Study of the arrangement of the functional domains along the yeast cytoplasmic aspartyl-tRNA synthetase

Aspartyl-tRNA synthetase from yeast (AspRS) was screened for functional domains by measuring the effect of two types of amino acid mutations on its catalytic properties: (a) insertion of a dipeptide or a tetrapeptide along the polypeptide chain, (b) deletion of various lengths from the enzyme C-terminal. It was shown that insertion mutations significantly affect the kinetic properties of AspRS only when occurring in the second quarter of the molecule and the two centrally located mutations even inactivate the enzyme completely. Analysis of kinetic data strongly suggests that, in fact, all the observed activity modifications result from alteration of the activation reaction rate constant, kappa cat only. This led to the conclusion that the domain involved in aspartic acid activation should be located in the second quarter of the molecule. Furthermore, a deletion mutant with a modification of the last five amino acid residues was isolated. This mutant is fully active in the activation step, but has lost 80% of the wild-type aminoacylation activity. This involvement of the C-terminus in acylation implies that it has to be folded towards strategic regions of the enzyme, thus favouring conformations required for catalysis or maintaining the tRNA in a functional position.     

Glycoconjugate pattern of membranes in the acinar cell of the rat pancreas

Summary Lectin-binding studies were performed at the ultrastructural level to characterize glycoconjugate patterns on membrane systems in pancreatic acinar cells of the rat. Five lectins reacting with different sugar moieties were applied to ultrathin frozen sections: concanavalin A (ConA): glucose, mannose; wheat-germ agglutinin (WGA): N-acetylglucosamine, sialic acid; Ricinus communis agglutinin I (RCA I): galactose; Ulex europaeus agglutinin I (UEA I): l-fucose; soybean agglutinin (SBA): N-acetylgalactosamine). Binding sites of lectins were visualized either by direct conjugation to colloidal gold or by the use of a three-step procedure involving additional immune reactions. The rough endoplasmic reticulum and the nuclear envelope of acinar cells was selectively labelled for ConA. The membranes of the Golgi apparatus bound all lectins applied with an increasing intensity proceeding from the cis-to the trans-Golgi area for SBA, UEA I and WGA. In contrast RCA I selectively labelled the trans-Golgi cisternae. The membranes of condensing vacuoles and zymogen granules were labelled for all lectins used although the density of the label differed between the lectins. In contrast the content of zymogen granules failed to bind SBA and WGA. Lysosomal bodies (membranes and content) revealed binding sites for all lectins used. The plasma membranes were heavily labelled by all lectins except for SBA which showed only a weak binding to the lateral and the apical plasma membrane. These results are in accordance to current biochemical knowledge of the successive steps in the glycosylation of membrane proteins. It could be demonstrated, that the cryo-section technique is suitable for the fine structural localisation of surface glycoconjugates of plasma membranes and internal membranes in pancreatic acinar cells using plant lectins.     

Degradation of synthetic lignins and some lignin monomers by the yeastRhodotorula glutinis

Rhodotorula glutinis degraded variously¹⁴C-labelled synthetic lignins in the presence of 0.1% glucose as co-substrate. Side chain-labelled DHP was degraded the most. While this yeastutilized vanillate and forulate for growth, sinapate/syringate were poorly or not degraded. Gallate, protocatechuate and acetate also supported growth. [carboxy-¹⁴C]Syringate was rapidly converted to¹⁴CO₂ by the yeast only in presence of glucose while [carboxy-¹⁴C]vanillate did not require any additional cosubstrate for mineralization. Ring-labelled vanillyl alcohol was also dagraded proving that the yeast could rapidly metabolize guaiacyl structures while syringyl structures required the presence of additional energy sources.

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Résumé Rhodotorula glutinis dégrade des lignines synthétiques marquées au¹⁴C de manière variée, en présence de 0.1% de glucose comme co-substrat. La DHP marquée sur la chaîne latérale est dégradée le plus. Alors que cette levure utilise le vanillate et le férulate pour sa croissance, le sinapate et le syringate sont peu ou prou dégradables. Le gallate, le protocatéchuate et l'acétate supportent également la croissance. Le syringate marqué au¹⁴C dans sa fonctioncarboxyle estrapidement convert en¹⁴CO₂ par le levure mais exclusivement en présence de glucose, tandis que le vanillate marqué au¹⁴C dans sa fonction carboxyle ne requiert aucun co-substrat additionel pour sa minéralisation. L'alcool vanillique marqué dans son cycle, est également dégradé démonstrant ainsi que la levure peut métaboliser rapidement des structures guaiacyliques tandis que les structures syringiques requièrent la présence de sources auxiliaires d'énergie.

     

Scopolin, a Biochemical Marker for Resistance to Thielaviopsis basicola in Callus and Crown-Gall Tissue Cultures of Tobacco

Scopolin concentration increased in tissue cultures where Thielaviopsis basicola growth ceased (primary and established callus cultures of the resistant tobacco cultivar Ky 170) while it decreased in tissue cultures where fungal growth persisted (primary and established callus cultures of the susceptible tobacco cultivar Ky 151 and crown-gall cultures of Ky 170 and Ky 151). The concentration of chlorogenic acid, scopoletin, and two other unknown soluble phenols varied after inoculation and no, correlation with tissue culture resistance could be established. Incorporation of Agrobacterium tumefaciens T-DNA in the genome of both cultivars induced a drastic reduction of scopolin in inoculated tissue cultures. T-DNA incorporation had less, influence on uninoculated tissues. Scopolin at concentrations found in tissue cultures was not toxic to the fungus.     

Resistance of Tobacco to Thielaviopsis basicola under Tissue Culture Conditions and Increased Susceptibility after Transformation with Agrobacterium tumefaciens T-DNA

Tobacco callus cultures from a Thielaviopsis basicola (Berk. et Br.) Ferraris resistant cultivar were less severely colonized than callus cultures from susceptible cultivars by the pathogen at all concentrations of kinetin and α-indoleacetic acid tested. However, at concentrations where these substances influenced the morphology of the callus cultures they also influenced the degree of colonization by the fungus. Incorporation of T-DNA of Agrobacterium tumefaciens induced susceptibility in tissue culture of the resistant cultivar but did not significantly modify the reaction of the susceptible cultivar tested.     

The diagnostic accuracy of sputum and urine cytology

The diagnostic accuracies of sputum and urinary cytology were examined in series spanning more than 20 years. The sensitivity and respiratory tract cytology in 1,289 patients with subsequently proven lung cancer was 69% while that or urine cytology in 860 patients with urinary tract cancer was 77%. The specificities were 96% for lung cancer and 97% for urinary tract cancer. Neither procedure was widely used for routine screening, but diagnostic cytology played an important part in providing a definite morphologic diagnosis in many of these cases. Urinary cytology was also very effective in the follow-up of patients with treated bladder cancer because of its high sensitivity for detecting carcinoma in situ.     

Regulation of bile acid synthesis via direct effects on the microsomal membrane

Rats treated with ethinylestradiol (5 mg kg-1 day-1 for 5 days) secrete de novo synthesized bile acids at a markedly reduced rate (-57%). Administration of the nonionic detergent Triton WR-1339 to estradiol-treated rats rapidly restored the rate of secretion of de novo synthesized bile acids to control levels. In contrast, when Triton was administered to control rats, the secretion rate of bile acids was unaffected. The reduction in bile acid synthesis displayed by estradiol-treated rats was similar to the 50% decrease in the activity of hepatic microsomal 7 alpha-hydroxylase. The activity of 7 alpha-hydroxylase was also restored to control levels by the administration of Triton to estradiol-treated rats. We examined the possibility that estradiol acts directly on the hepatic microsomes. Adding increasing amounts of estradiol to microsomes obtained from control rats resulted in decreasing activities of 7 alpha-hydroxylase. The inhibition by estradiol of 7 alpha-hydroxylase obtained in vitro occurred with amounts of estradiol that were found to accumulate in the liver via in vivo treatment. Double-reciprocal analysis showed that at and below 50 micrograms of estradiol/0.5 mg of protein uncompetitive inhibition was displayed. Additional experiments showed that adding Triton to microsomes obtained from estradiol-treated rats increased the activity of 7 alpha-hydroxylase to control levels. In contrast, Triton did not increase the activity of 7 alpha-hydroxylase when it was added to control microsomes. These data show for the first time that the estrogenic steroid estradiol acts directly on the microsomes and inhibits both the activity of 7 alpha-hydroxylase and the rate of bile acid synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of peptidoleukotriene-induced increases in pulmonary transvascular fluid filtration

We examined the effects of leukotrienes C4 (LTC4) and D4 (LTD4) (1 microgram) on the pulmonary vascular filtration coefficient, a measure of vessel wall conductivity to water, and the alterations in pulmonary vascular resistance (PVR) in isolated-perfused guinea pig lungs. We also assessed whether LTC4 and LTD4 increased the permeability to albumin in cultured monolayers of pulmonary artery endothelial cells. In Ringer-perfused and blood-perfused lungs, LTC4 resulted in increases in pulmonary arterial pressure (Ppa) and the pulmonary capillary pressure (Pcap) measured as the equilibration pressure after simultaneous pulmonary arterial and venous occlusions. Pulmonary venous resistance (Rv) increased to a greater extent than arterial resistance (Ra) in both Ringer-perfused and blood-perused lungs challenged with LTC4. The greater increase in PVR in blood-perfused lungs corresponded with a greater elevation of lung effluent thromboxane B2 (TxB2) concentration. The LTC4-stimulated increase in PVR was prevented by pretreatment with meclofenamate (10(-4) M). LTD4 also induced rapid increases in Ppa and Pcap in both Ringer-perfused and blood-perfused lungs; however, Ppa decreased before stabilizing at a pressure higher than base line. The increases in Rv with LTD4 were greater than Ra. The LTD4-stimulated increases in Ra and Rv also paralleled the elevation in TxB2 concentration. As with LTC4, the increases in Ppa, Pcap, PVR, and TxB2 concentration were greater in blood-perfused than in Ringer-perfused lungs. Pretreatment with meclofenamate reduced the magnitude of the initial increase in Ppa, but did not prevent the response.(ABSTRACT TRUNCATED AT 250 WORDS)