Biomedical subjects

A Hubberstey

Publications and source records attributed to A Hubberstey.

4 recordsLinked to original sources

PubMed2004

Sensitive genetic biomarkers for determining apoptosis in the brown bullhead (Ameiurus nebulosus).

Biomarkers are necessary for monitoring environmentally induced alterations at the molecular level in order to assess the impact of xenobiotic compounds on organism health. Apoptosis is a highly regulated cellular process that controls programmed cell death and is involved in tumor formation. Apoptosis thus may provide the basis for developing biomarkers for use in the field of ecotoxicology to monitor non-lethal levels of xenobiotic induced cellular stress and toxicity. This study shows that a brown bullhead (Ameiurus nebulosus) fibroblast cell line (BB-2) responds to known apoptotic inducers (staurosporine, cycloheximide, and tumor necrosis factor alpha (TNF-alpha)), as characterized by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP digoxigenin nick end-labelling (TUNEL). Furthermore, we characterized the apoptotic process using a series of newly identified bullhead genetic markers. Exposure to protein kinase C inhibitors altered the transcription of TF-cell apoptosis-related protein (TFAR)-15 and p23 with no effect on p53, inhibitor of apoptosis protein (IAP), or PNAS-2. Inhibition of protein synthesis caused a consistent reduction in the transcription of p53 and PNAS-2. This study demonstrates that our novel transcriptional markers are sensitive biomarkers for the study of the effects of xenobiotic chemicals on apoptosis in the brown bullhead.

Animals↗

PubMed2000

Skh1, the MEK component of the mkh1 signaling pathway in Schizosaccharomyces pombe.

We previously reported the identification of Mkh1, a MEK kinase in Schizosaccharomyces pombe that is required for cell wall integrity, and we presented genetic evidence that Pmk1/Spm1, a MAP kinase, functions downstream from Mkh1 in the same pathway. Here, we report the identification of Skh1, a MEK (MAP kinase kinase) in S. pombe. The sequence of Skh1 is nearly identical to that of the recently reported Pek1 sequence. We present biochemical and genetic evidence that Skh1 is the MEK component of the Mkh1-Spm1 MAP kinase cascade. Our yeast two-hybrid results indicate that Mkh1, Skh1, and Spm1 physically interact to form a ternary complex. Deletion of mkh1, skh1 or spm1 results in identical phenotypes, including sensitivity to (beta)-glucanase treatment, growth inhibition on media containing KCl, and filamentous growth on medium containing caffeine. Double mutant strains exhibit phenotypes that are identical to the single mutant strains. Furthermore, expression of an activated HA-Skh1(DD )protein suppressed these defects in mkh1(delta) cells, and overexpression of Spm1 suppressed these defects in skh1(delta) cells. We also show that HA-Spm1 is hyper-phosphorylated on tyrosine residues in cells co-expressing the activated HA-Skh1(DD) protein. Furthermore, we found the phosphorylated/activated form of GFP-HA-Spm1 at detectable levels in wild-type cells, but not at appreciable levels in mkh1(delta) or skh1(delta) cells expressing this fusion protein. Together, our results indicate that Mkh1, Skh1 and Spm1 constitute a MAPK cascade in fission yeast.

Amino Acid Sequence↗

PubMed1996

Mammalian CAP interacts with CAP, CAP2, and actin.

We previously identified human CAP, a homolog of the yeast adenylyl cyclase-associated protein. Previous studies suggest that the N-terminal and C-terminal domains of CAP have distinct functions. We have explored the interactions of human CAP with various proteins. First, by performing yeast two-hybrid screens, we have identified peptides from several proteins that interact with the C-terminal and/or the N-terminal domains of human CAP. These peptides include regions derived from CAP and BAT3, a protein with unknown function. We have further shown that MBP fusions with these peptides can associate in vitro with the N-terminal or C-terminal domains of CAP fused to GST. Our observations indicate that CAP contains regions in both the N-terminal and C-terminal domains that are capable of interacting with each other or with themselves. Furthermore, we found that myc-epitope-tagged CAP coimmunoprecipitates with HA-epitope-tagged CAP from either yeast or mammalian cell extracts. Similar results demonstrate that human CAP can also interact with human CAP2. We also show that human CAP interacts with actin, both by the yeast two-hybrid test and by coimmunoprecipitation of epitope-tagged CAP from yeast or mammalian cell extracts. This interaction requires the C-terminal domain of CAP, but not the N-terminal domain. Thus CAP appears to be capable of interacting in vivo with other CAP molecules, CAP2, and actin. We also show that actin co-immunoprecipitates with HA-CAP2 from mammalian cell extracts.

3T3 Cells↗

PubMed1995

Differential expression of CAP and CAP2 in adult rat tissues.

We previously reported the identification of the human CAP and CAP2 genes which encode proteins related to the yeast adenylyl cyclase (CYR)-associated CAP protein. The rat CAP homolog, MCH1, has also been previously cloned. We have cloned a cDNA encoding the rat homolog of CAP2. Rat CAP/MCH1 and CAP2 are 63% identical to each other. Using the reverse transcription-polymerase chain reaction (RT-PCR) method, we have examined CAP/MCH1 and CAP2 mRNA levels in various adult rat tissues. Our results show a dramatic difference in the pattern of expression of these two genes. Consistent with previous reports, we detected CAP/MCH1 mRNA in all tissues examined; however, levels vary substantially between tissues. In particular, we found that CAP/MCH1 mRNA are present at relatively high levels in spleen, testes and lung, at moderate levels in brain, kidney, liver and small intestine, and at significantly lower levels in heart, skeletal muscle and skin. We have also investigated the levels of CAP/MCH1 in rat tissues by immunoblotting with a polyclonal antibody raised against a human CAP::GST fusion protein. In general, we find that the CAP/MCH1 mRNA levels reflect the amount of CAP/MCH1 found in different tissues. In contrast, CAP2 transcripts were present at relatively high levels in testes, at moderate levels in brain, heart and skeletal muscle, at lower levels in lung, skin, kidney and small intestine, and were undetectable in liver or spleen. The differences between the sequences and expression patterns of CAP/MCH1 and CAP2 are significant and suggest that these proteins have distinct functional roles.

Adaptor Proteins, Signal Transducing↗