Maintenance of low cl concentrations in mesophyll cells of leaf blades of barley seedlings exposed to salt stress

The concentrations of vacuolar Na⁺ and Cl⁻ in the epidermal and mesophyll cells of the leaf blade and sheath of Hordeum vulgare seedlings (cv California Mariout and Clipper) were measured by means of quantitative electron probe x-ray microanalysis. A preferential accumulation of Cl⁻ in vacuoles of epidermal cells in both blade and sheath and a low level in mesophyll cells of the blade were evident in plants grown in full strength Johnson solution. The concentration of Cl⁻ in the mesophyll cells of the blade remained at a low level after exposure to 50 or 100 millimolar NaCl for 1 day or to 50 millimolar for 4 days, while at the same time the concentration of Cl⁻ in the epidermis and mesophyll of the sheath showed a dramatic increase. Clipper generally contained more Cl⁻ in the mesophyll cells of the blade than California Mariout. A greater accumulation of Na⁺ in the mesophyll of the sheath relative to that of the blade was only apparent after treatment with 100 millimolar NaCl for 1 day or 50 millimolar for 4 days. These results confirm the suggestion that sheath tissue is capable of accumulating excess Cl⁻ (and to a lesser extent Na⁺) and suggest that the site of regulation of Cl⁻ concentration in the barley leaf is located in the mesophyll cells of the blade.     

Lysophosphatidate acyltransferase activities in the microsomes from palm endosperm, maize scutellum, and rapeseed cotyledon of maturing seeds

Lysophosphatidate (LPA) acyltransferase (EC 2.3.1.51) in the microsomes from palm endosperm (Syagrus cocoides Martius), maize scutellum (Zea mays L.), and rapeseed cotyledon (Brassica napus L.) of maturing seeds were studied for their specificities toward the acyl moiety of the substrates lysophosphatidate and acyl coenzyme A (CoA). The LPA acceptor greatly influenced the acyl CoA specificity of the enzyme and vice versa. With 1-oleoyl-lysophosphatidate (LPA-18:1), the palm enzyme was equally active on oleoyl CoA and lauroyl CoA, whereas the maize and rapeseed enzymes were more active on oleoyl CoA than on lauroyl CoA. With 1-lauroyl-lysophosphatidate (LPA-12), which generated less activity than LPA-18:1, the palm enzyme was three times more active on lauroyl CoA than on oleoyl CoA. LPA-12 was an inactive substrate for the maize and rapeseed enzymes. The selectivity of the enzymes was also studied using a mixture of LPA-18:1 and LPA-12, as well as lauroyl CoA and oleoyl CoA. Under this selectivity condition and compared to the specificity condition, the enzymes from all the three seeds exerted stronger preference for oleoyl moiety in either the LPA or acyl CoA, and again, only the palm enzyme could act on LPA-12. Similar studies, although in lesser detail, showed that the enzymes from soybean and castor bean were similar to the maize and rapeseed enzymes in having little activity on substrates containing lauroyl moiety. The results demonstrate the importance of the acyl group in the sn-1 position of LPA in determining the acyl preference in the sn-2 position in phosphatidate synthesis. The palm enzyme appears to be the only one capable of synthesizing phosphatidates containing high amounts of lauric moieties.     

Two structural genes on different chromosomes are required for encoding the major subunit of human red cell glucose-6-phosphate dehydrogenase

Structural analysis revealed the existence of two types of subunits in human red cell glucose-6-phosphate dehydrogenase. The two subunits have the same COOH region consisting of 479 amino acid residues, but their NH2-terminal regions are different in size and sequence. The minor subunit can be fully encoded by the X-linked G6PD cDNA, but the NH2-terminal region of the major subunit cannot. The cDNA and the gene for the NH2-terminal region of the major subunit were cloned and characterized. Southern blot hybridization indicated that the gene for the NH2-terminal region is on chromosome 6, not on the X chromosome. Northern blot hybridization demonstrated an existence of two separate mRNA components, one for the COOH-terminal region and the other for the NH2-terminal region. Two separate structural genes, the X-linked and chromosome 6-linked genes, must be coresponsible for encoding the single chain subunit. Either cross-translation of two mRNAs, or transpeptidation, or some other mechanism must be involved in the synthesis of human red cell G6PD.     

Consequences of terbium (111) binding on the conformation and enzymatic activity of Guinea pig liver transglutaminase

Calcium ions are crucial for expression of transglutaminase activity. Although lanthanides have been reported to substitute for calcium in a variety of protein functions, they did not replace the calcium requirement during transglutaminase activity measurements. Furthermore, lanthanides strongly inhibited purified liver transglutaminase activity using either casein or fibrinogen as substrates. Terbium (III) inhibition of transglutaminase-catalyzed putrescine incorporation into casein was not reversed by the presence of 10–200 fold molar excess of calcium ions (Ki for Tb(III)=60 µM). Conformational changes in purified liver transglutaminase upon Tb(III) binding were evident from a biphasic effect of Tb(III) on transglutaminase binding to fibrin. Low concentrations of Tb(III) (1 µM to 10 µM inhibited the binding of transglutaminase to fibrin, whereas higher concentrations (20 µM to 100 µM promoted binding. Conformational changes in purified liver transglutaminase consequent to Tb(III) binding were also demonstrated by fluorescence spectroscopy due to Forster energy transfer. Fluorescence emission was stable to the presence of 200 mM NaCl and 100 mM CaCl₂ only partially quenched emission. Purified liver transglutaminase strongly bound to Tb(III)-Chelating Sepharose beads and binding could not be disrupted by 100 mM CaCl₂ solution. Our data suggest that Tb(III)-induced conformational changes in transglutaminase are responsible for the observed effects on enzyme structure and function. The potential applications of Tb(III)-transglutaminase interactions in elucidating the structure-function relationships of liver transglutaminase are discussed.     

Transformation ofAspergillus flavus to study aflatoxin biosynthesis

Aflatoxin contamination of agricultural commodities continues to be a serious problem in the United States. Breeding for resistant genotypes has been unsuccessful and detoxification of food sources is not economically feasible. New strategies for control may become apparent once more is known about the biosynthesis and regulation of aflatoxin. Although the biosynthetic pathway of aflatoxin has been extensively studied, little is known about the regulation of the individual steps in the pathway. We have developed a genetic transformation system forAspergillus flavus that provides a new and expedient approach to studying the biosynthesis of aflatoxin and its regulation. Through the use of this genetic transformation system, genes for aflatoxin biosynthesis can be identified and isolated by the complementation of aflatoxin negative mutants. In this paper we discuss molecular strategies for studying the regulation and biosynthesis of aflatoxin.     

Mn2+ reduces Yz + in manganese-depleted Photosystem II preparations

Manganese in the oxygen-evolving complex is a physiological electron donor to Photosystem II. PS II depleted of manganese may oxidize exogenous reductants including benzidine and Mn²⁺. Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Y_z ⁺, the tyrosine radical formed in PS II membranes under illumination. Kinetics were measured with membranes that did or did not contain the 33 kDa extrinsic polypeptide of PS II, whose presence had no effect on the reaction kinetics with either reductant. The rate of Y_z ⁺ reduction by benzidine was a linear function of benzidine concentration. The rate of Y_z ⁺ reduction by Mn²⁺ at pH 6 increased linearly at low Mn²⁺ concentrations and reached a maximum at the Mn²⁺ concentrations equal to several times the reaction center concentration. The rate was inhibited by K⁺, Ca²⁺ and Mg²⁺. These data are described by a model in which negative charge on the membrane causes a local increase in the cation concentration. The rate of Y_z ⁺ reduction at pH 7.5 was biphasic with a fast 400 s phase that suggests binding of Mn²⁺ near Y_z ⁺ at a site that may be one of the native manganese binding sites.Abbreviations PS II Photosystem II - Y_D tyrosine residue in Photosystem II that gives rise to the stable Signal II EPR spectrum - Y_z tyrosine residue in Photosystem II that mediates electron transfer between the reaction center chlorophyll and the site of water oxidation - ESR electron spin resonance - DPC diphenylcarbazide - DCIP dichlorophenolindophenol     

Studies on light absorption and photochemical activity changes in chloroplast suspensions and leaves due to light scattering and light filtration across chloroplast and vegetation layers

An experimental analysis is presented concerning the effect on relative light absorption by the two photosystems caused by (a) a highly light scattering environment (the detour effect) and (b) light filtration across successive chloroplast layers (the light attenuation effect). Both suspensions of isolated chloroplasts and leaves were employed.It is concluded that within a single spinach leaf these phenomena are likely to lead to only rather small increases in relative photosystem I absorption and activity with respect to photosystem II and will thus not exert a significant effect on non cyclic electron transport. On the contrary when light is filtrated across successive vegetation layers (shade light) significant increases in the relative PSI absorption and activity may be encountered.It is determined that the detour effect in mature leaves from a variety of plants increases overall photosynthetically useful light absorption by 35–40%.Abbreviations F_M maximal fluorescence - LHCP₂ light-harvesting chlorophyl a/b protein complex II - Q_A-primary quinone acceptor of photosystem II     

Evidence that vascular actions of PHI are mediated by a VIP-preferring receptor

Vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) are homologous neuropeptides which share vasodilatory properties. This paper addresses the question of whether PHI exerts its vascular action via a receptor distinct from that for VIP. Radioligand binding experiments were done using [Tyr(125I)10]VIP, [Tyr(125I)22]porcine PHI, [Tyr(125I)10]rat PHI and arterial preparations from rat, bovine and porcine species. The radioiodination of rat PHI by the lactoperoxidase-glucose oxidase method and analysis of the structure of the major radiolabeled derivatives were described. All the receptor binding experiments identified a VIP-preferring receptor irrespective of which radioligand or arterial preparation was utilized. VIP and PHI peptides demonstrated cross-desensitization in studies of relaxation of porcine coronary arterial strips in vitro. The present results favor the conclusion that the vascular actions of the PHI peptides are best explained by binding to a VIP-preferring receptor.     

Higher order structure is present in the yeast nucleus: autoantibody probes demonstrate that the nucleolus lies opposite the spindle pole body

A panel of sera from 892 autoimmune patients was screened by indirect immunofluorescence on mammalian cells. Seventy-three sera were identified that recognize the nucleolus. Three of these sera appear to stain the nucleolus in yeast, suggesting that they recognize highly conserved antigens. These three sera also immunoprecipitate mammalian U3 snRNA-containing particles, which reside in the nucleolus and have been implicated in rRNA processing. Double immunofluorescence experiments with anti-nucleolus and anti-tubulin antibodies revealed a novel form of non-random nuclear organization in yeast. The spindle pole body and the nucleolus — both of which are associated with the nuclear envelope — preferentially localize at opposite ends of the nucleus. Organization of these and other components into specific regions of the nucleus may be important for optimizing their proper function.