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B Herman

Publications and source records attributed to B Herman.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1995

The E5 oncoprotein of human papillomavirus type 16 inhibits the acidification of endosomes in human keratinocytes.

The human papillomavirus type 16 E5 oncoprotein possesses mitogenic activity that acts synergistically with epidermal growth factor (EGF) in human keratinocytes and inhibits the degradation of the EGF receptor in endosomal compartments after ligand-stimulated endocytosis. One potential explanation for these observations is that E5 inhibits the acidification of endosomes. This may be mediated through the 16-kDa component of the vacuolar proton-ATPase, since animal and human papillomavirus E5 proteins bind this subunit protein. Using a ratio-imaging technique to determine endosomal pH, we found that the acidification of endosomes in E5-expressing keratinocytes was delayed at least fourfold compared with normal human keratinocytes and endosomes in some cells never completely acidified. Furthermore, E5 expression increased the resistance of keratinocytes to protein synthesis inhibition by diphtheria toxin, a process dependent on efficient endosomal acidification. Finally, artificially inhibiting endosomal acidification with chloroquine during the endocytosis of EGF receptors in keratinocytes demonstrated many of the same effects as the expression of human papillomavirus type 16 E5, including prolonged retention of undegraded EGF receptors in intracellular vesicles.

Cells, Cultured↗
PubMed1995

Preservation of peristaltic reflex in hypertrophied ileum of guinea pig.

Chronic obstruction of the guinea pig ileum leads to distension and muscular hypertrophy, but how this affects passive biomechanical and nerve-mediated contractions and clearance known as peristaltic reflex is unclear. Ileum of controls had a diameter of 3.0 +/- 1.1 mm and a circular muscle thickness of 37.2 +/- 11.2 microns; 4 wk after placement of a nonconstricting Gore-Tex band, the ileum was distended to 10.0 +/- 0.19 mm, and its muscle had hypertrophied to 195.0 +/- 61.2 microns. Hypertrophied segments exceeded controls in capacity (e.g., 5.1 +/- 1.1 vs. 1.1 +/0 0.2 ml at 6 cm), compliance, and hysteresis. Threshold volumes and pressures that triggered the reflex were 3.3 +/- 1.3 ml and 3.1 +/- 0.01 mmHg in hypertrophied vs. 0.7 +/- 0.2 ml and 1.5 +/- 0.2 mmHg in controls. The diameter increase that triggered the reflex was 1.4 +/- 0.1 mm in hypertrophied segments and 0.6 +/- 0.1 mm in controls. Hypertrophied segments generated fewer contractions of virtually double the amplitude and failed to generate a pressure differential between up- and downstream sites as controls did. Hypertrophied segments generated larger stroke volumes and cumulative clearance than controls. The ratio of antegrade to retrograde clearance was similar in hypertrophied and control segments. The length of the occluding segment in hypertrophied preparations exceeded that of controls. Control contractions indented the antimesenteric border and propagated antegrade from their site of origin; bizarre writhing movements of hypertrophied segments made their contractions difficult to monitor. Thus distension and muscular hypertrophy do not interfere with the ability of the chronically obstructed guinea pig ileum to generate a peristaltic reflex at least as readily and as powerful and as effective in clearing the lumen as controls.

Animals↗
PubMed1995

Noninvasive pressure support ventilation in patients with acute respiratory failure. A randomized comparison with conventional therapy.

The benefit of noninvasive pressure support ventilation (NIPSV) in avoiding the need for endotracheal intubation and reducing morbidity and mortality associated with endotracheal intubation was evaluated in 41 patients who presented with acute respiratory failure not related to chronic obstructive pulmonary disease (COPD). Patients were randomly assigned to receive conventional therapy (n = 20) or conventional therapy plus NIPSV (n = 21). NIPSV was delivered to the patient by a face mask connected to a ventilator (Puritan-Bennett 7200a) set in inspiratory pressure support (IPS) mode. The mean levels of IPS, positive end-expiratory pressure (PEEP), and fraction of inspired oxygen (FIO2) were respectively 15 +/- 3 cm H2O, 4 +/- 2 cm H2O, and 57 +/- 22%. The rate of endotracheal intubation (62 vs 70%, p = 0.88), the length of ICU stay (17 +/- 19 days vs 25 +/- 23 days, p = 0.16), and the mortality rate (33 vs 50%, p = 0.46) were not different between patients treated with NIPSV and those treated conventionally. Post hoc analysis suggested that in patients with PaCO2 > 45 mm Hg (n = 17), NIPSV was associated with a reduction in the rate of endotracheal intubation (36 vs 100%, p = 0.02), in the length of ICU stay (13 +/- 15 days vs 32 +/- 30 days, p = 0.04), and in the mortality rate (9 vs 66%, p = 0.06). We conclude that NIPSV is of no benefit when used systematically in all forms of acute respiratory failure not related to COPD. A subgroup of patients, characterized by acute ventilatory failure and hypercapnia, may potentially benefit from this therapy and further studies are needed to focus on this aspect.

Acute Disease↗
PubMed1995

Progression of subcellular changes during chemical hypoxia to cultured rat hepatocytes: a laser scanning confocal microscopic study.

The aim of this study was to evaluate changes in the subcellular organelles of cultured hepatocytes by laser scanning confocal microscopy during chemical hypoxia with cyanide and iodoacetate, inhibitors of mitochondrial respiration and glycolysis, respectively. Parameter-specific fluorophores used were calcein for cell topography and membrane permeability, rhodamine-dextran for lysosomes, rhodamine 123 and tetramethylrhodamine methylester (TMRM) for mitochondrial membrane potential (delta psi) and propidium iodide for loss of cell viability. During the first 30 to 40 minutes of chemical hypoxia to cultured hepatocytes, numerous surface blebs formed and cell volume increased, but delta psi decreased relatively little. Subsequently, the nonspecific permeability of mitochondrial membranes increased, and mitochondria depolarized. These events were followed a few minutes later by disintegration of individual lysosomes. After a few more minutes, viability was lost as indicated by bleb rupture, gross plasma membrane permeability to calcein, and nuclear labeling with propidium iodide. Thus, the following sequence of intracellular events occurred during chemical hypoxia: adenosine triphosphate (ATP) depletion, bleb formation with cellular swelling, onset of a mitochondrial permeability transition, disintegration of lysosomes, plasma membrane failure from bleb rupture, and cell death. Any explanation of the pathophysiology of hypoxic injury must take into account this unique sequence of events.

Animals↗
PubMed1994

Isoform-specific induction of nuclear free calcium oscillations by platelet-derived growth factor.

Confocal laser scanning microscopy was used to analyze alterations in nuclear free calcium (Ca2+n) levels induced by platelet-derived growth factor (PDGF) isoforms in BALB/c3T3 fibroblasts loaded with the calcium-sensitive fluorescent indicator Fluo-3. Both AA-PDGF and BB-PDGF caused a transient increase in Ca2+n. Analysis of PDGF-induced Ca2+n alterations as a function of time revealed that BB-PDGF stimulation resulted in the generation of Ca2+n oscillations that diminished over time. The frequency of BB-PDGF-stimulated oscillations was modulated by extracellular Ca2+ and could not be mimicked by increasing intracellular inositol 1,4,5-trisphosphate levels in the absence of growth factor stimulation. Caffeine alone had no effect on Ca2+n levels, but exposure of cells to caffeine after BB-PDGF stimulation augmented Ca2+n oscillations, either by increasing the frequency or reinitiating preexisting oscillations. The genesis of these oscillations in Ca2+n appears to be in the region just outside of the nucleus, as perinuclear cytoplasmic free calcium (Ca2+i) increased just prior to Ca2+n. In contrast, AA-PDGF stimulation resulted in the generation of one or two irregular, transient Ca2+n spikes. Caffeine pretreatment followed by AA-PDGF stimulation resulted in Ca2+n oscillations very similar to those produced by BB-PDGF alone. Additionally, the AA-PDGF and BB-PDGF isoforms appeared to modulate distinct pools of cellular Ca2+, as BB-PDGF was still capable of inducing Ca2+n oscillations subsequent to prior induction of oscillations by AA-PDGF/caffeine. These PDGF isoform-specific changes in nuclear free Ca2+ could serve as a mechanism by which isoform-specific cellular signaling pathways may be manifested by the growth factors.

3T3 Cells↗
PubMed1994

Automatic detection of clustered, fluorescent-stained nuclei by digital image-based cytometry.

Automatic image-based cytometry (IC) can conveniently quantify the distributions of several specific, fluorescence-labeled molecules within individual, isolated cells of slide- or tissue-based specimens. However, many specimens contain clusters of cells or nuclei that are not detected as individual entities by existing automatic methods. We have developed analysis algorithms which detected individual nuclei occurring in clusters or as isolated nuclei. Specimens were labeled with a fluorescent DNA stain, imaged and the images were segmented into regions of nuclei and background. Clusters of nuclei, identified by their size and shape, were divided into individual nuclei by searching for dividing paths between nuclei. The paths, which need not be straight, possessed the highest average gradient per pixel. In addition, both high- and low-pass filtered images of the original image were analyzed. For each individual nucleus, one of the three segmented regions representing the nucleus (from either the original or one of two filtered images) was chosen as the final result, based on the closeness of the regions to average nuclear morphology. The algorithms correctly detected a high proportion of isolated (328/333) and clustered (254/271) nuclei when applied to images of 2 microns prostate and breast cancer sections. Thus, these algorithms should enable much more accurate detection and analyses of nuclei in intact specimens.

Algorithms↗
PubMed1994

Detection of human papillomavirus type 16/18 DNA in cervicovaginal cells by fluorescence based in situ hybridization and automated image cytometry.

Automatic fluorescence image cytometry (AFIC) is a fast, sensitive, and reliable approach for screening slide-based clinical specimens. In this study, we applied AFIC to identify cancer-associated human papillomavirus (HPV) genotypes 16 and 18 in individual cells of cervical smears using a sensitive fluorescence based in situ hybridization (FISH) assay. HPV sequences were labeled by FISH and the cells imaged using an epi-fluorescence microscope coupled to a low-light color CCD camera. Before application to clinical specimens, AFIC was assessed using fluorescent calibration beads and cervical cancer cell lines containing known numbers of integrated HPV genomes per nucleus. Assessment showed that our AFIC had a linear response, was quantitatively accurate, and had the sensitivity to detect one HPV genome per nucleus. After acquisition of images, computer algorithms identified every cell nucleus (via a fluorescent DNA counterstain) and quantified the FISH signal per nucleus. AFIC was employed to screen 27 patient specimens for HPV 16/18, of which 12 were positive. The HPV status of the specimens positively correlated with the pathological diagnosis, and since AFIC automatically and correctly located every cell, it was possible to directly compare morphology and HPV status in the same cell. In conclusion, the combination of FISH and AFIC is a sensitive and quantitative method to detect high risk HPV sequences in cervical smears.

Algorithms↗
PubMed1994

Additive effect on gas exchange of inhaled nitric oxide and intravenous almitrine bismesylate in the adult respiratory distress syndrome.

OBJECTIVE: To assess the additive effect of inhaled nitric oxide (NO) and intravenous almitrine bismesylate (ALM) on gas exchange. DESIGN: Prospective self-controlled study. SETTING: 3 medico-surgical intensive care units. PATIENTS: 17 patients with severe hypoxemia (PaO2/FIO2 ratio: 88 +/- 30 mmHg, venous admixture: 47 +/- 7%) and elevated mean pulmonary artery pressure (MPAP: 30 +/- 5 mmHg) due to adult respiratory distress syndrome (ARDS). INTERVENTIONS: 5 conditions were studied: 1) baseline, 2) 5 to 10 ppm of NO during 30 min, 3) discontinuation of NO during 30 min, 4) ALM infusion (0.5 mg/kg) during 30 min, 5) ALM infusion (0.5 mg/kg) during 30 min in combination with 5 to 10 ppm of NO. MEASUREMENT AND RESULTS: The PaO2/FIO2 ratio rose from 88 +/- 30 to 98 +/- 37 mmHg (NS) with NO alone, and from 92 +/- 25 to 130 +/- 56 mmHg (p < 0.01) with NO + ALM (p < 0.05 vs NO alone). Seven patients were considered as "NO-responders" (rise in PaO2/FIO2 ratio of 10 mmHg or more with NO); in this subgroup the PaO2/FIO2 ratio rose from 87 +/- 30 to 128 +/- 39 mmHg (p < 0.05) with NO alone, and from 93 +/- 20 to 169 +/- 51 mmHg (p < 0.01) with NO + ALM (p < 0.05 versus NO alone). MPAP decreased from 30 +/- 5 to 26 +/- 5 mmHg (p < 0.01) with NO alone, increased slightly from 28 +/- 5 to 31 +/- 5 mmHg (NS) with ALM alone and decreased to 27 +/- 5 mmHg (p < 0.05) with NO + ALM. CONCLUSIONS: NO + ALM had additive effects on gas exchange while decreasing MPAP in patients with ARDS. The effects of NO alone were small and non significant, except in a subgroup of 7 patients in whom the combination of both therapies had the more pronounced results.

Administration, Inhalation↗
PubMed1994

Distribution of electrical potential, pH, free Ca2+, and volume inside cultured adult rabbit cardiac myocytes during chemical hypoxia: a multiparameter digitized confocal microscopic study.

Exploiting the optical sectioning capabilities of laser scanning confocal microscopy and using parameter-specific fluorescent probes, we determined the distribution of pH, free Ca2+, electrical potential, and volume inside cultured adult rabbit cardiac myocytes during ATP depletion and reductive stress with cyanide and 2-deoxyglucose ("chemical hypoxia"). During normoxic incubations, myocytes exhibited a cytosolic pH of 7.1 and a mitochondrial pH of 8.0 (delta pH = 0.9 units). Sarcolemmal membrane potential (delta psi) was -80 mV, and mitochondrial delta psi was as high as -100 mV, yielding a mitochondrial protonmotive force (delta p) of -155 mV (delta P = delta psi - 60 delta pH). After 30 min of chemical hypoxia, mitochondrial delta pH decreased to 0.5 pH units, but mitochondrial delta psi remained essentially unchanged. By 40 min, delta pH was collapsed, and mitochondrial and cytosolic free Ca2+ began to increase. Mitochondrial and sarcolemmal delta psi remained high. as Ca2+ rose, myocytes shortened, hypercontracted, and blebbed with a 30% decrease of cell volume. After hypercontraction, extensive mitochondrial Ca2+ loading occurred. After another few minutes, mitochondrial depolarized completely and released their load of Ca2+. After many more minutes, the sarcolemmal permeability barrier broke down, and viability was lost. These studies demonstrate a sequence of subcellular ionic and electrical changes that may underlie the progression to irreversible hypoxic injury.

Animals↗
PubMed1994

Moderate and vigorous leisure-time physical activity and cardiovascular disease risk factors in West Germany, 1984-1991.

The relationship between leisure-time physical activity (LTPA) and cardiovascular disease (CVD) risk factors was analysed based on data of three cross-sectional National Health Surveys, carried out in West Germany between 1984 and 1991. A total of 7663 males and 7722 females, aged 25-69 years, were examined. A self-administered questionnaire was used to assess LTPA which was defined as the mean duration of time (in minutes) spent during the past week on 14 specified sports during the previous 3 months. The mean time for LTPA per week was 102 minutes for males and 70 minutes for females. No significant differences were observed in LTPA mean duration for the three surveys. Younger age and higher social class were strong predictors for greater LTPA. In multiple linear regression models, after adjustment for age and social class, LTPA was significantly associated with lower systolic and diastolic blood pressure (both sexes), total cholesterol (males only), cigarette smoking (males only) and body mass index (females only), and with higher HDL-cholesterol (both sexes). The data suggest that LTPA plays an important role if aiming at reducing CVD risk factors in the general population.

Adult↗
PubMed1994

ATP depletion rather than mitochondrial depolarization mediates hepatocyte killing after metabolic inhibition.

The importance of ATP depletion and mitochondrial depolarization in the toxicity of cyanide, oligomycin, and carbonyl cyanide m-cholorophenylhydrazone (CCCP), an uncoupler, was evaluated in rat hepatocytes. Oligomycin, an inhibitor of the reversible mitochondrial ATP synthase (F1F0-adenosinetriphosphatase), caused dose-dependent cell killing with 0.1 microgram/ml being the minimum concentration causing the maximum cell killing. Oligomycin also caused rapid ATP depletion without causing mitochondrial depolarization. Fructose (20 mM), a potent glycolytic substrate in liver, protected completely against oligomycin toxicity. CCCP (5 microM) also caused rapid killing of hepatocytes. Fructose retarded cell death caused by CCCP but failed to prevent lethal cell injury. Although oligomycin (1.0 microgram/ml) was lethally toxic by itself, in the presence of fructose it protected completely against CCCP-induced cell killing. Cyanide (2.5 mM), an inhibitor of mitochondrial respiration, caused rapid cell killing that was reversed by fructose. CCCP completely blocked fructose protection against cyanide, causing mitochondrial depolarization and rapid ATP depletion. In the presence of fructose and cyanide, oligomycin protected cells against CCCP-induced ATP depletion and cell death but did not prevent mitochondrial depolarization. In every instance, cell killing was associated with ATP depletion, whereas protection against lethal cell injury was associated with preservation of ATP. In conclusion, protection by fructose against toxicity of cyanide, oligomycin, and CCCP was mediated by glycolytic ATP formation rather than by preservation of the mitochondrial membrane potential. These findings support the hypothesis that inhibition of cellular ATP formation is a crucial event in the progression of irreversible cell injury.

Adenosine Triphosphate↗
PubMed1994

Introduction of a fast and sensitive fluorescent in situ hybridization method for single-copy detection of human papillomavirus (HPV) genome.

At present, in situ hybridization (ISH) is the only method for detection of specific genes in morphologically intact cells or tissue. We have developed a highly sensitive and quantitative fluorescence-based in situ hybridization (FISH) technique that can detect as few as one to five copies of the integrated human papillomavirus (HPV) type 16 genome in cervical cell lines, using digoxygenin tail-labeled oligonucleotides (Method 1). The entire procedure can be carried out in 4.5 hr through the elimination of some of the steps routinely used in other ISH protocols. We also compared the sensitivity of this new FISH method (Method 1) to four other FISH techniques: digoxigenin-labeled DNA probe (Method 2); fluorescein-15-d-ATP-labeled oligonucleotides (Method 3); fluorescein 15-d-ATP labeled DNA probe (Method 4); and biotin-DNA-labeled probe (Method 5), for their ability to detect HPV DNA in the HPV-positive human cervical cell lines CaSki (500 copies) and SiHa (1-5 copies), but not in C33-A and HT-3, which do not contain any copies of HPV. Our results indicate that Method 1 is more sensitive than the other methods employed. Method 1 was the only method that could reliably detect an HPV-16 genome in all SiHa cells. Our data suggest that the Method 1 FISH technique is highly sensitive and may therefore be of general use for detection and quantitation of a variety of viral genomes (including HIV), oncogenes, and drug-resistant genes, in a variety of morphologically intact cells and tissues.

Base Sequence↗
PubMed1993

Mitochondria as a source of reactive oxygen species during reductive stress in rat hepatocytes.

Cell killing, oxygen consumption, and hydroperoxide formation were determined in rat hepatocytes after glycolytic and respiratory inhibition. These conditions model the ATP depletion and reductive stress of anoxia ("chemical hypoxia"). Glycolysis was inhibited with iodoacetate, and mitochondrial electron transfer was blocked with sodium azide, cyanide, or myxothiazol. Cell killing, hydroperoxide formation, and inhibitor-insensitive oxygen consumption were greater after azide than after myxothiazol or cyanide. Desferrioxamine, an inhibitor of iron-catalyzed hydroxyl radical formation, delayed cell killing after each of the respiratory inhibitors. Anoxia also delayed cell killing during chemical hypoxia. However, during anoxic incubations, desferrioxamine did not delay the onset of cell death. These findings indicate that reactive oxygen species participate in lethal cell injury during chemical hypoxia. In isolated mitochondria, previous studies have shown that myxothiazol inhibits Q cycle-mediated ubisemiquinone formation in complex III (ubiquinol-cytochrome c oxidoreductase) and that ubisemiquinone can react with molecular oxygen to form superoxide. Decreased killing of hepatocytes with myxothiazol compared with azide suggests, therefore, that mitochondrial oxygen radical formation by complex III is involved in cell killing during reductive stress. In support of this hypothesis, myxothiazol reduced rates of cell killing and hydroperoxide formation in hepatocytes incubated with azide or cyanide. This mitochondrial mechanism for oxygen radical formation may be important in relative but not absolute hypoxia.

Animals↗
PubMed1993

Intracellular pH and Ca2+ homeostasis in the pH paradox of reperfusion injury to neonatal rat cardiac myocytes.

Ischemia is characterized by anoxia and a large decrease of tissue pH. After a critical period of ischemia, reperfusion precipitates irreversible injury. Previous work showed that reperfusion injury to cultured neonatal myocytes was precipitated by a rapid return to physiological pH, a "pH paradox" (Bond, J., B. Herman, and J. Lemasters. Biochem. Biophys. Res. Commun. 179: 798-803, 1991). The aim of this study was to measure intracellular pH (pHi) and cytosolic free Ca2+ during the pH paradox of reperfusion injury to cultured neonatal rat cardiac myocytes. pHi and free Ca2+ were measured by ratio imaging of 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein and fura 2 fluorescence. To simulate ATP depletion and acidosis of ischemia, myocytes were incubated with 20 mM 2-deoxyglucose plus 2.5 mM NaCN at pH 6.2. During simulated ischemia, pHi dropped to < 6.5 and subsequently remained constant. During this time, some blebbing but little hypercontraction occurred. After 3 or 4 h of simulated ischemia, inhibitors were removed and cells were incubated at pH 7.4 to simulate reperfusion. pHi began to increase, blebbing accelerated, and myocytes hypercontracted. As pHi increased, viability was lost. The same occurred if pH was increased but metabolic inhibitors were not removed. Monensin, a Na(+)-H+ ionophore, accelerated the increase of pH after reperfusion and hastened cell killing. Hypercontraction, blebbing, and loss of viability did not occur when inhibitors were removed at pH 6.2 or in the presence of dimethylamiloride, an inhibitor of Na(+)-H+ exchange. Protection was associated with maintenance of an acidotic pHi. Free Ca2+ progressively increased during simulated ischemia. After simulated reperfusion, free Ca2+ increased further.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗