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Biomedical subjects

A R Means

Publications and source records attributed to A R Means.

At least 181 records · Page 10Linked to original sources
PubMed1982

Evolution of a complex eukaryotic gene.

Our current efforts to understand the evolutionary origin of the ovomucoid gene are reviewed. Sequence analyses have suggested that introns were present in the primordial ovomucoid gene before birds and mammals diverged, about 300 million years ago. Our work suggests that the present ovomucoid gene has evolved from primordial ovomucoid gene by two separate intragenic duplications followed by the addition of a final segment which codes for a secretory signal sequence. The 3 domains of the secreted peptide and also the signal sequence are constructed by an apparent assembly of exons which code for individual peptide segments. The exact position of introns within the ovomucoid gene has been defined and the results support the theory that introns separate gene segments that code for functional domains of proteins and provide insight into the manner by which eukaryotic genes were constructed during the process of evolution.

Animals↗
PubMed1981

Gonadotropin releasing hormone stimulates calmodulin redistribution in rat pituitary.

Calcium (Ca2+) seems to have an informational role in many tissues. In particular, it fulfills the requirements of a second messenger for gonadotropin releasing hormone (GnRH)-stimulated luteinizing hormone (LH) release from the pituitary gonadotrope (see ref. 1 for review). Very little is known about the effect of this ion on intracellular targets or the mechanism by which Ca2+ mobilization stimulates LH release. One intracellular target for Ca2+ is calmodulin, a ubiquitous intracellular Ca2+ receptor that has been shown to modulate many cellular functions, including cyclic nucleotide and glycogen metabolism, protein phosphorylation, microtubule assembly and disassembly, a Ca2+ flux, and the activities of NAD kinase, tryptophan 5' monooxidase and phospholipase A2 (see refs 2-5 for reviews). We have now used a specific and sensitive radioimmunoassay to determine the quantity and distribution of calmodulin in the gonadotrope before and during GnRH-stimulated LH release. The data indicate that GnRH stimulates redistribution of calmodulin from the cytosol to the plasma membrane and suggest that the molecule may have a role in the mechanism of stimulus-secretion coupling.

Animals↗
PubMed1981

Steroid structural requirements for high affinity binding to human sex steroid binding protein (SBP).

The sex steroid binding protein (SBP) which binds androgens circulating in the blood of man has been examined to determine the structural requirements for high affinity binding. SBP was purified partially and the ability of each of more than 150 steroids to compete with [3H]dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) for binding to SBP was assessed. Binding was enhanced by reduction of the delta 4 double bond to 5 alpha-dihydro, addition of a methyl group at C-4 and in one case unsaturation at C-14,15. Affinity was always reduced by modifications of the C-17 beta hydroxy. Binding was also severely decreased by deletion of the keto moiety at C-3; however, relatively high affinity was retained by an alcohol or an unsubstituted pyrazole group at C-3. Certain alpha surface substitutions such as 17 alpha-ethinyl had limited effects on binding; whereas, other modifications such as 7 alpha-methyl or 17 alpha-methyl caused significant reduction in binding. Most modifications at C-2, 6, 9 or 11 also impaired affinity, and the 5 beta steroids had reduced affinity.

Androgen-Binding Protein↗
PubMed1981

Production and characterization of an antibody to myosin light chain kinase and intracellular localization of the enzyme.

A specific precipitating antibody against chicken gizzard myosin light chain kinase (MLCK) has been produced in rabbits. The antibody inhibited enzyme activity in a dose-dependent manner with 11 moles of antibody required to inhibit 80% of the activity of one mole MLCK. Crude homogenates from various chicken tissues were analyzed by SDS-polyacrylamide gel electrophoresis, and the proteins were transferred onto nitrocellulose sheets and reacted with antibody. In each case, the antibody bound to only one protein with a molecular weight of approximately 130,000. These data suggest the MLCK present in all types of muscle as well as non-muscle tissues is of the same molecular weight. Indirect immunofluorescent microscopy of non-muscle tissue culture cells revealed MLCK to be localized in the spindle apparatus and midbody of mitotic cells and on the stress fibers and in the nucleolus of interphase cells. The nucleolar localization was confirmed by electron microscopy and shown to be restricted to the fibrillar region. In myofibrils isolated from skeletal and cardiac muscle, anti-MLCK decorated the actin-containing I bands of the sarcomere. These results are consistent with the suggestion that MLCK and calmodulin are intermediates in the regulation of cell motility by calcium.

Animals↗
PubMed1981

Interactions of calmodulin with coated vesicles from brain.

Coated vesicles purified in the presence of calcium are enriched approximately 7-fold in calmodulin content relative to standard preparations isolated in the absence of free calcium. Radioiodinated calmodulin binds specifically to coated vesicles in vitro. Binding is saturable (Kd, 10 nM) and calcium dependent. Half-maximal binding occurs at 2.4 microM free Ca2+ whereas up to 1.2 mM Mg2+ has no effect on binding. Troponin C, a protein homologous to calmodulin, competes with binding of 125I-labeled calmodulin with 1/30th the affinity of native calmodulin. Chromatography of 2 M urea-solubilized coated vesicles on a calmodulin-Sepharose column demonstrated a Ca2+-dependent interaction of coated vesicle proteins and calmodulin. The properties of calmodulin binding to coated vesicles are comparable to those of calmodulin activities in other systems.

Animals↗
PubMed1981

Regulation of intracellular levels of calmodulin and tubulin in normal and transformed cells.

Transformation of mammalian tissue culture cells by oncogenic viruses results in a 2-fold increase in the intracellular concentration of calmodulin quantitated by radioimmunoassay. The two pairs of companion cell lines used in this study were the Swiss mouse 3T3/simian virus 40-transformed 3T3 cells and the normal rat kidney (NRK)/Rous sarcoma virus-transformed NRK cells. The increased intracellular levels of calmodulin in the transformed cells are due to a greater increase in the rate of synthesis (3-fold) relative to the change in the rate of degradation (1.4-fold). On the other hand, no increases were observed in tubulin levels as quantitated by a colchicine-binding assay. The lack of change in tubulin concentration was accounted for by a 2-fold increase in the rate of degradation that is compensated by a similar increase in the rate of synthesis. The consequence of such changes in both transformed cell types is a 2-fold increase in the calmodulin-to-tubulin protein ratio relative to that in their nontransformed counterparts.

Animals↗
PubMed1981

A cloned calmodulin structural gene probe is complementary to DNA sequences from diverse species.

Calmodulin mRNA has been partially purified from a total nucleic acid extract of the electroplax of Electrophorus electricus by oligo(dT)-cellulose chromatography and sucrose gradient centrifugation. A 9- to 10S fraction was determined to contain 39% calmodulin mRNA by translation in a reticulocyte lysate followed by immunoprecipitation with antibodies to calmodulin. Double-stranded cDNA was synthesized from the RNA fraction by using reverse transcriptase from avian myeloblastosis virus. The double-stranded cDNA was joined to pBR322 linearized by restriction endonuclease Pst I and used to transform Escherichia coli RRI. DNAs from 60 tetracycline-resistant cloned hybridized to [32P]cDNA synthesized from the partially purified calmodulin mRNA fraction. By direct DNA sequence analysis, one of these clones, pCM109, was shown to contain calmodulin-specific sequences corresponding to amino acid residues 93--148 of calmodulin or approximately 38% of the peptide-coding region of the calmodulin structural gene sequence. pCM109 was hybridized to DNA isolated from three vertebrate and one plant species by the procedure of Southern. Positive hybridization bands were noted regardless of the DNA source. These data suggest thaat calmodulin gene sequences are evolutionarily conserved, as has been shown to be the case for the primary amino acid sequence.

Animals↗
PubMed1981

Presence and indirect immunofluorescent localization of calmodulin in Paramecium tetraurelia.

In this paper we demonstrate the presence and localization of calmodulin, a calcium-dependent regulatory protein, in the ciliated protozoan Paramecium tetraurelia. Calmodulin is demonstrated by several criteria: (a) the ability of whole cell Paramecium extracts to stimulate mammalian phosphodiesterase activity, (b) the presence of an acidic, thermostable, 17,000-dalton polypeptide whose mobility shifts in SDS polyacrylamide gel electrophoresis in the presence of Ca2+, and (c) the affinity of antibodies against mammalian calmodulin for a Paramecium component as demonstrated by both indirect immunofluorescent localization and radioimmunoassay. Indirect immunofluorescence studies reveal that Paramecium calmodulin is distributed in three distinct regions of the cell, i.e., (a) large, spherical cytoplasmic organelles representing perhaps the food vacuoles or other vacuolar inclusions of the cell, (b) along the entire length of oral and somatic cilia, and (c) along a linear punctate pattern corresponding to the kinetics (basal bodies) of the cell.

Animals↗
PubMed1981

Regulation of protein kinase inhibitor by follicle-stimulating hormone in Sertoli cells in vitro.

The heat-stable protein inhibitor of cAMP-dependent protein kinase is specifically regulated by hormones in cultures of rat Sertoli cells maintained under completely defined conditions. Hormones that are known to elevate Sertoli cell cAMP concentrations, namely FSH and isoproterenol, produce a 4- to 5-fold increase in the specific activity of protein kinase inhibitor, whereas testosterone and LH have no effect. The stimulatory effects of FSH or isoproterenol on protein kinase inhibitor are completely mimicked by dibutyryl cAMP. The ability of FSH to stimulate protein kinase inhibitor is dependent upon the age of the animals used as a source for the Sertoli cells. FSH stimulates protein kinase inhibitor activity in cells from 9- and 16-day-old animals, but not in cells from 32-day-old animals. On the other hand, isoproterenol or dibutyryl cAMP will stimulate protein kinase inhibitor in cells from both young and old animals. FSH can stimulate protein kinase inhibitor activity in older cells only in the added presence of the phosphodiesterase inhibitor, 1-methyl-3-isobutyl xanthine. Using specific antibodies to protein kinase inhibitor, we have shown that this protein is regulated by hormones via preferential stimulation of de novo synthesis of the inhibitor. (Endocrinology 108: 427, 1981)

Aging↗
PubMed1981

Developmental changes in the hormonal regulation of rat testis Sertoli cell adenylyl cyclase.

The stimulatory effects of FSH on Sertoli cell functions such as cAMP accumulation, protein kinase activation, and RNA and protein synthesis wane during testis maturation. However, FSH receptors increase with age and addition of cAMP stimulates these biochemical events in Sertoli cells from animals of any age. In order to determine if this loss of responsiveness to FSH was due to an inability to stimulate adenylyl cyclase, the hormonal responsiveness of this enzyme was investigated as a function of testicular development. In agreement with intact cell studies, adenylyl cyclase activity was found to be stimulated by FSH 2- to 3-fold in homogenates of testes from immature (5-20 days of age) Sertoli cell-enriched rats, while no stimulation of the enzyme by FSH was observed in similar homogenates from Sertoli cell-enriched animals 20 days of age or older. The possibility of a decrease in enzyme sensitivity to the gonadotropin as a function of maturation ws ruled out by dose-response studies. Catalytic activity of the enzyme was retained with increasing animal age as evidenced by the ability of fluoride (10 mM) to stimulate basal activity 4-fold. Hormonal responsiveness of the Sertoli cell adenylyl cyclase of mature animals could be restored, however, either by addition of the nonmetabolizable guanosine 5'-triphosphate analog, 5'-guanylyl-imidodiphosphate to homogenates or by preparation of membrane particles. We found that 5'-guanylyl-imidodiphosphate selectively potentiated FSH effects on cyclase in testicular homogenates from mature animals while having no effect on the relative degree of hormone stimulation in homogenates from immature animals, and that in contrast to homogenates, testicular membrane preparations retain their FSH responsiveness upon animal maturation.

Adenylyl Cyclases↗