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Biomedical subjects

A R Means

Publications and source records attributed to A R Means.

At least 163 records · Page 9Linked to original sources
PubMed1984

Changes in calmodulin and its mRNA accompany reentry of quiescent (G0) cells into the cell cycle.

Release of CHO-K1 cells from plateau or stationary phase and reentry into the cell cycle is specifically and reversibly blocked at two distinct sites by the anticalmodulin drug W13. The first block occurs early during release while the cells are still at G0/G1, whereas the second occurs later in reentry during early S phase. As determined by radioimmunoassay, calmodulin levels undergo changes at three distinct steps in plateau-phase entry and release. First, the entry of exponentially growing cells into plateau phase is accompanied by an increase in the calmodulin level. The second change is a reduction in the calmodulin content of cells within the first hour following release from plateau phase. The third change is the subsequent increase in calmodulin levels, which precedes entry of the cells into S phase. Analysis of calmodulin mRNA levels by dot-blot hybridization demonstrates that the changes in calmodulin protein are preceded by changes in calmodulin mRNA. Furthermore, whereas a decrease in CaM mRNA is observed within the first hour following plateau release, no such decrease is observed for beta-actin mRNA, suggesting that this decrease may be selective for calmodulin. This selectivity is further substantiated by the fact that identical changes in calmodulin and calmodulin mRNA are observed in cells released from plateau by two different techniques. Taken together, these data suggest that calmodulin may play an important role in the reentry of cells into the cell cycle.

Animals↗
PubMed1984

Flagellar motility requires the cAMP-dependent phosphorylation of a heat-stable NP-40-soluble 56 kd protein, axokinin.

Using NP-40-treated dog sperm as a model, the stimulatory effect of cAMP upon reactivated flagellar motility has been shown to be dependent upon the cAMP-dependent phosphorylation of a heat-stable NP-40-soluble protein of 56 kd. Examination by two-dimensional polyacrylamide gel electrophoresis of NP-40 extract proteins phosphorylated with gamma-32P-ATP revealed a major cAMP-dependent phosphopeptide at 56 kd. This is the only cAMP-dependent phosphoprotein common to NP-40 extracts of all tissues that show cAMP-dependent stimulation of flagellar motility. These cells and tissues include sea urchin, dog, and human sperm, as well as dog trachea and retina. Moreover, this phosphoprotein is absent in nonstimulatory extracts from tissues such as skeletal muscle, brain, and liver. We conclude that the cAMP-dependent phosphorylation of the 56 kd peptide represents a major regulatory component of not only sperm but other types of axonemal motility as well.

Animals↗
PubMed1984

Calmodulin-microtubule association in cultured mammalian cells.

A Triton X-100-lysed cell system has been used to identify calmodulin on the cytoskeleton of 3T3 and transformed SV3T3 cells. By indirect immunofluorescence, calmodulin was found to be associated with both the cytoplasmic microtubule complex and the centrosomes. A number of cytoplasmic microtubules more resistant to disassembly upon either cold (0-4 degrees C) or hypotonic treatment, as well as following dilution have been identified. Most of the stable microtubules appeared to be associated with the centrosome at one end and with the plasma membrane at the other end. These microtubules could be induced to depolymerize, however, by micromolar Ca++ concentrations. These data suggest that, by interacting directly with the microtubule, calmodulin may influence microtubule assembly and ensure the Ca++-sensitivity of both mitotic and cytoplasmic microtubules.

Animals↗
PubMed1984

Estrogen stimulates the transient association of calmodulin and myosin light chain kinase with the chicken liver nuclear matrix.

Previous work has demonstrated that estrogen administration to immature chickens results in a rapid but transient increase in nuclear estrogen receptor content, a large portion of which is associated with the nuclear matrix. The present studies were undertaken to determine whether estrogen produced a more generalized change in the protein composition of the nuclear matrix. High-resolution two-dimensional gel analysis of the matrix revealed a very complex protein pattern, but several major qualitative differences were observed after estrogen treatment. To simplify the number of proteins evaluated, we examined the effects of estrogen on a subset of matrix proteins, namely, calmodulin and its binding proteins. Calmodulin was measured by radioimmunoassay and the binding proteins were detected by interaction of 125I-calmodulin with matrix proteins distributed on one-dimensional polyacrylamide gels. Calmodulin and two specific Ca2+-dependent calmodulin-binding proteins were found to be associated with matrix preparations. The two binding proteins exhibited apparent Mr of 200,000 and 130,000. The Mr 130,000 protein was identified as myosin light chain kinase on the basis of enzymatic activity and immunoreactivity with a specific antibody to this enzyme. Estrogen treatment of immature chickens did not alter the hepatic content of calmodulin. However, the steroid did result in an enrichment of the proportion of calmodulin and its two binding proteins associated with the nuclear matrix within 4 h after injection. The time course of these changes paralleled those previously documented for estrogen receptor. Taken together, these data are compatible with a role for calmodulin and myosin light chain kinase in the response of chicken liver cells to steroid hormones.

Animals↗
PubMed1984

Estrogen modulation of nuclear matrix-associated steroid hormone binding.

Estrogen regulation of the intranuclear distribution of estrogen-binding proteins has been examined in the chicken liver. Administration of estrogen [diethylstilbestrol (DES)] resulted in a rapid, dose-dependent increase in two types of estrogen-binding sites associated with the nuclear matrix. Type I appeared to be the estrogen receptor (ER; Kd for estradiol = 2.0 nM), whereas type II was equivalent to binding sites previously defined in rat uterine nuclei (Kd = 15 nM). The association of nuclear ER and type II sites with the nuclear matrix followed a time course corresponding to the maximal accumulation of nuclear ER in the liver. A 2-fold enrichment (per U protein) of nuclear ER in the matrix was obtained at a dose of 20 micrograms DES/0.2 kg compared to control values. Further enrichment was observed with higher doses of DES (200 and 2000 micrograms). The antiestrogens tamoxifen (tamoxifen-citrate) and zuclomiphine 2-[p-(chloro-1,2-diphenyl-vinyl)phenoxyl]citrate) were potent inhibitors (greater than or equal to 50%) of the estrogen-stimulated association of both ER and type II sites with the matrix. However, they did not increase total nuclear or matrix ER concentrations when administered alone. These data demonstrate that the association of ER with liver nuclear matrix is estrogen dependent and suggest that the biological effects of estrogen at the nuclear level may be mediated by this association.

Animals↗
PubMed1983

Chicken calmodulin genes. A species comparison of cDNA sequences and isolation of a genomic clone.

A cDNA library, prepared from poly(A+) mRNA isolated from chicken brain, was screened for calmodulin sequences using the cloned full length structural gene from Electrophorus electricus as probe (Lagacé, L., Chandra, T., Woo, S.L.C., and Means, A.R. (1983) J. Biol. Chem. 258, 1684-1688). Fifteen positive signals were detected among 4500 recombinant clones from which two overlapping clones (pCB12 and pCB15) were selected for subsequent DNA sequencing. The combined unique sequences of the two cDNA clones yielded 1395 base pairs and contained the entire coding region for calmodulin, 94 base pairs of the 5'-nontranslated region, and the entire 3'-nontranslated region of 857 base pairs. The derived amino acid sequence of chicken calmodulin is identical with that of the bovine or human protein. Compared to the eel, there is a single conservative amino acid substitution at position 74 which is occupied by Arg in the chicken and Lys in the eel. The overall nucleotide homology between the amino acid coding regions of chicken and eel calmodulin mRNA is 79%. However, the 5'- and 3'-nontranslated regions of the chicken and eel mRNA for calmodulin are highly diverged with sequence homologies of 21 and 29%, respectively. The cDNA clones were used as probes to determine the size and distribution of calmodulin mRNA in a variety of chicken tissues. In all tissues examined, two species of mRNA for calmodulin were detected at 1600 and 1900 nucleotides. Both mRNAs occurred in the cytoplasm with an abundance ratio of 4:1 for the 1600 and 1900 species, respectively. The two mRNAs appear to result from differential processing of transcripts from a single calmodulin gene. Screening of a chicken genomic phage library using pCB12 as a probe yielded a single positive designated CL-1 which contains a DNA insert of 13.5 kilobase pairs. Partial sequencing of CL-1 has confirmed the presence of sequences which code for calmodulin. A comparison of the restriction maps of CL-1 and pCB12 and pCB15 indicates that CL-1 contains at least 3 intervening sequences.

Amino Acid Sequence↗
PubMed1983

Identification of multiple species of calmodulin messenger RNA using a full length complementary DNA.

Poly(A) RNA from eel electroplax was used to construct a full length cDNA complementary to calmodulin (CaM) mRNA which was cloned in the PstI site of pBR322 DNA. Recombinant plasmids containing sequences complementary to CaM mRNA were identified by hybridization using a 32P-labeled CaM cDNA (pCM109). Nucleotide sequence analysis reveals that the clone from the plasmid pCM116 contains a 5' nontranslated region of 26 nucleotides, the entire coding region, and a 3' nontranslated region of 408 nucleotides. The amino acid sequence deduced from the nucleotide sequence is similar to those previously reported for CaM from other species. Comparison between the nucleotide sequence of the functional domains of the protein shows extensive homology between all four domains. pCM116 was utilized to determine and compare the populations of RNA present in different tissues. In eel electroplax, three species of cytoplasmic RNAs at 820, 1100, and 2000 nucleotides hybridize to the cDNA probe. The nucleus contains an additional CaM RNA molecule of 5500 nucleotides which may represent a primary transcript of the calmodulin gene. Sequence analysis of the 3' noncoding region of pCM116 reveals 3 possible polyadenylation sites (AATAAA) at positions 573, 580, and 855. The mRNA of 820 nucleotides was derived by polyadenylation at the first site whereas the mRNA of 1100 nucleotides was derived by poly(A) addition at position 855. These data are compatible with the idea that at least 2 of the 3 CaM mRNAs in eel electroplax tissue are derived from a single nuclear transcript by differential polyadenylation during processing.

Amino Acid Sequence↗
PubMed1983

Tissue-specific expression of a chicken calmodulin pseudogene lacking intervening sequences.

An eel calmodulin cDNA probe has been used to isolate a calmodulin gene from a chicken DNA library. Sequence analysis revealed this calmodulin gene (cCM1) to contain the nucleotides that code for 148 amino acids, a termination codon, and 486 residues of 3'-noncoding sequence before an A-A-T-A-A-A poly(A) addition signal. The amino acid sequence derived from these nucleotides is 87% homologous to that of bovine brain calmodulin. cCM1 is one of two calmodulin genes in the chicken genome but is unique in that it does not contain intervening sequences to interrupt the structural segments of the protein. This suggests that cCM1 originated as a processed gene copy derived from the other calmodulin gene, cCL1, a circumstance usually associated with pseudogenes. In contrast, cCM1 appears to be a functional member of a multigene family whose expression is specific for muscle cells.

Amino Acid Sequence↗
PubMed1983

Cyclic adenosine 3',5' monophosphate, calcium and protein phosphorylation in flagellar motility.

cAMP and calcium are two important regulators of sperm flagellar motility. cAMP stimulates sperm motility by activating cAMP-dependent protein kinase and catalyzing the phosphorylation of sperm proteins. The stimulation of sperm motility by cAMP appears to be at two different levels. Evidence has been presented to suggest that cAMP-dependent phosphorylations may be required in order for motility to be initiated. In addition, cAMP-dependent phosphorylation appears to modulate specific parameters of motility resulting in higher beat frequency or greater wave amplitude. Calcium, on the other hand, when elevated intracellularly to 10(-6) M or higher, inhibits flagellar motility. The calcium-binding protein, calmodulin, appears to mediate a large number of effects of calcium on motility. Evidence suggests that calcium-calmodulin may be involved at the level of the membrane to pump calcium out of the flagellum. In addition, calcium-calmodulin may be involved in the control of axonemal function by regulating dynein ATPase and myosin light chain kinase activities. The identification of cAMP-dependent protein kinase, calmodulin and myosin light chain kinase in the sperm head suggests that cAMP and calcium-dependent phosphorylations are also involved in the control of the fertilization process, i.e., the acrosome reaction, in a manner similar to that known for the control of stimulus/secretion coupling. Finally, the effects of cAMP on flagellar motility are mediated by protein phosphorylation while the effects of calcium on motility are also in part, mediated by effects on protein phosphorylation.

Adenosine Triphosphatases↗
PubMed1983

Follicle-stimulating hormone activation of glycogen phosphorylase in the Sertoli cell-enriched rat testis.

The potential role of glycogen phosphorylase in providing energy for the Sertoli cell-enriched testis has been investigated. This enzyme is detectable in testes from rats 6-54 days of age. Glycogen phosphorylase in isolated Sertoli cell-enriched testes is specifically stimulated by FSH. Maximal activation (2-fold) is obtained within 10 min after adding 0.5 micrograms FSH/ml to isolated immature testes (16 days old). There is only a 1.1-fold activation by FSH in testes from mature (34 days old) animals. The sensitivity to the gonadotropin can be restored by adding 1-methyl-3-isobutylxanthine, a phosphodiesterase inhibitor, with the FSH. Phosphorylase can be activated by effectors that mimic the actions of the two proposed mediators of FSH action, cAMP and Ca+2. Phosphorylase from testis of either age is maximally activated by an analog of cAMP, 8-bromo-cAMP. While phosphorylase is rapidly activated 1.4-fold by incubating isolated testis for 2 min with A23187, a Ca+2 ionophore, the age, time, and dose dependence of FSH activation are consistent with conversion mediated by cAMP. Phosphorylase was localized in cultured Sertoli cells by indirect immunofluorescence microscopy. Affinity-purified antiphosphorylase decorated cytoskeletal structures that resemble stress fibers, suggesting that phosphorylase may function in Sertoli cells to provide energy for cytoskeletal motility.

1-Methyl-3-isobutylxanthine↗
PubMed1982

The evolution of a complex eucaryotic gene.

Two thirds of the natural chicken ovomucoid gene has been sequenced, including all exons and the intron sequences surrounding all fourteen intron/exon junctions. The junctions sequences surrounding four of the introns are redundant: however, the sequences surrounding the other three introns contain no redundancies and thus the splicing sites at either end of these three introns are unambiguous. The splicing in all cases conforms to the GT-AG rule. We compare the structural organization of the ovomucoid gene with the ovomucoid protein sequence to examine theories of the evolution of ovomucoids as well as the origin of intervening sequences. This analysis suggests that the present ovomucoid gene evolved from a primordia ovomucoid gene by two separate intragenic duplications. Furthermore, sequence analyses suggest that introns were present in the primordial ovomucoid gene before birds and mammals diverged, about 300 million years ago. Finally, the positions of the introns within the ovomucoid gene support the theory that introns separate gene segments that code for functional domains of proteins and provide insight on the manner by which eucaryotic genes were constructed during the process of evolution.

Amino Acid Sequence↗
PubMed1982

Calmodulin and the cell cycle: involvement in regulation of cell-cycle progression.

Calmodulin levels are elevated twofold at late G1 and/or early S phases during the growth cycle of CHO-K1 cells. These levels are maintained throughout the remainder of the cell cycle unit cytokinesis. The G1 daughter cells then contain half the intracellular calmodulin level found prior to cell division. Elevation of calmodulin at the G1-S boundary is independent of the length of G1, and the increase in calmodulin appears to be related to progression into S phase. The importance of calmodulin for G1-S progression is suggested by the ability of the anticalmodulin drug W13 to elicit specific and reversible progression delays into and through S phase.

Animals↗
PubMed1982

Association of myosin light chain kinase with lymphocyte membrane-cytoskeleton complex.

A specific antibody against myosin light chain kinase (MLCK) was used to identify the presence of a Ca2+-calmodulin-activated MLCK in mouse 1-lymphoma cells. With a double immunofluorescence technique, MLCK was determined to be accumulated directly under Con A-capped structures in a manner similar to that of previously described accumulation of actomyosin. The lymphocyte MLCK was phosphorylated in the uncapped cell and, by immunoprecipitation with a specific MLCK antibody, was shown to possess a Mr of 130,000. The MLCK was also found to constitute a major fraction of the phosphoproteins present in the plasma membrane associated-cytoskeleton. Myosin light chain kinase catalyzed the phosphorylation of both endogenous lymphocyte myosin light chains and those from smooth and skeletal muscle. The enzyme activity was dependent on the presence of Ca2+-calmodulin and was inhibited by the calmodulin-binding drug, trifluoperazine. These data suggest that the membrane-cytoskeleton-associated MLCK activity may be important in regulation of the actinmyosin contraction which is believed to be required for the collection of surface receptors into capped structures.

Animals↗
PubMed1982

Regulation of protein phosphorylation and motility of sperm by cyclic adenosine monophosphate and calcium.

Motility and protein phosphorylation have been measured under identical experimental conditions in ejaculated dog sperm lysed with low concentrations of Triton X-100 and reactivated with [gamma-32P]ATP. Cyclic AMP stimulates motility and protein phosphorylation while calcium inhibits motility and the overall incorporation of phosphate into endogenous proteins. Analysis of 32P-labeled sperm proteins on 1- and 2-dimensional polyacrylamide gels demonstrates that an enhanced phosphorylation of a defined number of specific proteins is associated with cAMP-stimulated motility. A major axonemal proteins, namely tubulin, has been tentatively identified as a phosphoprotein subject to regulation by cAMP. The phosphorylation of tubulin is almost completely dependent upon cAMP and is not affected by microM calcium. On the other hand, the cAMP-dependent stimulated phosphorylation of the other sperm proteins still occurs, but in most instances at a reduced rate in the presence of calcium. Two high molecular weight (Mr) phosphoproteins (350,000 and 260,000 daltons) whose phosphorylation states are modified by cAMP and calcium also were identified. It is suggested that 1 or both these proteins may be high Mr subunits of dynein. The phosphorylation of 1 of these proteins is stimulated by cAMP, but not affected by calcium; the other is stimulated by cAMP and inhibited by calcium. Three major cAMP-independent phosphoproteins of Mr 98,000, 43,000 and 26,000 have been identified. The phosphorylation of the 98,000 Mr protein is markedly reduced by micromolar calcium and not restored by cAMP. Using anticalmodulin drugs to inhibit motility, we suggest that the inhibitory effects of calcium on flagellar motility may be mediated in part by calmodulin. We conclude that the regulation of flagellar motility in cAMP and calcium includes mechanisms involving the control of the phosphorylation state of sperm proteins, some of which may be axonemal components.

Animals↗