Search PubMed⌕ Search

Biomedical subjects

S Grimm

Publications and source records attributed to S Grimm.

34 records · Page 2Linked to original sources
PubMed1996

Bcl-2 down-regulates the activity of transcription factor NF-kappaB induced upon apoptosis.

Among the many target genes of the transcription factor NF-kappaB are p53 and c-myc, both of which are involved in apoptosis. This prompted us to investigate the role of NF-kappaB in this process. We report that NF-kappaB is potently activated upon serum starvation, a condition leading to apoptosis in 293 cells. Similar to Bcl-2, a transdominant-negative mutant of the NF-kappaB p65 subunit partially inhibited apoptosis, indicating a direct involvement of the transcription factor in induction of cell death. As expected, the p65 mutant suppresses kappaB-dependent gene expression. Surprisingly, transiently or stably overexpressed Bcl-2 had the same effect. The transcription inhibitory activity of the two proteins correlated with their cell death protective potential. Like Bcl-2, the related protein Bcl-xL but not Bcl-xS was able to suppress kB-dependent transcription. Bcl-2 inhibited NF-kappaB activity by an unusual mechanism. It did not prevent the release of IkappaB in the cytoplasm but down-modulated the transactivating potential of nuclear p65. These data show that NF-kappaB can participate in apoptosis. We suggest that at least part of the anti-apoptotic potential of Bcl-2 may be explained from a hitherto undiscovered activity of Bcl-2 in controlling nuclear gene expression.

Animals↗
PubMed1995

Synthesis, deprotection, analysis and purification of RNA and ribozymes.

Improvements in the synthesis, deprotection and purification of oligoribonucleotides are described. These advances allow for reduced synthesis and deprotection times, while improving product yield. Coupling times are reduced by half using 5-ethylthio-1H-tetrazole (S-ethyltetrazole) as the activator. Base and 2'-O-t-butyldimethylsilyl deprotection with methylamine (MA) and anhydrous triethylamine/hydrogen fluoride in N-methylpyrrolidinone (TEA.HF/NMP), respectively, requires a fraction of the time necessitated by current standard methods. In addition, the ease of oligoribonucleotide purification and analysis have been significantly enhanced using anion exchange chromatography. These new methods improve the yield and quality of the oligoribonucleotides synthesized. Hammerhead ribozymes synthesized utilizing the described methods exhibited no diminution in catalytic activity.

Base Sequence↗
PubMed1994

Inhibition of endothelial cell adhesion molecule expression with antisense oligonucleotides.

In response to inflammatory stimuli, expression of a group of proteins that bind circulating leukocytes (endothelial-leukocyte adhesion molecules) are induced on the luminal surface of vascular endothelium. A series of phosphorothioate oligonucleotides 18 to 21 bases in length were designed and synthesized to hybridize selectively to the mRNA, which encodes three such endothelial-leukocyte adhesion molecules; human intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Antisense oligonucleotides were identified that selectively inhibited ICAM-1, VCAM-1, and E-selectin expression in HUVEC. Oligonucleotides that hybridized to the 3'-untranslated region of either ICAM-1, VCAM-1, or E-selectin mRNAs promoted a selective reduction in the respective mRNA levels. In contrast, oligonucleotides that hybridized to 5'-untranslated sequences did not significantly reduce target mRNA levels, although they did promote a reduction in protein expression. With the use of flow cytometry to measure cell surface expression, ICAM-1 and E-selectin were selectively inhibited by their respective antisense oligonucleotide. At low concentrations of oligonucleotides, only VCAM-1 antisense oligonucleotides inhibited VCAM-1 expression. However, at an oligonucleotide concentration of 50 nM or greater, phosphorothioate oligonucleotides not predicted to hybridize to VCAM-1 mRNA also reduced VCAM-1 expression. The sequence-independent inhibition of VCAM-1 expression by phosphorothioate oligonucleotides could be the result of a perturbation in the transcriptional regulation of the VCAM-1 gene. ICAM-1, VCAM-1, and E-selectin antisense oligonucleotides reduced adhesion of HL-60 cells to TNF-activated HUVEC. These data demonstrate that phosphorothioate oligonucleotides are capable of selectively inhibiting the expression of ICAM-1, VCAM-1, and E-selectin in HUVEC.

Base Sequence↗
PubMed1994

Dose and load studies for subcutaneous and oral delivery of poly(lactide-co-glycolide) microspheres containing ovalbumin.

Poly(lactide-co-glycolide) microspheres containing different loads of OVA (0.05, 0.1, 0.5 and 1.0% w/w) were manufactured by a w/o/w emulsion/solvent evaporation method. Low load efficiencies of less than 20% were observed. Normal size distributions with mean volume diameters ranging from 3.7 to 4.7 microns were obtained for different batches. The in vitro release of OVA from different loaded microspheres showed an expected burst release with all batches. The in vivo dose study (1, 10, 25, 50 micrograms of OVA) was performed by subcutaneous and oral inoculation in mice by single (0 week) or double (0 and 3 weeks) administration of PLGA 50/50 microspheres containing 0.1% OVA. Subcutaneous administration showed an immune response (serum Ig levels by ELISA) statistically (Fisher's paired t-test; P < 0.05) above OVA saline negative controls at 3, 6 and 12 weeks after administration. Oral administration of microspheres produced statistically higher systemic immune responses at the higher doses. Single and double inoculation orally and subcutaneously produced similar serum antibody levels. The in vivo load study was performed by subcutaneous and oral administration to mice of 25 micrograms OVA contained in various loaded (0.05, 0.1, 0.5 and 1.0% w/w) microspheres. Serum immune responses at 3, 6, and 12 weeks after inoculation were statistically above OVA saline controls and were inversely proportional to the OVA load using either route. This observation suggested a relationship between the number of microspheres delivered and the in vivo serum response. Single subcutaneous administration of 0.05 or 0.1% OVA loaded PLGA 50/50 microspheres induced larger immune responses compared with complete Freund's adjuvant.

Administration, Oral↗
PubMed1994

Failure of the splicing variant p65 delta of the NF-kappa B subunit p65 to transform fibroblasts.

A naturally occurring splice variant of the p65 subunit of the inducible transcription factor NK-kappa B, called p65 delta, has recently been reported to transform rat-1 fibroblasts in transfection experiments. Criteria for transformation included focus formation, growth in soft agar and tumor formation in athymic mice. In the present study we provide evidence that p65 delta cannot transform either rat-1 or rat-2 cells although p65 delta was efficiently expressed and showed all transcriptional activities reported previously. Eleven rat-1 fibroblasts cells lines selected for stably overexpressing p65 delta also showed neither focus formation nor anchorage-independent growth in soft agar. No transformation of primary rat fibroblasts was evident when p65 delta was cotransfected with either ras or myc oncogenes, whereas a combination of the two oncogenes caused focus formation. We submit that the transforming potential of p65 delta is highly conditional and presumably restricted to a particular subclone of rat-1 cells.

Animals↗
PubMed1994

The chicken A-myb protein is a transcriptional activator.

c-myb encodes a trans-activator that is essential for the proliferation of most hematopoietic precursor cells but lacks a major role in non-hematopoietic cells. The recent identification of two myb-related genes (A-myb and B-myb) in several vertebrate species has raised the possibility that these genes perform c-myb like functions in non-hematopoietic cells. Here, we report the isolation and preliminary characterization of the chicken A-myb gene. Like other members of the myb family, the A-myb protein contains an N-terminally located repeat domain that is highly homologous to the DNA-binding domain of the c-myb protein. Unlike c-myb, A-myb is expressed in fibroblasts, suggesting that it functions in non-hematopoietic cells. We demonstrate that A-myb transactivates myb-responsive reporter genes. Furthermore, when stably expressed in a chicken macrophage cell line. A-myb causes activation of several myb-inducible endogenous genes. These findings show that A-myb encodes a transactivating member of the myb family whose functional properties resemble those of the c-myb and v-myb proteins. Thus, our results support the idea that A-myb is a functional equivalent of c-myb.

Acetyltransferases↗
PubMed1993

An invertebrate calcium-binding protein of the calbindin subfamily: protein structure, genomic organization, and expression pattern of the calbindin-32 gene of Drosophila.

Antisera against vertebrate calcium-binding proteins cross-react with Drosophila nervous and muscle tissue. We have used an antiserum against carp parvalbumin to isolate from a Drosophila head cDNA library immunopositive expression clones. Tissue in situ hybridization identified a clone that labeled specific neurons and muscles similar to the parvalbumin-like immunohistochemical staining pattern. Five independent cDNAs derive from an mRNA whose open reading frame codes for a 310 amino acid polypeptide. Sequence analysis identifies six EF-hand calcium-binding domains and reveals 42% and 37% homology to chicken calretinin and calbindin D-28k, respectively. Since the positions of 9 out of 10 introns within the ORF are conserved from the Drosophila gene to both vertebrate genes, we conclude that we have identified the first invertebrate member of the calbindin sub-family of calcium-binding protein genes of the EF-hand homolog family. The calbindin-32 gene (cbn) maps to 53E on the second chromosome. It is expressed through most of ontogenesis with a selective distribution in the nervous system and in a few small adult thoracic muscles. The cloning of a Drosophila homolog to vertebrate neuronal Ca(2+)-binding proteins opens new routes to study the so far largely elusive function of these brain molecules.

Amino Acid Sequence↗
PubMed1992

Functional antagonism between members of the myb family: B-myb inhibits v-myb-induced gene activation.

The oncogene v-myb and its cellular progenitor c-myb encode nuclear, DNA binding phosphoproteins that control the expression of certain target genes in immature hematopoietic cells. Here, we report the isolation of a myb-related chicken gene, chicken B-myb. We show that expression of B-myb, unlike that of c-myb, is not restricted to hematopoietic cells, suggesting that B-myb functions in a broader spectrum of cell types than c-myb. We have identified the authentic chicken B-myb protein as a nuclear protein of approximately 110 kDa. We show that the B-myb protein specifically recognizes v-myb binding sites in vitro and that binding is mediated by an N-terminally located DNA binding domain. Although B-myb protein recognizes myb binding sites, B-myb fails to transactivate several myb-responsive gene constructs as well as the endogenous myb-responsive gene mim-1. Instead, we find that B-myb represses v-myb- and c-myb-mediated activation of the mim-1 gene, most likely by competing with other myb proteins for binding sites. Our results raise the possibility that B-myb is an inhibitory member of the myb family.

Amino Acid Sequence↗
PubMed1991

Participation of Colorado pediatricians and family physicians in the Medicaid program.

The Pediatric Health Policy Group of the University of Colorado Health Sciences Center (Denver) surveyed 650 family physicians and 296 pediatricians in 1988, with 50% of family physicians and 48% of pediatricians responding. Half of the pediatricians in private practice and 35% of family physicians in private practice accepted all children who were Medicaid beneficiaries into their practice; 42% of pediatricians and 50% of family physicians accepted all non-Medicaid patients but only some new Medicaid patients; and 8% of pediatricians and 15% of family physicians accepted new non-Medicaid patients but no Medicaid patients. Practice location was associated with the level of Medicaid participation for these primary care physicians: Significantly more rural pediatricians and family physicians than those with urban practices accepted Medicaid patients. The average reimbursement level for these physicians was shown to be an important determinant of whether physicians would accept Medicaid patients. Nonparticipatory physicians were more concerned about excessive paperwork compared with physicians with limited participation. Among physicians with limited participation, family physicians and pediatricians both cited problems of excessive paperwork, reimbursement delays, and retroactive denials of payment as important deterrents to accepting Medicaid patients.

Colorado↗
PubMed1989

[Alternative methods: development and use of two "in vitro" models for endocrine studies].

Most endocrinological studies are normally performed on several animal groups: one group for the determination of hormonal levels in tissues and plasma under basal conditions, a second group for the same determinations after "in vivo" treatment of the animals (stimulation or inhibition of the endocrine activities) and two additional groups for morphological investigations. Surgical approaches belong not rarely to the "in vivo" treatment of the animals. Moreover, the results from the different animal groups have to be extrapolated. In order to overcome these drawbacks, we have developed two "in vitro" models by means of which we can study on the tissues from the same animal: 1. function and structure of the hypothalamus and 2. function and structure of isolated pituitary cells. By using these models we can considerably reduce the number of animals needed for the studies, replace the "in vivo" by the "in vitro" experiments and refine the methods avoiding, among others, the extrapolation of results.

Animals↗
PubMedDate not supplied

Crisis intervention response and long-term outcome: a pilot study.

We investigated whether process variables (therapeutic alliance and insight) measured at the termination of crisis intervention predict long-term treatment compliance and 2-year outcome. Thirty-seven consecutive depressed psychiatric patients assigned to outpatient crisis intervention (CCI) were assessed with both questionnaires and standardized instruments at intake, 1 week, and CCI termination (mean, 6 weeks). Thirty-one subjects (84%) were also evaluated at 1-year and 2-year follow-up. We found that working alliance and development of insight predicted positive global change and symptom improvement at 1 and 2 years' follow-up. Furthermore, the observed correlation between process measures and 2-year outcome was found to be independent of age, sex, symptoms severity at intake, improvement of symptoms at CCI termination, premorbid adjustment, DSM-III-R axis I/axis II diagnosis, and therapeutic alliance at intake.

Adult↗