Cellular & molecular biology research最新文献

Analysis of casein kinase II in organisms amenable to genetic manipulation is essential to elucidating the physiological function(s) of this ubiquitous protein kinase. This paper summarizes work from our laboratory on the enzyme from Saccharomyces cerevisiae. The biochemistry, molecular biology, and genetics of S. cerevisiae casein kinase II are reviewed and discussed.

A brief overview is presented of progress in the development of specific inhibitors of protein kinases CKI and CKII. Two promising classes of inhibitors, which have the ability to traverse cell membranes, are now known. One of these is based on halogenated benzimidazoles and 2-aza-benzimidazoles (benzotriazoles) and some of their nucleosides. The second embraces modified isoquinoline sulfonamides, several of which are known as inhibitors of other protein kinases. Both classes include analogs that permit discrimination between CKI and CKII. Ongoing research with halogenated benzotriazoles leads to inhibitors with Ki values below 1 microM. Also considered are nucleoside triphosphate analog inhibitors and their potential properties as donors, with illustrative examples from the field of nucleoside kinases, including the apparent existence of a dual-specific viral protein/nucleoside kinase. The role of cellular CKII and viral-encoded CKII-like activities in viral replication underlines the potential of CKII inhibitors as antiviral agents, exemplified by the case of vesicular stomatitis virus.

Rat liver cytosolic CKII shows heterogeneity resulting from association of the alpha/alpha'-subunits with the beta-subunit or with a phosphorylatable protein of 49 kDa (pp49). Preparations of pp49 were resolved into several spots by two dimensional analysis which might be derived from different degrees of phosphorylation. pp49 was phosphorylated in vitro by purified rat liver CKII and to a lower extent by purified rat brain protein kinase C. In all cases, phosphorylation of pp49 occurred exclusively on Ser. Phosphopeptide maps of phosphorylated pp49 confirmed that the phosphorylation by CKII or PKC takes place in different sites. Prior phosphorylation of pp49 by protein kinase C had no significant influence on the increase of the Km value for beta-casein of CKII, caused by pp49. A tryptic peptide from pp49 has been recently sequenced and antibodies against it had been raised. The antibodies were able to recognize pp49 in rat liver extracts as well as in HL-60 extracts what leads us to presume that this kind of interaction might exist in other species and tissues.

Thyroid hormone (T3) stimulates Na,K-ATPase activity and alpha and beta subunit mRNA abundances in myocardial cells in vivo and in vitro. In this study, we used transient transfection and nuclear run-on assays to determine whether T3 regulates the transcription rate of the Na,K-ATPase alpha 2 subunit gene. Primary cultures of neonatal rat cardiac myocytes were incubated with 100 nM T3 for 1, 3, and 6 d, and alpha 2 mRNA levels were measured by Northern blot hybridization analysis. There was no change in the abundance of alpha 2 mRNA by 1 d of T3 treatment, whereas a two- and threefold increase in alpha 2 mRNA was evident when cells were exposed to T3 for 3 and 6 d, respectively. A portion of the rat alpha 2 gene containing 1700 base pairs (bp) of 5'-flanking DNA sequence was isolated and fused to the firefly luciferase gene. Transient transfection experiments utilizing this chimeric gene showed no T3 trans-activation of reporter gene activity either in the absence or presence of cotransfected beta 1 or alpha 1 isoforms of rat T3 receptor (T3R). In contrast, cotransfection of T3R facilitated a strong stimulation of luciferase activity driven by a construct containing a single copy of a palindromic T3 response element (TRE). Nuclear run-on analysis indicated that the rate of transcription of the endogenous alpha 2 gene was enhanced 1.2-fold at 3 d of T3 treatment, and was not regulated at either 1 or 6 d. These results indicate that the T3-dependent increase in alpha 2 mRNA content at 6 d is mediated at a post-transcriptional level. Unexpectedly, we observed a T3-dependent three-to sixfold repression of alpha 2/luciferase expression in cardiac myocytes cotransfected with T3R. Deletion analysis of the 5' end of the alpha 2 gene revealed a negative TRE between nucleotides -354 and -100.

Genomic instability in the form of microsatellite alterations at nine loci on chromosomes 2p, 8p, 10p, 11p, and nm23-H1 locus on 17q21.3 were studied in sporadic colorectal tumors. Alterations in dinucleotide repeats in tumor DNA as larger allele, smaller allele, or loss of heterozygosity (LOH) were observed. Forty percent of tumor showed an RER+ phenotype. A significantly high number of alterations was detected at loci of chromosome 8p. The markers on chromosomes 2p, 10p, and 11p did not show such significant alteration. LOH was found to be associated with the nm23-H1 locus. No correlation was found between the age, site of tumor occurrence, or metastasis and the microsatellite instability.

Various proteins of cardiac myocytes are preprogrammed at a very early stage of heart development, but functional load (stretch, pressure) plays an important role in their expression under both physiological and pathological circumstances. Mechanical factors are also important in growth of vessels, particularly with respect to hypertrophy or hyperplasia of vascular smooth muscle. Their effect on growth of endothelial cells is less clear. Although they have been studied in cell culture, little is known about their involvement in capillary growth in vivo. Their possible role is considered in capillary growth in the normal adult heart where it was elicited by long-term administration of various vasodilators, by long-term bradycardia, or by increased inotropic action. Here the mechanical stimuli may act either by increased shear stress (resulting from increased velocity of flow in long-term dilatation) or by increasing vessel wall tension (in conjunction with increased diameters and/or stretch produced by increased inotropism). While the role of growth factors in the development of myocytes has been established, it is still questionable in capillary growth. It is also possible that various growth factors exert their effect on vessel growth by their vasoactive activity.

In mammalian cells, the transmembrane beta-amyloid peptide precursor (beta-APP) undergoes a complex series of alternative proteolytic processing steps that result in the secretion of varying proportions of its extra-cellular domain (protease nexin II) and beta-amyloid peptide. The protein is also reinternalized and degraded in the endosomal-lysosomal system. The relative efficiencies of these competing processes determine the yield of beta-amyloid peptide. Several proteases have been implicated in this complex processing pathway, although none has been identified to date. The yeast secretory system contains proteases homologous to mammalian pro-hormone convertases and is susceptible to genetic manipulation. We therefore investigated the expression and processing of the beta-amyloid peptide precursors (beta-APP-695 and beta-APP-751) in Saccharomyces cerevisiae transformed with human beta-APP cDNA's. beta-APP (695 or 751) cDNA either with its authentic signal sequence or the yeast-derived prepro-alpha-factor leader, was inserted into a glucose-regulated expression vector and transfected into a protease-deficient yeast strain. In all instances, expression of beta-APP was about 1% of total protein. Protease protection studies indicated that either the natural human signal sequence or the alpha-factor leader sequence targetted beta-APP to the endoplasmic reticulum and inserted it with the amino-terminal domain in the lumen. All of the beta-APP fused to the alpha-factor leader proceeded to the trans-Golgi, where Kex2 endopeptidase removed the leader and released the normal amino-terminus of beta-APP. About one-half of the beta-APP was also cleaved at the "alpha-secretase" site in the middle of the beta-peptide sequence, 12 residues before the membrane-spanning sequence. A fraction of the alpha-secretase-cleaved beta-APP appeared in the culture medium; however, most of it associated with the exterior of the cells. The carboxyl-terminal fragments formed by cleavage at the alpha-secretase site accumulated in the membranes. Other proteolytic processes generated membrane-associated carboxyl-terminal fragments that also resembled those found in mammalian cells. These results indicate that the secretory system of S. cerevisiae possesses proteases with specificities similar to the mammalian enzymes that process beta-APP.