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B A Burkhart

Publications and source records attributed to B A Burkhart.

11 recordsLinked to original sources
PubMed1999

Two posttranscriptional pathways that regulate p21(Cip1/Waf1/Sdi1) are identified by HPV16-E6 interaction and correlate with life span and cellular senescence.

The p21((Cip1/Waf1/Sdi1)) protein is a cyclin-dependent kinase inhibitor that is induced in normal human fibroblasts (NHF) following DNA damage, following serum stimulation, and at cellular senescence. Expression of the human papilloma virus 16 E6 oncoprotein in NHF cells results in the loss of the p21 protein, independent of mRNA level under most conditions. The p21 protein levels in NHF-E6 cells remained low following DNA damage or serum stimulation even though mRNA levels increased. In contrast, the p21 protein was transiently induced in NHF-E6 cells at the onset of cellular senescence. Expression of the E6 oncoprotein in transformed cells had no effect on p21 protein levels. This demonstrates that two posttranscriptional pathways regulate expression of p21 protein in NHF cells under different conditions. Disruption of posttranscriptional regulation is correlated with extension of life span, altered cell fate, and transformation.

Cell Transformation, Viral↗
PubMed1993

ENU-induced mutagenesis at a single A: T base pair in transgenic mice containing phi X174.

Transgenic mice containing the bacteriophage phi X174 am3 as a chromosomally integrated and recoverable marker for in vivo mutation have been produced to measure spontaneous and induced substitutions at an A:T base pair among single copies. phi X174 was chosen for its small size (5 kb), unique sequence, and the opportunity to take advantage of previously reported in vitro data on mutation and repair; the am3 site provides sequence specificity in a reversion assay for mutation of an A:T base pair. Inbred C57Bl/6 mice have been made homozygous for approximately 100 copies of the the phage sequence without any apparent detrimental effects on the homozygous individuals. Recoveries of phage from mouse tissues are in the range of 1-5 x 10(7) PFU per micrograms mouse DNA; both recovery and mutation are independent of endogenous CpG methylation. Background mutation frequencies are 2-4 x 10(-7) among phage recovered from liver, brain, spleen, and kidney. Adult mice were treated with 200 mg/kg N-ethyl-N-nitrosourea, and phage were recovered at 2 and 14 days after treatment. At 2 days after treatment we observed a slight increase only among phage isolated from the brain of one mouse out of four. At 14 days after ENU treatment, there were significant increases in mutation frequencies among phage recovered from the liver (6 x) and spleen (10 x). These results demonstrate (1) response of a single A:T base pair to alkylation-induced mutation in a nonexpressed gene, (2) the role of cell proliferation in somatic mutagenesis, and (3) provide a model for a transgenic approach for study of site-specific mutagenesis in vivo in higher eukaryotes.

Adenine↗
PubMed1991

Down-regulation of cdc2 in senescent human and hamster cells.

Senescent cells fail to respond to serum-induced signals for DNA synthesis. Because a central role for the p34cdc2 protein kinase is postulated in control of the cell cycle, we examined the status of this kinase in senescent cells and other growth-arrested cells. In growing human and Syrian hamster fibroblasts, three 35S-labeled proteins of 34-36 kDa were immunoprecipitated with p34cdc2 antiserum. Only the two slower migrating forms were phosphorylated as determined by 32P labelling. In senescent cells, which failed to incorporate [3H]thymidine, no p34cdc2 protein was synthesized and very little or no cdc2 mRNA was observed. When maintained for 48 h in 0.5% serum, young cells also retained only marginal cdc2 expression. After stimulation of low serum-arrested cells by addition of 10% serum, a time-dependent increase of cdc2 mRNA was observed, whereas serum stimulation of senescent cells did not increase cdc2 mRNA. In contrast to senescent and low serum-arrested cells, cdc2 mRNA was expressed at normal levels in cells partially growth arrested by isoleucine deficiency in G1, by aphidicolin at G1-S, by etoposide in G2, or by Colcemid in the M phase of the cell cycle, indicating that cdc2 down-regulation does not always occur upon growth arrest. Following transfection of a plasmid containing the human CDC2 gene into hamster cells, expression of human cdc2 failed to overcome the block to DNA synthesis in senescent cells. Although p34cdc2 was synthesized in the transfected cells, the multiple phosphorylated forms of the proteins were not observed. Taken together, these data support the concept that a chain of events leads to senescence. While p34cdc2 kinase may be one of the critical elements, other cell cycle controls are also involved.

Animals↗
PubMed1990

Two steroid 15 alpha-hydroxylase genes and a homologous gene family in mice.

Steroid 15 alpha-hydroxylase (P45015 alpha) activity is concomitant with the expression of two types of mRNA in the mouse liver. Two discrete genes, designated 15 alpha oh-1 and 15 alpha oh-2, that encode the two mRNAs were recovered from total genomic libraries of the inbred mouse strains 129/J and C57Bl/6J and identified by cDNA hybridization, restriction-site analysis and partial nucleotide sequence. Both genes are approx. 9 kb long and share significant homology, including flanking regions, over a region of at least 30 kb. The two distinct 15 alpha oh genes are members of a larger family of homologous genes and/or pseudogenes of unknown function. The most extensive sequence homology among family members in the 3' portion of the gene with progressively less homology toward the 5' end. The far 5' portions of 15 alpha oh-1 and 15 alpha oh-2 are very similar to one another but there is no observed homology with other genes of the family. The two 15 alpha oh genes and the homologous family have been localized to mouse chromosome 7 by somatic cell hybrid mapping. Analysis of a restriction fragment length polymorphism in recombinant inbred mice shows a close linkage of 15 alpha oh-1 and 15 alpha oh-2 with the Coh locus.

Animals↗
PubMed1989

In vitro electrophysiology of neurons in the lateral dorsal tegmental nucleus.

The lateral dorsal tegmental nucleus (LDT) provides ascending cholinergic projections to forebrain structures such as prefrontal cortex, septum, habenula, and thalamus, but relatively little is known of the physiology of LDT neurons. Intracellular recordings from LDT neurons in guinea pig brain slices found that most neurons fired action potentials either tonically or in bursts. The voltage dependent characteristics of the neurons suggest that a prolonged afterhyperpolarization due to an outward potassium current and a low-threshold calcium conductance contributed to these two modes of firing. Intracellular injections of Lucifer Yellow and subsequent staining for NADPH-diaphorase activity permitted positive identification of cholinergic neurons.

Action Potentials↗
PubMed1987

Reduction of dopamine receptor activity differentially alters striatal neuropeptide mRNA levels.

We have investigated the effect of dopamine receptor blockade on striatal proenkephalin mRNA and protachykinin mRNA by Northern gel analysis and by in situ hybridization histochemistry. Chronic haloperidol treatment resulted in a 3.5 fold increase in striatal proenkephalin mRNA and a 30% decrease in protachykinin mRNA (no apparent change in alpha-tubulin mRNA was observed). The changes in mRNA levels for protachykinin and proenkephalin were uniform throughout the caudate-putamen of the rat as determined by in situ hybridization histochemistry. The results imply that altering receptor-mediated neurotransmitter functions can lead to profound, specific, and long-lasting alterations in neuronal gene expression.

Animals↗
PubMed1985

Sexual dimorphism of testosterone 15 alpha-hydroxylase mRNA levels in mouse liver. cDNA cloning and regulation.

A cDNA library was constructed from 129/J female mouse liver poly(A)+ RNA immunoenriched for testosterone 15 alpha-hydroxylase (P-450(15)alpha) in the expression vector pUC9. Fifteen clones coding for P-450(15)alpha were identified by duplicate colony hybridization to nick-translated cDNAs synthesized from immunoenriched or depleted poly(A)+ RNA. Recombinant plasmid p 15 alpha-29 contained the largest cDNA (1.6 kilobases) and cross-hybridized strongly with all other clones. cDNAs representing two different transcripts were identified based on restriction map analysis. Clones p 15 alpha-29 and p 15 alpha-15 have identical internal restriction fragments generated by ClaI, BamHI, and StuI digests, but p15 alpha-15 has unique PstI and HindIII sites located within 150 base pairs of each other. This suggests P-450(15)alpha is transcribed from two genes. Northern hybridization of poly(A)+ RNA with nick-translated p15 alpha-29 or p15 alpha-15 showed a single size mRNA of 2.1 kilobases. The level of P-450(15)alpha mRNA was 6.6 times higher in 129/J female than in male mice. The difference between P-450(15)alpha mRNA levels in females and males was positively correlated with P-450(15)alpha protein detected by anti-P-450(15)alpha and testosterone 15 alpha-hydroxylase activity in microsomes. Sexual dimorphism in hepatic testosterone 15 alpha-hydroxylase activity is shown to result from differential mRNA regulation in females and males.

Animals↗
PubMed1983

A mouse model for beta-thalassemia.

A mutation that produces an absolute deficiency of normal beta-major globin polypeptides has been recovered from a DBA/2J male mouse. Most mice homozygous for the deficiency survived to adulthood and reproduced but were smaller at birth than their littermates and demonstrated a hypochromic, microcytic anemia with severe anisocytosis, poikilocytosis, and reticulocytosis and the presence of inclusion bodies in a high proportion of circulating erythrocytes. Mice heterozygous for the deficiency demonstrated a mild reticulocytosis but were not clinically anemic. Analysis of globin chain synthesis in vitro by 3H-leucine incorporation revealed that beta-globin synthesis was nearly normal (95%) in heterozygotes and about 75% of normal in deficiency homozygotes. Molecular characterization of the mutation by restriction analysis revealed a deletion of about 3.3 kb of DNA, including regulatory sequences and all coding blocks for beta-major globin. Based on genetic and hematological criteria, mice homozygous for the mutant allele, designated Hbbth-1, represent the first animal model of beta-thalassemia (Cooley's anemia), a severe genetic disease of humans.

Animals↗