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Biomedical subjects

C Rudolph

Publications and source records attributed to C Rudolph.

At least 19 recordsLinked to original sources
PubMed1999

Determination of copy number of c-Myc protein per cell by quantitative Western blotting.

The protooncogene c-Myc plays a key role in growth control, differentiation, and apoptosis. An abnormally high expression of c-myc has been found to be associated with many neoplasms. c-Myc gene expression is usually measured at the mRNA level. Few studies have been published on quantitative Myc protein determination. A major drawback of ELISA (enzyme-linked immunosorbent assay) methods is the uncertainty of the specificity of the antibody reaction. In contrast, antibody specificity can be easily controlled by Western/immunoblotting. Here we describe a method to quantify c-Myc protein in primary human IMR90 lung fibroblasts based on Western blotting. Using a high-resolution polyacrylamide gel, we were able to differentiate the cellular c-Myc protein (64 kDa) from a c-Myc internal standard (65 kDa). We determined both the total c-Myc protein content per cell and its distribution in the cytoplasmic and nuclear fractions. About 4000 c-Myc protein molecules were detected in the cytoplasmic fraction and 29,000 copies in the nuclear fraction for proliferating human lung fibroblasts IMR90. The ratio of nuclear (active) to cytoplasmic (inactive) c-Myc protein changed from 17:1 for proliferating cells to 2.5:1 for confluent cells.

Blotting, Western↗
PubMed1999

The msh2 gene of Schizosaccharomyces pombe is involved in mismatch repair, mating-type switching, and meiotic chromosome organization.

We have identified in the fission yeast Schizosaccharomyces pombe a MutS homolog that shows highest homology to the Msh2 subgroup. msh2 disruption gives rise to increased mitotic mutation rates and increased levels of postmeiotic segregation of genetic markers. In bandshift assays performed with msh2Delta cell extracts, a general mismatch-binding activity is absent. By complementation assays, we showed that S. pombe msh2 is allelic with the previously identified swi8 and mut3 genes, which are involved in mating-type switching. The swi8-137 mutant has a mutation in the msh2 gene which causes a truncated Msh2 peptide lacking a putative DNA-binding domain. Cytological analysis revealed that during meiotic prophase of msh2-defective cells, chromosomal structures were frequently formed; such structures are rarely found in the wild type. Our data show that besides having a function in mismatch repair, S. pombe msh2 is required for correct termination of copy synthesis during mating-type switching as well as for proper organization of chromosomes during meiosis.

Alleles↗
PubMed1998

The high mobility group domain protein Cmb1 of Schizosaccharomyces pombe binds to cytosines in base mismatches and opposite chemically altered guanines.

The mismatch-binding activity Cmb1 of Schizosaccharomyces pombe was enriched from wild type cells, and N-terminal sequencing enabled cloning of the respective gene. The deduced amino acid sequence of cmb1(+) contains a high mobility group domain, a motif that is common to a heterogeneous family of DNA-binding proteins. In crude protein extracts of a cmb1 gene-disruption strain, specific binding to C/T, C/A, and C/Delta was abolished. Weak binding to C/C revealed the presence of a second mismatch-binding activity, Cmb2. Cmb1, enriched from S. pombe and purified from Escherichia coli, bound specifically to C/C, C/T, C/A, T/T, and C/Delta but showed little or no affinity to other mismatches and small loops. Cmb1 recognizes 1,2 GpG intrastrand cross-links, produced by the chemotherapeutic drug cisplatin, when two cytosines are opposite the cross-linked guanines but not when other bases are present. Consistently, O6-methylguanine:C but not O6-methylguanine/T lesions were bound. Thus, cytosines in mismatches and opposite chemically modified guanines are the preferred target of Cmb1 recognition. cmb1 mutant cells are more sensitive to cisplatin than wild type cells, indicating a role of Cmb1 in repair of cisplatin-induced DNA damage.

Amino Acid Sequence↗
PubMed1998

Accelerated proliferative senescence of rat embryo fibroblasts after stable transfection of multiple copies of the c-Myc DNA-binding sequence.

The protooncogene c-myc positively regulates cellular proliferation whereas it exhibits negative effects on both cellular senescence and differentiation. Ectopic overexpression of c-myc in transfection experiments or titration of the c-myc mRNA by antisense oligonucleotides has demonstrated that small changes of the concentration of cellular c-myc mRNA or protein levels can be crucial for these processes. In view of the role of c-Myc as a transcription factor, most of these effects may be mediated via its binding to specific DNA sequences. Here we studied the cellular reactions after manipulating the cellular concentration of c-Myc DNA-binding sites. Multiple copies of the c-Myc-binding sequence GACCACGTGGTC or, alternatively, the control sequence GACCAGCTGGTC that displays only a poor affinity for c-Myc were stably introduced into the genome of rat embryo fibroblasts. Transfection with the c-Myc-binding sequence yielded much lower clone numbers and sizes than transfection with the control sequence. After polyclonal selection and further subcultivation cells transfected with c-Myc-binding sequence exhibited a reduced growth rate and achieved less than two-thirds of the cumulative population doublings before becoming senescent and irreversibly growth arrested compared to the controls. Southern blot analysis demonstrated that 30 binding sequences on average were integrated into the cellular genome. Our results can be interpreted as competition of the ectopically introduced c-Myc-binding sequences with the functional genomic ones and assume that a fairly low number of the latter exist in the normal cellular genome. Hence, only a low copy number of introduced c-Myc-binding sequences is sufficient to cause signs of accelerated proliferative senescence.

Animals↗
PubMed1998

Gastroesophageal reflux in patients with subglottic stenosis.

OBJECTIVES: To determine the incidence of gastroesophageal reflux in patients with subglottic stenosis (SGS) and to determine if upper esophageal reflux occurs in addition to lower esophageal reflux in these patients. DESIGN: Esophageal pH probe studies were reviewed in patients diagnosed as having SGS. SETTING: A tertiary care pediatric medical center. PATIENTS: All patients diagnosed as having SGS between January 1990 and July 1996 who had undergone monitoring with an overnight esophageal pH probe. Seventy-four patients qualified for the study. All 74 patients underwent lower probe testing, and 55 of the 74 underwent dual (upper and lower) probe testing. MAIN OUTCOME MEASURES: The percent of time a pH measurement of less than 4.0 was recorded in the upper and lower esophagus. A lower probe pH measurement of less than 4.0 more than 10% of the study time was considered high risk for developing reflux-associated pathologic symptoms. A lower probe pH measurement of less than 4.0 for 5% to 10% of the study time was considered a marginal risk for developing reflux-associated pathologic symptoms. Upper probe criteria for reflux-associated symptoms have not been established. Therefore, patients were grouped as having a pH of less than 4.0 in the upper esophagus for 0%, 0.1% to 0.9%, 1.0% to 1.9%, 2.0% to 3.0%, or more than 3% of the study time. RESULTS: Thirty-seven of the 74 patients who underwent lower probe testing had a pH of less than 4.0 more than 5% of the study time, and 24 had a pH of less than 4.0 more than 10% of the study time. Twelve of the 55 patients who underwent upper probe testing had no measurable reflux; 27 of the 55 had a pH of less than 4.0 more than 1% of the study time; 14 had a pH of less than 4.0 more than 2% of the study time, and 11 had a pH of less than 4.0 more than 3% of the study time. CONCLUSIONS: Gastroesophageal reflux is frequently present in patients with SGS. Gastric contents frequently reach the upper and lower esophagus in these patients. In addition, the high incidence of gastroesophageal reflux in these patients suggests that it may play a role in the development of SGS. The possible effect of gastroesophageal reflux on the surgical repair of SGS requires further study.

Adolescent↗
PubMed1998

Schizosaccharomyces pombe exo1 is involved in the same mismatch repair pathway as msh2 and pms1.

Besides the MutLS-like system, Schizosaccharomyces pombe has an additional pathway of mismatch repair. This minor pathway, producing short excision tracts, repairs C/C and, with lower efficiency, other mismatches also. We investigated the involvement of the exo1+, msh2+ and pms1+ genes in the two pathways. The exo1+ gene encodes a 5' to 3' exonuclease, while msh2+ and pms1+ are homologs of Escherichia coli mutS and mutL, respectively. Intragenic two-factor crosses showed that exo1+, msh2+ and pms1+ are involved in the major, but not in the C/C-correcting, pathway. Post-meiotic segregation frequencies and mitotic mutation rates in single and double mutants supported this finding. Furthermore, msh2 delta was epistatic over exo1 delta, and the ExoI enzyme is likely to be redundant with other exonucleases.

Carrier Proteins↗
PubMed1998

Effects of fiber laxatives and calcium docusate on regional water content and viscosity of digesta in the large intestine of the pig.

The aim of this study was to determine how bulk fibers and calcium docusate affect regional dehydration and digesta viscosity throughout the large intestine. Fifty-two pigs were fed a chow diet supplemented with a bulk laxative, placebo, or calcium docusate for three days, after which the pigs were sacrificed and the contents of the large bowel were analyzed. Digesta occurred as a continuum from liquid (cecum, 91.2% water content) to solid (rectum, 70.5% water content). The observed 20.7% difference in water content resulted in a 240-fold increase in viscosity. Half of this water is reabsorbed in the first 18% of the large bowel length where viscosity remains relatively low. Compared to placebo, calcium docusate and calcium polycarbophil had no significant effect on digesta water content or viscosity, polycarbophil exhibited significantly (P < 0.05) lower digesta viscosity in three bowel segments, and psyllium exhibited significantly (P < 0.01) lower viscosity in six bowel segments and higher water content in nine bowel segments. In conclusion, the majority of digesta dehydration occurs early in the proximal large bowel, while the greatest increases in viscosity occur in the distal bowel. Relatively small decreases in digesta water content result in large increases in digesta viscosity. Psyllium, and to a lesser extent polycarbophil, are able to resist dehydration, resulting in a softer digesta.

Animals↗
PubMed1998

Characterization of propagating contractions in proximal colon of ambulatory mini pigs.

The aim of this study was to characterize propagating contractions in the unprepared colon of freely ambulating mini pigs. A telemetric method was used to record colonic motility continuously for six consecutive days in a 40-cm segment of proximal colon. Propagating contractions occurred over a wide range of propagation rates (0.4-16.7 cm/sec), peak amplitudes (10-116 mm Hg) and pressure wave durations (5.3-40.0 sec). Propagating contractions were divided into two groups by duration and wave-form: short-duration symmetrical and long-duration asymmetrical. Short-duration (7.8 +/- 0.9 sec) symmetrical wave-from propagating contractions exhibited a higher frequency (27.9 +/- 2.6 events/day), more rapid propagation rate (3-16.7 cm/sec; mean +/- SEM: 4.9 +/- 1.7 cm/sec), and a lower peak amplitude (31.2 +/- 0.9 mm Hg) compared to long-duration (19.2 +/- 5.1 sec) asymmetrical propagating contractions, which were less frequent (6.1 +/- 0.7 events/day), slower in propagation rate (0.4-2 cm/sec; mean +/- SEM: 1.5 +/- 0.7 cm/sec), and higher in peak amplitude (51.6 +/- 2.4 mm Hg). The results show that propagating contractions occur over a wide spectrum, from short-duration, low-amplitude, rapidly propagating contractions to long-duration, high-amplitude, slowly propagating contractions.

Animals↗
PubMed1998

Classifying complex pediatric feeding disorders.

BACKGROUND: This study defines the multiple characteristics associated with complex pediatric feeding problems and determines the relative frequency of each classification in a population referred to an interdisciplinary feeding team. METHODS: The written reports from team evaluations on 103 children (64 males, 39 females; age range 4 months to 17 years) were reviewed. Prematurity and/or presence of developmental delay was coded. Identified factors related to current feeding problems were coded according to five categories: structural abnormalities, neurological conditions, behavioral issues, cardiorespiratory problems, metabolic dysfunction. RESULTS: Interrater reliability for the classification coding was 88%. Thirty-eight percent of the children had a history of pre- maturity and 74% were reported to have evidence of developmental delay. The following five categories or combinations were coded most frequently: structural-neurological-behavioral (30%), neurological-behavioral (27%), behavioral (12%), structural-behavioral (9%), and structural-neurological (8%). Overall, behavioral issues were coded more often (85%) than neurological conditions (73%), structural abnormalities (57%), cardiorespiratory problems (7%), or metabolic dysfunction (5%). CONCLUSIONS: Data analysis using this classification system revealed that the majority of children in this sample had a behavioral component to their complex feeding problem, regardless of concurrent physical factors. These findings suggest that complex pediatric feeding problems are biobehavioral conditions in which biological and behavioral aspects mutually interact, and both need to be addressed to achieve normal feeding.

Adolescent↗
PubMed1998

Evaluation of outcomes and cost-effectiveness of a community behavioral support and crisis response demonstration project.

A behavioral support and crisis response demonstration project authorized by the Minnesota Legislature in 1992 was evaluated. We described the demonstration program, its service users, and satisfaction and concerns with the program of service recipients, their families and careproviders, and county case managers. We also provided follow-up data on the outcomes of the first year service users and gave the service outcomes projected by case managers had the program not been established. These projected outcomes were validated by follow-up of a comparison group of persons unable to access the program's services. Cost-effectiveness was computed from costs of establishing and operating the demonstration program and the actual average costs of the services that were projected to otherwise have been used.

Adolescent↗
PubMed1996

[Clinical experiences with fiber optic intubation with the Bonfils intubation fiberscope].

In a prospective study 107 patients were analyzed regarding difficulty of intubation. One hundred and three were intubated with the retromolar fibrescope named after Bonfils. The report gives an account of the intubation technique and experiences with this device. Intubations, primarily assumed to be difficult, can be accomplished with its help without any problems. This is especially true in situations, in which the difficulty of intubation is diagnosed only after the induction of anaesthesia and relaxation of the patient. For this technique, a sufficient opening of the mouth for the introduction of the apparatus is necessary. The rigid fibrescope cannot compete with the flexible optic, but supplements the repertoire of aids for difficult intubations. It could open up new areas of indication for safe and gentle intubation.

Adolescent↗
PubMed1995

Lack of an effect of novel inhibitors with high specificity for protein kinase C on the action of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate on mouse skin in vivo.

The inhibitory effects of three novel staurosporine-derived compounds were tested with five different types of protein kinases, including protein kinase C (PKC). IC50 values of two of these compounds were found to be 300 to > 5000 times lower for PKC alpha beta gamma (a mixture of the PKC isoenzymes alpha, beta and gamma) than for any of the other protein kinases. The inhibitory action of the most selective inhibitor was tested also with the Ca(2+)-unresponsive PKC isoenzyme delta and was found to suppress PKC alpha beta gamma and PKC delta differentially. The highly specific PKC inhibitors are active both in cell culture and in vivo. They inhibit the PKC-catalyzed phosphorylation of the specific PKC substrate MARCKS in Swiss-3T3 fibroblasts and the okadaic acid-induced edema of the mouse ear. However, the more complex biological processes triggered by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate in mouse skin, such as inflammation, stimulation of cellular hyperproliferation and tumor promotion, remain largely unaffected upon topical application of these compounds.

3T3 Cells↗
PubMed1994

The mutator gene swi8 effects specific mutations in the mating-type region of Schizosaccharomyces pombe.

The swi8+ gene of Schizosaccharomyces pombe appears to be involved in the termination step of copy synthesis during mating-type (MT) switching. Mutations in swi8 confer a general mutator phenotype and, in particular, generate specific mutations in the MT region. Sequencing of the MT cassettes of the h90 swi8-137 mutant revealed three altered sites. One is situated at the switching (smt) signal adjacent to the H1 homology box of the expression locus mat1:1. It reduces the rate of MT switching. The alteration at the smt signal arose frequently in other h90 swi8 strains and is probably caused by gene conversion in which the sequence adjacent to the H1 box of mat2:2 is used as template. This change might be generated during the process of MT switching when hybrid DNA formation is anomalously extended into the more heterologous region flanking the H1 homology box. In addition to the gene conversion at mat1:1, two mutations were found in the H3 homology boxes of the silent cassettes mat2:2 and mat3:3.

Base Sequence↗
PubMed1993

Gö 6976, a selective inhibitor of protein kinase C, is a potent antagonist of human immunodeficiency virus 1 induction from latent/low-level-producing reservoir cells in vitro.

Human immunodeficiency virus (HIV-1) infection is followed by a period of latency or a low-level-persistent (LLP) state that results in an asymptomatic infection of the host. Productive viral expression may be triggered by a variety of activators including mitogens, antigens, and cytokines. Protein kinase C (PKC) has been shown to be important in the intracellular cascade of signals induced by such activators. With U1 and ACH-2 cell lines representative of an HIV-1 postintegration state, the effect of Gö 6976, a synthetic inhibitor of PKC was tested. Gö 6976 is a nonglycosidic indolocarbazole found to potently inhibit HIV-1 induction by Bryostatin 1, tumor necrosis factor alpha, and interleukin 6. Gö 6976 effectively blocks viral transcription induced by Bryostatin 1 or tumor necrosis factor alpha that leads to the inhibition of intracellular viral protein synthesis and viral shedding. Gö 6976 also blocks interleukin 6-mediated posttranscriptional induction of viral proteins. The IC50 of Gö 6976 shows a 12- to 60-fold more potent effect than for H-7, another PKC inhibitor with a similar mechanism. The inhibitory effect is reduced when Gö 6976 is not added before or within 1 hr of induction by the potent PKC activator Bryostatin 1. However, U1 cells can be grown for long periods in a nontoxic concentration of Gö 6976 (300 nM), which confers virtual inhibition of HIV-1 induction without the development of resistance. Results indicate that inhibition of HIV-1 proviral induction from latent/low-level-producing infectious states with potent PKC inhibitors like Gö 6976 may represent an additional and promising antiviral approach.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1993

Novel indolocarbazole protein kinase C inhibitors prevent reactivation of HIV-1 in latently infected cells.

Suppression of human immunodeficiency virus-1 (HIV-1) reactivation in latently infected cells by protein kinase C (PKC) inhibitors has been described. Based on an initial finding with the indolocarbazole inhibitor Gö 6976 we have examined several members of this new class of potent and specific PKC inhibitors with respect to their ability to prevent the PKC-mediated induction of HIV-1 replication in the latently infected U1 cell line. Two of these compounds strongly inhibited not only PMA-induced release of p24-antigen and infectious virus particles into the supernatant (50% inhibition at 0.04-0.35 microM) but also TNF-alpha-mediated HIV-1 reactivation in the same concentration range. Significant lower toxicities compared to Gö 6976 were observed for the new compounds, with 50% cytotoxic concentrations at 5.2 microM for Gö 7775 and 3.4 microM for Gö 7716. This resulted in selectivity indices which were 10-20-times higher compared to the reference compound Gö 6976 and were comparable to those of registered anti-AIDS drugs. No anti-HIV-1 activity was observed for a closely related indolocarbazole analogue with no inhibitory activity in the PKC in vitro enzyme assay. This study demonstrates the important role of PKC in reactivation of HIV-1 in latently infected cells and points to the potential of indolocarbazoles to preserve the latent state of HIV-1 infection.

Antineoplastic Agents↗