Aurintricarboxilic acid as a tool for investigating the template-bound and unbound forms of RNA polymerase I in permeabilized cells

Several polypeptide hormones of apparently diverse structure and function have a number of similarities which suggest that there may be common features in their mechanism of action. These hormones are all composed of a single linear sequence of about 30 amino acids; their hydrophobic amino acids are regularly spaced at every third or fourth amino acid residue, allowing them to form amphipathic structures which can interact with phospholipids; a fragment at or near their N-terminus is required for biological activity. These hormones include glucagon, beta-endorphin, parathyroid hormone and calcitonin. A model is proposed in which all regions of the hormone bind to the receptor with comparable affinity except for a small segment which, when intact, triggers a conformational change in the receptor resulting in a further stabilization of the hormone-receptor complex. The activity of partial sequences and chemically modified forms of beta-endorphin, parathyroid hormone and glucagon are discussed in relation to this model.     

Hydrogen fluoride saccharification of cellulose and xylan: Isolation of α-d-glucopyranosyl fluoride and α-d-xylopyranosyl fluoride intermed

Termination of the hydrogen fluoride solvolysis of cellulose and of xylan by instantaneous neu- tralization enabled the identification of the solvolysi     

Metabolic regulation of apical sodium permeability in toad urinary bladder in the presence and absence of aldosterone

In the present study, further evidence was adduced for energy-dependent regulation of passive apical transport of Na in toad bladder epithelium. In potassium-depolarized preparations studied by current-voltage analysis, additions of pyruvate or glucose to the media of substrate-depleted bladders evoked proportionate increases in the transepithelial Na current and in apical Na permeability. These responses were large in aldosterone pretreated hemibladders and almost absent in the aldosterone-depleted preparations or when hormonal action was blocked by spironolactone or cycloheximide. The substrate-induced increases in apical Na permeability were fully reversed by appropriate metabolic inhibitors, i.e. 2-deoxyglucose and oxythiamine. Moreover, the inhibitory effect of 2-deoxyglucose was bypassed by the addition of pyruvate to the serosal medium. Thus apical Na permeability is clearly sensitive to the supply of cellular energy. The possibility that changes in intracellular free Na activity may mediate metabolic regulation of apical Na permeability was evaluated by prolonged exposure to Na-free mucosal and serosal media, with and without inhibition of the Na/K-pump by ouabain. The stimulatory and inhibitory effects of pyruvate, 2-deoxyglucose and oxythiamine on Na currents and Na conductances were preserved under these circumstances. Furthermore, reduction of serosal Ca to a minimal level of 3 microM, was without effect on the response to metabolic inhibition. These experiments demonstrate the existence of Na-independent metabolic regulation of apical Na transport and imply that neither basal-lateral nor mitochondrial Na/Ca exchange is required for this regulatory process under the imposed conditions. The possibility that a Na-independent, Ca transport mechanism in mitochondria or endoplasmic reticulum may be involved in metabolic regulation of apical Na transport, however, remains to be evaluated.     

Tissue-specific processing and polarized compartmentalization of clone-produced cholinesterase in microinjectedXenopus oocytes

1. To approach the involvement of tissue-specific elements in the compartmentalization of ubiquitous polymorphic proteins, immunohistochemical methods were used to analyze the localization of butyrylcholinesterase (BuChE) in Xenopus oocytes microinjected with synthetic BuChEmRNA alone and in combination with tissue-extracted mRNAs. 2. When injected alone BuChEmRNA efficiently directed the synthesis of small membrane-associated accumulations localized principally on the external surface of the oocyte's animal pole. Tunicamycin blocked the appearance of such accumulations, suggesting that glycosylation is involved in the transport of nascent BuChE molecules to the oocyte's surface. Coinjection with brain or muscle mRNA, but not liver mRNA, facilitated the formation of pronounced, tissue-characteristic BuChE aggregates. 3. These findings implicate tissue-specific mRNAs in the assembly of the clone-produced protein and in its nonuniform distribution in the oocyte membrane or extracellular material.     

Block in the elongation of protein synthesis in rabbit reticulocyte by action of the ionophore Valinomycin

In this paper we show that ribosomes isolated from cells inhibited for protein synthesis by the antibiotic ionophore Valinomycin can still support incorporation of [¹⁴C]-leucine into polypeptides in a cell-free system. The extent of this functional integrity depends upon the concentration of Valinomycin used and whether it acted as an ionophore or not. We demonstrate that the antibiotic acts on protein synthesis at the level of elongation and has no action either at the initiation level or as activating an RNase. Moreover, we show that it can inhibit protein synthesis at concentrations where its action as an ionophore cannot be detected.     

Antagonism of the B subunit of DNA gyrase eliminates plasmids pBR322 and pMG110 from Escherichia coli.

The constructed plasmid pBR322 and the native plasmid pMG110 were eliminated (cured) from growing Escherichia coli cells by the antagonism of the B subunit of the bacterial enzyme DNA gyrase. The antagonism may be by the growth of cells (i) at semipermissive temperatures in a bacterial mutant containing a thermolabile gyrase B subunit or (ii) at semipermissive concentrations of coumermycin A1, an antibiotic that specifically inhibits the B subunit of DNA gyrase. The kinetics of plasmid elimination indicate that plasmid loss occurs too rapidly to be explained solely by the faster growth of that plasmid-free bacteria and, therefore, represents interference with plasmid maintenance.     

Superoxide dismutase: A possible protective agent against sunscald in tomatoes (lycopersicon esculentum mill.)

Superoxide dismutase (SOD, EC 1.15.1.1) was concentrated from mature-green tomato fruits by gel chromatography. The enzyme was inhibited by cyanide but not by chloroform-ethanol, and appears to contain zinc and lesser amounts of copper. SOD-activity levels were high in immature green fruits, declined to a minimum in the mature-green and breaker stages known to be most susceptible to sunscald damage, increased again until the fruits were pink, and finally decreased through the red-ripe and overripe stages to the level of the mature-green fruit. When tolerance to sunscald damage was induced in mature-green fruits by controlled temperature treatment and samples of the fruits were challenged at various times during this process with a combined heat-and-light treatment known to cause sunscald, SOD activity was found to be inversely related to the susceptibility of the fruit to sunscald damage. It is suggested that superoxide is involved in sunscald injury to tomatoes and that tolerance is acquired through increases in SOD activity. Possibly SOD acts as a general protective agent against photodynamic damage to green tissues in plants that have become conditioned as the result of normal diurnal temperature fluctuations.