A C-terminal di-leucine motif and nearby sequences are required for NH4+-induced inactivation and degradation of the general amino acid permease, Gap1p, of Saccharomyces cerevisiae

The general amino acid permease, Gap1, of Saccharomyces cerevisiae is very active in cells grown on proline as the sole nitrogen source. Adding NH₄⁺ to the medium triggers inactivation and degradation of the permease via a regulatory process involving Npi1p/Rsp5p, a ubiquitin–protein ligase. In this study, we describe several mutations affecting the C-terminal region of Gap1p that render the permease resistant to NH₄⁺-induced inactivation. An in vivo isolated mutation ( gap1 ^pgr  ) causes a single Glu→Lys substitution in an amino acid context similar to the DXKSS sequence involved in ubiquitination and endocytosis of the yeast α-factor receptor, Ste2p. Another replacement, substitution of two alanines for a di-leucine motif, likewise protects the Gap1 permease against NH₄⁺-induced inactivation. In mammalian cells, such a motif is involved in the internalization of several cell-surface proteins. These data provide the first indication that a di-leucine motif influences the function of a plasma membrane protein in yeast. Mutagenesis of a putative phosphorylation site upstream from the di-leucine motif altered neither the activity nor the regulation of the permease. In contrast, deletion of the last eleven amino acids of Gap1p, a region conserved in other amino acid permeases, conferred resistance to NH₄⁺ inactivation. Although the C-terminal region of Gap1p plays an important role in nitrogen control of activity, it was not sufficient to confer this regulation to two NH₄⁺-insensitive permeases, namely the arginine (Can1p) and uracil (Fur4p) permeases.     

Structural and biological roles of glycosylations in pulmonary angiotensin I-converting enzyme

We enzymatically deglycosylated pig lung angiotensin I-convertingenzyme (ACE) to study the involvement of its glycanic chainsin its physicochemical and catalytic properties. The effectsof endoglycosidases F₂ and H, and of N-glycanase were assessedby ACE mobility in SDS-PAGE. N-Glycanase only was completelyeffective with or without previous denaturation, leading toa shift in ACE M_r from 172 to 135 kDa; endoglycosidase F₂ producedthe same shift but only without previous denaturation. DeglycosylatedACE had the same kcat as native ACE for the substrate hippuryl-histidyl-leucine,and an identical Stokes radius as measured by size-exclusionhigh performance liquid chromatography. Neuraminidase had noeffect on ACE Stokes radius but slightly decreased its kcatwhich could be related to variations in ionization of the activesite. The isoelectric point of ACE, as, determined by isoelectricfocusing, increased from 4.5–4.8 to 5.0–5.3 aftereither endoglycosidase F₂ or neuraminidase digestion, but stillwith microheterogeneities which thus did not seem to be relatedto ACE glycans. Deglycosylated ACE did not bind onto agaroselectinsin contrast to native ACE which bound strongly to concanavalinA showing interactions involving oligomannosidic or biantennaryand sialylated N-acetyl-lactosaminic isoglycans. Finally, tunicamycin,an inhibitor of N-glycosylation, did not modify ACE secretionby endothelial cells. Thus, ACE glycans have no drastic effectson structural and biological properties of the protein, butthey may have a functional role on intracellular targeting ofboth secreted and membrane-bound ACE isoforms, also for theprotection of the soluble plasma form against hepatic lectinsand the maintenance of its hydrosolubility. converting enzyme (peptidyldipeptidase EC.3.4.15.1) endothelium glycosidases lectins     

A spatial model of interspecific competition and selective predation: The case of the two Hippolais

Mutual exclusion between congeneric species has been observed such as the case of the grey and red squirrels in Great Britain and the case of the twoHippolais warbler speciesHippolais icterina andH. polyglotta in Europe. This process can lead to the formation of an extinction wave which propagates. Two main assumptions are tested, competition and selective predation. The aim of this work is to present spatial models of these two processes. The animals of two species are assumed to move on a two dimensional array of spatial patches with local interactions of competition or of selective predation between them. We focus on the case of mutual exclusion. Initially, the two competing species occupy complementary areas in an array of spatial patches with a small common zone. Numerical simulations show that under particular conditions, one species gets extinct and the other invades the whole set of spatial patches. These simulations show that with time, the length of the overlapping zone stabilizes and moves at a constant velocity. The limit length of the overlapping band and the velocity of the extinction wave are found to be functions of the parameters of the models. We relate this general model to the case of two sibling species of birds:H. icterina andH. polyglotta.     

Functional Morphology of the Introvert and Digestive System of Myzostoma cirriferum (Myzostomida)

Myzostoma cirriferum feeds by diverting food particles carried by the ambulacral grooves of its comatulid host Antedon bifida. When searching for food, the myzostome uses its protrusible introvert to fulfil two major functions: sensory perception and the capture of food particles. The digestive system is composed of four parts, viz. a pharynx, that is contained within the introvert, a stomach, a series of paired caeca and an intestine that lie in the myzostome's trunk. The pharynx is supplied with a thick muscle which, thanks to peristaltic movements, carries food particles from the mouth to the stomach. Both stomach and caecal cells are able to absorb dissolved nutriments and to store lipids, whereas intestinal cells are only capable of absorption. Due to the beating of their cilia, stomach cells also carry food particles into the caecal lumen, where they are subjected to endocytosis and intracellular digestion by caecal cells. Undigested food fragments eventually gather in a very large, apical vacuole, and the cell apices containing vacuoles are eliminated into the caecal lumen by an apocrinal process. Detached cell apices reach the stomach, where they are embedded in a matrix, together forming a spindle-shaped faecal mass that is expelled through the postero-ventral anus. The observed digestive process—entailing the regular elimination of the apical part of the caecal digestive cells—appears to be unique among the Spiralia.     

Time linkages between pollination onsets of different taxa over an 11-year period in Perugia,Central Italy

Times of pollination of different taxa in the atmosphere of Perugia (Central Italy) over an 11-year period (1982–1992) were recorded and analysed by means of a 7-day volumetric Hirst-type pollen trap. For some taxa, the pollination period varied from year to year from a chronological and/or quantitative point of view. Several taxa showed a linkage in their starting dates of pollination. Knowledge of this kind of linkage allows us to build a forecasting model.     

Synthesis and platelet aggregation inhibition activity of a series of enantiomeric bicyclo[3.2.0]heptane-6-oximinoacetic acids (1)

Opening of racemic epoxide (3) with (3S)- or (3R)-dimethyl-3-(dimethyl-t-butylsilyloxy)oct-1-ynyl aluminum gave two regioisomers, which were separated chromatographically. The separated regioisomers, themselves mixtures of chromatographically inseparable diastereoisomers, were converted into their dicobalthexacarbonyl complexes, which were easily resolved and isolated by chromatography. The individual diastereoisomers were deprotected to give bicyclo[3.2.0]heptan-3-ones, whose absolute stereochemistry was assigned using circular dichroism. One of these compounds, (1R,2R,3S,5R,3'S)-3-(3'-hydroxyoct-1'-ynyl)-bicyclo[3.2.0]++ +heptan-2-ol-6- oximinoacetic acid (11a) was 4.5 times more potent than PGE1 in inhibiting the ADP-induced aggregation of human platelets. The next most potent compound in this series was the "ent-15-epi" compound (11b), which was 0.034 times the potency of PGE1 in the platelet aggregation assay.     

A chlorinated phenylpyrrole antibiotic fromActinoplanes

Two chlorophenyl-containing antibiotics have been isolated from a strain ofActinoplanes (ATCC 33002). Antibiotic A 15104 Y is a chlorinated phenylpyrrole compound whose structure has been confirmed by chemical synthesis. Antibiotic A 15104 Z is a chlorophenol derivative for which a structural formula is proposed on the basis of its physicochemical properties. A 15104 Y shows antimicrobial activity against Gram-positive, Gram-negative, and acid-fast bacteria, yeasts, fungi, and protozoa, while A 15104 Z possesses a low activity against Gram-positive bacteria andTrichophyton. A 15104 Y has a weak activity in curing experimentally infected mice, at a dose that is one-fifth the LD₅₀ for the same species. This is the first example of a chloropyrrole derivative isolated from an actinomycete.     

Evaluation by electron microscopy of the integrity of iodinated plasmalipoproteins

Summary Iodination of proteins and lipoproteins is a widely used in vitro labelling procedure in metabolic, autoradiographic and various other studies. However, all available iodination techniques have involved the possible damage to the proteins by self-irradiation, oxidizing agents, the alkaline milieu or by the introduction of iodine into the molecular structure itself. To evaluate the integrity of iodinated lipoproteins, we observed the electron microscopic appearance of normal and iodinated rabbit very low density lipoproteins (VLDL) by negative staining with phosphotungstic acid. Iodination up to a molar iodine/protein ratio of 2.89 did not result in any change of shape, size or aggregating tendency of the particles. No stacks or disk-like particles like those of various hyperlipoproteinemic states were found. We conclude that electron microscopy is a valuable tool in assessing the morphological appearance of lipoprotein iodination, but it should be complemented by other techniques.     

Further studies on bilitranslocase,a plasma membrane protein involved in hepatic organic anion uptake

Bilitranslocase, a plasma membrane protein involved in bilirubin and other organic anion uptake by the liver, exhibits a high molecular weight (170 000) when isolated in the presence of deoxycholate. This value is decreased to approx. 100 000 if deoxycholate is not included in the isolation medium. Both preparations can be resolved into two kinds of subunit, α and β, of 37 000 and 35 500, respectively, by reduction with 2-mercaptoethanol and addition of sodium dodecyl sulfate. Under these conditions the two subunits are still capable of high-affinity sulfobromophthalein binding and, despite the presence of the detergent, may be isolated by preparative polyacrylamide gel electrophoresis still associated with the dye. It may be suggested that the physiological subunit composition of bilitranslocase is α₂-β.     

Lysis protein S of phage lambda functions in Saccharomyces cerevisiae.

The lambda S lysis gene was cloned into a Saccharomyces cerevisiae expression vector under GAL1 control. Induction with galactose in S. cerevisiae terminated cell growth and prevented colony formation. Several membrane proteins immunoreactive with anti-S antibody accumulated in the membranes, indicating that sodium dodecyl sulfate-resistant oligomers of S are formed, similar to those observed in the membranes of Escherichia coli cells killed by expression of the S gene. These observations suggest that the S gene product functions as a cytotoxic protein in the yeast cytoplasmic membrane as it does in the bacterial membrane.