SUMOylation of nonstructural 5A protein regulates hepatitis C virus replication.

Viruses exploit cellular SUMOylation machinery to favour their own propagation. We show that NS5A is a target protein of small ubiquitin-like modifier (SUMO) and is SUMOylated at lysine residue 348. We demonstrated that SUMOylation increased protein stability of NS5A by inhibiting ubiquitylation, and SUMOylation was also required for protein interaction with NS5B. These data imply that SUMO modification may contribute to HCV replication. Indeed, silencing of UBC9 impaired HCV replication in Jc1-infected cells, and HCV replication level was also significantly reduced in SUMO-defective subgenomic replicon cells. Taken together, these data indicate that HCV replication is regulated by SUMO modification of NS5A protein. We provide evidence for the first time that HCV exploits host cellular SUMO modification system to favour its own replication.

Activation of estrogen receptor ß reduces blood-brain barrier breakdown following ischemic injury.

Estrogen receptors (ERs) play important roles in estrogen-mediated neuroprotection. However, their effects on blood-brain barrier (BBB) disruption with vasogenic edema after ischemic stroke have not been determined. We evaluated a role for ERß in the brain without effects in the peripheral reproductive organs for the amelioration of vasogenic edema following ischemic stroke. Transient focal ischemic stroke was induced in ovariectomized female C57BL/6 mice (age 10-11weeks) that were treated with the ERß-selective agonist diarylpropionitrile (DPN). BBB breakdown as determined by the extravasation of endogenous immunoglobulin G (IgG), vasogenic edema, and the infarct volume was significantly reduced by DPN compared to vehicle. Protein expressions of endothelial tight junction proteins (occludin and claudin-5) and the water channel protein aquaporin 4 in the ischemic cortex were not changed by DPN. However, protein levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1α (HIF-1α), a transcription factor that increases VEGF expression, were significantly decreased in the ischemic cortex by DPN. These results suggest that ERß contributes to the reduction of vasogenic edema caused by BBB breakdown via the inhibition of HIF-1α and VEGF following ischemic stroke.

Nanosized titanium dioxide enhanced inflammatory responses in the septic brain of mouse.

Nanosized titanium dioxide (TiO(2)) is used widely in various everyday products and can be applied to the medical field for diagnostic or therapeutic tools. However, its neurobiological responses have not been defined completely in the brain. To evaluate the acute inflammatory response to TiO(2) particles of two different sizes in normal and septic brains, male C57BL/6 mice were given intraperitoneal injections of fine (<1 microm) or ultrafine (21 nm) TiO(2), 30 min after vehicle or lipopolysaccaride (LPS). In the normal brain, neither fine nor ultrafine TiO(2) induced inflammation. However, in the brains of LPS-exposed mice, ultrafine TiO(2) significantly elevated proinflammatory cytokine interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) mRNAs, and IL-1beta protein levels. Also ultrafine TiO(2) increased the levels of reactive oxygen species and activated microglia 24 h after LPS challenge. In BV2 microglial cells stimulated with LPS, ultrafine TiO(2) enhanced TNF-alpha production and augmented nuclear factor-kB binding activity. These findings suggest that nanosized TiO(2) promotes an exaggerated neuroinflammatory responses by enhancing microglial activation in the pre-inflamed brain, in part.

Direct application of the PCR restriction analysis method for identifying NTM species in AFB smear-positive respiratory specimens.

The early differentiation of Mycobacterium tuberculosis from non-tuberculous mycobacteria (NTM) and the identification of NTM species are crucial for the proper management of patients with smear-positive sputum. We evaluated the usefulness of a polymerase chain reaction restriction analysis (PRA) method based on the rpoB gene for identifying NTM species in a study of 121 smear-positive respiratory specimens with presumed NTM. The PRA method amplified mycobacterial DNA in 72 specimens (60%) and differentiated NTM species correctly in 68 (94%). The PRA method could be a useful and rapid method for identifying NTM species in smear-positive respiratory specimens when urgent clinical decisions are required.

Ibuprofen protects ischemia-induced neuronal injury via up-regulating interleukin-1 receptor antagonist expression.

The inflammatory response accompanies and exacerbates the developing injury after cerebral ischemia. Ibuprofen, a non-steroidal anti-inflammatory drug, has been shown to attenuate injuries in animal models of various neurological diseases. In the present study, we investigated ibuprofen's neuroprotective effects in rats exposed to transient forebrain ischemia and in cultures exposed to oxygen glucose deprivation (OGD). Rats treated with ibuprofen after transient forebrain ischemia displayed long-lasting protection of CA1 hippocampal neurons. There were selective increases in interleukin-1 receptor antagonist gene and protein expression in ibuprofen-treated OGD microglia. Furthermore, treatment with ibuprofen in neuron/microglia co-cultures increased the number of surviving HC2S2 neurons against OGD whereas IL-1ra neutralizing antibody reversed the ibuprofen-induced neuroprotection. The data indicate that ibuprofen-induced IL-1ra secretion is involved in neuroprotection against ischemic conditions.

A neuroprotective role of extracellular signal-regulated kinase in N-acetyl-O-methyldopamine-treated hippocampal neurons after exposure to in vitro and in vivo ischemia.

In response to cerebral ischemia, neurons activate survival/repair pathways in addition to death cascades. Activation of cyclic AMP-response-element-binding protein (CREB) is linked to neuroprotection in experimental animal models of stroke. However, a role of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MAPK/ERK or MEK), an upstream kinase for CREB, and its relation to CREB phosphorylation in neuroprotection in cerebral ischemia has not been delineated. Previously, we reported that N-acetyl-O-methyldopamine (NAMDA) significantly protected CA1 neurons after transient forebrain ischemia [J Neurosci 19 (1999b) 87.8]. The current study is to investigate whether NAMDA-induced neuroprotection occurs via the activation of ERK and its downstream effector, CREB. NAMDA induced ERK1/2 and CREB phosphorylation with increased survival of HC2S2 hippocampal neurons subjected to oxygen-glucose deprivation. These effects were reversed by U0126, a MEK kinase inhibitor. Similarly, animals treated with NAMDA following ischemia showed increased ERK and CREB phosphorylation in the CA1 subregion of the hippocampus during early reperfusion period with increased number of surviving neurons examined 7 days following ischemia. The NAMDA-induced neuroprotection was abolished by U0126 administered shortly after reperfusion. The results showed that the ERK-CREB signaling pathway might be involved in NAMDA-induced neuroprotection following transient global ischemia and imply that the activation of the pathway in neurons may be an effective therapeutic strategy to treat stroke or other neurological syndromes.

Inhibitory effect of a naphthazarin derivative, S64, on heat shock factor (Hsf) activation and glutathione status following hypoxia.

The presence of hypoxic cells in solid tumors has long been considered a problem in cancer treatment. Resistance of hypoxic cells to ionizing radiation and anticancer drugs has in part been attributed to changes in altered gene expression by hypoxia. We previously reported an activation of heat shock factor (Hsf) in murine tumor RIF cells following hypoxia and suggested that a subsequent accumulation of heat shock protein(s) (Hsp) is likely to contribute to the malignant progression of hypoxic tumor cells (Baek et al., 2001). In this study, we showed that hypoxia induced a DNA-binding activity of Hsf and activation of hsp70 gene expression in colon cancer Clone A cells, and that a naphthazarin derivative, S64, significantly inhibited the hypoxia-inducible hsp70 gene expression in Clone A cells. We also showed that S64 significantly reduced the cellular glutathione levels in this cell line. Considering the proposed effects of Hsp and glutathione on radiation and chemotherapy sensitivity, we suggest that the inhibitory effects of S64 on Hsf activation and cellular glutathione levels have potentially important clinical implications. We believe that the previously reported in vitro and in vivo anti-tumor effect of S64 (Song et al., 2000a, 2001) might be attributed, at least in part, to its effect on Hsf activation and/or glutathione depletion. We also believe that the detailed molecular mechanisms underlying the effects of S64 on Hsf and glutathione level following hypoxia deserve a more rigorous future study, the results of which could offer novel strategy to manipulate the resistance mechanisms of solid tumors.

Preferential elevation of Prx I and Trx expression in lung cancer cells following hypoxia and in human lung cancer tissues.

Transient/chronic microenvironmental hypoxia that exists within a majority of solid tumors has been suggested to have a profound influence on tumor growth and therapeutic outcome. Since the functions of novel antioxidant proteins, peroxiredoxin I (Prx I) and II, have been implicated in regulating cell proliferation, differentiation, and apoptosis, it was of our special interest to probe a possible role of Prx I and II in the context of hypoxic tumor microenvironment. Since both Prx I and II use thioredoxin (Trx) as an electron donor and Trx is a substrate for thioredoxin reductase (TrxR), we investigated the regulation of Trx and TrxR as well as Prx expression following hypoxia. Here we show a dynamic change of glutathione homeostasis in lung cancer A549 cells and an up-regulation of Prx I and Trx following hypoxia. Western blot analysis of 10 human lung cancer and paired normal lung tissues also revealed an elevated expression of Prx I and Trx proteins in lung cancer tissues. Immunohistochemical analysis of the lung cancer tissues confirmed an augmented Prx I and Trx expression in cancer cells with respect to the parenchymal cells in adjacent normal lung tissue. Based on these results, we suggest that the redox changes in lung tumor microenvironment could have acted as a trigger for the up-regulation of Prx I and Trx in lung cancer cells. Although the clinical significance of our finding awaits more rigorous future study, preferential augmentation of the Prx I and Trx in lung cancer cells may well represent an attempt of cancer cells to manipulate a dynamic redox change in tumor microenvironment in a manner that is beneficial for their proliferation and malignant progression.

Cerebral activation and distribution of inducible hsp110 and hsp70 mRNAs following focal ischemia in rat.

A potential function for inducible heat shock protein 70 (hsp70i) expression in the pathophysiology of ischemic brain has been well documented. The recently cloned hsp70 superfamily member, hsp110, was shown to be highly expressed in the brain and suggested to have a similar functional property as members of the hsp70 family. In this study, as an initial step to probe for its physiological significance in the ischemic brain, cerebral activation and distribution of hsp110 mRNA was comparatively evaluated with that of hsp70i mRNA by in situ hybridization. A rat focal cerebral ischemia model was employed to examine the distribution and localization of hsp110 and hsp70i mRNAs in both affected (ipsilateral) and unaffected (contralateral) hemispheres of the same animal. Our results demonstrated a significant accumulation of hsp110 as well as hsp70i mRNAs following ischemia; although the magnitude and kinetics of induction differ slightly, spatial expression profiles of hsp110 and hsp70i mRNAs were highly correlated in the affected region. In control brain, limited hybridization signal was observed with 3'-untranslated region (UTR) containing hsp110 probe, suggesting a possible existence of inducible hsp110 and a selective recognition of our 3'-UTR containing probe for the inducible hsp110 mRNA species. Subsequent 2D western analysis with Hsp110 specific Ab was consistent with our view, which resolved constitutive and inducible immunostained spots in rat ischemic brain. Considering a regulatory similarity as well as previously documented structural and functional similarities between hsp110 and hsp70i, we propose that coordinated cerebral activation of hsp110 and hsp70i is likely to be of significant relevance in the context of pathophysiology of ischemic brain. Further study is required to characterize the genetic and biochemical nature of rat inducible hsp110 identified in the current study.

Role of protein kinase Cdelta in transmitting hypoxia signal to HSF and HIF-1.

An hypoxic microenvironment is an important modulator of gene expression in many pathophysiological conditions. In this study, we show a coordinate activation of heat shock transcription factor (HSF) and hypoxia-inducible factor-1 (HIF-1) in RIF tumor cells by hypoxia. Since heat shock protein (hsp) and angiogenic factor genes that are regulated by HSF and HIF-1 are thought to contribute to the malignant progression of hypoxic tumor cells, it was of our major interest to identify the components that are responsible for the activation of both HSF and HIF-1. Our finding that a bioflavonoid quercetin (QCT), a well known inhibitor of hsp gene expression, significantly inhibited the transcriptional activation of HSF and HIF-1 strongly suggests that QCT-sensitive molecule(s) is involved in the transcriptional activation of HSF and HIF-1 by hypoxia. Our results revealed that PCKalpha, delta and epsilon isoforms are expressed in RIF cells, but only PKCdelta was specifically translocated to the membrane by hypoxia. Our results also revealed that the translocation of PKCdelta was completely abrogated by QCT. Moreover, inhibiting the PKCdelta activation, either pharmacologically with phorbol 12-myristate 13-acetate or with bisindolymaleimide II or genetically by transient transfection of a dominant negative PKCdelta, significantly inhibited the transcriptional activation of HSF and HIF-1 by hypoxia. These results strongly substantiate a view that the PKCdelta isozyme is the QCT-sensitive molecule that plays an important role in transmitting hypoxia signals to both HSF and HIF-1. Here we show that the membrane translocation of PKCdelta is dependent on the activation of phosphoinositol 3-kinase (PI3K). Treatment with PI3K inhibitor, wortmannin or LY294002, abrogated not only PKCdelta translocation but the subsequent transcriptional activation of HSF and HIF-1 by hypoxia. Together, our study shows that the PKCdelta isozyme acts as a shared component in transmitting hypoxia-induced signals to both HSF and HIF-1, and that the upstream regulator of PKCdelta is PI3K.

Detection of rifampin-resistant Mycobacterium tuberculosis in sputa by nested PCR-linked single-strand conformation polymorphism and DNA sequencing.

Either PCR-mediated single strand conformation polymorphism (SSCP) analysis or DNA sequencing of rpoB DNA (157 bp) can be used as a rapid screening method for the detection of mutations related to the rifampin resistance of Mycobacterium tuberculosis. However, due to the nonspecific amplification of rpoB DNA from nontuberculous mycobacteria these methods cannot be directly applied to clinical specimens such as sputa. We developed a nested PCR method that can specifically amplify the rpoB DNA of M. tuberculosis on the basis of rpoB DNA sequences of 44 mycobacteria. Nested PCR-linked SSCP analysis and the DNA sequencing method were applied directly in order to detect M. tuberculosis and determine its rifampin susceptibility in 56 sputa. The results obtained by nested PCR-SSCP and DNA sequencing were concordant with those of conventional drug susceptibility testing and DNA sequencing performed with culture isolates.

Early c-Fos induction after cerebral ischemia: a possible neuroprotective role.

The role of c-Fos in neurodegeneration or neuroprotection after cerebral ischemia is controversial. To investigate whether early c-Fos induction after ischemia is associated with neuroprotection, rats were subjected to 10 minutes of transient forebrain ischemia and c-Fos expression was examined. Resistant dentate granule cells and neurons in CA2-4 displayed more robust immunoreactivity than vulnerable neurons in the CA1 region of hippocampus during early hours of reperfusion. By 6 hours after reperfusion, c-Fos immunoreactivity was greatly diminished in all areas of the hippocampus. Administration of N-acetyl-O-methyldopamine (NAMDA), a compound previously shown to protect CA1 neurons against ischemia, increased c-Fos immunoreactivity in the CA1 vulnerable region at 6 hours after ischemia and protected SK-N-BE(2)C neurons from oxygen glucose deprivation. Further in vitro study showed that NAMDA potentiated phorbol-12 myristate-13 acetate (PMA)-induced c-Fos expression, AP1 binding activity, and late gene expression determined by chloramphenicol acetyltransferase (CAT) activity from AP1 containing tyrosine hydroxylase promoter-CAT fusion gene in SK-N-BE(2)C neurons. In vivo and in vitro results showed that a neuroprotectant, NAMDA, in concert with another stimulus (for example, ischemia or PMA) up-regulates c-Fos expression and suggested that the early rise of NAMDA-induced c-Fos expression in vulnerable CA1 neurons may account for neuroprotection by means of up-regulating late gene expression for survival.

Repression of proinflammatory cytokine and inducible nitric oxide synthase (NOS2) gene expression in activated microglia by N-acetyl-O-methyldopamine: protein kinase A-dependent mechanism.

Excessive proinflammatory cytokine and NO production by activated microglia play a role in neurodegenerative disorders. To investigate whether the neuroprotectant N-acetyl-O-methyldopamine (NAMDA) downregulates genes associated with microglial activation, we measured gene expression of TNF-alpha, IL-1beta, inducible nitric oxide synthase (NOS2), and an associated cofactor synthesis gene, GTP cyclohydrolase I (GTPCH) in LPS-stimulated microglia cells in the presence or absence of NAMDA. The temporal pattern of cytokine gene expression showed that LPS (0.2 microg/ml) increased TNF-alpha and IL-1beta gene expression at 1 and 3 h, which was repressed by cotreatment of NAMDA. Similarly, LPS also induced GTPCH and NOS2 gene expression at 3 and 6 h, and cotreatment of NAMDA repressed the induction with parallel reduction of nitrite, an oxidative metabolite of nitric oxide. Since transcription factor NF-kappaB is involved in regulating expression of these genes, the effects of NAMDA on NF-kappaB nuclear translocation and DNA binding in immunostimulated microglia were investigated. We found that neither LPS-induced NF-kappaB translocation nor DNA binding activity was affected by cotreatment with NAMDA in BV-2 microglia. On the other hand, NAMDA increased intracellular cAMP levels and potentiated LPS-induced phosphorylated cAMP-responsive element binding protein (pCREB) expression. Treatment with adenosine 3'5'-cyclic monophosphothioate, a specific inhibitor of cAMP-dependent protein kinase (PKA), reversed not only NAMDA-induced pCREB upregulation but also NAMDA-induced repression of TNF-alpha and IL-1beta gene transcription. The data demonstrate that NAMDA represses LPS-induced proinflammatory cytokines gene expression via a cAMP-dependent protein kinase pathway. Thus, repressing proinflammatory cytokines and NOS2 gene expression in activated microglia by NAMDA may provide new therapeutic strategies for ischemic cerebral disease as well as other neurodegenerative diseases.

Preoperative triglycerides predict post-coronary artery bypass graft survival in diabetic patients: a sex analysis.

OBJECTIVE: Hypertriglyceridemia is commonly observed in association with diabetes. Despite cross-sectional studies and isolated longitudinal analyses in patients without coronary artery disease, the suggestion that triglyceride levels are relevant to subsequent cardiovascular events in the setting of diabetes remains controversial. This study evaluates the predictive value of serum triglyceride levels on mortality in post-coronary artery bypass graft (CABG) diabetic patients with subsequent analysis by sex. RESEARCH DESIGN AND METHODS: This longitudinal observational study involving a large metropolitan hospital consists of 1,172 diabetic post-CABG patients (792 men and 380 women) with lipid data collected between the years 1982 and 1992. Cox proportional hazards regression models were used to estimate the risk of mortality and cardiac events associated with triglyceride levels in the highest quartile (> 2.90 mmol/l for men and > 3.12 mmol/l for women). RESULTS: Elevated preoperative serum triglyceride values in post-CABG subjects with diabetes were correlated with increased overall mortality (hazard ratio [HR] 1.26, 95% CI 1.00-1.59). The greatest influence of triglyceride levels was observed on overall (1.89, 1.30-2.73) and event-free survival (1.49, 1.06-2.08) in women. High triglyceride values were also modestly related to risk of cardiac events in diabetic men (1.28, 0.99-1.66). CONCLUSIONS: These data suggest that increased preoperative triglyceride levels predict increased late mortality and cardiac event risk in diabetic post-CABG patients, more strongly in women than in men.

Measurement of glutathione oxidation and 8-hydroxy-2'-deoxyguanosine accumulation in the gerbil hippocampus following global ischemia.

Involvement of oxidative stress in ischemia/reperfusion-induced brain damage has been suggested. However, experimental support of this suggestion was limited partly because sensitive indices to assess oxidative consequences of ischemic brain damage were few. We have established biochemical assay systems to assess oxidative brain damage following ischemia. Mongolian gerbil brains were subjected to global ischemia/reperfusion, and the hippocampi were analyzed for oxidative damage by measuring temporal changes in glutathione and 8-ohdG following ischemia. Under oxidative stress, glutathione is known to be oxidized and subsequently depleted from cells. Therefore, glutathione content and its redox status can serve as sensitive indicators of oxidative damage. The accumulation of 8-ohdG has also been recognized as an excellent marker for oxidative DNA damage. The reduced and oxidized glutathione were measured by HPLC method following derivatization with 2,4-dinitrofluorobenzene. The 8-ohdG in DNA hydrolyzate was measured by HPLC with electrochemical detection. While total glutathione content decreased, glutathione oxidation ratio and 8-ohdG accumulation increased over a period of 30 min of reperfusion following ischemia. The results demonstrated that glutathione content, its oxidation ratio, and the accumulated 8-ohdG could be utilized as sensitive indices for the assessment of oxidative brain damage.

Ultra-rapid freezing of human multipronuclear zygotes using electron microscope grids.

Developmental capacity of human multipronuclear (PN) zygotes cryopreserved using an ultra-rapid freezing method and electron microscope (EM) grids was studied. Multipronuclear zygotes obtained from a human IVF programme were used as an alternative to normal 2PN zygotes; they were divided into 3PN or >or =4PN zygotes and their in-vitro development and cryo-injury were compared according to PN number. EFS30, which consisted of 30% ethylene glycol, 18% Ficoll, 0. 5 mol/l sucrose and 10% fetal bovine serum with added modified Dulbecco's phosphate buffered saline was used as the freezing solution. After ultra-rapid freezing and thawing 85.5% of multipronuclear zygotes survived. A comparison of cleavage rates between the control and cryopreserved groups showed no significant difference (3PN; 81.3 and 85.4% and > or =4PN; 90.0 and 95.7% respectively). Comparing the in-vitro development after thawing up to blastocyst formation on day 5 after IVF, the outcome of the frozen 3PN group (22.0%) was not different from that of control 3PN group (38.5%), while the outcome of the frozen > or =4PN group (4.5%) was significantly lower than that of control > or =4PN group (44.4%) (P < 0.05).

Reduced glutathione oxidation ratio and 8 ohdG accumulation by mild ischemic pretreatment.

A critical role of oxidative stress has been implicated in ischemic brain damage. Mild ischemic pretreatment and/or synthesis of heat shock proteins (HSPs) has been suggested to protect against oxidative brain damage. However, experimental support of this suggestion have proven to be difficult partly because sensitive indices to assess oxidative consequences of ischemic brain damage were few. In this study, we have attempted to establish biochemical assay systems to quantitate oxidative brain damage following ischemia. We produced experimental brain ischemia in the Mongolian gerbil (Meriones unguiculatus) and examined the hippocampus for ischemic brain damage. The results obtained from ischemic gerbil hippocampus demonstrated that oxidative brain damage can be quantitated by determining glutathione oxidation ratio together with the accumulation of the oxidative DNA damage product, 8-hydroxy-2'-deoxyguanosine (8 ohdG). Our results also demonstrated a role for mild ischemic pretreatment and synthesis of HSPs against oxidative brain damage. We showed that mild 2-min ischemic pretreatment reduced the degree of both glutathione oxidation ratio and 8 ohdG accumulation in gerbil hippocampus subsequent to 10 min ischemic challenge. We also showed that the accumulation of HSP70 was closely associated with the reduction of oxidative brain damage. To our knowledge, this is the first report to investigate glutathione redox states and oxidative DNA damage levels to evaluate a protective role of mild ischemic pretreatment and HSP synthesis following brain ischemia. Our data validate the previous suggestions and provide new additional data that argue for the protective role of mild ischemic pretreatment and HSP70 synthesis against oxidative brain damage.

Role of small heat shock protein HSP25 in radioresistance and glutathione-redox cycle.

Expression of heat shock proteins (HSPs) has been shown to protect mammalian cells exposed to a variety of stress stimuli. Among various HSPs, small HSPs from diverse species were shown to protect cells against oxidative stress. Here, we show that the overexpression of the mouse small hsp gene, hsp25, provides protection against ionizing radiation. Our results demonstrate that the radiation survival of the L929 cells stably transfected with hsp25 was enhanced compared with that of the parental or vector transfected control, L25#1 cells. Our results also demonstrate that the radiation-induced apoptosis was reduced in HSP25 overexpressors. A detailed analysis of glutathione composition of those clones that overexpressed HSP25 revealed the increases of the glutathione pool, which primarily resulted from the increase of reduced glutathione. Our data suggest that higher content of GSH in HSP25 overexpressors was because of a faster reduction of oxidized glutathione (GSSG) to GSH rather than an increased de novo synthesis of GSH. The activities of glutathione reductase (GRd) and glutathione peroxidase (GPx) were greater in HSP25 overexpressors but the activity of gamma-glutamylcysteine synthetase was similar between the transfectants and the control cells. Consistent with our view, a steady state ratio of the GSH/GSSG was greater in the transfectants in comparison with the control L25#1 cells. A difference in the relative ratio became more significant after exposure to the ionizing radiation. To our knowledge, this study provides the first experimental evidence in support of the hypothesis that small HSP plays a key role in radioresistance by modulating the metabolism of glutathione. Based on the results obtained from the current investigation, we propose that HSP25 helps facilitate the glutathione-redox cycle and therefore, enhances glutathione utilization and maintains the cellular glutathione pool in favor of the reduced states.

Oxidative damage in tissues of rats exposed to cigarette smoke.

Cigarette smoke is known to contain high concentrations of free radicals and oxidants. To examine the oxidative effect of cigarette smoking, we subjected rats to inhalation of cigarette smoke, and measured cellular free glutathione, the degree of protein S-thiolation, and 8-oxo-2'-deoxyguanosine (oxo8dG) in DNA. Inhalation of the cigarette smoke for 30 days, three times a day, resulted in a significant decrease of the total free glutathione contents in tissues, especially in the lung. Elevated levels of oxidized glutathione and protein S-thiolation were observed in the lung but not in other tissues. Increased contents of oxo8dG in DNA were found in all tissues analyzed. When rats were treated with buthionine sulfoximine (BSO, 80 mg/kg/day) to deplete glutathione, the oxidative effect of cigarette smoking was greatly potentiated. The effect of glutathione depletion was most evident in the lung. Cigarette smoking for only 7 days resulted in extreme depletion of the glutathione both in the lungs and in the liver of BSO-treated rats. Furthermore, oxo8dG in DNA increased markedly, especially in lung. The results verified that the lung is a primary target of cigarette smoke-induced oxidative damage, and cigarette smoke exerts its oxidative effects on the rest of the entire organs eventually. Our results indicate that glutathione plays crucial roles in protecting proteins and DNA from oxidation caused by cigarette smoking.

Purification and molecular cloning of calobin, a thrombin-like enzyme from Agkistrodon caliginosus (Korean viper).

A thrombin-like enzyme, calobin, has been purified to homogeneity from the venom of Agkistrodon caliginosus by a procedure involving Bio-Gel P-100, Mono S, and Pro-RPC. The enzyme was identified as a monomer with a molecular weight of 34,000 on SDS-PAGE, and its isoelectric point was 6.2. Calobin acted on fibrinogen to form fibrin with a specific activity of 226 NIH equivalent units, and also exhibited arginine esterase activity. The enzyme predominantly cleaved the alpha-chain of fibrinogen with little degradation of the beta-chain. It contained abundant asparagine/aspartic acid residues, but very few tyrosine or methionine residues. The proteolytic activity of the enzyme with TAME as a substrate was higher than that of thrombin. However, it showed neither lysine esterase nor caseinolytic activity. The enzyme activity was strongly inhibited by PMSF, and moderately by benzamidine and soybean trypsin inhibitor, indicating it is a serine protease. On the other hand, the enzyme activity was not inhibited by hirudin or aprotinin. cDNA (1.6 kb) for calobin has been cloned from an A. caliginosus cDNA library. The cDNA sequence indicates that calobin is synthesized as a pre-zymogen of 262 amino acids, including a putative secretory signal peptide of 18 amino acids and a proposed zymogen peptide of 6 amino acid residues. The cDNA sequence encodes a 238-amino acid residue molecule exhibiting strong amino acid sequence homology to those of ancrod, batroxobin, and flavoxobin isolated from other snake venoms. Calobin contains 12 cysteine residues. As judged on alignment of the amino acid sequences of other thrombin-like enzymes (batroxobin, ancrod, and flavoxobin), calobin constitute the formation of six disulfide bridges. Amino acid residues, His43, Asp88, and Ser182, which are thought to be the catalytic active site are highly conserved. As calobin is a glycoprotein, its possible glycosylation site, Asn-X-Thr, is located at amino acid residues 81-83.