Cellular & molecular biology research最新文献

Transforming growth factor-beta (TGF-beta) has been implicated to participate in heart development. The receptors which transduce the signal(s) mediated by TGF-beta ligand binding have only recently been cloned. One of the most prominent effects of TGF-beta is inhibition of cell proliferation, a process that is tightly regulated during heart development. Using the developing rat ventricle as a model system, we have determined the steady state expression patterns for the Type I, II, and III TGF-beta receptors (TGF-beta Rs). Using RNA isolated from ventricular chambers on day 18 of gestation through the ninth postnatal week of age, we detected a modest increase in expression levels for Type I and Type III TGF-beta Rs. In contrast, steady state transcript levels for the Type II TGF-beta R showed a profound developmental increase from nearly undetectable levels at the fetal ages examined to high levels during the first postnatal week of age. Immunoelectron microscopic localization of Type II TGF-beta R confirmed that the 3-week-old ventricular myocyte, as well as nonmyocytes, contained immunoreactive material. Immunoreactivity was found at both the cell surface as well as intracellular compartment. Regional variations (right ventricle, left ventricle, or septum) in the expression pattern of several markers of heart development, but not the TGF-beta R's, were found in RNA obtained from 3-week-old postnatal animals. These data suggest that "downstream" effectors of TGF-beta-mediated stimulation are modulated in a developmental, regional-specific manner in the neonatal/mature myocardium by the level of bioactive TGF-beta.

A key feature of the copy control in the 2 micron circle plasmid of Saccharomyces cerevisiae is its ability to amplify when the copy number drops below the steady state value. The Flp protein encoded by the plasmid is an essential component of the amplification mechanism. A central regulatory event in amplification involves the phosphorylation/dephosphorylation of Tyr-343 of Flp. Tyrosine phosphorylation is achieved by a transesterification mechanism involving a specific phosphodiester within the 2 micron circle. The dephosphorylation is also a transesterification reaction that uses a specific 5'-OH (generated during tyrosine phosphorylation) as the phosphoryl acceptor. A sum of four phosphorylation/dephosphorylation reactions, coordinated in sets of two, is thought to invert the relative directions of a pair of replication forks. This allows more than one copy of the plasmid to be made from a single replication initiation event. In this paper we discuss the structural features of the Flp active site that control and coordinate the transesterification reactions required for amplification.

Requirement of protein kinase CK2 during cell cycle was examined by specific perturbation of CK2 in the intact cell by antisense-oligodeoxynucleotides and microinjection of antibodies. When quiescent human primary lung fibroblasts (IMR-90) were exposed before growth stimulation to oligodeoxynucleotides complementary to the translation start region of mRNAs encoding subunit alpha or beta, a significant inhibition of growth stimulation by epidermal growth factor or serum was observed. The inhibition was reversible and decreased or abolished with mutated antisense-oligodeoxynucleotides. The inhibitory effect coincided with a decrease of CK2 protein (immunostaining with beta subunit antibody) at entry into and during the first several hours of the cell cycle. Injection of beta-specific monoclonal and polyclonal antibodies into IMR-90 cells caused significant inhibition of growth stimulation. The inhibition was reversible, not observed with control antibodies, and strongly reduced by coinjection of CK2 holoenzyme. Cytoplasmic injection inhibited up to 50-60% and was effective at two intervals within the first 2 h and at 12-16 h poststimulation, i.e., at G0/G1 phase transition and at G1/S boundary, respectively. The inhibition at G0/G1 transition is paralleled by an inhibition of cytoplasmic-nuclear translocation of beta subunit protein. Injection of beta antibodies into the nucleus inhibited growth stimulation by as much as 80-85% and was effective for the first 6 h poststimulation, i.e., at G0/G1 phase transition and progression through the adjoining early G1 phase. Nuclear as well as cytoplasmic injections performed during S phase affected neither DNA synthesis nor cell division.(ABSTRACT TRUNCATED AT 250 WORDS)

The enzyme termed nowadays protein kinase CK2 was first described in liver extracts (as a mixture with protein kinase CK1), using casein as artificial substrate, by Burnett and Kennedy (1954). In 1960 it was shown that such casein/phosvitin phosphorylating activity was ubiquitous and distinct from phosphorylase kinase, i.e., the only other protein kinase known at that time. CK1 and CK2 were distinguished from each other at the end of the sixties, and during the seventies CK2 was purified to homogeneity in several laboratories and thoroughly characterized as far as its subunit structure (alpha 2 beta 2), site specificity, and in vitro responsiveness to various effectors were concerned. The first endogenous substrate for CK2 (eIF-3) was described in 1976, but it was during the eighties that it became clear that CK2 is a pleiotropic protein kinase committed with the phosphorylation of a myriad of cellular targets. More than 100 CK2 substrates are known, sharing typical phosphoacceptor sites specified by multiple acidic residues on the C terminal side of Ser/Thr. The definition of the primary structure of CK2 catalytic subunit, in 1987, definitely included CK2 in the big family of eukariotic protein kinases. The growing interest for CK2 is accounted for by its unusual properties, by the increasing number of its substrates, and by several coincidental arguments suggesting that this pleiotropic protein kinase plays a fundamental role in cellular regulation. A major and intriguing problem concerning CK2 is its apparent lack of regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

cDNA clones coding for the alpha and beta subunits of protein kinase 2 (CK2) in zebrafish (Danio rerio) have been isolated. Sequencing of the cDNA clones has demonstrated that one contains the complete coding sequence for the beta subunit of CK2 while the alpha clone is truncated and lacks 183 nucleotides of the 5' coding region. Comparison of the deduced amino acid sequences shows an extremely high degree of evolutionary sequence conservation of these two proteins. Northern analysis of the mRNAs coding for the alpha subunit indicates that this messenger is present in 1 h embryos as a 3.6 Kb and a 1.9 Kb species, both of which decrease in 24-h embryos. In the case of beta, the major mRNA species of approximately 1.7 Kb maintains its level during the period of embryogenesis studied. In situ hybridization of early embryos, using antisense RNAs against alpha and beta mRNAs demonstrates temporal and tissue specific expression patterns. The alpha mRNA decreases after blastula, when it is evenly distributed. The beta mRNA is maintained at high levels between 4 and 24 h of development, showing in 18 h embryos a higher concentration in the developing neural tube and in the embryonic optic and otic vesicles.

Many of the effects of growth factors or hormones are mediated through the activation of protein kinase cascades. In this regard, it is well established that the activity of several protein kinases can be dramatically increased when cells are treated with a variety of stimuli. Since 1987, there have been several reports demonstrating that the activity of casein kinase II (CKII) can be acutely increased by hormones or growth factors. However, these are a number of discrepancies regarding the activation of CKII. In this study, we have examined CKII activities in extracts prepared from cells following treatment with stimuli that had been previously shown to elicit dramatic increases in CKII activity. Human WI.38 diploid lung fibroblasts were stimulated with serum or a variety of other stimuli including insulin, platelet-derived growth factor, fibroblast growth factor, epidermal growth factor, or phorbol myristate acetate. Human A431 epidermal carcinoma cells were similarly treated with epidermal growth factor. No reproducible increases in CKII activity were observed in response to any of these treatments. By comparison, a dramatic increase in kinase activity towards a synthetic peptide based on phosphorylation sites within the ribosomal S6 protein was consistently measured. Our observations indicate that CKII is not regulated in a similar manner by growth factors as are the protein kinases of the MAP kinase cascade, e.g., MAP kinase itself or ribosomal protein S6 kinase.

The U7 small nuclear ribonucleoprotein (snRNP) is an essential component of the endonucleolytic cleavage reaction which leads to the production of mature 3'-ends of histone premRNAs. We have examined the relative amount and the structure of the U7 snRNP, as assayed by sensitivity to micrococcal nuclease, during the cell cycle in human HeLa and WI-38 cells. Using an RNase A protection assay, we find no change in the steady state levels of U7 throughout the cell cycle. Similarly, the sensitivity of U7 to micrococcal nuclease remained unchanged in both cell types. Contact inhibited WI-38 cells, that are deemed to have left the cell cycle and entered a quiescent state, displayed similar levels of U7 to cells in S and G1 phases of the cell cycle, however, the U7 snRNA was slightly more resistant to micrococcal nuclease. Histone 3' end mRNA processing was also assayed in HeLa cell cycle phase-specific extracts. In marked contrast to previous observations in extracts prepared from the rodent cell line, C3H10T1/2, (Hoffmann and Birnstiel, 1990), we find that the 3' end processing reaction remained constant throughout the cell cycle.

Bradycardia has been shown to be beneficial for the normal and ischaemic heart because it improves diastolic perfusion and oxygen supply demand balance. Experimentally, a chronically induced decrease in heart rate, either by electrical pacing or pharmacological means, was found previously to increase myocardial capillary supply in normal rabbit and rat hearts. These studies have been extended to a larger mammal, the pig, in which a direct bradycardia (approximately 30% decrease in heart rate) was induced by electrical pacing for 4-5 weeks. There was no evidence of heart hypertrophy and capillary density was found to be significantly increased in the left, but not right, ventricle. Cardiac function during dobutamine inotropic challenge was better in pig hearts which had been paced bradycardially. They performed greater stroke work-higher stroke flow output at lower heart rate--for similar coronary blood flow, thus demonstrating an improved economy of flow utilisation. Heart rate reduction may facilitate capillary growth in the absence of cardiac hypertrophy by prolonging diastolic perfusion, and/or mechanical stretch of vessels due to increased stroke volume capacity. In either case, capillaries would be exposed to increased wall tension which could trigger angiogenesis.

Several genes critical to the regulation of melanin production in mammals have recently been cloned and characterized. They map to the albino, brown, and slaty loci in mice, and encode proteins with similar structures and features, but with distinct catalytic capacities. The albino locus encodes tyrosinase, an enzyme with three distinct melanogenic functions, the brown locus encodes TRP1 (tyrosinase-related protein-1), and the slaty locus encodes TRP2, another tyrosinase related-protein. TRP2 functions as DOPAchrome tautomerase, an enzyme that preserves the carboxylic acid content of melanins, which would be spontaneously lost in its absence, while TRP1 is able to oxidize the DHICA produced by TRP2. In this study we have used three different systems (immune-affinity purified melanogenic enzymes, mutant melanocytes, and transfected cells) to examine the enzymatic interactions of these proteins, and their stabilization in a complex which significantly increases their physiological half-life. When extrapolated to the melanocyte, our results demonstrate the catalytic functions of these proteins and suggest how they might stably interact within a melanogenic complex in the melanosome to regulate the quantity and quality of melanin synthesized.

We have identified a novel protein tyrosine phosphatase gene from an embryonal rat brain by reverse transcription-based polymerase chain reaction. Its transcription is specific to brain and is developmentally regulated, as it is expressed at high levels in embryonal and neonatal stages but scarcely in an adult. Southern blot analysis indicates that the gene exists as a single copy and is conserved among various species. Homology search of the deduced amino acid sequence suggests that this gene belongs to the same family of the membrane-type tyrosine phosphatase gene of Drosophila (DPTP10D), whose expression is specific to an central nervous system of fly embryo.