SAAS bulletin, biochemistry and biotechnology最新文献

RNA extracted from rabbit leukocytes and reticulocytes was reverse transcribed and used in the Polymerase Chain Reaction technique along with primers designed to amplify the coding sequence of rabbit cytochrome b5. The resultant amplified products were subcloned and analyzed. Sequencing confirmed that leukocyte and liver cDNAs are homologous and encode the membrane-bound form of the protein. In contrast, reticulocytes exhibit a highly similar, but different mRNA which encodes the smaller, soluble cytochrome b5. This is the first example of a cytochrome b5 sequence from a tissue other than liver, erythrocyte or reticulocyte.

Possible role of methylation of proteins in platelet activation was examined in this study. Electropermeabilized platelets incorporated radioactivity in the presence of [methyl-3H]-S-adenosylmethionine. Thrombin, PDBu and GTP gamma S increased incorporation of radioactivity in a time-dependent manner. In other experiments, 23 kD membrane proteins incorporated radioactivity in the presence of [methyl-3H]-S-adenosylmethionine and platelet cytosol. Using rap specific antisera the 23 kD methylated proteins were characterized as low Mr G proteins, known as rap1 proteins. N-Acetyl-S-farnesyl-L-cysteine (AFC), an inhibitor of the methyltransferase, inhibited carboxyl methylation of platelet rap1 proteins and also inhibited platelet aggregation and mobilization of cytosolic calcium induced by a variety of agonists in a concentration-dependent manner. Inhibition of methylation of rap1 proteins as well as inhibition of platelet activation by AFC suggests that methylation and consequently translocation of rap1 proteins to plasma membrane may be important for agonist-induced signal transduction in human platelets.

The proliferation of normal mammalian cells is regulated by external growth factors. The complex array of biochemical events triggered by these factors is believed to be modulated by changes in gene expression and leads to cell growth. The long range goals of this research are to identify, isolate, and characterize the genes that modulate cell growth and to determine the functions of their protein products. Previously, several genes were identified whose expression was characterized as growth-regulated; their cognate cytoplasmic transcripts were induced when quiescent cells were stimulated with serum. Predicated on the fact that only 1% of active genes respond to mitogenic stimulation, we hypothesize that these growth-regulated genes are functionally involved in the mitogenic process. We have identified one of these growth regulated genes by sequence analysis to be annexin II, a major substrate of tyrosine kinases. We have determined that the expression of this gene is growth-regulated and that its membrane/cytoskeleton association changes as a function of mitogenic stimulation.

Mutant strains containing APH(3')-II were constructed via site-directed mutagenesis of the cloned gene and by random mutagenesis of a strain containing the APH(3')-II gene on a conjugative plasmid. Substitutions at highly conserved amino acid residues produced APH(3') enzymes which in general showed reduced activity and conferred reduced levels of resistance to their substrates. Substitutions at Tyr 218 altered substrate specificity for the enzymes. Random mutagenesis produced plasmid-borne mutations conferring amikacin resistance. Two of these mutations appeared to be localized to the APH(3')-II structural gene.

Escherichia coli has a carbon-phosphorus (C-P) lyase with a broad substrate specificity, whose synthesis is induced many hundred fold during phosphate (Pi) limitation. Fourteen genes for phosphonate metabolism comprise the phnC-to-phnP gene cluster: three gene products (PhnC, PhnD, and PhnE) comprise a binding protein-dependent phosphonate transporter, which also transports Pi and phosphate esters; two gene products (PhnF and PhnO) may have a role in gene regulation; and nine gene products (PhnG, PhnH, PhnI, PhnJ, PhnK, PhnL, PhnM, PhnN, and PhnP) may comprise a C-P lyase enzyme complex. Phosphonate biodegradation via a C-P lyase appears to be limited by the specificity of the PhnCDE transporter and not by the specificity of the C-P lyase. These interpretations are based on results from a combination of molecular genetic and molecular biological studies on phosphonate metabolism in E. coli.

Clinical success in the treatment of tumors with chemotherapy has significantly improved over the past several years. However, treatment failures due to drug resistance of cancer cells has remained a major problem. The classical form of multiple drug resistance is perhaps also the most common type of drug resistance, and represents the overexpression of a transmembrane glycoprotein pump (P-170) that mediates the efflux of a spectrum of structurally and functionally unrelated drugs. Here, we discuss recent evidence that support the concept that the total phenomenon of multiple drug resistance (MDR) involves several other mechanisms in addition to that underlying "classical" MDR. These include the action of other energy-dependent membrane efflux pumps, elevated levels of GSH for drug conjugation and detoxification to facilitate export, enhanced DNA repair facility, gene amplification and oncogene activation. The combination of mechanisms used by any particular cell line is variable and suggests that many of these mechanisms are independent. Successful reversal of drug resistance appears to require the identification of relevant operative resistance mechanisms. An example is the competitive inhibition of P-170 with verapamil, quinine and tamoxifen. A broadly successful strategy for killing drug-resistant cancer cells, however, could be based on either selective energy depletion of cancer cells or the permeabilization of tumor cells with an effective bypass of efflux pumps, since many mechanisms of drug resistance entail the energy-dependent export of toxins. The latter approach may be achieved via membrane lipid modifications or the introduction of membrane pores by biological or physical (electroporation) means.

The tissue restricted and developmental potentiation of transcription by chicken alpha-skeletal actin promoter regions fused to the reporter gene chloramphenicol acetyl transferase (CAT) were characterized in transgenic mice. Six of eight expressing transgenic mouse lines containing the chicken alpha-skeletal actin promoter fused to CAT resulted in preferential transgene transcription in skeletal muscle tissue, similar to the endogenous mouse alpha-skeletal actin gene. Two of the eight lines departed from the preferred pattern of skeletal muscle expression with primary expression of the transgene in the heart, a tissue containing primarily cardiac actin isoforms. Developmentally, a transition from embryonic heart to fetal and neonatal skeletal muscle expression was produced by the transgene promoter, a pattern of regulation similar to that of the endogenous alpha-skeletal actin gene. Instances of departure of transgene expression from the endogenous gene implied the existance of higher order muscle gene regulatory mechanisms.

Acetoacetate in Escherichia coli is metabolized via the combined enzymatic action of a CoA-transferase and a thiolase. Growth of E. coli on short chain fatty acids such as butyrate and valerate is also predicated upon the expression of these enzymes. The genes for these enzymes (atoDAB) are arranged in an operon and are coordinately transcribed in response to the inducer acetoacetate. A positive regulatory element, the product of the atoC gene, regulates expression of the operon. The atoC gene lies adjacent to the atoDAB operon and all the ato genes have been cloned as a single 6.2 kbp restriction fragment (kindly provided by Dr. Lauren Sallus Jenkins). We have isolated a series of mutant E. coli strains with altered regulatory properties that are either inducible by an alternate substrate, or that show constitutive expression of the atoDAB genes. The -10 and -35 regions upstream of the atoDAB operon poorly match consensus sequences. In addition, the transcriptional start is preceded by a catabolite activator protein binding site (CAP site), as well as a putative binding site for the atoC gene product as represented by a region of dyad symmetry.

The development of tumor drug resistance is the major obstacle to successful systemic chemotherapy. Therefore, devising methods for reversing drug resistance is a high priority and could lead to significant improvements in cancer treatment. The mechanisms of tumor drug resistance are manifold and are not well understood. The phenomenon of multidrug resistance (MDR) represents the development of resistance to most drugs, regardless of their chemical structure. Several types of MDR are known, for example, the overexpression of a cell membrane glycoprotein (P-170), increased activity of glutathione S-transferase, elevated levels of glutathione (GSH), and alterations in topoisomerase action. A partial reversal of tumor drug resistance has been achieved by the use of competitive inhibitors for the function of glycoprotein P-170, or by the inhibition of GSH synthesis; however, this strategy has not been substantially successful for improving the response of human tumors to clinical therapy. We have recently used electroporation, in conjunction with the cytotoxic drug, cisplatin (cDDP), in an attempt to circumvent drug resistance in cDDP-resistant mouse tumor cells (RIF/Ptr1). Electroporation is the application of a high-voltage electric shock which is known to create transient pores in plasma membranes of cultured cells. Electroporation plus cDDP treatment increased intracellular cDDP concentration and reversed cellular resistance to cDDP-induced cell killing.

We have purified and characterized a novel exoribonuclease that was isolated from the mitochondria of Saccharomyces cerevisiae. The enzyme degraded RNA in a 3' to 5' direction and was dependent on nucleotide triphosphates for activity. All eight of the standard ribo- and deoxyribonucleotide triphosphates supported activity with an apparent Km ranging from 20 to 90 uM. The enzyme also exhibited an RNA-dependent ATPase activity. Evidence suggests that in vivo the enzyme may associate with mitochondrial factors which can alleviate the dependence on nucleotide triphosphates for enzymatic activity. A model is discussed for the role of the enzyme in regulating the turnover of mitochondrial RNAs.