation includes immunohistochemistry, image analysis of living and fixed cells and electron microscopy were executed. Pretreatment with the adenosine deaminase considerably increased the hypoxic damage in the cardiomyocytes indicating the importance of extracellular adenosine. Blocking adenosine receptors with selective A1 and A3 receptor antagonists abolished the protective effects of adenosine. A1R and A3R activation during the hypoxic insult delays onset of irreversible cell injury and collapse of mitochondrial membrane potential as assessed using DASPMI fluorochrom. Cardioprotection induced by the A1R agonist, CCPA, was abolished by an A1R antagonist, DPCPX, and was not affected by an A3R antagonist, MRS1523. Cardioprotection caused by the A3R agonist, Cl-IB-MECA, was antagonized completely by MRS1523 and only partially by DPCPX. Activation of both A1R and A3R together was more efficient in protection against hypoxia than by each one alone. Our study indicates that activation of either A1 or A3 adenosine receptors in the rat can attenuate myocyte injury during hypoxia. Highly selective A1R and A3R agonists may have potential as cardioprotective agents against ischemia or heart surgery.
TL;DR: In conclusion, quercetin shows both antihypertensive and antioxidant properties in this model of genetic hypertension.
Abstract: The effects of chronic administration of an oral daily dose of quercetin (10 mg Kg−1), the most abundant dietary flavonoid, were investigated on hepatic oxidative status in spontaneously hypertensive rats and normotensive Wistar Kyoto rats. Decreased liver glutathione peroxidase activity, increased liver total glutathione levels and increased both hepatic and plasmatic malondialdehyde concentrations were observed in spontaneously hypertensive rats when compared to Wistar Kyoto rats. In spontaneously hypertensive rats, treatment with quercetin for 5 weeks reduced blood pressure, increased glutathione peroxidase activity and reduced both plasma and hepatic malondialdehyde levels. However, none of these effects were observed in Wistar Kyoto rats. In conclusion, quercetin shows both antihypertensive and antioxidant properties in this model of genetic hypertension.
TL;DR: The results suggest that PTP1B is a target protein for the insulin receptor tyrosine kinase and PTP2B can regulate its own phosphatase activity by maintaining the balance between its phosphorylated and dephosphorylated state.
Abstract: We have previously reported a direct in vivo interaction between the activated insulin receptor and protein-tyrosine phosphatase-1B (PTP1B), which leads to an increase in PTP1B tyrosine phosphorylation. In order to determine if PTP1B is a substrate for the insulin receptor tyrosine kinase, the phosphorylation of the Cys 215 Ser, catalytically inactive mutant PTP1B (CS-PTP1B) was measured in the presence of partially purified and activated insulin receptor. In vitro, the insulin receptor tyrosine kinase catalyzed the tyrosine phosphorylation of PTP1B. 53% of the total cellular PTP1B became tyrosine phosphorylated in response to insulin in vivo. Tyrosine phosphorylation of PTP1B by the insulin receptor was absolutely dependent upon insulin-stimulated receptor autophosphorylation and required an intact kinase domain, containing insulin receptor tyrosines 1146, 1150 and 1151. Tyrosine phosphorylation of wild type PTP1B by the insulin receptor kinase increased phosphatase activity of the protein. Intermolecular transdephosphorylation was demonstrated both in vitro and in vivo, by dephosphorylation of phosphorylated CS-PTP1B by the active wild type enzyme either in a cell-free system or via expression of the wild type PTP1B into Hirc-M cell line, which constitutively overexpress the human insulin receptor and CS-PTP1B. These results suggest that PTP1B is a target protein for the insulin receptor tyrosine kinase and PTP1B can regulate its own phosphatase activity by maintaining the balance between its phosphorylated (the active form) and dephosphorylated (the inactive form) state.
TL;DR: Results indicate that Cr-induced DNA interstrand cross links, and not DNA-Cr-GSH crosslinks, are the principal lesions responsible for blocking DNA replication.
Abstract: Hexavalent chromium (Cr (VI)) is reduced intracellularly to Cr (V), Cr (IV) and Cr (HI) by ascorbate (Asc), cysteine and glutathione (GSH). These metabolites induce a spectrum of genomic DNA damage resulting in the inhibition of DNA replication. Our previous studies have shown that treatment of DNA with Cr (III) or Cr (VI) plus Asc results in the formation of DNA-CrDNA crosslinks (Cr-DDC) and guanine-specific arrests of both prokaryotic and mammalian DNA polymerases. GSH not only acts as a reductant of Cr (VI) but also becomes crosslinked to DNA by Cr, thus, the focus of the present study was to examine the role of GSH in Cr-induced DNA damage and polymerase arrests. Co-incubation of Cr (III) with plasmid DNA in the presence of GSH led to the crosslinking of GSH to DNA. GSH co-treatment with Cr (III) also led to a decrease in the degree of Cr-induced DNA interstrand crosslinks relative to Cr(III)alone, without affecting total Cr DNA binding. DNA polymerase arrests were observed following treatment of DNA with Cr (III) alone, but were markedly reduced when GSH was added to the reaction mixture. Pre-formed polymerase-arresting lesions (Cr-DDC) were not removed by subsequent addition of GSH. Treatment of DNA with Cr (VI), in the presence of GSH, resulted in crosslinking of GSH to DNA, but failed to produce detectable DNA interstrand crosslinks or polymerase arrests. The inhibitory effect of GSH on Cr-induced polymerase arrest was further confirmed in human genomic DNA using quantitative PCR (QPCR) analysis. Treatment of genomic DNA with Cr (III) resulted in a marked inhibition of the amplification of a 1.6 kb target fragment of the p53 gene byTagpolymerase. This was almost completely prevented by co-treatment with GSH and Cr (III). These results indicate that Cr-induced DNA interstrand crosslinks, and not DNA-Cr-GSH crosslinks, are the principal lesions responsible for blocking DNA replication. Moreover, the formation of DNA-Cr-GSH crosslinks may actually preclude the formation of the polymerase arresting lesions. (Mol Cell Biochem222:173-182, 2001)
TL;DR: The radioresponse of antioxidant enzymes seemed to be significantly different in the liver of tumorburdened mice compared to controls, and the enhanced activities might be due to relatively more damage caused by radiation.
Abstract: Since the radiation dose tolerance of normal tissues/organs away from the site of tumor influences the success of radiation therapy of cancer, and antioxidant status is likely to be one of the factors to determine the tolerance; the radioresponse of antioxidant enzymes has been examined in the liver as a representative distant organ in the tumorbearing mice.
TL;DR: The results indicate that chronic alterations in thyroid status specially affect oxidative damage to lipids in skeletal muscle, with a probably stronger effect on mitochondrial membranes, whereas the cytosolic redox potential and DNA are better protected possibly due to homeostatic compensatory reactions on the long‐term.
Abstract: While the biochemical literature on free radical metabolism is extensive, there is little information on the endocrine control of tissue oxidative stress, and in the case of thyroid hormones it is mainly limited to liver tissue and to short‐term effects on a few selected biochemical parameters. In this investigation, chronic hypothyroidism and hyperthyroidism were successfully induced in mice, and various oxidative‐stress‐related parameters were studied in skeletal muscle. In vivo and in vitro lipid peroxidation significantly increased in hyperthyroidism and did not change in the hypothyroid state. The fatty acid composition of the major phospholipid classes was affected by thyroid hormones, leading to a significant decrease in total fatty acid unsaturation both in hypothyroid and hyperthyroid muscle in phosphatidylcholine and phosphatidylethanolamine fractions. In cardiolipin, however, the double bond content significantly increased as a function of thyroid status, leading to a 2.7 fold increase in the peroxidizability index from euthyroid to hyperthyroid muscle. Cardiolipin content was also directly and significantly related to thyroid state across the three groups. Glutathione system was not modified by thyroid state. The oxidative damage marker 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine did not change in mitochondrial DNA, and decreased in genomic DNA both in hypothyroid and hyperthyroid muscle. The results indicate that chronic alterations in thyroid status specially affect oxidative damage to lipids in skeletal muscle, with a probably stronger effect on mitochondrial membranes, whereas the cytosolic redox potential and DNA are better protected possibly due to homeostatic compensatory reactions on the long‐term.
TL;DR: The findings suggest that the Th2 cell response induced by melatonin in antigen sensitised mice neither dependent on endogenous opioid system nor is modulated through the central or peripheral benzodiazepine receptors.
Abstract: Chronic administration of melatonin for 5 days to antigen-primed mice increased the production of pro-inflammatory cytokine IL-10 but decreased the secretion of anti-inflammatory cytokine TNF-alpha. These results further confirm that melatonin activates Th2-like immune response. Whether melatonin-mediated Th2 response is dependent on opioid or central and peripheral benzodiazepine receptors was also examined. Hence, melatonin was administered to antigen-sensitised mice with either naltrexone (a mu opioid receptor antagonist) or flumazenil (a central benzodiazepine receptor antagonist) or PK11195 (a peripheral benzoidiazepine receptor antagonist). No significant difference in melatonin-induced Th2 cell response was observed by naltrexone, flumazenil or PK11195 treatment. These findings suggest that the Th2 cell response induced by melatonin in antigen sensitised mice neither dependent on endogenous opioid system nor is modulated through the central or peripheral benzodiazepine receptors.
TL;DR: The process of directed evolution is reviewed and its application to improve substrate specificity, activity, enantioselectivity and thermal stability is reviewed.
Abstract: Directed evolution is a new process for developing industrially viable biocatalysts This technique does not require a comprehensive knowledge of the relationships between sequence structure and function of proteins as required by protein engineering It mimics the process of Darwinian evolution in a test tube combining random mutagenesis and recombination with screening or selection for enzyme variants that have the desired properties Directed evolution helps in enhancing the enzyme performance both in natural and synthetic environments This article reviews the process of directed evolution and its application to improve substrate specificity, activity, enantioselectivity and thermal stability
TL;DR: Results indicate that homocysteine stimulates MCP-1 expression in endothelial cells leading to enhanced monocyte chemotaxis in patients with hyperhomocysteinemia.
Abstract: Hyperhomocysteinemia has been identified as an independent risk factor for atherosclerosis. The infiltration of monocytes into the arterial wall is one of the key events during atherogenesis. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that stimulates the migration of monocytes into the intima of the arterial wall. The mechanism by which increased monocyte infiltration occurs in atherosclerotic lesions in patients with hyperhomocysteinemia has not been delineated. The objective of the present study was to investigate the effect of homocysteine on MCP-1 production in endothelial cells. Cells were incubated with homocysteine. The secretion of MCP-1 protein was significantly increased (195% as compared to the control) in cells treated with pathological concentrations of homocysteine. Such effect was accompanied by an increased expression of MCP-1 mRNA (176% as compared to the control) in endothelial cells which resulted in enhanced monocyte chemotaxis. The p38 MAP kinase as well as other members of the p38 MAP kinase pathway, including MKK3, MKK6, ATF-2 and Elk-1, were activated in homocysteine-treated cells. Homocysteine-induced MCP-1 expression and subsequent monocyte chemotaxis were blocked by a p38 MAP kinase inhibitor (SB203580) suggesting that the p38 MAP kinase pathway might be involved in homocysteine-induced MCP-1 expression in endothelial cells. In contrast, staurosporine, a protein kinase C inhibitor, had no effect on homocysteine-induced MCP-1 expression. In conclusion, our results indicate that homocysteine stimulates MCP-1 expression in endothelial cells leading to enhanced monocyte chemotaxis.
TL;DR: The results suggest that when iron is orally administered, free radicals are formed at the site of absorption causing damage to the GI tract mucosa.
Abstract: Free radical mediated effects on the gastrointestinal (GI) tract were studied by supplementing 8 mg of iron orally for 15 days to groups of both control (C+) and iron deficient (D+) rats. They were compared with their respective unsupplemented groups C and D. Incorporation of 3H-thymidine into the isolated mucosal cells, as a measure of cell turn over, was lowered significantly in both the D+ and C+ groups compared to their respective controls D and C. It was observed that a single dose of 8 mg of iron given orally to control rats could cause apoptosis of GI tract mucosal cells as shown by the ladder pattern of DNA on electrophoresis. Continuous administration of the same dose of iron for a period of 15 days resulted in necrosis of the GI tract absorptive surface in D+ and C+ rats. In addition to this, a reduction of microvillus height in C+ and complete erosion of the same in D+ were observed by the transmission electron microscopy. EPR spectroscopy identified production of hydroxyl and methoxyl radicals in both the luminal and mucosal contents in the GI tract of rats. These results suggest that when iron is orally administered, free radicals are formed at the site of absorption causing damage to the GI tract mucosa.