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B Herman

Publications and source records attributed to B Herman.

At least 19 recordsLinked to original sources
PubMed1997

Role of ICE-like proteases in endothelial cell hypoxic and reperfusion injury.

Because of its location between blood and tissue, the endothelium is particularly vulnerable to hypoxic/reperfusion injury, but the mechanisms responsible for this injury are not known. A number of recent findings suggest that hypoxia and reperfusion injures neuronal cells via apoptosis. Apoptosis has recently been shown to depend on the activation of a class of proteases with homology to Interleukin-1 beta converting enzyme (ICE) protease. Therefore, we examined the effect of specific inhibitors of ICE-like proteases on hypoxic and reperfusion injury in cultured EAhy926 endothelial cells. Pretreatment of cells with ICE inhibitor II (Ac-YVAD-CMK), ICE inhibitor III (Z-Asp-2,6-dichlorobenzoyloxy-methylketone-Z-Asp-CH2-DCB+ ++), or ICE inhibitor IV (Ac-YVKD-CHO) (all at 10-100 microM) did not protect cells from hypoxic injury. However, pretreatment of cells with ICE inhibitor III and to a lesser extent with ICE inhibitor II, but not with ICE inhibitor IV, protected cells from reperfusion injury. The protective effect of ICE inhibitor III was not dependent upon pH, but was associated with decreased release of arachidonic acid from cells. These findings suggest that reperfusion injury to EAhy926 endothelial cells involves ICE-like proteases. The identity of the protease(s) is not known but it does not appear to be a YAMA-type protease based upon ICE inhibitor specificity. Our data also indicate that a potential target of this protease is phospholipase A2 (PLA2).

Arachidonic Acid↗
PubMed1997

A sex difference in short-term survival after initial acute myocardial infarction. The MONICA-Bremen Acute Myocardial Infarction Register, 1985-1990.

AIMS: To assess the difference between men and women as regards fatality shortly after acute myocardial infarction, and the relationship of patient characteristics. METHODS AND RESULTS: One thousand seven hundred and ten male and 563 female patients, 25-69 years of age and hospitalized with a first acute myocardial infarction occurring from 1985 to 1990, were included in the population-based World Health Organization MONICA-Bremen Acute Myocardial Infarction Register. Patient information, including short-term survival status, was obtained from the medical records of the seven Bremen hospitals with internal medicine departments and municipal death certificate files. The unadjusted 28-day fatality rate after acute myocardial infarction was higher among women than among men (23.1% vs 16.1% respectively: P < 0.001). Adjusting for the older age of women did not eliminate the difference completely (females; 20.9%, males: 16.8%; P = 0.041). Controlling for previous use of inotropic medicine and diuretics, during-the-event receipt of thrombolysis and platelet inhibitors, and age in logistic regression analyses resulted in a similar 28-day mortality risk after acute myocardial infarction for both sexes (female/male odds ratio = 1.13, 95% confidence interval = 0.86 1.50; P = 0.389). CONCLUSIONS: Sex was not an independent predictor of early acute myocardial infarction fatality. Our data suggest that the excess mortality risk in women can be explained by sex differences in age, pre-infarction cardiac impairment, and treatment during the coronary event.

Adult↗
PubMed1997

Mitochondrial permeability transition in hepatocytes induced by t-BuOOH: NAD(P)H and reactive oxygen species.

Tert-butyl hydroperoxide (t-BuOOH) induces the mitochondrial permeability transition (MPT) in hepatocytes, leading to cell death. Using confocal microscopy, we visualized pyridine nucleotide oxidation and reactive oxygen species (ROS) formation induced by t-BuOOH. Reduced mitochondrial pyridine nucleotides (NADH and NADPH) were imaged by autofluorescence. Mitochondrial membrane potential, ROS, onset of MPT, and cell death were monitored with tetramethylrhodamine methyl ester (TMRM), dichlorofluorescin, calcein, and propidium iodide, respectively. t-BuOOH rapidly oxidized mitochondrial NAD(P)H. Oxidation was biphasic, and the second slower phase occurred during mitochondrial ROS generation. Subsequently, MPT took place, mitochondria depolarized, and cells died. beta-Hydroxybutyrate, which reduces mitochondrial NAD+, delayed cell killing, but lactate, which reduces cytosolic NAD+, did not. Trifluoperazine, which inhibits MPT, did not block the initial oxidation of NAD(P)H but prevented the second phase of oxidation, partially blocked ROS formation, and preserved cell viability. The antioxidants, deferoxamine and diphenylphenylenediamine, also prevented the second phase of NAD(P)H oxidation. They also blocked ROS formation nearly completely and stopped cell killing. Both antioxidants also prevented the mitochondrial permeability transition and subsequent mitochondrial depolarization. In conclusion, NAD(P)H oxidation and ROS formation are critical events promoting MPT in oxidative injury and death of hepatocytes.

Animals↗
PubMed1996

Mitochondrial free calcium transients during excitation-contraction coupling in rabbit cardiac myocytes.

Mitochondrial free Ca2+ may regulate mitochondrial ATP production during cardiac exercise. Here, using laser scanning confocal microscopy of adult rabbit cardiac myocytes co-loaded with Fluo-3 to measure free Ca2+ and tetramethylrhodamine methylester to identify mitochondria, we measured cytosolic and mitochondrial Ca2+ transients during the contractile cycle. In resting cells, cytosolic and mitochondrial Fluo-3 signals were similar. During electrical pacing, transients of Fluo-3 fluorescence occurred in both the cytosolic and mitochondrial compartments. Both the mitochondrial and the cytosolic transients were potentiated by isoproterenol. These experiments show directly that mitochondrial free Ca2+ rises and falls during excitation-contraction coupling in cardiac myocytes and that changes of mitochondrial Ca2+ are kinetically competent to regulate mitochondrial metabolism on a beat-to-beat basis.

Aniline Compounds↗
PubMed1996

Down-regulation of HCW9 mRNA in rat hepatocytes during chemical hypoxia involves both transcriptional and posttranscriptional mechanisms.

HCW9 cDNA encodes a rat protein with 95% homology to mouse phospholipase A2 activating protein (PLAP). Its mRNA, which is substantially decreased in rat hepatocytes during chemical hypoxic injury, was found to be expressed in all rat tissues examined, including liver, heart, brain, spleen, lung, skeletal muscle, kidney, and testis. To elucidate the mechanisms responsible for this hypoxia-induced down-regulation of HCW9 mRNA levels, the transcription rate and half-life of HCW9 mRNA were measured. Nuclear run-off assays revealed a 54-57% inhibition in the transcription rate of HCW9 gene during chemical hypoxic injury. The half-life of HCW9 mRNA decreased from approximately 15 min under normoxic conditions to approximately 7 min during chemical hypoxic injury. These findings suggest that HCW9 expression in rat hepatocytes is regulated at both the transcriptional and posttranscriptional levels during chemical hypoxia.

Animals↗
PubMed1996

The pH paradox in ischemia-reperfusion injury to cardiac myocytes.

During myocardial ischemia, a large reduction of tissue pH develops, and tissue pH returns to normal after reperfusion. In recent studies, we evaluated the role of pH in ischemia/reperfusion injury to cultured cardiac myocytes and perfused papillary muscles. Acidosis (pH < or = 7.0) protected profoundly against cell death during ischemia. However, the return from acidotic to normal pH after reperfusion caused myocytes to lose viability. This worsening of injury is a 'pH paradox' and was mediated by changes of intracellular pH (pH(i)), since manipulations that caused pH(i), to increase more rapidly after reperfusion accelerated cell killing, whereas manipulations that delayed the increase of pH(i) prevented loss of myocyte viability. Specifically, inhibition of the Na+/H+ exchanger with dimethylamiloride or HOE694 delayed the return of physiologic pH(i) after reperfusion and prevented reperfusion-induced cell killing to both cultured myocytes and perfused papillary muscle. Dimethylamiloride and HOE694 did not reduce intracellular free Ca2+ during reperfusion. By contrast, reperfusion with dichlorobenzamil, an inhibitor of Na+/Ca2+ exchange, decreased free Ca2+ but did not reduce cell killing. Thus, the pH paradox is not Ca(2+)-dependent. Our working hypothesis is that ischemia activates hydrolytic enzymes, such as phospholipases and proteases, whose activity is inhibited at acidotic pH. Upon reperfusion, the return to normal pH releases this inhibition and hydrolytic injury ensues. Increasing pH(i) may also induce a pH-dependent mitochondrial permeability transition and activate the myofibrillar ATPase, effects that increase ATP demand and compromise ATP supply. In conclusion, acidotic pH is generally protective in ischemia, whereas a return to physiologic pH precipitates lethal reperfusion injury to myocytes.

Animals↗
PubMed1996

Coronary heart disease mortality, morbidity, and case fatality in five east and west German cities 1985-1989. Acute Myocardial Infarction Register Teams of Augsburg, Bremen, Chemnitz, Erfurt, and Zwickau.

Cardiovascular mortality (CVD; International Classification of Diseases [ICD] 390-458) is higher in East than in West Germany, but the differences in official coronary heart disease mortality (CHD; ICD 410-414) are not so pronounced. The aim of this study was to validate the official mortality statistics based on the five German AMI registers and to analyze whether these mortality differences are due to differences in the attack rates of acute myocardial infarction (AMI) or to differences in the 28-day case fatality rates. This comparison includes the MONICA study cities of Augsburg and Bremen, both in West Germany, as well as the cities of Chemnitz, Erfurt, and Zwickau in East Germany (former the German Democratic Republic). The rates were calculated on the basis of all MONICA cases of definite AMI or coronary death aged 35 to 64 years occurring in the respective study populations between 1985 and 1989. All study populations except women in Augsburg showed higher coronary death rates compared to the rates based on the official cause of death statistics (ICD 410-414), but this difference was significant only for men in Chemnitz. In men there were no significant differences in the register-based coronary death rates between these urban areas (160/100,000 in Zwickau to 170/100,000 in Chemnitz) nor in the AMI attack rates (327/100,000 in Augsburg to 363/100,000 in Chemnitz), and consequently no significant center differences in the overall 28-day case fatality. However, the prehospital case fatality was significantly higher in Erfurt (34%) than in Bremen (27%). There were no significant differences in the AMI attack rates in women as well (60/100,000 in Chemnitz to 70/100,000 in Bremen and Erfurt), but the overall 28-day case fatality showed a clear gradient from the East (61-71%) to the West German cities (48-56%) and therefore also the register-based coronary death rates (38-50/100,000 and 34-38/100,000, respectively). However, the higher 28-day case fatality in women found in the MONICA registers in East compared to West Germany is not reflected in the CHD mortality statistics because of a stronger underestimation of the official mortality rates and in East than in West Germany, in particular in women. Nevertheless, the total mortality rates and in most cases also the CVD mortality rates were in women significantly higher in the East German compared to the West German cities. The East German official preunification CHD mortality data cannot be used for national and international comparisons. The results of the MONICA AMI registers in East and West Germany indicate, furthermore, the need to improve coronary care in women in the eastern part of the country. Nevertheless, because of the relatively high AMI attack rate in both parts of Germany primary prevention must generally be intensified.

Adult↗
PubMed1996

Contribution of pH-dependent group II phospholipase A2 to chemical hypoxic injury in rat hepatocytes.

Numerous studies have suggested that enhanced membrane phospholipid degradation contributes to hypoxic and ischemic injury. Recently, acidosis has been found to potently protect against hypoxic and ischemic injury. To investigate the interrelationships of these two events in hypoxic injury, we studied the role of a pH-dependent group II phospholipase A2 (PLA2, E.C. 3.1.1.4) in chemical hypoxic injury in rat hepatocytes. Northern blot analysis of RNA extracted from normoxic and hypoxic rat hepatocytes with group II rat liver PLA2-specific oligonucleotide cDNA probes revealed a 0.9 kb transcript whose abundance was significantly increased in rat hepatocytes within 15 min after initiation of chemical hypoxia and remained high until cells lost viability. Immunofluorescence staining of hepatocytes with polyclonal antibodies that recognize group II PLA2 demonstrated a substantial increase in the level of PLA2 protein in hypoxic rat hepatocytes within 30 min of initiation of hypoxic injury. Treatment of hepatocytes with group II PLA2-specific anti-sense DNA oligonucleotides: 1) abolished accumulation of PLA2 protein in hypoxic rat hepatocytes as assessed by immunofluorescence staining with anti-PLA2 antibodies; 2) decreased the enzymatic activity of PLA2 manifested as decreased arachidonic acid release in hypoxic hepatocytes; and 3) significantly delayed cell death evoked by chemical hypoxia as indicated by a decrease in propidium iodide uptake. These findings suggest that pH-dependent group II PLA2 plays an important role in chemical hypoxic injury of rat hepatocytes.

Animals↗
PubMed1996

Structure-function comparisons of the proapoptotic protein Bax in yeast and mammalian cells.

Expression of the proapoptotic protein Bax under the control of a GAL10 promoter in Saccharomyces cerevisiae resulted in galactose-inducible cell death. Immunofluorescence studies suggested that Bax is principally associated with mitochondria in yeast cells. Removal of the carboxyl-terminal transmembrane (TM) domain from Bax [creating Bax (deltaTM)] prevented targeting to mitochondrial and completely abolished cytotoxic function in yeast cells, suggesting that membrane targeting is crucial for Bax-mediated lethality. Fusing a TM domain from Mas70p, a yeast mitochondrial outer membrane protein, to Bax (deltaTM) restored targeting to mitochondria and cytotoxic function in yeast cells. Deletion of four well-conserved amino acids (IGDE) from the BH3 domain of Bax ablated its ability to homodimerize and completely abrogated lethality in yeast cells. In contrast, several Bax mutants which retained ability to homodimerize (deltaBH1, deltaBH2, and delta1-58) also retained at least partial lethal function in yeast cells. In coimmunoprecipitation experiments, expression of the wild-type Bax protein in Rat-1 fibroblasts and 293 epithelial cells induced apoptosis, whereas the Bax (deltaIGDE) mutant failed to induce apoptosis and did not associate with endogenous wild-type Bax protein. In contrast to yeast cells, Bax (deltaTM) protein retained cytotoxic function in Rat-1 and 293 cells, was targeted largely to mitochondria, and dimerized with endogenous Bax in mammalian cells. Thus, the dimerization-mediating BH3 domain and targeting to mitochondrial membranes appear to be essential for the cytotoxic function of Bax in both yeast and mammalian cells.

Animals↗
PubMed1996

The presence of human papillomavirus-16/-18 E6, p53, and Bcl-2 protein in cervicovaginal smears from patients with invasive cervical cancer.

Cervical cancer is the second leading cause of death from cancer in women worldwide, and recent epidemiological studies have strongly implicated the sexually transmitted human papillomavirus (HPV) as a causative agent. The ability of high-risk HPVs to contribute to malignant progression seems to depend on expression of the viral E6 and E7 oncogenes. The E6 oncoprotein forms a complex with the cellular tumor suppressor protein p53, leading to degradation of p53 via ubiquitin-dependent proteolysis. Thus, E6 expression results in the loss of p53 function in cells, including stimulation of apoptosis and inhibition of the expression of the antiapoptotic protein bcl-2. Recently, we found increased bcl-2 expression in cervical carcinoma cell lines containing mutated or E6-inactivated p53 (X. L. Liang, S. Mungal, A. Ayscue, J. D. Meissner, P. Wodnicki, G. Gordon, S. Lockett, and B. Herman. J. Cell. Biochem., 57: 509-520, 1995). Based on these findings, we examined Papanicolaou smears from 94 women with varying degrees of cervical disease for the presence or absence of p53, HPV-16/18 E6, and bcl-2 proteins using immunofluorescence microscopy. Our findings indicate that there is a statistically significant, inverse association between the presence of p53 and invasive cervical disease [odds ratio (OR), 0.3; 95% confidence interval (CI), 0.1-0.7]. Moreover, the odds of being diagnosed with an invasive stage of cervical cancer were 3.7 times higher (95% CI, 1.6-8.8) for women positive for the E6 protein and 17 times higher (95% CI, 5.5-58.3) for women positive for the bcl-2 protein compared with women negative for E6 and bcl-2. Women with invasive cervical cancer were also 4.59 times more likely to test positive for the presence of more than one marker (95% CI, 1.8-11.8). Chi(2) analysis demonstrated a strong association between the presence of E6 and bcl-2 (P < 0.001) as well as between the presence of E6 of bcl-2 and diagnosis (P = 0.015 and < 0.001, respectively). In the multivariate analysis, the presence of bcl-2 (OR, 18.8; 95% CI, 5.5-67.8) and age at diagnosis (> or = 50 years; OR, 7.8; 95% CI, 2.5-24.5) showed significant association with Invasive cervical disease. These findings indicate that: (a) the presence of the bcl-2 protein is strongly associated with the development of invasive cervical disease: (b) the pattern of the presence of high-risk HPV-E6, p53, and bcl-2 proteins may be useful for identifying women at increased risk for the development of invasive cervical cancer; and (c) a defect in apoptosis may partially underlie the development of cervical cancer.

Adult↗
PubMed1995

Cloning of a rat cDNA encoding a novel LIM domain protein with high homology to rat RIL.

A complementary DNA (1392 bp) encoding a protein with high homology to rat reversion-induced LIM (RIL) protein was cloned from rat hepatocytes by differential screening of a subtractive (normoxic minus hypoxic) lambda GEM-2 cDNA library. This cDNA clone, denoted CLP-36, encodes a 327-amino-acid (aa) protein that contained a restrictively conserved LIM (a Cys-rich domain with consensus aa sequence C-X2-C-X17-19-H-X2-C-X2-C-X2-C-X16-20-C-X2-C/H/D that was initially identified in homeodomain proteins, Lin-11 [Freyd et al., Nature 344 (1990) 876-879], Isl-1 [Karlsson et al., Nature 344 (1990) 879-882] and Mec-3 [Way et al., Cell 54 (1988) 5-16] in the C-terminal portion (aa 192-327). It shared an overall 45.1% identity to a rat LIM domain RIL protein [Kiess et al., Oncogene 10 (1995) 61-68], with 62.0% identity in the N terminus (aa 1-89) and 50.0% identity in the C terminus (aa 188-327). The rat CLP-36 mRNA is expressed most abundantly in heart, lung and liver, moderately in spleen and skeletal muscle, and at extremely low levels (if at all) in testis and brain tissues. Northern blotting analysis of total RNA extracted from normoxic or hypoxic rat hepatocytes, with a fragment of clone CLP-36 as probe, demonstrated a single band with a mobility corresponding to a size of 1.4 kb, whose level was significantly decreased during chemical hypoxia.

Amino Acid Sequence↗
PubMed1995

Cloning of a rat cDNA encoding a protein with high homology to mouse phospholipase A2-activating protein.

A complementary DNA (2510 bp) encoding a protein with high homology to mouse phospholipase A2-activating protein (PLAP) was cloned from rat hepatocytes by differential screening of a subtractive (normoxic minus hypoxic) lambda GEM-2 cDNA library. This cDNA clone, denoted as HCW9, encodes a 647-amino-acid (aa) protein with 96.1% identity in the N-terminal portion (255 aa) to mouse PLAP [Clark et al., Proc. Natl. Acad. Sci. USA 88 (1991) 5418-5422]. Four cDNA clones were further isolated by screening a primary normoxic lambda GEM-2 cDNA library with a fragment of the HCW9 clone as probe. Partial sequencing of these clones revealed that all of the four clones were identical to clone HCW9. Northern blotting analysis of total RNA extracted from normoxic or hypoxic rat hepatocytes, with two different fragments of clone HCW9 as probes, demonstrated a single band with a mobility corresponding to a size of 2.5 kb, whose level was significantly decreased during chemical hypoxia.

Amino Acid Sequence↗
PubMed1995

Contribution of the mitochondrial permeability transition to lethal injury after exposure of hepatocytes to t-butylhydroperoxide.

We have developed a novel method for monitoring the mitochondrial permeability transition in single intact hepatocytes during injury with t-butylhydroperoxide (t-BuOOH). Cultured hepatocytes were loaded with the fluorescence probes, calcein and tetramethylrhodamine methyl ester (TMRM). Depending on loading conditions, calcein labelled the cytosolic space exclusively and did not enter mitochondria or it stained both cytosol and mitochondria. TMRM labelled mitochondria as an indicator of mitochondrial polarization. Fluorescence of two probes was imaged simultaneously using laser-scanning confocal microscopy. During normal incubations, TMRM labelled mitochondria indefinitely (longer than 63 min), and calcein did not redistribute between cytosol and mitochondria. These findings indicate that the mitochondrial permeability transition pore ('megachannel') remained closed continuously. After addition of 100 microM t-BuOOH, mitochondria filled quickly with calcein, indicating the onset of mitochondrial permeability transition. This event was accompanied by mitochondrial depolarization, as shown by loss of TMRM. Subsequently, the concentration of ATP declined and cells lost viability. Trifluoperazine, a phospholipase inhibitor that inhibits the permeability transition in isolated mitochondria, prevented calcein redistribution into mitochondria, mitochondrial depolarization, ATP depletion and cell death. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, also rapidly depolarized mitochondria of intact hepatocytes but did not alone induce a permeability transition. Trifluoperazine did not prevent ATP depletion and cell death after the addition of CCCP. In conclusion, the permeability transition pore does not 'flicker' open during normal incubation of hepatocytes but remains continuously closed. Moreover, mitochondrial depolarization per se does not cause the permeability transition in intact cells. During oxidative stress, however, a permeability transition occurs quickly which leads to mitochondrial depolarization and cell death.

Adenosine Triphosphate↗
PubMed1995

Bcl-2 protooncogene expression in cervical carcinoma cell lines containing inactive p53.

Bcl-2 protein expression has been found to block apoptosis and its overexpression has been implicated in lymphoid malignancies where the chromosomal translocation t(14;18) is present. In this study we investigated bcl-2 transcription and protein expression in cultured cervical carcinoma cell lines and keratinocytes. Western blotting and immunofluorescence microscopy demonstrated bcl-2 expression in the cytoplasm of 4 out of 5 cervical carcinoma cell lines examined (HeLa, CaSki, C-33A, and HT-3, but not SiHa). Bcl-2 protein expression was undetectable in normal keratinocytes. None of the cell lines examined demonstrated chromosomal translocation or rearrangement at the major breakpoint-cluster region (MBR) of the bcl-2 gene using either Southern blot or polymerase chain reaction (PCR) analyses. Northern blot analysis demonstrated low levels of bcl-2 transcription in HeLa, CaSki, and C-33A cell lines while reverse transcriptase (RT)-PCR demonstrated bcl-2 transcription in all cervical carcinoma cell lines which had bcl-2 protein expression. Thus, these data suggest that bcl-2 expression occurs in cervical carcinoma cell lines in the absence of chromosomal translocation or rearrangement of the bcl-2 gene. However, each of these cervical carcinoma cell lines contains inactive p53, either due to mutation (C-33A and HT-3) or via complexation and degradation with human papillomavirus (HPV) 16/18 E6 protein (HeLa and CaSki). Thus, functional p53, which can induce apoptosis in certain cells, is not present in these cervical cells which have increased bcl-2 expression. Increased bcl-2 expression under conditions of p53 inactivation may provide cells with a selective advantage for survival and consequently play a role in the development of cervical carcinogenesis.

Female↗
PubMed1995

Analysis of simulated and experimental fluorescence recovery after photobleaching. Data for two diffusing components.

Fluorescence recovery after photobleaching has been a popular technique to quantify the lateral mobility of membrane components. A variety of analysis methods have been used to determine the lateral diffusional mobility, D. However, many of these methods suffer from the drawbacks that they are not able to discern two-component diffusion (i.e., three-point fit), cannot solve for two components (linearization procedures), and do not perform well at low signal-to-noise. To overcome these limitations, we have adopted the approach of fitting fluorescence recovery after photobleaching curves by the full series solution using a Marquardt algorithm. Using simulated data of one or two diffusing components, determinations of the accuracy and reliability of the method with regard to extraction of diffusion parameters and the differentiation of one- versus two-component recovery curves were made under a variety of conditions comparable with those found in actual experimental situations. The performance of the method was also examined in experiments on artificial liposomes and fibroblast membranes labeled with fluorescent lipid and/or protein components. Our results indicate that: 1) the method was capable of extracting one- and two-component D values over a large range of conditions; 2) the D of a one-component recovery can be measured to within 10% with a small signal (100 prebleach photon counts per channel); 3) a two-component recovery requires more than 100-fold greater signal level than a one-component recovery for the same error; and 4) for two-component fits, multiple recovery curves may be needed to provide adequate signal to achieve the desired level of confidence in the fitted parameters and in the differentiation of one- and two-component diffusion.

3T3 Cells↗