An improved purification procedure and properties of casein kinase II from brain

A simple and short purification procedure applicable to casein kinase II has been developed, for fully characterizing the enzyme from calf cerebral cortex cytosol. The procedure consists of four chromatographic steps: DEAE-cellulose, phosphocellulose, phosvitin-Sepharose and ATP-agarose which yields 87% pure casein kinase II. The purified enzyme shows three major bands with apparent molecular masses of 42, 38, and 27 kDa by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and is self-autophosphorylated on its 27 kDa polypeptide. The enzyme shows all the characteristics described for casein kinase II from other sources: it is independent of cyclic nucleotides, calcium/phospholipids, and double-stranded poly(I).poly(C); it can utilize both ATP and GTP as phosphoryl donors and can phosphorylate both casein and phosvitin but not histone. The kinetic studies establish that theK _m for ATP is 12.5 M and 25.1 M when using phosvitin and casein respectively as phosphoryl acceptors. TheK _m for phosvitin is 0.91 mg/ml and for casein 1.43 mg/ml, while theV _max is 315 nmol/min/per mg protein and 479 nmol/min/per mg protein for phosvitin and casein respectively. The activity of the kinase is highly stimulated by KCl or NaCl, and almost completely inhibited by heparin concentrations of 1 g/ml (92%). This inhibition is reduced to only 33% in the presence of optimal KCl concentrations (150 mM). Spermine stimulates enzyme activity, whilst hemin produces a slight inhibition.     

Water Balance in Cucumis Plants, Measured by Nuclear Magnetic Resonance, I

In this paper we demonstrate the study of plant water balanceby the non-invasive measurement of tissue water content andwater flow using proton nuclear magnetic resonance (NMR). Sapvelocity and flux were measured independently in the presenceof an excess of stationary tissue water. The instrumentationdescribed allows automated and unattended measurement of flow-and water content-variables in a well-defined region of theplant over periods of several days, with a time resolution betweensuccessive measurements of c. 5 s. Using this apparatus theeffect of changes in light intensity (day/night rhythm) andrelative humidity on stem tissue water content as well as onthe velocity and flux of xylem sap in the stem were investigatedin a cucumber plant. The results are in agreement with predictionsfrom a simple model for plant water balance, which is basedon water potential, flow rate and resistance to flow. As longas only transpiration is varied, flow rate and water content(or potential) are affected in opposite ways as demonstratedin this paper. In contrast, the model predicts that changesin uptake (resulting from changes in, for example, root resistance)will induce changes in water content and flow in the same direction.An experimental verification of this prediction is given ina subsequent paper, where, in addition, the NMR results arecompared to those obtained with a dendrometer. Key words: Water balance model, Cucumis sativus L., flow, water content, NMR, water balance measurement     

Chromosome-mediated iron uptake system in pathogenic strains of Vibrio anguillarum.

We describe in this work a new iron uptake system encoded by chromosomal genes in pathogenic strains of Vibrio anguillarum. This iron uptake system differs from the plasmid-encoded anguibactin-mediated system present in certain strains of V. anguillarum in several properties. The siderophore anguibactin is not utilized as an external siderophore, and although characteristic outer membrane proteins are synthesized under iron-limiting conditions, these are not related to the plasmid-mediated outer membrane protein OM2 associated with ferric anguibactin transport. Furthermore, the siderophore produced by the plasmidless strains may be functionally related to enterobactin as demonstrated by bioassays with enterobactin-deficient mutants, although its behavior under various chemical treatments suggested major differences from that siderophore. Hybridization experiments suggested that the V. anguillarum chromosome-mediated iron uptake system is unrelated genetically to either the anguibactin or enterobactin-associated iron assimilation systems.     

Functional heterogeneity of GEF-free initiation factor 2 purified from suckling and adult rat brain

The functional behavior of initiation factor 2 was studied in purified preparations from the brains of suckling (4-12-day-old) and adult (60-day-old) rats. Adult eIF2 has lower GDP and GTP affinity than suckling eIF2, even in the presence of a large excess of GTP, whereas suckling eIF2 has a lower capacity to bind GTP. Since these two factors are free of guanine nucleotide exchange factor (GEF), and ribosomal fractions show an age-dependent difference in GEF activity, the observed functional heterogeneity may be due to a different ratio in eIF2 species (eIF2-GDP, eIF2(P)).

Developing brain Protein synthesis Initiation factor 2 Guanine nucleotide exchange factor     

Epitopes recognized by the neutralizing antibodies of an HIV-1-infected individual.

Sera from individuals infected by HIV-1 usually neutralize multiple viral isolates. To determine the extent to which these neutralizing antibodies recognize a principal neutralizing determinant in the V3 region of the envelope protein gp120 (amino acids 308-332), one broadly neutralizing serum was fractionated by affinity chromatography on immobilized peptide columns. Antibodies that neutralize one isolate (HTLV-IIIMN) were substantially but not completely absorbed by the peptide corresponding to a portion of its V3 determinant, whereas the antibodies that neutralize two other isolates (HTLV-IIIB and HTLV-IIIRF) were not absorbed by homologous peptides corresponding to their neutralizing determinants. Neutralizing antibodies also failed to be absorbed by full length envelope protein gp160 and by two other envelope peptides previously reported to be broadly neutralizing epitopes (amino acids 254-274 and 735-752). We conclude that the infected individual had raised a type-restricted neutralizing response targeted at a linear epitope in the V3 region, and that broad neutralization resulted from recognition of epitopes not yet identified.     

Leishmania infantum Nicolle, 1908 from southern Spain: Characterisation of the strains from human visceral and cutaneous leishmaniasis and from sandflies; with a numerical analysis of the isoenzymatic data

This study presents the results of the characterisation of 17 strains ofLeishmania by isoenzyme electrophoresis from a focus of leishmaniasis in southern Spain: two from human visceral leishmaniasis, four from human cutaneous leishmaniasis and 11 from sandflies. The 17 strains are grouped in 6 zymodemes characterised by their variability as regards to the electrophoretic mobility of the enzymes MDH, G6PD, NP and ME. Thus, we confirm the high intraspecific variability ofLeishmania (L.) infantum in a focus of southern Spain, as already suggested by previous studies. Zymodemes GR-15 and GR-17 are also described for the first time in Spain, and they characteristically possess the same relative electrophoretic mobility in the enzyme ME (93). Sixteen zymodemes of theL. infantum complex found in southern Spain were numerically analysed on the basis of the enzymatic profiles of 122Leishmania strains characterised from this area.     

TASTE GROUP THRESHOLDS AND SENSORY EVALUATION OF SPANISH WINE VINEGARS

Wine vinegar is a product obtained from wine acidification which contains at least 5% by wt. of acetic acid, in general without any additives or colorings.
Aspects studied in this work include: the determination of the taste group thresholds (geometric mean of the individual best-estimate thresholds "BET") of two different acids (citric and acetic acids) in aqueous solution and spanish vinegars produced from table and sherry wines. The results obtained suggest that wine vinegar can be considered something more than just an acidulant agent.
In order to evaluate differences among wine vinegars, discriminant tests for twenty-five spanish vinegars (sherry, table and flavored vinegars) were applied. Six of the twelve attributes freely chosen by assessors allowed grouping of the spanish wine vinegars according to their sensory aspects.     

Two related low-temperature-inducible genes of Arabidopsis encode proteins showing high homology to 14-3-3 proteins,a family of putative kinase regulators

We have isolated two Rare Cold-Inducible (RCI1 and RCI2) cDNAs by screening a cDNA library prepared from cold-acclimated etiolated seedlings of Arabidopsis thaliana with a subtracted probe. RNA-blot hybridizations revealed that the expression of both RCI1 and RCI2 genes is induced by low temperature independently of the plant organ or the developmental stage considered. However, RCI1 mRNA accumulates faster and at higher levels than the RCI2 one indicating that these genes have differential responsiveness to cold stress. Additionally, when plants are returned to room temperature, RCI1 mRNA decreases faster than RCI2. In contrast to most of the cold-inducible plant genes characterized, the expression of RCI1 and RCI2 is not induced by ABA or water stress. The nucleotide sequences of RCI1 and RCI2 cDNAs predict two acidic polypeptides of 255 and 251 amino acids with molecular weights of 29 and 28 kDa respectively. The alignment of these polypeptides indicates that they have 181 identical amino acids suggesting that the corresponding genes have a common origin. Sequence comparisons reveal no similarities between the RCI proteins and any other cold-regulated plant protein so far described. Instead, they demonstrate that the RCI proteins are highly homologous to a family of proteins, known as 14-3-3 proteins, which are thought to be involved in the regulation of multifunctional protein kinases.