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Biomedical subjects

H Mueller

Publications and source records attributed to H Mueller.

At least 37 records · Page 2Linked to original sources
PubMed1997

Selective modulation of protein kinase A and protein kinase C activities in epidermal growth factor (EGF)-stimulated MCF-7 breast cancer cells.

In human MCF-7 breast cancer cells, both protein kinase A (PKA) and different members of the protein kinase C (PKC) family are stimulated upon binding of epidermal growth factor (EGF) to cell surface receptors. Selective stimulation of calcium-dependent PKCs with 10(-6) to 10(-9) M Thymeleatoxin significantly increased the proliferation rate of MCF-7 cells over 5 days in culture. This stimulation was blocked by the PKC antagonist Chelerythrine. In contrast, selective activation of PKA by addition of 1 mM dibutyryl cyclic AMP (dBcAMP) did not affect the proliferation rate of MCF-7 cells. Similarly, activation of the adenylate cyclase by 1 microM Forskolin and inhibition of PKA by the cyclic AMP analogue Rp-cAMPS did not modulate the proliferation rate of these cells. Activation of PKC stimulated the expression of the immediate early gene c-fos but c-myc expression was not significantly enhanced. On the other hand, PKA activation increased both c-myc and c-fos expression in MCF-7 cells. These results suggest that PKA activation and c-myc expression are not obligatory for proliferation of MCF-7 cells.

Alkaloids↗
PubMed1996

Factors limiting the enrollment of women in a randomized coronary artery disease trial. The Asymptomatic Cardiac Ischemia Pilot Study (ACIP) Investigators.

BACKGROUND AND HYPOTHESIS: Although it is recognized that women have been underrepresented in clinical trials of cardiovascular disease, the reasons for their limited enrollment have not been elucidated. METHODS: A prospective tracking system was established in the Asymptomatic Cardiac Ischemia Pilot study (ACIP) to monitor recruitment and identify protocol issues that interfered with the recruitment of women. Patients with stress test evidence for ischemia during the course of routine clinical care were screened for asymptomatic ischemia with an ambulatory electrocardiogram (ECG). RESULTS: Those with at least one episode of asymptomatic ischemia and angiographic evidence of coronary artery disease suited for revascularization could be randomized. Women comprised only 17% of the 1,820 patients screened for asymptomatic ischemia, and only 14% of the 558 patients randomized. The limited number of women screened for ischemia was largely due to the limited number of women (25% of all patients) found to have test evidence for ischemia or coronary artery disease suited for revascularization during the course of routine clinical care. Once patients were identified as having ischemia on stress test and ambulatory ECG, the major difference in eligibility was the difference in disqualifying angiograms, occurring 21/2 times as frequently in women as in men (p < 0.001). CONCLUSION: The percentage of women recruited was lower than the prevalence of ischemic heart disease in the general population because at participating centers (1) women were found to have ischemia less often than men during the course of routine clinical care, and (2) screening tests for ischemia were less predictive of protocol-defined coronary disease in women than in men.

Analysis of Variance↗
PubMed1996

Sequence analysis of hepatitis GB virus C (GBV-C) isolates from 14 patients.

In 1995, a new human hepatitis virus belonging to the family Flaviviridae was described and designated hepatitis GBV-C. To investigate variations within the genome of GBV-C and to study the relationship of GBV-C to GBV-A/B or hepatitis C virus (HCV), we established a detection system using reverse transcriptase polymerase chain reaction (RT-PCR) of the putative helicase region (NS3). So far, isolates derived from 14 different GBV-C-positive sera were analyzed (GBV-C/S3-36), showing 80.1-89.4% (mean: 85%) identical nucleotides. The deduced amino acid sequences revealed 97.3% homology. Nucleotide sequences of GBV-C/S3-36 revealed about 60% identity to GBV-A as well as to HCV, but only 56% identity to GBV-B. Amino acid sequences revealed 73.4 and 68.6% similarity to GBV-A and GBV-B, respectively, but a slightly higher percentage of 78.5% to HCV sequences. Thus, according to the putative GBV-C helicase sequence, a subtyping of GBV-C into different genotypes may be necessary.

Adult↗
PubMed1996

Tumour necrosis factor and interferon are selectively cytostatic in vitro for hormone-dependent and hormone-independent human breast cancer cells.

Since experimental studies have shown that tumour necrosis factor-alpha (TNF-alpha) has potent anti-tumour activity that can be potentiated with cytokines, we tested the efficacy of TNF-alpha with interferon-gamma (IFN-gamma) on different human breast cancer cell lines, particularly comparing hormone-dependent and -independent phenotypes. TNF-alpha inhibited the growth of hormone-dependent human MCF-7, ZR-75-1 and T47-D breast cancer cells with a half maximal concentration of 0.25 nM. In contrast, the growth of hormone-independent cells MDA-MB-231 and HS578T was not affected by TNF-alpha alone, but a synergistic inhibition was observed when using IFN-gamma and TNF-alpha together. The mRNA for the proto-oncogene C-MYC, as an intracellular indicator of cell activation, was significantly increased in MCF-7 cells in the presence of TNF-alpha. In MDA-MB-231 cells this mRNA was increased only in the presence of both TNF-alpha and IFN-gamma, without a change in the number of surface TNF receptors. These findings indicate that TNF-alpha treatment in combination with IFN-gamma may provide a successful approach to overcome the cellular heterogeneity of advanced breast tumours.

Breast Neoplasms↗
PubMed1996

Allergenic epitopes of bovine alpha S1-casein recognized by human IgE and IgG.

B-cell epitopes of bovine alpha S1-casein, one of the major allergens of cow's milk, were identified by a screening method based on synthetic peptides. According to the known amino acid sequence of alpha S1-casein, a set of 188 overlapping sequential decapeptides shifted by one amino acid was manually synthesized on polyethylene pins by the 9-fluorenyl-methoxycarbonyl (Fmoc) method. Peptides were screened by an enzyme-linked immunosorbent assay (ELISA) specific for human IgE and IgG. Bound antibodies were detected by successive incubation with up to three polyclonal antibodies, the last one conjugated to horseradish peroxidase. Tested sera were from 15 patients with acute clinical reactions to cow's milk and IgE-specific reactions to bovine alpha-casein in the ELISA and immunoblot. Sera from 10 healthy subjects without remarkable reactions to cow's milk proteins were used as controls. All sera from allergic subjects showed reactions with three regions of alpha S1-casein, corresponding to amino acids 19-30, 93-98, and 141-150. Furthermore, individual sera showed reactions with other parts of the protein. No essential differences in the epitope specificity of IgE and IgG were found. Inhibition of IgE binding to alpha S1-casein with soluble synthetic peptides confirmed the results and revealed peptide CN-2 as the most inhibiting one.

Adolescent↗
PubMed1995

Selective regulation of steroid receptor expression in MCF-7 breast cancer cells by a novel member of the heregulin family.

A 52 kDa heregulin secreted by estrogen receptor (ER)-negative human breast cancer cells induced rapid growth of ER-positive MCF-7 breast cancer cells with a stimulatory effect observed at 10(-11)M. This heregulin down-regulated the message for ER in MCF-7 cells within 24 hours after stimulation. Similarly the ER protein was down-regulated within 24 to 48 hours after stimulation of cells. However, this down-regulation occurred without activation of the ER, since the progesterone receptor (PR) level of cells stimulated with the 52 kDa heregulin did not increase over the time period measured. As a control, estradiol down-regulated and activated ER as shown by a pronounced increase in PR content of MCF-7 cells. This finding indicates an important role of this heregulin in the down-regulation of ER in estrogen-dependent human breast cancer cells.

Breast Neoplasms↗
PubMed1994

Isolation of a heregulin-like growth factor secreted by estrogen receptor-negative MDA-MB-231 human breast cancer cells that stimulates estrogen receptor-positive cells.

A 52 kDa peptide growth factor secreted by the estrogen receptor (ER)-negative human breast cancer cell line MDA-MB-231 was purified to homogeneity. It induced transient membrane ruffling, lamellipodia formation, cell motility and proliferation exclusively of ER-positive human breast cancer cells. Partial sequencing revealed a high homology to the protein family of heregulins. However, the obtained amino acid sequences of the new factor were not completely identical to any of the members of the heregulin family. This finding together with the observation that the successful purification protocol was significantly different from that used to isolate other members of the heregulin family indicate the isolation of a novel heregulin-like proliferation factor for ER-positive human breast cancer cells.

Amino Acid Sequence↗
PubMed1994

Expression of Ca(2+)-binding proteins of the S100 family in malignant human breast-cancer cell lines and biopsy samples.

In order to examine whether the expression of calcium-binding proteins of the S100 family may correlate with the transformation grade of human mammary-tumor cells, we studied the expression patterns of 4 members of this family (CACY, CAPL, S100L, S100 alpha/beta) in human breast-cancer cell lines. Each S100 protein is shown to be individually regulated in the human breast-cancer cell lines we studied, but it appears that the expression levels of S100 proteins do not strictly correlate with prognostic factors or the tumorigenicity of the cells. However, 2 aggressive cell lines, MDA-MB-231 and HS-578T, show elevated expression of CAPL. We show that methylation may account for partial regulation of the S100 genes, whereas neither genomic rearrangements in the S100 gene cluster region nor gene dosis effects seem to influence their expression pattern in MDA-MB-231 and MCF-7 cells. On the basis of our genomic analyses, we can localize the gene for S100L within 5 kb upstream of S100E, thus extending the S100 gene cluster by one gene. A series of primary breast tumors was collected and tested for expression of CAPL, CACY and S100 alpha/beta. The results show that all human breast-cancer tissues tested express CACY, whereas the presence of CAPL is more restricted. There is a significant correlation between enhanced expression of CAPL and presence of the invasivity marker urokinase-type plasminogen activator (uPA). This observation suggests that CAPL may play an important role in the acquisition of metastatic potential of human mammary epithelial cells.

Breast Neoplasms↗
PubMed1994

Differential signal transduction of epidermal-growth-factor receptors in hormone-dependent and hormone-independent human breast cancer cells.

In breast cancer, hormone dependency is inversely correlated with the number of surface epidermal-growth-factor (EGF) receptors on the tumor cells. In vitro, EGF stimulated only hormone-dependent immortalized human breast cancer cells to grow with an increased rate whereas hormone-independent cells were not affected by EGF. The number of EGF surface receptors is about 5-10-times smaller on hormone-dependent cells than on hormone-independent cells. Two cell lines representing the two cell types were used to demonstrate the signal-transduction capabilities of the EGF receptors. The two cell lines were the hormone-dependent MCF-7 cells and the hormone-independent MDA-MB-231 cells. Incubation at 37 degrees C for 15 min with 10(-8) M EGF increased the surface EGF-receptor density substantially on MCF-7 cells (50%) and reduced the number of these receptors on MDA-MB-231 cells to about 65% of the control. Both cell lines internalized a fluorescein-isothiocyanate-labeled EGF with similar kinetics. EGF triggered tyrosine phosphorylation of several targets in isolated MCF-7 cell membranes. One of these targets was shown by immunoprecipitation to be the EGF receptor. In MDA-MB-231 cell membranes, the EGF receptor was demonstrated to be the main target for tyrosine phosphorylation. The mRNA expression of the immediate early proto-oncogene c-fos was stimulated by EGF only in MCF-7 cells. In contrast, the mRNA of the EGF receptors was stimulated by EGF in both cell lines. These results demonstrate that, although EGF-binding sites are present on both cell lines, their signal-transduction capacity and activities are substantially different and resulted in a divergent response of the two cell types to EGF.

Blotting, Northern↗
PubMed1994

Growth factor receptors and their ligands.

The understanding of the signal transduction cascade involving growth factors and their receptors is one major key for diagnostic and therapeutic improvements in human neoplasms. Using receptor autoradiography, an inverse relationship for the incidence of somatostatin receptors (SSR) and epidermal growth factor receptors (EGFR) was found in gliomas [1]. In the majority of low grade gliomas, SSR were present but EGFR were absent. In contrast, EGFR were present in most glioblastomas, but no SSR were detected. Recently, the amplification of the EGFR gene and its overexpression was demonstrated to be associated with the development of glioblastomas. Several independent reports revealed that 40-50% of tumors show amplified EGFR [2-4]. The frequency of EGFR amplification was directly associated with tumor malignancy. In addition, amplified EGFR levels indicate a bad prognosis and shorter overall survival [5]. Recent analysis of the EGFR gene in tumors has shown that regions of this gene frequently undergo alteration. Hence, not only amplification but also mutation may be the cause of the increased malignancy in EGFR overexpressing cells [6].

Brain Neoplasms↗
PubMed1994

Impact of age on clinical outcome and postlytic management strategies in patients treated with intravenous thrombolytic therapy. Results from the TIMI II Study. TIMI II Investigators.

BACKGROUND: Few thrombolytic studies have assessed whether patient age is an indication for routine postlytic cardiac catheterization and revascularization or evaluated the impact of age on 1-year outcome differences after acute myocardial infarction. METHODS AND RESULTS: A secondary analysis of 3339 patients enrolled in the TIMI II trial was performed to identify differences in clinical and coronary angiographic findings and 1-year cardiac event rates among 841 patients < 50 years old, 1639 patients 50 to 64 years old, and 859 patients 65 to 75 years old. Differences in 1-year clinical outcome were assessed among patients randomly assigned to an invasive or a conservative postlytic strategy within each age group. The percentages of patients with a prior history of myocardial infarction, angina, congestive heart failure, hypertension, or diabetes mellitus or an infarction complicated at the time of study entry by shock, pulmonary edema, hypotension, rales more than one third of lung fields, or atrial fibrillation as well as the percentage of female patients (all P < .001) increased with age. Fewer older patients (65 to 75 years) received early (ie, < or = within 2 hours after symptom onset) treatment with recombinant tissue-type plasminogen activator (rTPA), and fewer were eligible for random assignment to immediate or deferred beta-blocker therapy (P = .01). The location of the infarct-related artery and the percentage of patients with patent (ie, TIMI flow grade 2 or 3) or "complete" (ie, TIMI flow grade 3) infarct-related artery flow did not vary with age. The percentage of patients with multivessel disease was greatest in the older patients (P = .001). Cumulative 1-year mortality was low in the youngest patients (2.8%; 99% confidence interval [CI], 1.6% to 4.7%) regardless of whether the infarct location was anterior (3.7%) or nonanterior (1.6%). The highest 1-year mortality occurred in the older patients (13.6%; 99% CI, 10.9% to 16.9%), particularly when the infarct location was anterior (18%). The 42-day rates of reinfarction (P = .85), death (P = .95), or death or reinfarction (P = .99) were similar in patients assigned to the invasive or conservative postlytic treatment strategy, regardless of age group. CONCLUSIONS: Among patients with acute myocardial infarction treated with intravenous rTPA, heparin, and aspirin, there were age-related differences in time to treatment with thrombolytic therapy, use of beta-blockers, extent of coronary artery disease, and 1-year cardiac event rates. Routine use of cardiac catheterization and coronary revascularization does not improve immediate or 1-year outcome in terms of mortality or reinfarction compared with a more conservative strategy in young, middle-aged, or elderly patients similar to those enrolled in TIMI II.

Adult↗
PubMed1994

Intensive vascular training in stage IIb of peripheral arterial occlusive disease. The additive effects of intravenous prostaglandin E1 or intravenous pentoxifylline during training.

In a randomized open study, the combination of either prostaglandin E1 (PGE1) or pentoxifylline with controlled vascular training was compared with vascular training alone in patients with peripheral arterial occlusive disease in stage IIb. Forty-four patients were randomly assigned to treatment either of intensive vascular training alone (n = 15) or in combination with either i.v. pentoxifylline (200 mg over 2 hours BID, n = 15) or PGE1 (40 micrograms over 2 hours BID, n = 14). The basic therapy was a well-defined routine for vascular training, which was identical for all groups. The duration of therapy was 4 weeks. In all three test groups, there was a significant increase in the walking distance. There was a 119% increase in symptom-free walking distance in the exercise-only group. In comparison with exercise alone, the additional administration of pentoxifylline produced no greater effect; the increase was 105%. In contrast, administration of PGE1 combined with exercise achieved a remarkable improvement of 604%. Between-group comparison revealed the significant superiority of treatment with PGE1 (P < .05). During the 1-year follow-up, there was a reduction in the walking performance in all groups, albeit of variable extent. In the exercise-only and the pentoxifylline groups, the maintained increase in walking distance was only 30% compared with baseline values before the beginning of therapy. In the PGE1 group, on the other hand, the maintained improvement was 149%. Nine of 14 patients were still in stage IIa of peripheral arterial occlusive disease 1 year after PGE1 therapy.

Alprostadil↗
PubMed1992

c-fos, c-jun and c-myc expressions are not growth rate limiting for the human MCF-7 breast cancer cells.

Insulin-like growth factor I (IGF-I) was 3 times more potent in pagating MCF-7 cell proliferation than epidermal growth factor (EGF). IGF-I stimulated c-fos mRNA expression about 5 times less than EGF. Both growth factors were equipotent in inducing c-jun and c-myc mRNA expressions. The protein level of c-Myc correlated with the mRNA level. IGF-I and EGF stimulated the transcriptional activity dependent on the phorbol 12-myristate 13-acetate-responsive element (TRE) to the same extent, when measured by the chloramphenicol acetyl transferase activity of a transiently transfected multiple TRE construct. These results strongly indicate that the expression levels of the measured proto-oncogenes do not correlate with the increase of growth stimulation by IGF-I and EGF and are not growth rate limiting for the human MCF-7 breast cancer cells.

Breast Neoplasms↗
PubMed1992

Reversal of inhibitory pathways in neutrophils by protein kinase antagonists: a rational approach to the restoration of depressed cell function?

Neutrophil functions are sensitive to both stimulatory and inhibitory pathways. For example, the endogenous hormones histamine, prostaglandin E1, adenosine, and catecholamine were found to inhibit the oxidant responses of human neutrophils by formyl peptide to 6.2, 16.8, 11.4, and 15.4%, respectively, of the initial response. The inhibition of cell function is mimicked by dibutyryl cAMP and forskolin, consistent with a pathway involving cAMP and an A kinase. Because of likely roles of kinases in both stimulatory and inhibitory pathways, we evaluated the potential for regulating either pathway by kinase inhibitors. Preincubation of intact neutrophils with membrane-permeable but nonspecific protein kinase antagonists blocked the isoproterenol-mediated inhibition of superoxide generation. The isoquinoline sulfonamides H-7, H-8, and H-9 at 100 microM reversed inhibition to 60.1, 66.6, and 70.9%, respectively, of the response of control cells. H-9 also antagonized the inhibition of superoxide production induced by other agents that regulate intracellular cAMP (prostaglandin E1, histamine, adenosine, forskolin, and dibutyryl cAMP). A synthetic peptide used as a specific but impermeable protein kinase A antagonist restored superoxide production inhibited by isoproterenol and cAMP up to 70% in electroporated cells. A small number of proteins are targets of cAMP-dependent phosphorylation in electroporated cells, and phosphorylation is inhibited in the presence of the peptide inhibitor. Taken together, these data show that a peptide inhibitor and isoquinoline sulfonamides reverse the inhibition of the respiratory burst in neutrophils evoked by the inhibitory pathways. Drugs that reverse the effect of endogenous inhibitors of neutrophil activation (by restoring cell function) have important therapeutic implications in restoring cell functions in patients whose cell functions are depressed under physiological conditions.

Cell Membrane Permeability↗
PubMed1992

Mechanisms of early death despite thrombolytic therapy: experience from the Thrombolysis in Myocardial Infarction Phase II (TIMI II) study.

Mechanisms of death among patients who died within 18 h of enrollment in the Thrombolysis in Myocardial Infarction Phase II (TIMI II) study were analyzed. Of 3,339 patients enrolled, 32 died within the 1st 4 h and 31 died within the subsequent 14 h. Thirteen of the 63 patients had shock at enrollment; 22 had advanced hemodynamic compromise without shock and 28 initially had minimal to no compromise. Prior infarction was present in 16 patients (25%). Pump failure was responsible for 39 early deaths (62%), ventricular rupture for 10 (16%), arrhythmia for 8 (13%) and complications of therapy for 6 (10%). Nine of 720 patients randomized to immediate intravenous beta-adrenergic blocking agent therapy had an early death compared with 6 of 714 assigned to deferred beta-blocker therapy. Thus, mortality is highest in the early hours after myocardial infarction, even in patients treated with thrombolytic therapy and is most frequently due to pump failure. These results imply that efforts to reduce mortality during this critical time period should be directed at prevention, limitation or palliation of early pump failure.

Adrenergic beta-Antagonists↗
PubMed1991

Rap1A is a substrate for cyclic AMP-dependent protein kinase in human neutrophils.

The Ras-related protein, Rap1B, has previously been shown to serve as a PKA substrate in vitro and to be phosphorylated by cAMP elevating agents in human platelets. We have purified a Rap1 protein that serves as a PKA substrate from human neutrophils, and we now identify this protein as Rap1A. A 23-kDa protein that co-migrated with recombinant Rap1A was phosphorylated in electroporated human neutrophils upon stimulation by cAMP in the presence of [gamma-32P]ATP. This protein could be immunoprecipitated by the Rap1A/B-specific antibody, R61. The 23-kDa phosphoprotein was monitored during the purification of Rap1 from neutrophil membrane extracts and was shown to copurify with Rap1 during the DEAE Sephacel, heptylamine Sepharose, and MonoQ chromatography steps utilized. The purified protein was phosphorylated to an extent of 1 mol phosphate/mol GTP gamma S bound. This protein was identified as Rap1A by: 1) amino acid sequence analysis; and 2) immunoblotting with a Rap1A-specific antibody. The amino acid phosphorylated on Rap1A by PKA was a serine residue. The site of phosphorylation was indicated by carboxypeptidase digestion and confirmed using a mutant recombinant Rap1A lacking the relevant serine (serine-180). Rap1A, not Rap1B, appears to be the major 23-kDa PKA substrate in human neutrophils. It is possible that Rap1A plays a role in human neutrophils in mediating the inhibitory effects of cAMP-elevating agents upon chemoattractant-stimulated cell activation.

Electrophoresis, Gel, Two-Dimensional↗