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C H Clarke

Publications and source records attributed to C H Clarke.

At least 19 recordsLinked to original sources
PubMed2000

Antibodies to the estrogen receptor-alpha modulate rapid prolactin release from rat pituitary tumor cells through plasma membrane estrogen receptors.

Antibodies (Abs) raised against the estrogen receptor-alpha (ERalpha) were used to investigate the role of ERalpha proteins located at the plasma membrane in mediating the rapid, estrogen-stimulated secretion of prolactin (PRL) from rat pituitary GH(3)/B6/F10 cells. Exposure of the cells to 1 nM 17beta-estradiol (E(2)) significantly increased PRL release after 3 or 6 min. When ERalpha Abs that bind specifically to ERalpha but are too large to diffuse into cells were tested for activity at the cell membrane, Ab R4, targeted to an ERalpha hinge region sequence, increased PRL release in a time- and concentration-dependent fashion. Ab H151, directed against a different hinge region epitope, decreased PRL release and blocked the stimulatory action of E(2). Abs raised against the DNA binding domain (H226) or the carboxyl terminus (C542) were not biologically active. When each Ab was examined for recognition of ERalpha on the cell surface by immunocytochemistry, all except H151 generated immunostaining in aldehyde-fixed cells. In live cells, however, Ab H151 but not Ab R4 blocked the membrane binding of fluorescently tagged E(2)-BSA. Overall, the data indicate that plasma membrane ERalpha proteins mediate estrogen-stimulated PRL release from GH(3)/B6/F10 cells. These results may also convey information about conformationally sensitive areas of the membrane form of ERalpha involved in rapid, nongenomic responses to estrogens.

Amino Acid Sequence↗
PubMed2000

Perimembrane localization of the estrogen receptor alpha protein in neuronal processes of cultured hippocampal neurons.

There is clear evidence of rapid, nongenomic responses to estrogen in a variety of neuronal model systems. To address the question of whether some of these rapid estrogen signals might be transduced by the classical estrogen receptor (ER) alpha or a closely related protein in nontransformed neurons, we undertook the present study using isolated fetal rat hippocampal neurons. Several antibodies developed to detect ERalpha were tested in this system and showed positive membrane staining in nonpermeabilized neurons. MC-20, an affinity purified anti-ERalpha, rabbit polyclonal IgG antibody which does not recognize ERbeta was selected to carry out the majority of the experiments. When permeabilized, the hippocampal neurons exhibited low levels of nuclear staining for ERalpha, but abundant labeling for ERalpha throughout the entire cell including the neurites. In addition to traditional immunocytochemistry controls, incubation of neurons for 24 h in the presence of 10 microM antisense oligonucleotide directed against the translation start site of ERalpha reduced ERalpha immunoreactivity throughout the neurons providing further evidence that the immunostaining was specific for ERalpha. Confocal and conventional microscopy demonstrated that the antigen was predominately extranuclear and localization of ERalpha in the neurites suggests that the receptor is in close proximity to the plasma membrane. This localization is consistent with a role for ERalpha as a transducer of rapid, nongenomic estrogen responses in hippocampal neurons.

Amino Acid Sequence↗
PubMed1998

Major histocompatibility class II-mediated signal transduction is regulated by the protein-tyrosine phosphatase CD45.

Major histocompatibility complex class II molecules and the B cell antigen receptor (BCR) transduce similar signals when cross-linked by ligand. Therefore, studies were conducted to determine whether the protein tyrosine phosphatase CD45 regulates signaling via these transmembrane receptors in an analogous manner. Cross-linking of either class II molecules or the BCR on CD45-positive K46-17micromlambda B lymphoma cells was observed to induce activation of the Src family protein- tyrosine kinase Lyn, tyrosine phosphorylation of Syk and phospholipase Cgamma, and the production of inositol 1,4,5-trisphosphate leading to intracellular mobilization as well as extracellular influx of Ca2+. In the absence of CD45, cross-linking of either class II molecules or the BCR failed to induce activation of Lyn. Syk was inducibly phosphorylated on tyrosine in a normal manner, whereas phospholipase Cgamma exhibited a high basal level of tyrosine phosphorylation that was not significantly increased upon stimulation. Nevertheless, phospholipase Cgamma appeared to be functional because CD45-negative cells produced elevated levels of inositol 1,4,5-trisphosphate following stimulation through class II or the BCR. Regardless of this, CD45-negative cells exhibited Ca2+ mobilization responses that were greatly diminished and transient in nature. Whereas little or no mobilization of Ca2+ was observed in response to class II cross-linking, CD45-deficient cells mobilized Ca2+ from intracellular stores but not the extracellular environment in response to BCR cross-linking. These results demonstrate that CD45 regulates both Src family kinase activation and Ca2+ mobilization associated with class II- and BCR-mediated signal transduction.

Animals↗
PubMed1996

Amino-terminal processing of the catalytic subunit from Na(+)-K(+)-ATPase.

The first five amino acids of the catalytic alpha 1-subunit predicted from its cDNA are not found in purified mammalian Na(+)-K(+)-ATPase, suggesting co- or posttranslational cleavage. To facilitate evaluation of amino-terminal structure and the cleavage process, we developed a site-directed antibody (anti-VGR) specific for the first nine residues of nascent alpha 1 from rat. In immunoblots of polypeptides generated by in vitro translation, anti-VGR detected a prominent band with a mobility appropriate for the alpha 1-subunit (100 kDa). Immunoblots of total protein from various rat organs, however, revealed no significant binding, implying that virtually all the alpha 1-subunit expressed in vivo was modified. We also assessed amino-terminal structure in various heterologous expression systems. Binding of anti-VGR was observed in Escherichia coli transformed with a vector containing an alpha 1/troponin fusion protein and in insect cells infected with baculovirus containing full-length alpha 1 or alpha 1T. This suggests that modification of the introduced alpha 1 in these expression systems was absent or different from that in mammals. In contrast, green monkey kidney cells (COS-1) transfected with alpha 1 did not reveal significant binding of the antibody, indicating that the introduced isoform was processed appropriately. These results demonstrate that the structure of the alpha 1-subunit's amino terminus differs among various expression systems. The results further imply that efficient co- or posttranslational processing of nascent alpha 1 is conserved among various organs within the rat, yet the required modification enzymes are not present in distant phyla.

Amino Acid Sequence↗
PubMed1996

Decreased GAP-43 accumulation in neurite tips of cultured hippocampal neurons by acrylamide.

The critical axonopathy- producing effect of acrylamide (ACR) is hypothesized to be an interruption of fast vesicular transport resulting in a deficiency of distal axonal proteins. This study used the accumulation of growth associated protein- 43 (GAP-43) in neurite tips of cultured hippocampal neurons as a model to examine the effect of an ACR block of fast transport on the eventual protein concentration within the distal axon. Twenty-four hour incubation with ACR produced a dose-dependent reduction with 0.5mM and 1.0mM concentrations in the percent of neurites with a terminal GAP-43 accumulation. No change in cell size (area), cell body GAP-43 fluorescence, distal neurite fluorescence, absolute tip fluorescence or neurite length were observed. Another known inhibitor of transport, colchicine (COL) also caused a significant decrease in the percent of neurites with a GAP-43 accumulation; propionamide (PA), a non-neurotoxic analogue of ACR, had no effect on protein accumulation. These findings support the hypothesis that the disruption of fast transport by ACR can lead to depletion of vital proteins in the distal axon.

Acrylamide↗
PubMed1990

Specificity of Escherichia coli mutD and mutL mutator strains.

The products of the mutD and mutL genes of Escherichia coli are involved in proofreading by DNA polymerase III and DNA adenine MTase (Dam)-dependent mismatch repair, respectively. We have used the plasmid-borne bacteriophage P22 mnt gene as a target to determine the types of mutations produced in mutL25 and mutD5 strains. Of 60 mutations identified from mutL25 cells, 52 were transition mutations and of these the AT----GC subset predominated (40 out of 52). The majority of AT----GC mutations were found at the same three sites (hotspots). In contrast, transversion mutations (47 out of 76) were found about twice as frequently as transitions (28 out of 76) from mutD5 bacteria. Two hotspots were identified but at different sites than those in the mutL25 cells. These results suggest that the proofreading function of DNA polymerase III primarily repairs potential transversion mutations while Dam-dependent mismatch repair rectifies potential transition mutations.

Coliphages↗
PubMed1990

Specificity of antimutagens against chemical mutagens in microbial systems.

Procedures have been developed which enable the study of antimutagenic specificity of certain antimutagenic chemicals against chemical mutagens/carcinogens. Modifications of the Ames Salmonella assay, the Bacillis subtilis rec assay of Kada and co-workers, and the Luria-Delbrück fluctuation test, along with procedures we have developed utilizing E. coli K12 strain ND160 developed by Dworkin, all are employed in these studies. Using these procedures, a number of naturally-occurring compounds and/or their derivatives have been shown to produce antimutagenic specificity either against changes at different specific genetic loci or against activity of specific chemical mutagens such as nitrofurazone, ethyl methanesulfonate, or caffeine. Compounds that demonstrate this activity include cinnamaldehyde, chlorophyllin, an extract of Glycyrrhiza glabra, spermine, and mixtures of guanosine and cytidine. The data demonstrate that some antimutagens act specifically against spontaneous mutations, while others inhibit the development of chemically-induced mutations at specific loci. These results have potential application to the prevention of chemical toxicological damage.

Bacteria↗
PubMed1989

Antimutagenic specificity against spontaneous and nitrofurazone-induced mutations in Escherichia coli K12ND160.

Escherichia coli K12ND-160 possesses characteristics which make it highly suitable for studies on mutagenic/antimutagenic specificity and has been used to investigate such effects for several agents. Nitrofurazone (NFZ) was used as a mutagen at non-lethal concentrations; it causes mutation from fucose sensitivity to resistance (FucS----FucR) and from inability to utilize melibiose to melibiose utilization (Mel(-)----Mel+), but does not induce mutations from deoxygalactose sensitivity to resistance (DGAS----DGAR). Caffeine is mutagenic for reversions from lactose non-utilization to utilization (Lac(-)----Lac+) and mutations from FucS----FucR, but it is antimutagenic for mutations from 6-azauracil sensitivity to resistance (AzaUS----AzaUR) and Mel(-)----Mel+ reversions and produces no effect on spontaneous DGAS----DGAR mutations. Added guanosine and cytidine (G + C at 100 micrograms/ml each) exert antimutagenic activity against spontaneous Lac(-)----Lac+ reversion, but not against caffeine-induced Lac(-)----Lac+ reversions; a strong antimutagenic effect on spontaneous Mel(-)----Mel+ reversion is also observed. The addition of G + C does not result in either mutagenic or antimutagenic effects against spontaneous or NFZ-induced FucS----FucR or DGAS----DGAR mutations; it is, however, strongly mutagenic for AzaUS----AzaUR mutations. The 'natural antimutagen', chlorophyllin, is antimutagenic for DGAS----DGAR mutations, but fails to demonstrate such activity against spontaneous or caffeine-induced Lac(-)----Lac+ reversion, spontaneous or NFZ-induced Mel(-)----Mel+ reversion, or spontaneous or NFZ-induced FucS----FucR mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Caffeine↗
PubMed1988

Antimutagens against spontaneous and induced reversion of a lacZ frameshift mutation in E. coli K-12 strain ND-160.

Isopropyl-beta-D-thiogalactoside (IPTG), at 0.5 or 1.0 mM, is shown to reduce spontaneous, and to virtually abolish caffeine- or quinacrine-induced reversions of the lacZ frameshift mutation in E. coli ND160. Guanosine at 200 micrograms/ml and Co2+ at 15 micrograms/ml had an erratic partial antimutagenic effect on spontaneous lac+ reversions. All 3 agents reduce caffeine-induced (500 micrograms/ml) mutagenesis. Spermine (250 micrograms/ml) also reduces quinacrine-induced Lac+ reversion frequencies in this system.

Caffeine↗
PubMed1985

Pattern analysis of 5S rRNA.

Some 200 different 5S rRNA sequences from eubacteria, chloroplasts, mitochondria, archaebacteria, and eukaryotes were analyzed for evolutionary kinship relationships and associated sequential features. Group-specific occupation schemes for the 149 positions of an overall alignment were established. Eubacterial, archaebacterial, and intermediate occupation schemes all yield a strongly biased base triplet pattern in one of the three possible reading frames strongest for eubacterial, chloroplastic, and archaebacterial, but still detectable for mitochondrial and eukaryotic cytoplasmic sequences. The frequency of triplets decays in the order RNY greater than RNR greater than YNY greater than YNR; R being a purine (guanine or adenine), Y is a pyrimidine (cytosine or uracil), and N is any base. A strong preference for guanine or cytosine was found in all triplet positions. The effects show no exceptions and are clearly above the level of statistical fluctuations.

Archaea↗
PubMed1983

The consequences of base-pair substitution mutations in AT- and GC-rich bacteria.

The likely consequences, in terms of premature stop codons, detectable missense mutants, silent missense mutants, and degenerate codon changes, have been determined for all 12 individual base substitution changes. This has been done for the full, 61 sense codon, genetic code and also for the much more limited codon availabilities of AT- or GC-rich DNA. The specificities and outcomes of individual base substitutions are likely to be rather different at AT- or GC-rich extremes, and also from the situation at an intermediate DNA base-ratio where all 61 sense codons are available. In particular, at DNA base-ratio extremes many mutations will be to non-utilized codons, which may well act as nonsense mutants. These in turn will give novel classes of suppressor-containing revertants. Even in bacteria with intermediate DNA base-ratios, particular codons for a given amino acid may be favoured, over alternatives, because their use maximizes, or minimizes, the mutational consequences of one, or more, base substitution changes.

Adenine↗