Analysis of gene expression in apoptosis of human lymphoma U937 cells induced by heat shock and the effects of α-phenyl <Emphasis Type="Italic">N-tert</Emphasis>-butylnitrone (PBN) and its derivatives

Hyperthermia, a modality of cancer therapy, has been known as a stress to induce apoptosis. However, the molecular mechanism of heat shock-induced apoptosis, especially on roles of intracellular oxidative stress, is not fully understood. First, when human lymphoma U937 cells were treated with heat shock (44C, 30 min), the fraction of apoptosis, revealed by phosphatidylserine externalization, increased gradually and peaked at 6 hr after the treatment. In contrast, intracellular superoxide formation increased early during the heat shock treatment and peaked at 30 min after the treatment. When the cells were treated with heat shock in the presence of -phenyl-N-tert-butylnitrone (PBN) and its derivatives, which are potent antioxidants, the DNA fragmentation was inhibited in an order according to the agents hydrophobicity. PBN showing the highest inhibitory effects suppressed not only intracellular superoxide formation but also various apoptosis indicators. cDNA microarray was employed to analyze gene expression associated with heat shock-induced apoptosis, and the time-course microarray analysis revealed 5 groups showing changes in their pattern of gene expression. Among these genes, c- jun mRNA expression showed more than 40 fold increase 2 hr after heat treatment. The expression level of c-jun mRNA verified by quantitative real-time PCR was about 20 fold increase, and c- jun expression was similarly suppressed by PBN and its derivatives. These results suggest that the change of c- jun expression is an excellent molecular marker for apoptosis mediated by intracellular oxidative stress induced by heat shock.     

Essential Role of TGF-β/Smad Pathway on Statin Dependent Vascular Smooth Muscle Cell Regulation

Background

The 3-hydroxy-3-methylglutaryl CoA reductase inhibitors (also called statins) exert proven beneficial effects on cardiovascular diseases. Recent data suggest a protective role for Transforming Growth Factor-β (TGF-β) in atherosclerosis by regulating the balance between inflammation and extracellular matrix accumulation. However, there are no studies about the effect of statins on TGF-β/Smad pathway in atherosclerosis and vascular cells.

Methodology

In cultured vascular smooth muscle cells (VSMCs) statins enhanced Smad pathway activation caused by TGF-β. In addition, statins upregulated TGF-β receptor type II (TRII), and increased TGF-β synthesis and TGF-β/Smad-dependent actions. In this sense, statins, through Smad activation, render VSMCs more susceptible to TGF-β induced apoptosis and increased TGF-β-mediated ECM production. It is well documented that high doses of statins induce apoptosis in cultured VSMC in the presence of serum; however the precise mechanism of this effect remains to be elucidated. We have found that statins-induced apoptosis was mediated by TGF-β/Smad pathway. Finally, we have described that RhoA inhibition is a common intracellular mechanisms involved in statins effects. The in vivo relevance of these findings was assessed in an experimental model of atherosclerosis in apolipoprotein E deficient mice: Treatment with Atorvastatin increased Smad3 phosphorylation and TRII overexpression, associated to elevated ECM deposition in the VSMCs within atheroma plaques, while apoptosis was not detected.

Conclusions

Statins enhance TGF-β/Smad pathway, regulating ligand levels, receptor, main signaling pathway and cellular responses of VSMC, including apoptosis and ECM accumulation. Our findings show that TGF-β/Smad pathway is essential for statins-dependent actions in VSMCs.     

Simultaneous Increases in Proliferation and Apoptosis of Vascular Smooth Muscle Cells Accelerate Diabetic Mouse Venous Atherosclerosis

Aims

This study was designed to demonstrate simultaneous increases in proliferation and apoptosis of vascular smooth muscle cells (VSMCs) leading to accelerated vein graft remodeling and to explore the underlying mechanisms.

Methods

Vein grafts were performed in non-diabetic and diabetic mice. The cultured quiescent VSMCs were subjected to mechanical stretch stress (SS) and/or advanced glycosylation end products (AGEs). Harvested vein grafts and treated VSMCs were used to detect cell proliferation, apoptosis, mitogen-activated protein kinases (MAPKs) activation and SM-α-actin expression.

Results

Significantly thicker vessel walls and greater increases in proliferation and apoptosis were observed in diabetic vein grafts than those in non-diabetic. Both SS and AGEs were found to induce different activation of three members of MAPKs and simultaneous increases in proliferation and apoptosis of VSMCs, and combined treatment with both had a synergistic effect. VSMCs with strong SM-α-actin expression represented more activated JNKs or p38MAPK, and cell apoptosis, while the cells with weak SM-α-actin expression demonstrated preferential activation of ERKs and cell proliferation. In contrast, inhibition of MAPKs signals triggered significant decreases in VSMC proliferation, and apoptosis. Treatment of the cells with RNA interference of receptor of AGEs (RAGE) also resulted in significant decreases in both proliferation and apoptosis.

Conclusions

Increased pressure-induced SS triggers simultaneous increases in proliferation and apoptosis of VSMCs in the vein grafts leading to vein arterializations, which can be synergistically accelerated by high glucose-induced AGEs resulting in vein graft atherosclerosis. Either SS or AGEs and their combination induce simultaneous increases in proliferation and apoptosis of VSMCs via different activation of three members of MAPKs resulting from different VSMC subtypes classified by SM-α-actin expression levels.     

Sustained Akt/PKB activation and transient attenuation of c-jun N-Terminal kinase in the inhibition of apoptosis by IGF-1 in vascular smooth muscle cells

Characteristics of hVSMC apoptosis and its inhibition by insulin-like growth factor-1 (IGF-1) remain unclear. Also unclear is whether a balance in hVSMCs exists whereby c-Jun N-terminal stress kinases (JNK) promote apoptosis while extracellular signal-regulated (ERK1/2) MAP kinases inhibit cell death. In this study, we examined the involvement of Akt/PKB and its upstream kinase, PDK1 and whether JNK activation correlated with human and rat VSMC apoptosis induced by staurosporine and by c-myc, respectively. We observed a strong, sustained JNK activation (and c-Jun phosphorylation), which correlated with VSMC apoptosis. IGF-1 (13.3 nM), during apoptosis inhibition, transiently inhibited JNK activity at 1 h in a phosphatidylinositol 3-kinase (PI3-K)- and MEK-ERK-dependent manner, as wortmannin (100 nM) or PD98059 (30 M) partially attenuated the IGF-1 effect. PKC down-regulation had no effect on JNK inhibition by IGF-1. While IGF-1 alone produced a strong phosphorylation of Akt/PKB in hVSMCs up to 6 h, it was notably stronger and more sustained during ratmyc and hVSMCs apoptosis inhibition. Further, whereas transient expression of phosphorylated Akt protected VSMCs from apoptosis by nearly 50%, expression of dominant interfering alleles of Akt or PDK1 strongly inhibited IGF-1-mediated VSMC survival. These results demonstrate for the first time that transient inhibition of a pro-apoptotic stimulus in VSMCs may be sufficient to inhibit a programmed cell death and that sustained anti-apoptotic signals (Akt) elicited by IGF-1 are augmented during a death stimulus. Furthermore, PI3-K and ERK-MAPK pathways may cooperate to protect VSMCs from cell death.This work was supported by a grant from the Nebraska cancer and Smoking Related Disease Program, Department of Health, Nebraska, and National Institutes of Health Grants R01HL070885 (D.K.A.) and R01HL073349 (D.K.A.).     

Profound negative regulatory effects by resveratrol on vascular smooth muscle cells: a role of p53-p21(WAF1/CIP1) pathway

We investigated the role of resveratrol, a polyphenol rich in red wine, in cell cycle progression and apoptosis of vascular smooth muscle cells (VSMCs). Resveratrol inhibited the growth of human aortic VSMCs at concentrations as low as 1 microM. This was due to the profound dose-dependent inhibition of DNA synthesis by resveratrol. DNA synthesis was more effectively inhibited when cells were pretreated with resveratrol. Resveratrol caused a dose-dependent increase in intracellular p53 and p21(WAF1/CIP1) levels. At lower concentrations (6.25-12.5 microM), resveratrol effectively blocked cell cycle progression of serum-stimulated VSMCs without inducing apoptosis, while the higher concentration of resveratrol (25 microM) selectively induced apoptosis in the same VSMCs. Intriguingly, however, the same high concentration of resveratrol could not induce apoptosis in quiescent VSMCs. These differential biological effects of resveratrol on quiescent and proliferating VSMCs suggest that resveratrol may be capable of selectively eliminating abnormally proliferating VSMCs of the arterial walls in vivo.     

Intracellular Ca2+ regulating proteins in vascular smooth muscle cells are altered with type 1 diabetes due to the direct effects of hyperglycemia

Background

Diminished calcium (Ca²⁺) transients in response to physiological agonists have been reported in vascular smooth muscle cells (VSMCs) from diabetic animals. However, the mechanism responsible was unclear.

Methodology/Principal Findings

VSMCs from autoimmune type 1 Diabetes Resistant Bio-Breeding (DR-BB) rats and streptozotocin-induced rats were examined for levels and distribution of inositol trisphosphate receptors (IP₃R) and the SR Ca²⁺ pumps (SERCA 2 and 3). Generally, a decrease in IP₃R levels and dramatic increase in ryanodine receptor (RyR) levels were noted in the aortic samples from diabetic animals. Redistribution of the specific IP₃R subtypes was dependent on the rat model. SERCA 2 was redistributed to a peri-nuclear pattern that was more prominent in the DR-BB diabetic rat aorta than the STZ diabetic rat. The free intracellular Ca²⁺ in freshly dispersed VSMCs from control and diabetic animals was monitored using ratiometric Ca²⁺ sensitive fluorophores viewed by confocal microscopy. In control VSMCs, basal fluorescence levels were significantly higher in the nucleus relative to the cytoplasm, while in diabetic VSMCs they were essentially the same. Vasopressin induced a predictable increase in free intracellular Ca²⁺ in the VSMCs from control rats with a prolonged and significantly blunted response in the diabetic VSMCs. A slow rise in free intracellular Ca²⁺ in response to thapsigargin, a specific blocker of SERCA was seen in the control VSMCs but was significantly delayed and prolonged in cells from diabetic rats. To determine whether the changes were due to the direct effects of hyperglycemica, experiments were repeated using cultured rat aortic smooth muscle cells (A7r5) grown in hyperglycemic and control conditions. In general, they demonstrated the same changes in protein levels and distribution as well as the blunted Ca²⁺ responses to vasopressin and thapsigargin as noted in the cells from diabetic animals.

Conclusions/Significance

This work demonstrates that the previously-reported reduced Ca²⁺ signaling in VSMCs from diabetic animals is related to decreases and/or redistribution in the IP₃R Ca²⁺ channels and SERCA proteins. These changes can be duplicated in culture with high glucose levels.     

Critical Role of Exogenous Nitric Oxide in ROCK Activity in Vascular Smooth Muscle Cells

Objective

Rho-associated kinase (ROCK) signaling pathway has been shown to mediate various cellular functions including cell proliferation, migration, adhesion, apoptosis, and contraction, all of which may be involved in pathogenesis of atherosclerosis. Endogenous nitric oxide (NO) is well known to have an anti-atherosclerotic effect, whereas the exogenous NO-mediated cardiovascular effect still remains controversial. The purpose of this study was to evaluate the effect of exogenous NO on ROCK activity in vascular smooth muscle cells (VSMCs) in vitro and in vivo.

Methods

VSMCs migration was evaluated using a modified Boyden chamber assay. ROCK activities were measured by Western blot analysis in murine and human VSMCs and aorta of mice treated with or without angiotensin II (Ang II) and/or sodium nitroprusside (SNP), an NO donor.

Results

Co-treatment with SNP inhibited the Ang II-induced cell migration and increases in ROCK activity in murine and human VSMCs. Similarly, the increased ROCK activity 2 weeks after Ang II infusion in the mouse aorta was substantially inhibited by subcutaneous injection of SNP.

Conclusions

These findings suggest that administration of exogenous NO can inhibit ROCK activity in VSMCs in vitro and in vivo.     

Involvement of reactive oxygen species and caspase 3 activation in arsenite-induced apoptosis

Recent studies indicate that arsenic may generate reactive oxygen species to exert its toxicity. However, the mechanism is still unclear. In this study, we demonstrate that arsenite is able to induce apoptosis in a concentration- and time-dependent manner; however, arsenate is unable to do so. An increase of intracellular peroxide levels was accompanied with arsenite-induced apoptosis, as demonstrated by flow cytometry using DCFH-DA. N-Acetyl-L -cysteine (a thiol-containing antioxidant), diphenylene iodonium (an inhibitor of NADPH oxidase), 4,5-dihydro-1,3-benzene disulfonic acid (a selective scavenger of O) and catalase significantly inhibit arsenite-induced apoptosis and intracellular fluorescence intensity. In contrast, allopurinol (an inhibitor of xanthine oxidase), indomethacin (an inhibitor of cyclooxygenase), superoxide dismutase, or PDTC had no effect on arsenite-induced cell death. Activation of CPP32 activity, PARP (a DNA repair enzyme) degradation, and release of cytochrome c from mitochondria to the cytosol are involved in arsenite-induced apoptosis, and Bcl-2 antagonize arsenite-induced apoptosis by a mechanism that interferes in the activity of CPP32. These results lead to a working hypothesis that arsenite-induced apoptosis is triggered by the generation of hydrogen peroxide through activation of flavoprotein-dependent superoxide-producing enzymes (such as NADPH oxidase), and hydrogen peroxide might play a role as a mediator to induce apoptosis through release of cytochrome c to cytosol, activation of CPP32 protease, and PARP degradation. J. Cell. Physiol. 177:324–333, 1998. © 1998 Wiley-Liss, Inc.     

Superoxide levels and function of cerebral blood vessels after inhibition of CuZn-SOD

The goal of this study was to examine the role of endogenous copper/zinc (CuZn)-superoxide dismutase (SOD) on superoxide levels and on responses of cerebral blood vessels to stimuli that are mediated by nitric oxide (acetylcholine) and cyclooxygenase-dependent mechanisms (bradykinin and arachidonic acid). Levels of superoxide in the rabbit basilar artery were measured using lucigenin-enhanced chemiluminescence (5 microM lucigenin). Diethyldithiocarbamate (DDC; 10 mM), an inhibitor of CuZn-SOD, increased superoxide levels by approximately 2.4-fold (P < 0.05) from a baseline value of 1.0 +/- 0.2 relative light units x min(-1) x mm(-2) (means +/- SE). The diameter of cerebral arterioles (baseline diameter, 99 +/- 3 microm) was also measured using a closed cranial window in anesthetized rabbits. Topical application of DDC attenuated responses to acetylcholine, bradykinin, and arachidonate, but not nitroprusside. For example, 10 microM arachidonic acid dilated cerebral arterioles by 40 +/- 5 and 2 +/- 2 microm under control conditions and after DDC, respectively (P < 0.05). These inhibitory effects of DDC were reversed by the superoxide scavenger 4,5-dihydroxy-1,3-benzenedisulfonic acid (10 mM). Arachidonate increased superoxide levels in the basilar artery moderately under normal conditions and this increase was greatly augmented in the presence of DDC. These findings suggest that endogenous CuZn-SOD limits superoxide levels under basal conditions and has a marked influence on increases in superoxide in vessels exposed to arachidonic acid. The results also suggest that nitric oxide- and cyclooxygenase-mediated responses in the cerebral microcirculation are dependent on normal activity of CuZn-SOD.     

Angiotensin II-induced activation of p21-activated kinase 1 requires Ca2+ and protein kinase C{delta} in vascular smooth muscle cells

ANG II promotes remodeling of vascular smooth muscle cells (VSMCs) in cardiovascular diseases. It has been shown to activate p21-activated kinase (PAK)1, a critical component of signaling pathways implicated in growth and migration. However, the detailed signaling mechanism by which ANG II induces PAK1 activation in VSMCs remains unclear. Therefore, we have examined the mechanism required for activation of PAK1 by ANG II in VSMCs. ANG II, through activation of the ANG II type 1 receptor, rapidly promotes phosphorylation of PAK1 in VSMCs via a pathway independent of transactivation of the epidermal growth factor receptor. Using selective agonists and inhibitors, we demonstrated that mobilization of intracellular Ca²⁺ and PKC activation are required for ANG II-induced PAK1 phosphorylation. Rottlerin, a PKC inhibitor, significantly blocked ANG II-induced PAK1 phosphorylation. Further support for this notion was provided through infection of VSMCs with adenovirus encoding a dominant-negative (dn)PKC, which also markedly reduced phosphorylation of PAK1 by ANG II. In this pathway, Ca²⁺ acts upstream of PKC because a Ca²⁺ ionophore rapidly induced PKC phosphorylation at Tyr311 and Ca²⁺-dependent PAK1 phosphorylation was blocked by rottlerin. In addition, dnPYK-2, dnRac, and antioxidants inhibited ANG II-induced PAK1 phosphorylation, suggesting that PYK-2, Rac, and reactive oxygen species are involved in the upstream signaling. Finally, dnPAK1 markedly inhibited ANG II-induced protein synthesis in VSMCs. These data provide a novel signaling pathway by which ANG II may contribute to vascular remodeling. vascular remodeling; signal transduction     

Roles of dopa decarboxylase and phenoloxidase in the melanization of the tobacco hornworm and their control by 20-hydroxyecdysone

Summary WhenManduca sexta larvae are allatectomized 5 h before head capsule slippage (HCS) in the final larval molt, the new larval cuticle contains granules that melanize 3 h before ecdysis when the ecdysteroid titer falls (Curtis et al. 1984). In both the epidermis and hemolymph of these allatectomized larvae dopamine was higher than dopa prior to and at the time of melanization. Dopamine also increased in the new cuticle as melanization began. Dopa decarboxylase (DDC) activity increased in the epidermis, cuticle, and fat body beginning 16 h after HCS, with a two-fold greater increase in the epidermis of allatectomized larvae. Both -MDH and -fluoromethyl-dopa inhibited epidermal DDC activity and inhibited melanization in vitro when dopa was used as a precursor. Addition of dopamine to the medium allowed melanization in the presence of the inhibitors. All these results indicate that dopamine is likely the primary precursor of cuticular melanin. The diphenoloxidase in the premelanin granules was activated in vivo between 19 and 21 h after HCS and was found to prefer dopamine to dopa and not to convert tyrosine to melanin. The activation of the prophenoloxidase was inhibited by 20-hydroxyecdysone (20-HE), both in vivo and in vitro, if hormone was given by 16 h after HCS. Infusion of 1.2 g/ml 20-HE into allatectomized larvae for 24 h from HCS prevented both the increase in DDC activity and the activation of the premelanin granules. Although the larvae ecdysed after a 15 h delay, melanization never occurred.Abbreviations -MDH L-3-(3,4 dihydroxyphenyl)-2-hydrazine-methylpropionic acid - -FM-dopa R-S--fluoromethyl-dopa - DCC dopa decarboxylase - 20-HE 20-hydroxyecdysone - JH juvenile hormone - HCS head capsule slippage     

Differential regulation and recovery of intracellular Ca2+ in cerebral and small mesenteric arterial smooth muscle cells of simulated microgravity rat

Background

The differential adaptations of cerebrovasculature and small mesenteric arteries could be one of critical factors in postspaceflight orthostatic intolerance, but the cellular mechanisms remain unknown. We hypothesize that there is a differential regulation of intracellular Ca²⁺ determined by the alterations in the functions of plasma membrane Ca_L channels and ryanodine-sensitive Ca²⁺ releases from sarcoplasmic reticulum (SR) in cerebral and small mesenteric vascular smooth muscle cells (VSMCs) of simulated microgravity rats, respectively.

Methodology/Principal Findings

Sprague-Dawley rats were subjected to 28-day hindlimb unweighting to simulate microgravity. In addition, tail-suspended rats were submitted to a recovery period of 3 or 7 days after removal of suspension. The function of Ca_L channels was evaluated by patch clamp and Western blotting. The function of ryanodine-sensitive Ca²⁺ releases in response to caffeine were assessed by a laser confocal microscope. Our results indicated that simulated microgravity increased the functions of Ca_L channels and ryanodine-sensitive Ca²⁺ releases in cerebral VSMCs, whereas, simulated microgravity decreased the functions of Ca_L channels and ryanodine-sensitive Ca²⁺ releases in small mesenteric VSMCs. In addition, 3- or 7-day recovery after removal of suspension could restore the functions of Ca_L channels and ryanodine-sensitive Ca²⁺ releases to their control levels in cerebral and small mesenteric VSMCs, respectively.

Conclusions

The differential regulation of Ca_L channels and ryanodine-sensitive Ca²⁺ releases in cerebral and small mesenteric VSMCs may be responsible for the differential regulation of intracellular Ca²⁺, which leads to the altered autoregulation of cerebral vasculature and the inability to adequately elevate peripheral vascular resistance in postspaceflight orthostatic intolerance.     

Synthesis of 5-methyl phenanthridium derivatives: A new class of human DOPA decarboxylase inhibitors

DOPA decarboxylase (DDC) is responsible for the decarboxylation of l-DOPA and related aromatic amino acids and correlates closely with a number of clinical disorders. Sanguinarine, a natural quaternary benzophenanthridine alkaloid (QBA), was reported to be inhibitor of rat DDC and possessed a different inhibitory mechanism. In this study, several natural QBAs were assayed as human DDC inhibitors for the first time. A series of 5-methyl phenanthridium derivatives that contain the basic core structure of QBAs were also synthesized and evaluated as human DDC inhibitors. The title compounds still possessed DDC inhibitory potential. Among the synthesized compounds, 2-hydroxyl-8-methoxy-5-methylphenanthridinium chloride (11k) showed good inhibitory activity with an IC₅₀ value of 0.12 mM. Preliminary structure–activity relationship indicated that DDC inhibitory potential of 5-methyl phenanthridium derivatives correlated with the π-electro densities on CN double bond of iminium cation. The hydroxyl group on compound 11k possibly contributed to the formation of hydrogen bond between DDC and the inhibitor.     

Heat shock protein 27 plays a protective role in thoracic aortic dissection by promoting cell proliferation and inhibiting apoptosis

Background

Thoracic aortic dissection (TAD) is one of the most severe aortic diseases. The study aimed to explore the potential role of heat shock protein 27 (HSP27) in the pathogenesis of TAD using an in vitro model of oxidative stress in vascular smooth muscle cells (VSMCs).

Methods

HSP27 was analyzed in aortic surgical specimens from 12 patients with TAD and 8 healthy controls. A lentiviral vector was used to overexpress HSP27 in rat aortic VSMCs. Cell proliferation and apoptosis were measured under oxidative stress induced by H₂O₂.

Results

HSP27 expression was significantly higher in aortic tissue from patients with TAD and VSMCs in the aortic media were the main cell type producing HSP27. Elevated oxidative stress was also detected in the TAD samples. Overexpression of HSP27 significantly attenuated H₂O₂-induced inhibition of cell proliferation. Furthermore, HSP27 was found to decrease H₂O₂-induced cell apoptosis and oxidative stress.

Conclusions

These results suggest that HSP27 expression promotes VSMC viability, suppresses cell apoptosis, and confers protection against oxidative stress in TAD.

     

Inactivation of intracellular copper-zinc superoxide dismutase by copper chelating agents without glutathione depletion and methemoglobin formation

The copper chelator N,N'-diethyldithiocarbamate (DDC), is often used to inactivate intracellular copper-zinc superoxide dismutase in erythrocytes. However, in studies with red cells we found that the compound also reacted with oxyhemoglobin to produce oxygen radicals in addition to generating lipid peroxidation products, oxidized N,N'-diethyldithiocarbamate, methemoglobin, and sulfhemoglobin. Moreover, intracellular glutathione was depleted and vital cellular enzymes were susceptible to inactivation. We, and others, have confirmed these findings in nonerythrocytic cell lines. Thus, cells exposed to DDC are severely damaged before studies on the effects of added putative superoxide producing compounds can be performed with them. In this report, we have systematically investigated other copper chelators for their ability to inactivate intracellular copper-zinc superoxide dismutase without producing the deleterious effects mentioned above. Catechol, triethylenetetramine, and tetraethylenepentamine were found to be such agents when erythrocytes were dialyzed in the cold against dilute solutions of these chelators. In addition, with a myeloid leukemic cell line (HL-60), triethylenetetramine inhibited SOD without causing significant GSH oxidation. Examination of the affinity constants of chelators active against purified copper-zinc superoxide dismutase indicated that an affinity binding constant (log K1) between 12.6 and 13.8 was required for the chelator to successfully remove copper from the enzyme.     

Ethanol-Induced Apoptosis in Hepatoma Cells Proceeds via Intracellular Ca Elevation, Activation of TLCK-Sensitive Proteases, and Cytochrome c Release

Ethanol is known to induce apoptosis in hepatocytes. However, intracellular signaling events of ethanol-induced death are still only partially understood. We studied such processes in ethanol-induced apoptosis in HepG2 cells as a model system for human liver cells. We determined the incidence of apoptosis by DNA fragmentation and tested the effects of various known inhibitors. Ethanol induces apoptosis in HepG2 cells in a dose- and time-dependent manner as well as in rat primary hepatocytes. This effect was not mediated through the death receptor CD95 and the tumor necrosis factor receptors. It was efficiently inhibited by the caspase inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zVAD-fmk), the Ca²⁺ chelator EGTA, and the serine protease inhibitor N-p-tosyl- -lysine chloromethyl ketone (TLCK). Upon ethanol treatment, the intracellular calcium ion concentration was increased and cytochrome c was released from the mitochondria, and caspases were activated. EGTA and TLCK could inhibit cytochrome c release from the mitochondria. Furthermore, overexpression of Bcl-x_L saved cells from ethanol-induced apoptosis. These data suggest that ethanol-induced apoptosis in liver cells is initiated by the intracellular Ca²⁺ elevation in the cytoplasm and activation of TLCK-sensitive serine proteases. Our data provide new insight into ethanol-induced apoptosis in liver cells and may lead to therapeutic strategies to prevent liver damage.     

The Genetics of Dopa Decarboxylase and a-Methyl Dopa Sensitivity in Drosophila melanogaster

Dopa decarboxylase (DDC) in the Diptera is an enzyme involvedin sclerotinization of the cuticle in the epidermis and theproduction of neurogenic amines in the central nervous system.Its appearance in the epidermis at pupariation is induced bythe molting hormone ecdysone. The dietary administration ofthe analog inhibitor -methyl dopa (a MD) was used to isolateresistant and hypersensitive mutants. Two of three dominantresistant strains isolated increase DDC activity 35-70%. Forboth the increase is due to mutations between rdo (53) and pr(54.5) on the left arm of the 2nd chromosome (2L). The veryhighly resistant strain which does not affect DDC in any wayis located at 54.0 on 2L. Twelve dominant, l(2)amd^H —MD hypersensitive alleles located immediately to the right ofhk (53.9) on 2L have been recovered. All are recessive lethalsand exhibit some intracistronic complementation, and none ofthem, not even heteroallelic heterozygotes, affect DDC in anyway. The.recovery and analysis of 16 overlapping deficienciespermitted the localization of a DDC dosage effect to bands 37B10-C7on 2L; a region which includes the l(2)amd locus. Subsequentlyeight DDC deficient lethal alleles were recovered in this elevenband region which as heterozygotes reduce activities to 28–53% of controls. Some heteroallelic heterozygotes exhibit intracistroniccomplementation; most with viabilities 5% and with a mutantphenotype probably derived from inadequately sclerotinized cuticle.These Ddc alleles are within 0.004 Map Units to the right ofl(2)amd. None as Ddc/CyO heterozygotes are sensitive to -MD,and complementation occurs between the Ddc alleles and the l(2)amdalleles both on the basis of viability and DDC activity. Althoughthe protein product mutated by the l(2)amd alleles has not yetbeen identified, it seems likely that the two groups of mutantsare functionally related. Finally, the Ddc structural mutantsreduce DDC activity in the central nervous system as well asthe epidermis.     

Chemical imaging of fresh vascular smooth muscle cell response by epi‐detected stimulated Raman scattering

An understanding of deformation of cardiovascular tissue under hemodynamic load is crucial for understanding the health and disease of blood vessels. In the present work, an epi‐detected stimulated Raman scattering (epi‐SRS) imaging platform was designed for in situ functional imaging of vascular smooth muscle cells (VMSCs) in fresh coronary arteries. For the first time, the pressure‐induced morphological deformation of fresh VSMCs was imaged with no fixation and in a label‐free manner. The relation between the loading pressure and the morphological parameters, including angle and length of the VSMCs, were apparent. The morphological responses of VMSCs to drug treatment were also explored, to demonstrate the capability of functional imaging for VSMCs by this method. The time‐course imaging revealed the drug induced change in angle and length of VSMCs. The present study provides a better understanding of the biomechanical framework of blood vessels, as well as their responses to external stimulations, which are fundamental for developing new strategies for cardiovascular disease treatment.